HER3 antibody-drug conjugates and uses thereof

Abstract

The present invention provides anti-Her3 antibody-drug conjugates that specifically bind to Her3 and compositions comprising the same. Methods of using the antibody-drug conjugates of the present invention and uses thereof are also provided.

Description

[Technical field]

[0001] The present invention provides an antibody-drug conjugate that specifically binds to Her3 and a composition comprising the same. Methods of using the antibody-drug conjugate of the present invention and uses thereof are also provided. [Background technology]

[0002] The epidermal growth factor receptor (EGFR) is a large transmembrane glycoprotein with a molecular weight of approximately 170 kDa, and is a member of the ErbB receptor family. The EGFR receptor itself is a tyrosine kinase that forms a dimer after binding to ligands such as EGF and TNF-α, and activates downstream signals (pathways such as MAPK, PI3K, and Stat) through phosphorylation transmission, thereby maintaining cell proliferation and promoting cell division and proliferation. Because ErbB family receptors are conserved, EGFR can also form heterodimers with other proteins of the same family (Her2, Her3, Her4, etc.), thereby controlling cell proliferation more broadly.

[0003] Her3 is a member of the ErbB family and plays an important role in cell proliferation, tumor metastasis and drug resistance. Drugs targeting EGFR and Her2 have shown great clinical benefits in mitigating various cancers, but previous efforts to develop anti-Her3 antibodies for cancer therapy have repeatedly failed, suggesting that Her3-only therapy and its pending approach may not be sufficient to suppress tumor growth.

[0004] Antibody-drug conjugates (ADCs) are composed of three parts: an antibody or its antigen-binding fragment (targeting), a linker, and a small molecule drug. The antibody or its antigen-binding fragment is linked to a physiologically active small molecule drug, such as a cytotoxin having cytotoxicity, via a cleavable or non-cleavable linker. This effectively utilizes the specificity of the antibody or its antigen-binding fragment to a target cell (target cell), or the binding specificity to a highly expressed antigen, and the high efficiency of the small molecule drug, thereby reducing or avoiding the side effects of toxicity to non-target cells. That is, the antibody-drug conjugates for tumors can accurately target tumor cells and reduce the impact on non-tumor cells compared with conventional tumor chemotherapy drugs.

[0005] There remains a need in the art for anti-Her3 antibody-drug conjugates with superior affinity, specificity, and the like. Summary of the Invention

[0006] In one aspect, the present application provides an anti-Her3 antibody-drug conjugate, an isomer thereof, a pharma- ceutically acceptable salt thereof, or a mixture thereof, wherein the anti-Her3 antibody-drug conjugate has the formula (I): Ab-(LMD) p (I) wherein: L and M are linker units; D is a cytotoxic drug; p represents the average number of connections, and p is selected from an integer or decimal number of 1 to 10, preferably an integer or decimal number of 3 to 8; The Ab is an anti-Her3 antibody or an antigen-binding fragment thereof, and comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 of the amino acid sequences shown in SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3, respectively, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 of the amino acid sequences shown in SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6, respectively.

[0007] In some embodiments, the present specification describes an anti-Her3 antibody-drug conjugate, an isomer thereof, a pharma- ceutically acceptable salt thereof, or a mixture thereof, the anti-Her3 antibody-drug conjugate having the formula (I-1):

[0008] [ka]

[0009] [During the ceremony, L and M are linker units; p represents the average number of connections, and n is selected from an integer or decimal number of 1 to 10, preferably an integer or decimal number of 3 to 8; Ab is an anti-Her3 antibody or an antigen-binding fragment thereof, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 of the amino acid sequences shown in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, respectively, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3 of the amino acid sequences shown in SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, respectively. It has the structure:

[0010] In some embodiments, the anti-Her3 antibody or antigen-binding fragment thereof described herein is (I) a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO: 7, or a heavy chain variable region having at least 95%, 96%, 97%, 98% or 99% identity thereto, and a light chain variable region having an amino acid sequence as set forth in SEQ ID NO: 8, or a light chain variable region having at least 95%, 96%, 97%, 98% or 99% identity thereto; or (II) a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO:9, or having at least 95%, 96%, 97%, 98%, or 99% identity thereto, and a light chain variable region having an amino acid sequence as set forth in SEQ ID NO:8, or having at least 95%, 96%, 97%, 98%, or 99% identity thereto; or (III) a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO: 10, or a heavy chain variable region having at least 95%, 96%, 97%, 98% or 99% identity thereto, and a light chain variable region having an amino acid sequence as set forth in SEQ ID NO: 8, or a light chain variable region having at least 95%, 96%, 97%, 98% or 99% identity thereto. Includes.

[0011] In some embodiments, the anti-Her3 antibody or antigen-binding fragment thereof described herein is (I) a heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 7 and a light chain variable region having the amino acid sequence shown in SEQ ID NO: 8; or (II) a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 9 and a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 8; or (III) a heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 10 and a light chain variable region having the amino acid sequence shown in SEQ ID NO: 8 Includes.

[0012] In some embodiments, the anti-Her3 antibody or antigen-binding fragment described herein is a murine antibody or fragment thereof, a chimeric antibody or antigen-binding fragment, a humanized antibody or antigen-binding fragment, or a fully human antibody or antigen-binding fragment.

[0013] In some embodiments, the anti-Her3 antibodies or antigen-binding fragments thereof described herein are humanized antibodies or fragments thereof.

[0014] In some embodiments, the anti-Her3 antibody or antigen-binding fragment thereof described herein is selected from a Fab, Fab', Fab'-SH, Fv, scFv, F(ab')2, sdAb, bispecific antibody, or linear antibody.

[0015] In some embodiments, the anti-Her3 antibodies described herein are monoclonal antibodies.

[0016] In some embodiments, the antibodies described herein are of the IgG1, IgG2, IgG3, or IgG4 type.

[0017] In some embodiments, the antibodies described herein are of the IgG1 type.

[0018] In some embodiments, the anti-Her3 antibodies, or antigen-binding fragments thereof, described herein are (1) a heavy chain having an amino acid sequence as set forth in SEQ ID NO:11, or a heavy chain having at least 95%, 96%, 97%, 98% or 99% identity thereto, and a light chain having an amino acid sequence as set forth in SEQ ID NO:12, or a light chain having at least 95%, 96%, 97%, 98% or 99% identity thereto; or (2) a heavy chain having an amino acid sequence as set forth in SEQ ID NO: 13, or a heavy chain having at least 95%, 96%, 97%, 98% or 99% identity thereto, and a light chain having an amino acid sequence as set forth in SEQ ID NO: 12, or a light chain having at least 95%, 96%, 97%, 98% or 99% identity thereto; or (3) A heavy chain having the amino acid sequence shown in SEQ ID NO: 14, or a heavy chain having at least 95%, 96%, 97%, 98% or 99% identity thereto, and a light chain having the amino acid sequence shown in SEQ ID NO: 12, or a light chain having at least 95%, 96%, 97%, 98% or 99% identity thereto. Includes.

[0019] In some embodiments, the anti-Her3 antibodies, or antigen-binding fragments thereof, described herein are (1) a heavy chain having the amino acid sequence set forth in SEQ ID NO: 11 and a light chain having the amino acid sequence set forth in SEQ ID NO: 12; or (2) a heavy chain having the amino acid sequence set forth in SEQ ID NO: 13 and a light chain having the amino acid sequence set forth in SEQ ID NO: 12; or (3) a heavy chain having the amino acid sequence shown in SEQ ID NO: 14 and a light chain having the amino acid sequence shown in SEQ ID NO: 12 Includes.

[0020] In some embodiments, described herein is an anti-Her3 antibody-drug conjugate, an isomer thereof, a pharma- ceutically acceptable salt thereof, or a mixture thereof, wherein: M is -L 2 -L 3 -XL 1 - and; L 2 is -O- or -S-; L 3 is -(C(R 1a )(R 1b )) m -, m is selected from 0, 1, 2 or 3, where L 3 If it contains a methylene unit, L 3 may be replaced by -C(O)- or -C(S)-; L 1 is -(C(R 2a )(R 2b )) n -, n is selected from 1, 2 or 3, where L 1 may contain methylene units, L 1 may be replaced by -C(O)- or -C(S)-; X is selected from 3- to 6-membered saturated carbocyclyl, 3- to 6-membered saturated heterocyclyl, or a single bond, where 3- to 6-membered saturated carbocyclyl and 3- to 6-membered saturated heterocyclyl are selected from 0, 1, 2, or 3 R 3a optionally replaced by; Here, each R 1a , each R 1b , each R 2a , each R 2b , and each R 3a is independently hydrogen, halogen, or C optionally substituted with R 1-6 may be an aliphatic group, Each R can independently be hydrogen or halogen.

[0021] In some embodiments, the 3-6 membered saturated heterocyclyl described herein contains 1-3 heteroatoms selected from N, O, and S.

[0022] In some embodiments, the L 3 , and X is not a single bond.

[0023] In some embodiments, the L 2 In some embodiments, L 2 is -S-.

[0024] In some embodiments, described herein is an anti-Her3 antibody-drug conjugate, an isomer thereof, a pharma- ceutically acceptable salt thereof, or a mixture thereof, wherein the linker unit M is L 2 is connected at its end to a linker unit L, 1 It is linked to a cytotoxic drug D at its terminus.

[0025] In some embodiments, the present specification describes an anti-Her3 antibody-drug conjugate, an isomer thereof, a pharma- ceutically acceptable salt thereof, or a mixture thereof, wherein L 3 is a single bond, -C(R 1a )(R 1b )- or -C(R 1a )(R 1b )C(R 1a )(R 1b )- is selected from Here, each R 1a and each R 1b are independently hydrogen, halogen, or C optionally substituted with R 1-6 may be an aliphatic group, wherein each R may independently be hydrogen or halogen.

[0026] In some embodiments, the present specification describes an anti-Her3 antibody-drug conjugate, an isomer thereof, a pharma- ceutically acceptable salt thereof, or a mixture thereof, wherein L 3 is a single bond, -C(R 1a )(R 1b )- or -C(R 1a )(R 1b )C(R 1a )(R 1b )- is selected from Here, each R 1a and each R 1b can be independently hydrogen, halogen, CH3, or CH2CH3.

[0027] In some embodiments, the present specification describes an anti-Her3 antibody-drug conjugate, an isomer thereof, a pharma- ceutically acceptable salt thereof, or a mixture thereof, wherein L 3 is selected from -CH2-, -CH(CH3)-, -C(CH3)2-, -CH2CH2-, -CH(CH3)CH2-, or -C(CH3)2CH2-.

[0028] In some embodiments, the present specification describes an anti-Her3 antibody-drug conjugate, an isomer thereof, a pharma- ceutically acceptable salt thereof, or a mixture thereof, wherein L 3 is selected from the following:

[0029] [ka]

[0030] In some embodiments, the present specification describes an anti-Her3 antibody-drug conjugate, an isomer thereof, a pharma- ceutically acceptable salt thereof, or a mixture thereof, 3 is selected from a single bond.

[0031] In some embodiments, the present specification describes an anti-Her3 antibody-drug conjugate, an isomer thereof, a pharma- ceutically acceptable salt thereof, or a mixture thereof, wherein L 1 is -C(R 2a )(R 2b )-, -C(R 2a )(R 2b )C(O)-, or -C(O)-; Here, each R 2a and each R 2b are independently hydrogen, halogen, or C optionally substituted with R 1-6 may be an aliphatic group, where each R can independently be hydrogen or halogen.

[0032] In some embodiments, the present specification describes an anti-Her3 antibody-drug conjugate, an isomer thereof, a pharma- ceutically acceptable salt thereof, or a mixture thereof, wherein L 1 is -C(R2a )(R 2b )-, -C(R 2a )(R 2b )C(O)-, or -C(O)-; Here, each R 2a and each R 2b can be independently hydrogen, halogen, CH3, or CH2CH3.

[0033] In some embodiments, the present specification describes an anti-Her3 antibody-drug conjugate, an isomer thereof, a pharma- ceutically acceptable salt thereof, or a mixture thereof, wherein L 1 is selected from -CH2-, -CH2C(O)-, -CH(CH3)C(O)-, or -C(O)-.

[0034] In some embodiments, the present specification describes an anti-Her3 antibody-drug conjugate, an isomer thereof, a pharma- ceutically acceptable salt thereof, or a mixture thereof, wherein L 1 is selected from -C(O)-.

[0035] In some embodiments, described herein is an anti-Her3 antibody-drug conjugate, an isomer thereof, a pharma- ceutically acceptable salt thereof, or a mixture thereof, wherein X is 0, 1, 2, or 3 R 3a or a single bond, Here, each R 3a are independently hydrogen, halogen, or C optionally substituted with R 1-6 may be an aliphatic group, wherein each R may independently be hydrogen or halogen.

[0036] In some embodiments, described herein is an anti-Her3 antibody-drug conjugate, an isomer thereof, a pharma- ceutically acceptable salt thereof, or a mixture thereof, wherein X is 0, 1, 2, or 3 R 3a or a single bond, Here, each R 3acan be independently hydrogen, halogen, CH3, or CH2CH3.

[0037] In some embodiments, described herein is an anti-Her3 antibody-drug conjugate, an isomer thereof, a pharma- ceutically acceptable salt thereof, or a mixture thereof, wherein X is cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, or a single bond.

[0038] In some embodiments, described herein is an anti-Her3 antibody-drug conjugate, an isomer thereof, a pharma- ceutically acceptable salt thereof, or a mixture thereof, wherein X is a single bond. In some embodiments, described herein is an anti-Her3 antibody-drug conjugate, an isomer thereof, a pharma- ceutically acceptable salt thereof, or a mixture thereof, wherein X is cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl, preferably cyclobutyl.

[0039] In some embodiments, described herein is an anti-Her3 antibody-drug conjugate, an isomer thereof, a pharma- ceutically acceptable salt thereof, or a mixture thereof, wherein X is

[0040] [ka]

[0041] and preferably, X is:

[0042] [ka]

[0043] In some embodiments, described herein is an anti-Her3 antibody-drug conjugate, an isomer thereof, a pharma- ceutically acceptable salt thereof, or a mixture thereof, wherein -M- is:

[0044] [ka]

[0045] where: L 2 is -O- or -S-; X is 0, 1, 2 or 3 R 3a selected from 3-6 membered saturated carbocyclyl optionally substituted with; L 1 is -C(R 2a )(R 2b )-, -C(R 2a )(R 2b )C(O)-, or -C(O)-; Here, each R 2a , each R 2b , or each R 3a are independently hydrogen, halogen, or C optionally substituted with R 1-6 may be an aliphatic group, wherein each R may independently be hydrogen or halogen.

[0046] In some embodiments, described herein is an anti-Her3 antibody-drug conjugate, an isomer thereof, a pharma- ceutically acceptable salt thereof, or a mixture thereof, wherein -M- is:

[0047] [ka]

[0048] where: L 2 is -O- or -S-; X is 0, 1, 2 or 3 R 3a is selected from 3- to 6-membered saturated carbocyclyl optionally substituted with Here, each R 3a are independently hydrogen, halogen, or C optionally substituted with R 1-6 may be an aliphatic group, wherein each R may independently be hydrogen or halogen.

[0049] In some embodiments, described herein is an anti-Her3 antibody-drug conjugate, an isomer thereof, a pharma- ceutically acceptable salt thereof, or a mixture thereof, wherein -M- is:

[0050] [ka]

[0051] where: L 2 is -O- or -S-; X is selected from cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl.

[0052] In some embodiments, described herein is an anti-Her3 antibody-drug conjugate, an isomer thereof, a pharma- ceutically acceptable salt thereof, or a mixture thereof, wherein -M- is:

[0053] [ka]

[0054] where: L 2 is -O- or -S-; X is selected from the following:

[0055] [ka]

[0056] In some embodiments, described herein is an anti-Her3 antibody-drug conjugate, an isomer thereof, a pharma- ceutically acceptable salt thereof, or a mixture thereof, wherein -M- is:

[0057] [ka]

[0058] where: L2 is -O- or -S-; L 3 is -C(R 1a )(R 1b )-or-C(R 1a )(R 1b )C(R 1a )(R 1b )-selected from; L 1 is -C(R 2a )(R 2b )-, -C(R 2a )(R 2b )C(O)-, or -C(O)-; Here, each R 1a , each R 1b , each R 2a , or each R 2b are independently hydrogen, halogen, or C optionally substituted with R 1-6 may be an aliphatic group, wherein each R may independently be hydrogen or halogen.

[0059] In some embodiments, described herein is an anti-Her3 antibody-drug conjugate, an isomer thereof, a pharma- ceutically acceptable salt thereof, or a mixture thereof, wherein -M- is:

[0060] [ka]

[0061] where: L 2 is -O- or -S-; L 3 is -C(R 1a )(R 1b )-or-C(R 1a )(R 1b )C(R 1a )(R 1b )- is selected from Here, each R 1a and each R 1b are independently hydrogen, halogen, or C optionally substituted with R 1-6may be an aliphatic group, wherein each R may independently be hydrogen or halogen. In some embodiments, described herein is an anti-Her3 antibody-drug conjugate, an isomer thereof, a pharma- ceutically acceptable salt thereof, or a mixture thereof, wherein -M- is:

[0062] [ka]

[0063] where: L 2 is -O- or -S-; L 3 is selected from -CH2-, -CH(CH3), -C(CH3)2, -CH2CH2-, -CH(CH3)CH2-, or -C(CH3)2CH2-.

[0064] In some embodiments, described herein is an anti-Her3 antibody-drug conjugate, an isomer thereof, a pharma- ceutically acceptable salt thereof, or a mixture thereof, wherein -M- is:

[0065] [ka]

[0066] where: L 2 is -O- or -S-; L 3 is selected from the following:

[0067] [ka]

[0068] In some embodiments, described herein is an anti-Her3 antibody-drug conjugate, an isomer thereof, a pharma- ceutically acceptable salt thereof, or a mixture thereof, wherein the linker unit M is L 2 It is linked to a linker unit L at its terminus.

[0069] In some embodiments, described herein is an anti-Her3 antibody-drug conjugate, an isomer thereof, a pharma- ceutically acceptable salt thereof, or a mixture thereof, wherein -M- is selected from the following:

[0070] [ka]

[0071] In some embodiments, described herein is an anti-Her3 antibody-drug conjugate, an isomer thereof, a pharma- ceutically acceptable salt thereof, or a mixture thereof, wherein L is -L a -L b -L c - and -L a -teeth,

[0072] [ka]

[0073] where: W is -(C(R wa )(R wb )) wn - and Y is -(OCH2CH2) yn -O yp - and Z is -(C(R za )(R zb )) zn and where wn is 1, 2, 3 or 6; Each methylene unit of W is independently -Cyr-, -N(R wx )C(O)-, -C(O)N(R wx )- or -C(O)-; where yn is 0, 4 or 8, and yp is 0 or 1; where zn is 1, 2 or 3; Each methylene unit of Z is independently -Cyr-, -N(R zx )C(O)-, -C(O)N(R zx)- or -C(O)-; -Cyr- is a 3- to 10-membered saturated carbocyclylene, where -Cyr- is unsubstituted or independently represents 1 to 3 substituents R cx Replaced with; Here, each R wa , each R wb , each R za , each R zb , each R wx , each R zx and each R cx are independently hydrogen, halogen, -OR r , or R r Optionally replaced by C 1-6 is an aliphatic group, Here, each R r are independently hydrogen, halogen or C 1-6 is an aliphatic group; -L b - is a peptide residue consisting of 2 to 7 amino acids, -L b - is a peptide residue formed from amino acids selected from the group consisting of phenylalanine, glycine, alanine, valine, citrulline, lysine, serine, glutamic acid and aspartic acid; -L c -teeth,

[0074] [ka]

[0075] where R L1 and R L2 are each independently hydrogen, halogen, -OH and C 1-6 aliphatic groups.

[0076] In some embodiments, described herein is an anti-Her3 antibody-drug conjugate, an isomer thereof, a pharma- ceutically acceptable salt thereof, or a mixture thereof, wherein L is -L a -L b -L c - and -L a -teeth,

[0077] [ka]

[0078] where: W is -(C(R wa )(R wb )) wn - and Y is -(OCH2CH2) yn -O yp - and Z is -(C(R za )(R zb )) zn and where wn is 1, 2, 3 or 6; Each methylene unit of W is independently -Cyr-, -N(R wx )C(O)-, -C(O)N(R wx )- or -C(O)-; where yn is 0, 4 or 8, and yp is 0 or 1; where zn is 1, 2 or 3; Each methylene unit of Z is independently -Cyr-, -N(R zx )C(O)-, -C(O)N(R zx )- or -C(O)-; -Cyr- is a 3- to 10-membered saturated carbocyclylene, where -Cyr- is unsubstituted or independently represents 1 to 3 substituents R cx Replaced with; Here, each R wa , each R wb , each R za , each R zb , each R wx , each R zx and each R cx are independently hydrogen, halogen, -OR r , or R r Optionally replaced by C 1-6 is an aliphatic group, Here, each R r are independently hydrogen, halogen or C 1-6 is an aliphatic group; -Lb -teeth,

[0079] [ka]

[0080] is selected from the group consisting of -L c -teeth,

[0081] [ka]

[0082] where R L1 and R L2 are each independently hydrogen, halogen, -OH and C 1-6 aliphatic groups.

[0083] In some embodiments, the present specification describes an anti-Her3 antibody-drug conjugate, an isomer thereof, a pharma- ceutically acceptable salt thereof, or a mixture thereof, wherein -L a -teeth,

[0084] [ka]

[0085] and preferably

[0086] [ka]

[0087] It is. In some embodiments, the present specification describes an anti-Her3 antibody-drug conjugate, an isomer thereof, a pharma- ceutically acceptable salt thereof, or a mixture thereof, wherein -L a - is as follows:

[0088] [ka]

[0089] In some embodiments, the present specification describes an anti-Her3 antibody-drug conjugate, an isomer thereof, a pharma- ceutically acceptable salt thereof, or a mixture thereof, wherein -L b -teeth,

[0090] [ka]

[0091] and preferably

[0092] [ka]

[0093] In some embodiments, the present specification describes an anti-Her3 antibody-drug conjugate, an isomer thereof, a pharma- ceutically acceptable salt thereof, or a mixture thereof, wherein -L c - is as follows:

[0094] [ka]

[0095] In some embodiments, described herein are anti-Her3 antibody-drug conjugates, their isomers, their pharma- ceutically acceptable salts, or mixtures thereof, wherein the linker unit L is L a Linked to Ab at the end, L c It is linked to a linker unit M at its end.

[0096] In some embodiments, described herein is an anti-Her3 antibody-drug conjugate, an isomer thereof, a pharma- ceutically acceptable salt thereof, or a mixture thereof, wherein L is:

[0097] [ka]

[0098] In some embodiments, provided herein is an anti-Her3 antibody-drug conjugate, an isomer thereof, a pharma- ceutically acceptable salt thereof, or a mixture thereof, wherein L is:

[0099] [ka]

[0100] In some embodiments, the present specification describes an anti-Her3 antibody-drug conjugate, an isomer thereof, a pharma- ceutically acceptable salt thereof, or a mixture thereof, wherein the anti-Her3 antibody-drug conjugate has the formula (II-1) or (II-2):

[0101] [ka]

[0102] wherein: Ab, L 2 , X, p, and L 3 are each as defined in any one of the embodiments herein. In some embodiments, the present specification describes an anti-Her3 antibody-drug conjugate, an isomer thereof, a pharma- ceutically acceptable salt thereof, or a mixture thereof, wherein the anti-Her3 antibody-drug conjugate has the formula (II-1) or (II-2):

[0103] [ka]

[0104] wherein: p represents the average number of connections, and p is selected from an integer or decimal number of 1 to 10, preferably an integer or decimal number of 3 to 8; Ab is as defined in any one of the embodiments herein; L 2 is -O- or -S-; X is 0, 1, 2 or 3 R3a selected from 3-6 membered saturated carbocyclyl optionally substituted with; L 3 is -C(R 1a )(R 1b )-or-C(R 1a )(R 1b )C(R 1a )(R 1b )- is selected from Here, each R 1a , each R 1b , or each R 3a are independently hydrogen, halogen, or C optionally substituted with R 1-6 may be an aliphatic group, wherein each R may independently be hydrogen or halogen.

[0105] In some embodiments, the present specification describes an anti-Her3 antibody-drug conjugate, an isomer thereof, a pharma- ceutically acceptable salt thereof, or a mixture thereof, wherein the anti-Her3 antibody-drug conjugate has the formula (II-1) or (II-2):

[0106] [ka]

[0107] wherein: p represents the average number of connections, and p is selected from an integer or decimal number of 1 to 10, preferably an integer or decimal number of 3 to 8; Ab is as defined in any one of the embodiments herein; L 2 is -O- or -S-; X is selected from cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl; L 3 is selected from -CH2-, -CH(CH3)-, -C(CH3)2-, -CH2CH2-, -CH(CH3)CH2-, or -C(CH3)2CH2-.

[0108] In some embodiments, the present specification describes an anti-Her3 antibody-drug conjugate, an isomer thereof, a pharma- ceutically acceptable salt thereof, or a mixture thereof, wherein the anti-Her3 antibody-drug conjugate has the following structural formula:

[0109] [ka]

[0110] [ka]

[0111] [ka]

[0112] [ka]

[0113] where: p represents the average number of connections, and p is selected from an integer or decimal number of 1 to 10, preferably an integer or decimal number of 3 to 8; Ab is as defined in any one of the embodiments herein.

[0114] In yet another aspect, the present invention provides an anti-Her3 antibody-drug conjugate, an isomer thereof, a pharma- ceutically acceptable salt thereof, or a mixture thereof, wherein the anti-Her3 antibody-drug conjugate is

[0115] [ka]

[0116] [ka]

[0117] [ka]

[0118] [ka]

[0119] [ka]

[0120] where: p represents the average number of connections, and is selected from an integer or decimal number of 1 to 10, and preferably an integer or decimal number of 3 to 8.

[0121] In the present invention, hu3F8-2 and hu3F8-3 are anti-Her2 antibodies. The anti-Her2 antibody hu3F8-2 comprises the heavy chain amino acid sequence shown in SEQ ID NO: 13 and the light chain amino acid sequence shown in SEQ ID NO: 12, and the anti-Her2 antibody hu3F8-3 comprises the heavy chain amino acid sequence shown in SEQ ID NO: 14 and the light chain amino acid sequence shown in SEQ ID NO: 12.

[0122] In some embodiments, the average number of connections p described herein can be an integer or decimal number between 2 and 8. For example, the average number of connections n can be an integer or decimal number between 3 and 8. For example, the average number of connections n can be an integer or decimal number between 1-2, 2-3, 3-4, 4-5, 5-6, 6-7, 7-8, 8-9, or 9-10.

[0123] In yet another aspect, the present invention provides a pharmaceutical composition comprising an anti-Her3 antibody-drug conjugate described herein, an isomer thereof, a pharma- ceutically acceptable salt thereof, or a mixture thereof, and a pharma- ceutically acceptable carrier or excipient.

[0124] In yet another aspect, the present invention provides the use of an anti-Her3 antibody-drug conjugate, an isomer thereof, a pharma- ceutically acceptable salt thereof, or a mixture thereof, or a pharmaceutical composition as described herein, in the preparation of a medicament for treating and / or preventing a Her3-mediated disease or condition, preferably, the disease or condition is cancer.

[0125] In yet another aspect, the present invention provides a method for treating and / or preventing a Her3-mediated disease or condition, the method comprising administering to a subject in need thereof an anti-Her3 antibody-drug conjugate, an isomer thereof, a pharma- ceutically acceptable salt thereof, or a mixture thereof, or a pharmaceutical composition as described herein, preferably, the disease or condition is cancer.

[0126] In yet another aspect, the present invention provides an anti-Her3 antibody-drug conjugate, an isomer thereof, a pharma- ceutically acceptable salt thereof, or a mixture thereof, or a pharmaceutical composition as described herein, for treating and / or preventing a Her3-mediated disease or condition, preferably, the disease or condition is cancer.

[0127] In some embodiments, the cancer described herein is selected from lung cancer, kidney cancer, urinary tract cancer, colorectal cancer, prostate cancer, glioblastoma multiforme, ovarian cancer, pancreatic cancer, breast cancer, melanoma, liver cancer, bladder cancer, gastric cancer, and esophageal cancer.

[0128] In yet another aspect, the present invention provides a pharmaceutical combination comprising an antibody-drug conjugate described herein or a pharma- ceutically acceptable salt thereof, or a pharmaceutical composition described herein, and one or more additional therapeutic agents.

[0129] In yet another aspect, the present invention provides a kit comprising an antibody-drug conjugate described herein or a pharmaceutical composition described herein. [Brief description of the drawings]

[0130] [Figure 1] FIG. 1 shows that murine 3F8 specifically binds to SP2 / 0-HER3 cells. [Diagram 2] FIG. 2 shows the binding affinity of murine 3F8 to human HER3, HER2 and EGFR as measured by ELISA. [Diagram 3] FIG. 3 shows the ability of mouse 3F8 to recognize human and monkey HER3, as measured by ELISA. [Figure 4] FIG. 4 shows that murine 3F8 inhibited NRG1-induced phosphorylation of HER3. [Diagram 5] FIG. 5 shows that murine 3F8 was rapidly taken up by cells with different surface HER3 levels. [Figure 6] FIG. 6 shows that anti-HER3 antibodies inhibited tumor growth in a BT474 subcutaneous xenograft model. [Figure 7] FIG. 7 shows [89Zr]Zr-ch3F8 imaging in gastric PDX model GAS078. [Figure 8] Figure 8 shows representative [89Zr]Zr-ch3F8 imaging in six PDX models. [Figure 9A] Figures 9A-9C show that hu3F8 retains binding affinity after heat, acid, and repeated freeze-thaw stress tests. Figure 9A, three clones of hu3F8 were incubated at pH 3.5 for 0, 2, 4, and 6 hours and subjected to ELISA to measure binding affinity. [Figure 9B] FIG. 9B, three hu3F8 clones were incubated at 40° C. for different days and subjected to ELISA to measure binding affinity. [Figure 9C] FIG. 9C, three clones of hu3F8 were repeatedly freeze-thawed for 3 or 5 cycles and then subjected to ELISA to measure binding affinity. [Figure 10] FIG. 10 shows the internalization activity of antibody-drug conjugates. [Figure 11A] Figures 11A-11C show in vitro growth inhibition tests of HER3 target tumor cells by ADC-1-X1 and ADC-1-X2. Figure 11A shows HCC1569 tumor cells that highly express Her3. [Figure 11B]FIG. 11B shows MDA-MB-453 tumor cells that highly express Her3. [Figure 11C] FIG. 11C shows MDA-MB-231 tumor cells that do not express Her3. [Figure 12A] Figures 12A-12B show the in vivo antitumor effects of antibody-drug conjugates ADC-1-X1 and ADC-1-X2 in HCC1569 tumor-bearing mice. Figure 12A shows the change in tumor volume. [Figure 12B] FIG. 12B shows tumor growth inhibition. [Figure 13A] Figures 13A-13B show the in vivo antitumor effects of antibody-drug conjugates ADC-1-X1 and ADC-1-X2 in Colo205 tumor-bearing mice. Figure 13A shows the change in tumor volume. [Figure 13B] FIG. 13B shows tumor growth inhibition. [Figure 14] FIG. 14 shows the in vivo antitumor effect of antibody-drug conjugate ADC-1-X2 in NCI-H441 lung cancer cell-bearing mice. [Figure 15] FIG. 15 shows the in vivo antitumor efficacy of antibody-drug conjugate ADC-1-X2 in mice bearing human lung cancer subcutaneous xenografts of LU1542. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0131] Hereinafter, embodiments of the present invention will be described with reference to specific examples. Other advantages and effects of the present invention will be readily apparent to those skilled in the art from the disclosure of this specification.

[0132] Definition of Terms Unless otherwise indicated, embodiments of the present invention employ conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, all of which are within the skill of the art.

[0133] In order to facilitate the understanding of the present invention, some technical and scientific terms are specifically defined as follows. Unless otherwise defined herein, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention belongs. For definitions and terms in the art, those skilled in the art can refer, in particular, to Current Protocols in Molecular Biology (Ausubel). The abbreviations for amino acid residues are the standard three-letter and / or one-letter codes used in the art to indicate one of the 20 commonly used L-amino acids. As used herein (including the claims), singular forms include their plural forms unless the context clearly dictates otherwise.

[0134] The term "about" generally refers to a variation of 0.5% to 10% above or below the stated value, for example, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5% or 10% above or below the stated value.

[0135] The term "human epidermal growth factor receptor 3 (Her3)", also known as the receptor tyrosine protein kinase ERBB-3 (ERBB3), is a member of the EGFR / erBB family. Unlike other erBB family members Her2 and EGFR, Her3 does not possess kinase activity by itself. Therefore, Her3 must bind to kinase-active members EGFR or Her2 to heterodimerize and trigger downstream activities. Upon binding to its natural ligand NRG1, Her3 undergoes conformational changes, heterodimerization, phosphorylation, and signaling to activate MAPK, PI3K / Akt, and PLCγ.

[0136] The term "antibody" refers to any form of antibody having the desired biological activity, and is therefore used in the broadest sense, specifically including, but not limited to, monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), humanized antibodies, fully human antibodies, chimeric antibodies, and camelized single domain antibodies.

[0137] The term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous antibody population; that is, the antibodies that make up the population are identical except for possible minor naturally occurring mutations. Monoclonal antibodies are highly specific, targeting a single antigenic epitope. In contrast, conventional (polyclonal) antibody preparations usually contain a large number of antibodies that are directed to (or specific for) different epitopes. The modifier "monoclonal" refers to the character of the antibody as being obtained from a substantially homogeneous antibody population and does not imply any particular method of production of the antibody.

[0138] The term "full-length antibody" refers to an immunoglobulin molecule that, when present in nature, consists of four peptide chains: two heavy chains (H) (total length about 50-70 kDa) and two light chains (L) (total length about 25 kDa) linked together by disulfide bonds. Each heavy chain consists of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region (abbreviated herein as CH). The heavy chain constant region consists of three domains (CH1, CH2, and CH3). Each light chain consists of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region consists of one domain, CL. The VH and VL regions are further divided into highly variable complementarity determining regions (CDRs) and more conservative regions called framework regions (FRs) spaced apart by the CDRs. Each VH or VL region is composed of three CDRs and four FRs, arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains constitute the binding domain that interacts with the antigen. The constant region of the antibody can mediate the binding of the immunoglobulin to host tissues or factors, including the binding of various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system.

[0139] The term "CDR" refers to the complementarity determining region in an antibody variable sequence. There are three CDRs in each of the heavy and light chain variable regions, which are named HCDR1, HCDR2, and HCDR3 in the heavy chain variable region, and LCDR1, LCDR2, and LCDR3 in the light chain variable region. Precise amino acid sequence boundaries of the variable region CDRs of the antibody of the present invention can be determined using the methods of Chothia (Chothia et al., (1989) Nature 342: 877-883; Al-Lazikani et al., Standard conformations for the canonical structures of immunoglobulins, Journal of Molecular Biology, 273, 927-948 (1997)), which are based on the three-dimensional structure of the antibody and the topology of the CDR loops; Kabat (Kabat et al., Sequences of Proteins of Immunological Interest, 4th edition, US Department of Health and Human Services, National Institutes of Health (1987)), which are based on antibody sequence variability; AbM (University of Bath), Contact (University College London), the International ImMunoGeneTics database (IMGT) (1999 Nucleic Acids Research, 27, 209-212), and the North American Statistical Method (NASTM) (Nucleic Acids Research, 27, 209-212), which is based on affinity propagation clustering using a large number of crystal structures. CDR definitions can be determined using any of a number of well-known schemes. The boundaries of the CDRs of the antibodies disclosed herein can be determined by one of skill in the art according to any scheme in the art (e.g., different assignment systems or combinations).

[0140] It should be noted that the boundaries of the CDRs of the variable regions of the same antibody determined by different assignment systems may be different, i.e., the CDR sequences of the variable regions of the same antibody defined by different assignment systems are different. Thus, when defining an antibody having a specific CDR sequence defined in the present invention, the scope of the antibody also includes antibodies whose variable region sequences consist of the specific CDR sequences, but whose claimed CDR boundaries differ from the specific CDR boundaries defined in the present invention due to the application of a different scheme (e.g., a different assignment system or combination).

[0141] The term "antigen-binding fragment" of an antibody ("parent antibody") includes fragments or derivatives of antibodies that generally contain at least a fragment of the antigen-binding or variable region (e.g., one or more CDRs) of the parent antibody and retain at least a portion of the binding specificity of the parent antibody. Examples of antibody binding fragments include, but are not limited to, Fab, Fab', F(ab')2 and Fv fragments; bispecific antibodies; linear antibodies; single-chain antibody molecules such as scFv; and nanobodies and multispecific antibodies formed by antibody fragments. A binding fragment or derivative generally retains at least 10% of its antigen-binding activity, expressed on a molar basis. Preferably, a binding fragment or derivative retains at least 20%, 50%, 70%, 80%, 90%, 95% or 100% or more of the antigen-binding affinity of the parent antibody. It is also intended that antigen-binding fragments of an antibody contain conservative or non-conservative amino acid substitutions (referred to as "conservative variants" or "function-conservative variants" of an antibody) that do not significantly alter its biological activity.

[0142] A "chimeric antibody" is an antibody that has the variable domains of a first antibody and the constant domains of a second antibody, where the first and second antibodies are from different species. Typically, the variable domains are obtained from an antibody of an experimental animal such as a rodent (the "parent antibody") and the constant domain sequences are obtained from a human antibody, making the resulting chimeric antibody less likely to induce a harmful immune response in a human subject compared to the parent rodent antibody.

[0143] "Humanized antibody" refers to antibody forms that contain both human and non-human (such as mouse and rat) antibody sequences. In general, a humanized antibody will contain substantially all of at least one, and typically two, variable domains, with all or substantially all of the hypervariable loops corresponding to those of a non-human immunoglobulin and all or substantially all of the framework regions (FR) being those of a human immunoglobulin sequence. The humanized antibody may optionally also contain at least a portion of a human immunoglobulin constant region (Fc).

[0144] The term "fully human antibody" refers to an antibody that consists entirely of human immunoglobulin sequences. A fully human antibody may contain mouse glycosylation if produced in a mouse, a mouse cell, or a hybridoma derived from a mouse cell. Similarly, a "mouse antibody" refers to an antibody that consists entirely of mouse immunoglobulin sequences. Alternatively, a fully human antibody may contain rat glycosylation if produced in a rat, a rat cell, or a hybridoma derived from a rat cell. Similarly, a "rat antibody" refers to an antibody that consists entirely of rat immunoglobulin sequences.

[0145] The term "isotype" of an antibody refers to the type of antibody (e.g., IgM, IgE, and IgG (such as IgG1, IgG2, or IgG4)) that is provided by the heavy chain constant region genes. Isotypes also include modified versions of any of these types that have been modified to alter Fc function, e.g., to enhance or diminish effector function or binding to an Fc receptor.

[0146] The term "Fc region" is used to define a C-terminal region of an immunoglobulin heavy chain consisting of at least a portion of the constant region. This term includes native sequence Fc regions and variant Fc regions. In some embodiments, the Fc region of a human IgG heavy chain extends from Cys226 or Pro230 to the carboxyl terminus of the heavy chain. However, the C-terminal lysine (Lys447) of the Fc region may or may not be present (numbering in this paragraph is according to the EU numbering system, also known as the EU index, Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991).

[0147] The term "cross-reaction" refers to binding with antigenic fragments of the same target molecule from human, monkey and / or murine (mouse or rat). Thus, "cross-reaction" should be understood as an inter-species reaction with the same molecule X expressed in different species. The cross-reaction specificity of a monoclonal antibody recognizing human Her3R and the cross-reaction specificity of monkey and / or murine (mouse or rat) Her3R can be determined by FACS analysis.

[0148] The term "affinity" or "binding affinity" refers to the inherent binding affinity that reflects the interaction between members of a binding pair. The affinity of a molecule X to a partner Y can generally be expressed by the equilibrium dissociation constant (KD), which is the ratio of the dissociation rate constant to the association rate constant (kdis and kkon, respectively). Affinity can be measured by common methods known in the art. One specific method for measuring affinity is the ForteBio kinetic binding assay described herein.

[0149] The term "does not bind to" a protein or cell means that it does not bind to the protein or cell or does not bind with high affinity, i.e., K D is 1.0×10-6 M or more, preferably 1.0×10 -5 M or more, and more preferably 1.0×10 -4 M or more, 1.0×10 -3 M or more, and even more preferably 1.0×10 -2 It means that it binds to a protein or cell of M or more.

[0150] The "high affinity" of an IgG antibody is defined as the K D is 1.0×10 -6 M or less, preferably 5.0×10 -8 M or less, preferably 1.0×10 -8 M or less, 5.0×10 -9 M or less, more preferably 1.0×10 -9 For other antibody subtypes, "high affinity" binding may be different. For example, "high affinity" binding for the IgM subtype is defined as binding of 10 -6 M or less, preferably 10 -7 M or less, more preferably 10 -8 K below M D Refers to...

[0151] The term "cytotoxic agent" generally refers to a toxic drug. A cytotoxic agent is a chemical molecule in tumor cells that is potent enough to inhibit the normal growth of tumor cells. At high enough concentrations, a cytotoxic agent can kill tumor cells. Cytotoxic agents include toxins, such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, radioisotopes (e.g., At 211 , I 131 , I 125 , Y 90 , Re 186 , Re 188 , Sm 153 , Bi 212 , P 32or a radioactive isotope of Lu), a toxic drug, a chemotherapy drug, an antibiotic and a nuclease; for example, the cytotoxic drug may be a toxic drug, including but not limited to a camptothecin derivative, such as the camptothecin derivative exatecan (chemical name: (1S,9S)-1-amino-9-ethyl-5-fluoro-2,3-dihydro-9-hydroxy-4-methyl-1H,12H-benzo[de]pyrano[3',4':6,7]imidazo[1,2-b]conolin-10,13(9H,15H)-dione).

[0152] The term "linker unit" or "linker structure" generally refers to a chemical structure fragment or bond that is linked at one end to a ligand and at the other end to a cytotoxic drug or to another linker before being linked to a cytotoxic drug. Direct or indirect linkage to a ligand may mean that the group is directly linked to the ligand via a covalent bond, or may be linked to the ligand via a linker structure. For example, the linker structure may be a structure shown as -Lax-Lb-Lc- and / or -La-Lb-Lc- as described herein. For example, the linker structure may be a chemical structure fragment or bond that consists of an acid-labile linker structure (e.g., hydrazone), a protease-sensitive (e.g., peptidase-sensitive) linker structure, a photolabile linker structure, a dimethyl linker structure, or a disulfide-containing linker structure.

[0153] The term "optionally linked to other molecular moieties" generally means that the structure is not linked to other chemical structures, or that the structure is linked (e.g., via a chemical bond or linker structure) to one or more other chemical structures that are different from the structure (e.g., a ligand described herein).

[0154] The term "ligand-drug conjugate" generally means that a ligand is linked to a biologically active cytotoxic drug via a stable linking unit. In this application, a "ligand-drug conjugate" can be an antibody-drug conjugate (ADC), which means that a monoclonal antibody or antibody fragment is linked to a biologically active cytotoxic drug via a stable linking unit.

[0155] In this application, the term "methylene" generally refers to a residue obtained by removing two hydrogen atoms from a group having one carbon atom. The methylene may be substituted or unsubstituted, substituted or unsubstituted. The term "alkylene" generally refers to a saturated linear or branched aliphatic hydrocarbon group having two residues derived from a parent alkane by removing two hydrogen atoms from the same carbon atom or two different carbon atoms, which may be a linear or branched group containing 1 to 20 carbon atoms, such as alkylene containing 1 to 12 carbon atoms (e.g., 1 to 6 carbon atoms). Non-limiting examples of alkylene include methylene (-CH-), 1,1-ethylidene (-CH(CH)-), 1,2-ethylidene (-CHCH)-, 1,1-propylidene (-CH(CHCH)-), 1,2-propylidene (-CHCH(CH)-), 1,3-propylidene (-CHCHCH-), 1,4-butylidene (-CHCHCHCHCH-), 1,5-butylidene (-CHCHCHCHCHCH-), etc. Alkylene groups can be substituted or unsubstituted, substituted or unsubstituted. For example, if substituted, the substitution with a substituent may occur at any available attachment point, and the substituent is preferably independently selected from one or more of alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocyclyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, and oxo, which may be, for example, hydrogen, protium, deuterium, tritium, halogen, -NO2, -CN, -OH, -SH, -NH2, -C(O)H, -CO2H, -C(O)C(O)H, -C(O)CH2C(O)H, -S(O)H, -S(O)2H, -C(O)NH2, -SON2NH2, -OC(O)H, -N(H)SO2H, or C(O)NH2. 1-6 It may be an aliphatic group.

[0156] The term "arylene" generally refers to a group having two residues derived by removing two hydrogen atoms from the same carbon atom or from two different carbon atoms of an aromatic ring. The term "aromatic ring" refers to a 6- to 14-membered all-carbon monocyclic or fused polycyclic ring (i.e., rings sharing adjacent pairs of carbon atoms) having a conjugated pi-electron system, and may be 6- to 10-membered, such as benzene and naphthalene. The aromatic ring may be fused to a heteroaryl, heterocyclyl, or cycloalkyl ring, and the ring connected to the parent structure is an aryl ring. The aryl may be substituted or unsubstituted, and if substituted, the substituents are preferably one or more groups independently selected from the group consisting of alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, and heterocycloalkylthio.

[0157] In this application, the term "heteroarylene" generally refers to a group having two residues derived by removing two hydrogen atoms from the same carbon atom or from two different carbon atoms of a heteroaromatic ring. The term "heteroaromatic ring" refers to a heteroaromatic system consisting of 1 to 4 heteroatoms and 5 to 14 ring atoms, where the heteroatoms may be selected from the group consisting of oxygen, sulfur and nitrogen. The heteroaryl may be 5 to 10 members, 5 or 6 members, such as furanyl, thienyl, pyridinyl, pyrrolyl, N-alkylpyrrolyl, pyrimidinyl, pyrazinyl, imidazolyl and tetrazolyl. The heteroaryl ring may be fused to an aryl, heterocyclyl or cycloalkyl ring, where the ring connected to the parent structure is a heteroaryl ring. Heteroarylene may be optionally substituted or unsubstituted, and when substituted, the substituents are preferably one or more groups independently selected from the group consisting of the following groups: alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, and heterocycloalkylthio.

[0158] The term "heterocyclylene" generally refers to a 3- to 7-membered monocyclic ring structure, a fused 7- to 10-membered bicyclic heterocyclic ring structure, or a bridged 6- to 10-membered bicyclic heterocyclic ring structure, which is stable and non-aromatic. These ring structures may be saturated or partially saturated and contain one or more heteroatoms in addition to carbon atoms, which may be selected from the group consisting of oxygen, sulfur, and nitrogen. For example, it may contain 1 to 4 heteroatoms as defined above. When used to refer to an atom on a heterocyclic ring structure, the term "nitrogen" may include nitrogen that has undergone a substitution reaction. Heterocyclylene may be substituted or unsubstituted.

[0159] The term "carbocyclylene" generally refers to a group having two residues derived by removing two hydrogen atoms from the same carbon atom or from two different carbon atoms of a carbocycle. The term "carbocycle" generally refers to a saturated or partially unsaturated monocyclic or polycyclic cyclic hydrocarbon, containing 3 to 20 carbon atoms, containing 3 to 12 carbon atoms, containing 3 to 10 carbon atoms, containing 3 to 8 carbon atoms, for example, 3 to 6 carbon atoms. Non-limiting examples of monocyclic carbocycles include cyclopropane, cyclobutane, cyclopentane, cyclopentene, cyclohexane, cyclohexene, cyclohexadiene, cycloheptane, cycloheptatriene, cyclooctane, etc., and polycyclic carbocycles include spirocarbocycles, fused carbocycles, bridged carbocycles, etc. For example, "3-6 membered saturated carbocyclyl" includes cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclopropylene, cyclobutylene, cyclopentylene, or cyclohexylene. Carbocyclyl and carbocyclylene may be substituted or unsubstituted, e.g., one or more halogens, C 1-6 C substituted with aliphatic groups or halogens 1-6 It may be substituted with an aliphatic group.

[0160] In this application, the term "partially unsaturated" generally means that the ring structure contains at least one double or triple bond between the ring molecules. The term "partially unsaturated" encompasses ring structures having multiple sites of unsaturation, but is not intended to include aromatic or heteroaromatic rings as defined herein. The term "unsaturated" means that the moiety has one or more degrees of unsaturation.

[0161] The term "halogen" generally refers to fluorine, chlorine, bromine or iodine and may be, for example, fluorine or chlorine.

[0162] The term "aliphatic group" generally refers to a linear, branched or cyclic hydrocarbon having 1 to 12 carbon atoms, which may be either fully saturated or have one or more units of unsaturation, but which are not aromatic. For example, suitable aliphatic groups may include substituted or unsubstituted linear, branched or cyclic alkyl, alkenyl, alkynyl and mixtures thereof, such as (cycloalkyl)alkyl, (cycloalkenyl)alkyl or (cycloalkyl)alkenyl. For example, aliphatic groups may have 1 to 12, 1 to 8, 1 to 6, 1 to 4 or 1 to 3 carbon atoms. For example, "C 1-6 The term "aliphatic group" refers to an aliphatic group as described above having 1 to 6 carbon atoms, and includes, but is not limited to, linear, branched, or cyclic alkyl, alkenyl, or alkynyl having 1 to 6 carbon atoms, such as methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, tert-butyl, pentyl, hexyl, and the like.

[0163] In this application, the term "optional" or "optionally" generally means that the subsequently described event or circumstance may, but need not, occur, and that the description includes instances in which the event or circumstance occurs or does not occur. For example, "a heterocyclyl group optionally substituted with alkyl" means that alkyl may, but need not, be present, and that both cases in which the heterocyclyl group is substituted with alkyl and cases in which it is not substituted are included herein.

[0164] The term "substituted" generally means that one or more hydrogen atoms in a group, for example up to 5 (for example, 1 to 3) hydrogen atoms, are each independently replaced with the corresponding number of substituents. The substituents are only their possible chemical positions, and a person skilled in the art would be able to determine (by experiment or theory) possible or impossible substituents without undue effort. For example, an amino or hydroxyl having free hydrogen may be unstable when bonded to a carbon atom having an unsaturated (such as olefinic) bond.

[0165] In this application, the term "zero or more (e.g., zero or at least one, zero or one, or zero) methylene units are substituted" generally refers to a structure that consists of one or more methylene units. One or more of the methylene units may be unsubstituted or substituted with one or more groups that are not methylene (e.g., NHC(O)-, -C(O)NH-, -C(O)-, -OC(O)-, -C(O)O-, -NH-, -O-, -S-, -SO-, -SO2-, -PH-, -P(=O)H-, -NHSO2-, -SONH-, -C(=S)-, -C(=NH)-, -N=N-, -C=N-, -N=C-, or -C(=N2)-).

[0166] One or more hydrogen atoms in the group, for example up to 5 (for example, 1 to 3) hydrogen atoms, are each independently replaced with the corresponding number of substituents. Substituents are present only at their possible chemical positions, and a person skilled in the art would be able to determine (by experiment or theory) possible or impossible substituents without undue effort. For example, an amino or hydroxyl having free hydrogen may be unstable when bonded to a carbon atom having an unsaturated (such as olefinic) bond.

[0167] The term "pharmaceutical acceptable salt" generally refers to a salt of the drug conjugate disclosed herein. Such salts may be safe and / or effective when used in mammals and have the required biological activity, and the antibody-drug conjugates disclosed herein may form salts with acids. Non-limiting examples of pharmaceutical 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, mesylate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, and the like.

[0168] The term "solvate" or "solvate" generally refers to a pharma- ceutically acceptable solvate formed by a drug conjugate disclosed herein with one or more solvent molecules, non-limiting examples of which include water, ethanol, acetonitrile, isopropanol, DMSO, and ethyl acetate.

[0169] The term "drug loading" generally refers to the average amount of cytotoxic drug loaded per ligand and may also be expressed as a ratio of cytotoxic drug to antibody, where the cytotoxic drug loading may range from 0 to 12 (e.g., 1 to 10) cytotoxic drugs per ligand (Ab). In an embodiment of the present application, the drug loading is expressed as Na and may illustratively be an average of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. The drug loading per ADC molecule after the coupling reaction may be characterized by conventional methods such as UV / visible spectroscopy, mass spectrometry, ELISA assay, and HPLC.

[0170] A "pharmaceutically acceptable carrier" refers to an ingredient in a pharmaceutical formulation or composition, other than an active ingredient, that is non-toxic to a subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0171] The terms "comprise", "include", "contain" or "containing" are generally intended to include the features expressly specified without the exclusion of other elements. The terms "greater than" and "less than" generally refer to a situation in which the number itself is inclusive.

[0172] A "subject" includes a human or non-human animal. The term "non-human animal" includes all vertebrates, e.g., mammals and non-mammals, such as non-human primates, sheep, dogs, cats, horses, cows, chickens, amphibians, reptiles, etc. As used herein, the term "cyno" refers to a cyno mole monkey.

[0173] Administration "in combination with" one or more other therapeutic agents includes simultaneous (concomitant) and sequential administration in any order.

[0174] "Therapeutically effective amount", "therapeutically effective dose" and "effective amount" refer to an amount of the Her3 antibody or antigen-binding fragment thereof of the present invention that is effective when administered to a cell, tissue or subject, alone or in combination with other therapeutic agents, to prevent or ameliorate one or more symptoms of a disease or condition, or to prevent the onset of a disease or condition. A therapeutically effective amount also refers to an amount of the antibody or antigen-binding fragment thereof sufficient to cause an improvement in symptoms, e.g., an amount to treat, cure, prevent or ameliorate an associated pathology, or an amount to promote the treatment, cure, prevention or amelioration of such a pathology. When an active ingredient is administered to an individual alone, the therapeutically effective amount refers to the amount of that ingredient alone. When administered in combination, the therapeutically effective amount refers to the combined amount of active ingredients that results in a therapeutic effect, regardless of whether the active ingredients are administered in combination, sequentially, or simultaneously. An effective amount of a therapeutic agent increases a diagnostic indicator or parameter by at least 10%, typically at least 20%, preferably at least about 30%, more preferably at least 40%, and most preferably at least 50%.

[0175] As used herein, the term "cancer" refers to a group of cells that exhibits abnormally high levels of proliferation and growth. Cancers can be benign (also referred to as benign tumors), pre-malignant or malignant. Cancer cells are solid cancer cells or leukemic cancer cells. As used herein, the term "tumor" refers to one or more cells that make up a cancer. As used herein, the term "tumor growth" refers to the proliferation or growth of one or more cells that make up a cancer, resulting in a corresponding increase in size or extent of the cancer.

[0176] Anti-Her3 antibody This application discloses a novel anti-HER3 antibody that can effectively inhibit tumor cell proliferation in vitro and in vivo and has good safety. The HER3 antibody 3F8 was identified in a mouse hybridoma immunized with SP2 / 0 cells overexpressing human HER3. 3F8 recognizes human and monkey HER3 with nanomolar binding affinity and is highly selective over other ERBB family members. Furthermore, cells with different HER3 levels can rapidly and effectively uptake HER3. HER3 is considered to be an essential property for an ideal ADC drug. PET imaging studies have demonstrated that HER3 is expressed in tumors in PDX models [ 89 Zr]Zr-3F8 was shown to accumulate significantly in the nucleus of the nucleoids, suggesting that 3F8 may be an effective vector for cytotoxicity in tumor cells. 3F8 and hu3F8 maintain the binding affinity, selectivity, and tumor suppression properties described above in PDX models. hu3F8 has good developability and shows little change in binding affinity, aggregation, or post-translational modifications in stress tests involving repeated freeze-thaw treatments, acid incubation, and storage at 40°C.

[0177] The terms "anti-Her3 antibody," "anti-Her3," "Her3 antibody," or "antibody that binds to Her3" of the present invention refer to an antibody that can bind to the Her3 protein or a fragment thereof with sufficient affinity so as to be used as a diagnostic and / or therapeutic agent targeting Her3.

[0178] The anti-Her3 antibodies or antigen-binding fragments thereof described herein include any one of the anti-Her3 antibodies or antigen-binding fragments thereof described in Application No. PCT / CN2021 / 099998, the disclosure of which is incorporated herein by reference in its entirety. In some embodiments, the CDR sequence of an antibody used in a drug conjugate, composition, use or method of the present invention consists of the CDR sequence of antibody hu3F8-2 described in PCT / CN2021 / 09998. In some embodiments, the variable region sequence of an antibody used in a drug conjugate, composition, use or method of the present invention consists of the variable region sequence of antibody hu3F8-1 described in PCT / CN2021 / 09998. In some embodiments, the variable region sequence of an antibody used in a drug conjugate, composition, use or method of the present invention consists of the variable region sequence of antibody hu3F8-2 described in PCT / CN2021 / 099998. In some embodiments, the variable region sequence of an antibody used in a drug conjugate, composition, or use of the invention consists of the variable region sequence of antibody hu3F8-3, as described in PCT / CN2021 / 09998.

[0179] Non-limiting exemplary antibodies used in the examples herein are selected from humanized antibodies hu3F8-1, hu3F8-2 and hu3F8-3 described in PCT / CN2021 / 099998, and their amino acid sequences are shown in the table below.

[0180] As used herein, hu3F8-1, hu3F8-2, and hu3F8-3 correspond to clone-1, clone-2, and clone-3 of PCT / CN2021 / 099998, respectively.

[0181] [Table 1]

[0182] To produce the antibodies of the present invention, any suitable method for producing antibodies can be employed. Any suitable form of Her3 can be used as an immunogen (antigen) for antibody production. By way of example and not limitation, any Her3 variant or fragment thereof can be used as an immunogen. In some embodiments, hybridoma cells producing mouse monoclonal anti-human Her3 antibodies can be produced by methods well known in the art. Antibodies from rodents (e.g., mice) can induce undesirable immunogenicity of the antibodies when used as therapeutics in vivo. Repeated use of these antibodies induces an immune response against the therapeutic antibodies in the human body. Such immune responses can result in at least a loss of therapeutic efficacy and in severe cases, can cause fatal allergic reactions. One method to reduce the immunogenicity of rodent antibodies is to create chimeric antibodies by fusing mouse variable regions to human constant regions (Liu et al., (1987) Proc. Natl. Acad. Sci. USA 84: 3439-43). However, preserving the murine variable regions intact in chimeric antibodies may still induce adverse immunogenicity in patients. Grafting complementarity determining region (CDR) loops of murine variable regions onto human frameworks (i.e., humanization) has been used to further minimize murine sequences (Jones et al., (1986) Nature 321: 522; Verhoeyen et al., (1988) Science 239: 1534).

[0183] In some embodiments, chimeric or humanized antibodies of the invention can be prepared based on the sequences of prepared murine monoclonal hybridoma antibodies. DNA encoding immunoglobulin heavy and light chains can be obtained from the murine hybridoma of interest and engineered to consist of non-murine (e.g., human) immunoglobulin sequences using standard molecular biology techniques.

[0184] In some embodiments, the chimeric Her3 antibodies described herein can be prepared by operably linking hybridoma-derived immunoglobulin heavy and light chain variable regions to human IgG constant regions using methods known in the art (see, for example, U.S. Patent No. 4,816,567 to Cabilly et al.), thereby obtaining chimeric heavy and chimeric light chains. In some embodiments, the chimeric antibodies of the invention comprise a constant region that can be selected from any human IgG subtype, such as IgG1, IgG2, IgG3, and IgG4, preferably IgG4.

[0185] In some embodiments, chimeric Her3 antibodies of the invention can be obtained by "mixing and matching" chimeric light chain expression plasmids and chimeric heavy chain expression plasmids and transfecting expressing cells. Her3 binding of such "mixed and matched" antibodies can be tested using the binding assays described above and other conventional binding assays (e.g., ELISA).

[0186] In the humanized antibodies described herein, the murine CDR regions can be inserted into human germline framework regions using methods known in the art (see U.S. Patent Nos. 5,225,539 to Winter et al., and 5,530,101, 5,585,089, 5,693,762, and 6,180,370 to Queen et al.).

[0187] In some embodiments, the amino acid changes include amino acid deletions, insertions, or substitutions. In some embodiments, the anti-Her3 antibodies or antigen-binding fragments thereof of the present invention include antibodies that are mutated by amino acid deletions, insertions, or substitutions, but still have at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequences of the above antibodies (particularly in the CDR regions defined in the above sequences). In some embodiments, the antibodies of the present invention do not have 1, 2, 3, 4, or 5 or more amino acid mutations (deletions, insertions, or substitutions) in the CDR regions when compared to the CDR regions set forth in a particular sequence.

[0188] In some embodiments, one or more amino acid modifications can be introduced into the Fc region of an antibody provided herein to generate an Fc region variant. The Fc region variant can consist of a human Fc region sequence (such as a human IgG1, IgG2, IgG3, or IgG4 Fc region) that contains an amino acid modification (such as a substitution) at one or more amino acid positions.

[0189] In some embodiments, it may be necessary to produce antibodies that have been modified by cysteine ​​engineering, for example "sulfo-MAbs," in which one or more residues of the antibody are replaced with cysteine ​​residues.

[0190] In some embodiments, the antibodies provided herein can be further modified to include other non-protein moieties that are known in the art and readily available. Suitable moieties for derivatization of antibodies include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include, but are not limited to, polyamino acids (homopolymers or random copolymers), glucans or poly(n-vinylpyrrolidone) polyethylene glycol, propylene glycol homopolymer, polypropylene oxide / ethylene oxide copolymer, polyoxyethylated polyols (such as glycerol), polyvinyl alcohol, and mixtures thereof, including, but not limited to, polyethylene glycol (PEG), ethylene glycol / propylene glycol copolymer, carboxymethylcellulose, glucan, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dioxane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymer, and the like.

[0191] Drug Conjugates The present application provides anti-Her3 antibody-drug conjugates, their isomers, their pharma- ceutically acceptable salts, or mixtures thereof, which may have one or more effects selected from the group consisting of: (1) having inhibitory activity against in vitro proliferation of tumor cells; (2) having targeting inhibition; (3) having plasma stability; (4) having in vivo tumor inhibitory effect; (5) having bystander effect; (6) having the ability to inhibit transport by transporters; (7) having in vivo tumor targeting ability; and (8) having good safety in vivo.

[0192] The present application provides an anti-Her3 antibody-drug conjugate, an isomer thereof, a pharma- ceutically acceptable salt thereof, or a mixture thereof, wherein the anti-Her3 antibody-drug conjugate has formula (I): Ab-(LMD) p (I) wherein: L and M are linker units; D is a cytotoxic drug; p represents the average number of connections, and p is selected from an integer or decimal number of 1 to 10, preferably an integer or decimal number of 3 to 8; The Ab is an anti-Her3 antibody or antigen-binding fragment thereof, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 of the amino acid sequences set forth in SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3, respectively, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 of the amino acid sequences set forth in SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6, respectively; preferably, the anti-Her3 antibody or antigen-binding fragment thereof described herein comprises: (I) a heavy chain variable region of the amino acid sequence set forth in SEQ ID NO:7 and a light chain variable region of the amino acid sequence set forth in SEQ ID NO:8; or (II) a light chain variable region of the amino acid sequence set forth in SEQ ID NO:9. or (III) a heavy chain variable region of the amino acid sequence set forth in SEQ ID NO: 10 and a light chain variable region of the amino acid sequence set forth in SEQ ID NO: 8; more preferably, the anti-Her3 antibody or antigen-binding fragment thereof described herein comprises (1) a heavy chain of the amino acid sequence set forth in SEQ ID NO: 11 and a light chain of the amino acid sequence set forth in SEQ ID NO: 12; or (2) a heavy chain of the amino acid sequence set forth in SEQ ID NO: 13 and a light chain of the amino acid sequence set forth in SEQ ID NO: 12; or (3) a heavy chain of the amino acid sequence set forth in SEQ ID NO: 14 and a light chain of the amino acid sequence set forth in SEQ ID NO: 12.

[0193] In some embodiments, described herein is an anti-Her3 antibody-drug conjugate, an isomer thereof, a pharma- ceutically acceptable salt thereof, or a mixture thereof, wherein the anti-Her3 antibody-drug conjugate has the formula (I-1):

[0194] [ka]

[0195] wherein: L and M are linker units; p represents the average number of connections, and n is an integer or decimal number selected from 1 to 10, preferably an integer or decimal number selected from 3 to 8; The Ab is an anti-Her3 antibody or antigen-binding fragment thereof, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 of the amino acid sequences set forth in SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3, respectively, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 of the amino acid sequences set forth in SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6, respectively; preferably, the anti-Her3 antibody or antigen-binding fragment thereof described herein comprises: (I) a heavy chain variable region of the amino acid sequence set forth in SEQ ID NO:7 and a light chain variable region of the amino acid sequence set forth in SEQ ID NO:8; or (II) a light chain variable region of the amino acid sequence set forth in SEQ ID NO:9. or (III) a heavy chain variable region of the amino acid sequence set forth in SEQ ID NO: 10 and a light chain variable region of the amino acid sequence set forth in SEQ ID NO: 8; more preferably, the anti-Her3 antibody or antigen-binding fragment thereof described herein comprises (1) a heavy chain of the amino acid sequence set forth in SEQ ID NO: 11 and a light chain of the amino acid sequence set forth in SEQ ID NO: 12; or (2) a heavy chain of the amino acid sequence set forth in SEQ ID NO: 13 and a light chain of the amino acid sequence set forth in SEQ ID NO: 12; or (3) a heavy chain of the amino acid sequence set forth in SEQ ID NO: 14 and a light chain of the amino acid sequence set forth in SEQ ID NO: 12.

[0196] In some embodiments, described herein is an anti-Her3 antibody-drug conjugate, an isomer thereof, a pharma- ceutically acceptable salt thereof, or a mixture thereof, wherein: M is -L 2 -L 3 -XL 1 - and; L 2 is -O- or -S-; L 3 is -(C(R 1a )(R 1b )) m -, m is selected from 0, 1, 2 or 3, where L 3 If it contains a methylene unit, L 3 may be replaced by -C(O)- or -C(=S)-; preferably, L 3 is a single bond, -C(R 1a)(R 1b )- or -C(R 1a )(R 1b )C(R 1a )(R 1b )-; more preferably, L 3 is selected from a single bond, -CH2-, -CH(CH3), -C(CH3)2, -CH2CH2-, -CH(CH3)CH2-, or -C(CH3)2CH2-; L 1 is -(C(R 2a )(R 2b )) n -, n is selected from 1, 2 or 3; L 1 When L may contain methylene units, 1 may be replaced by -C(O)- or -C(=S)-; preferably, L 1 is -C(R 2a )(R 2b )-, -C(R 2a )(R 2b )C(O)-, or -C(O)-; more preferably, L 1 is selected from -CH2-, -CH2C(O)-, -CH(CH3)C(O)-, or -C(O)-; X is selected from 3- to 6-membered saturated carbocyclyl, 3- to 6-membered saturated heterocyclyl, or a single bond, where 3- to 6-membered saturated carbocyclyl and 3- to 6-membered saturated heterocyclyl are selected from 0, 1, 2, or 3 R 3a and preferably, X is 0, 1, 2, or 3 R 3a or a single bond; more preferably, X is a 3- to 6-membered saturated carbocyclyl or a single bond; Here, each R 1a , each R 1b , each R 2a , each R 2b , and each R 3a are independently hydrogen, halogen, or C optionally substituted with R 1-6 may be an aliphatic group, wherein each R may independently be hydrogen or halogen.

[0197] In some embodiments, described herein is an anti-Her3 antibody-drug conjugate, an isomer thereof, a pharma- ceutically acceptable salt thereof, or a mixture thereof, wherein -M- is:

[0198] [ka]

[0199] is selected from the group consisting of L 2 is -O- or -S-; preferably, L 2 is -O-; X is 0, 1, 2, or 3 R 3a preferably, X is selected from cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl; more preferably, X is cyclopropyl; more preferably, X is cyclobutyl; more preferably, X is cyclohexyl; L 1 is -C(R 2a )(R 2b )-, -C(R 2a )(R 2b )C(O)-, or -C(O)-; preferably, L 1 is -C(O)-, Here, each R 2a , each R 2b , or each R 3a are independently hydrogen, halogen, or C optionally substituted with R 1-6 may be an aliphatic group, wherein each R may independently be hydrogen or halogen.

[0200] In some embodiments, described herein is an anti-Her3 antibody-drug conjugate, an isomer thereof, a pharma- ceutically acceptable salt thereof, or a mixture thereof, wherein -M- is:

[0201] [ka]

[0202] is selected from the group consisting of L 2 is -O- or -S-; preferably, L 2 is -O-; preferably, L 2 is -S-; L 3 is -C(R 1a )(R 1b )-or-C(R 1a )(R 1b )C(R 1a )(R 1b )-; preferably, L 3 is selected from -CH2-, -CH(CH3), -C(CH3)2, -CH2CH2-, -CH(CH3)CH2-, or -C(CH3)2CH2-; preferably, L 3 is selected from -CH2-, -CH(CH3), and -CH2CH2-.

[0203] L 1 is -C(R 2a )(R 2b )-, -C(R 2a )(R 2b )C(O)-, or -C(O)-; preferably, L 1 is -C(O)-; Here, each R 1a , each R 1b , each R 2a , or each R 2b are independently hydrogen, halogen, or C optionally substituted with R 1-6 may be an aliphatic group, wherein each R may independently be hydrogen or halogen.

[0204] In some preferred embodiments, described herein is an anti-Her3 antibody-drug conjugate, an isomer thereof, a pharma- ceutically acceptable salt thereof, or a mixture thereof, wherein -M- is selected from the following:

[0205] [ka]

[0206] In some embodiments, described herein is an anti-Her3 antibody-drug conjugate, an isomer thereof, a pharma- ceutically acceptable salt thereof, or a mixture thereof, wherein L is -L a -L b -L c - and -L a -teeth,

[0207] [ka]

[0208] where W is -(C(R wa )(R wb )) wn -, where wn is 1, 2, 3, or 6, and each methylene unit of W is independently -Cyr-, -N(R wx )C(O)-, -C(O)N(R wx )-, or -C(O)-; preferably, W is -(C(R wa )(R wb ))2- or -(C(R wa )(R wb ))3-; preferably, W is -CH2CH2CH2- or -CH2CH2-; Y is -(OCH2CH2) yn -O yp -, where yn is 0, 4 or 8 and yp is 0 or 1; preferably, Y is a single bond; Z is -(C(R za )(R zb )) zn where zn is 1, 2 or 3, and each methylene unit of Z is independently -Cyr-, -N(R zx )C(O)-, -C(O)N(R zx )-, or -C(O)-, where -Cyr- is a 3- to 10-membered saturated carbocyclylene, where -Cyr- is unsubstituted or independently substituted by 1 to 3 substituents R cxPreferably, Z is substituted with -(C(R wa )(R wb ))2C(O)- or -(C(R wa )(R wb ))3C(O)-; preferably Z is -CH2CH2CH2C(O)- or CH2CH2C(O)-; Here, each R wa , each R wb , each R za , each R zb , each R wx , each R zx and each R cx are independently hydrogen, halogen, -OR r , or R r Optionally replaced by C 1-6 is an aliphatic group, Here, each R r are independently hydrogen, halogen or C 1-6 is an aliphatic group; Preferably, -L a -teeth,

[0209] [ka]

[0210] [ka]

[0211] and preferably

[0212] [ka]

[0213] and -L b -teeth,

[0214] [ka]

[0215] and preferably, -L b -teeth,

[0216] [ka]

[0217] and -L c -teeth,

[0218] [ka]

[0219] where R L1 and R L2 are each independently hydrogen, halogen, -OH, and C 1-6 aliphatic groups; preferably, -L c - is as follows:

[0220] [ka]

[0221] In some particularly preferred embodiments, described herein are anti-Her3 antibody-drug conjugates, isomers thereof, pharma- ceutically acceptable salts thereof, or mixtures thereof, wherein L is:

[0222] [ka]

[0223] In some embodiments, the present specification describes an anti-Her3 antibody-drug conjugate, an isomer thereof, a pharma- ceutically acceptable salt thereof, or a mixture thereof, wherein the anti-Her3 antibody-drug conjugate has the formula (II-1) or (II-2):

[0224] [ka]

[0225] wherein: L 2 is -O- or -S-; preferably, L 2 is -O-; preferably, L 2 is -S-; X is 0, 1, 2 or 3 R 3a Preferably, X is selected from cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl; L 3 is -C(R 1a )(R 1b )-or-C(R 1a )(R 1b )C(R 1a )(R 1b )-, where each R 1a , each R 1b , or each R 3a are independently hydrogen, halogen, or C optionally substituted with R 1-6 R may be an aliphatic group, where each R may independently be hydrogen or halogen; preferably, L 3 is selected from -CH2-, -CH(CH3)-, -C(CH3)2-, -CH2CH2-, -CH(CH3)CH2-, or -C(CH3)2CH2-; p represents the average number of connections, and n is selected from an integer or decimal number of 1 to 10, preferably an integer or decimal number of 3 to 8; The Ab is an anti-Her3 antibody or antigen-binding fragment thereof, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 of the amino acid sequences set forth in SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3, respectively, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 of the amino acid sequences set forth in SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6, respectively; preferably, the anti-Her3 antibody or antigen-binding fragment thereof described herein comprises: (I) a heavy chain variable region of the amino acid sequence set forth in SEQ ID NO:7 and a light chain variable region of the amino acid sequence set forth in SEQ ID NO:8; or (II) a light chain variable region of the amino acid sequence set forth in SEQ ID NO:9. or (III) a heavy chain variable region of the amino acid sequence set forth in SEQ ID NO: 10 and a light chain variable region of the amino acid sequence set forth in SEQ ID NO: 8; more preferably, the anti-Her3 antibody or antigen-binding fragment thereof described herein comprises (1) a heavy chain of the amino acid sequence set forth in SEQ ID NO: 11 and a light chain of the amino acid sequence set forth in SEQ ID NO: 12; or (2) a heavy chain of the amino acid sequence set forth in SEQ ID NO: 13 and a light chain of the amino acid sequence set forth in SEQ ID NO: 12; or (3) a heavy chain of the amino acid sequence set forth in SEQ ID NO: 14 and a light chain of the amino acid sequence set forth in SEQ ID NO: 12.

[0226] In some embodiments, the average number of connections p described herein can be an integer or decimal number between 2 and 8. For example, the average number of connections n can be an integer or decimal number between 3 and 8. For example, the average number of connections n can be an integer or decimal number between 1-2, 2-3, 3-4, 4-5, 5-6, 6-7, 7-8, 8-9, or 9-10.

[0227] Pharmaceutical Compositions and Formulations In yet another aspect, the present invention provides a pharmaceutical composition comprising an anti-Her3 antibody-drug conjugate described herein, an isomer thereof, a pharma- ceutically acceptable salt thereof, or a mixture thereof, and a pharma- ceutically acceptable carrier or excipient.

[0228] It should be understood that the anti-Her3 antibody-drug conjugates or isomers thereof, pharma- ceutically acceptable salts thereof or mixtures thereof, or pharmaceutical compositions thereof provided herein can be incorporated into suitable carriers, excipients and other agents in a formulation for combined administration, thus providing improved transfer, delivery, tolerance, and the like.

[0229] The term "pharmaceutical composition" refers to a formulation that is capable of presenting the biological activity of the active ingredient contained therein in an effective form and that does not contain additional ingredients that are unacceptably toxic to the subject to which the formulation is administered.

[0230] Pharmaceutical formulations containing the anti-Her3 antibodies described herein can be prepared by mixing the anti-Her3 antibody-drug conjugate of the present invention or a pharma- ceutical acceptable salt thereof having the desired purity, preferably in the form of an aqueous solution or a lyophilized formulation, with one or more optional pharmaceutical adjuvants (Remington's Pharmaceutical Sciences, 16th edition, Osol, A. Ed. (1980)).

[0231] The pharmaceutical composition or formulation of the present invention may further comprise one or more additional active ingredients as required for the particular indication being treated, preferably active ingredients with complementary activities that do not adversely affect each other. In some embodiments, the additional active ingredients are chemotherapeutic agents, immune checkpoint inhibitors, growth inhibitors, antibiotics, or various known antitumor or anticancer agents, preferably present in combination in amounts effective for the intended purpose. In some embodiments, the pharmaceutical composition of the present invention also comprises a composition of a polynucleotide encoding an anti-Her3 antibody.

[0232] In yet another aspect, the present invention provides a pharmaceutical combination comprising an antibody-drug conjugate described herein or a pharma- ceutically acceptable salt thereof, or a pharmaceutical composition described herein, and one or more additional therapeutic agents.

[0233] In yet another aspect, the present invention provides a kit comprising an antibody-drug conjugate or an isomer thereof described herein, a pharma- ceutically acceptable salt thereof or a mixture thereof, or a pharmaceutical composition described herein, preferably further comprising a drug delivery device.

[0234] Medical Use In yet another aspect, the present invention provides the use of an antibody-drug conjugate as described herein, an isomer thereof, a pharma- ceutically acceptable salt thereof, or a mixture thereof, or a pharmaceutical composition as described herein, in the preparation of a medicament for treating and / or preventing a Her3-mediated disease or condition, wherein preferably, the disease or condition is cancer.

[0235] In yet another aspect, the present invention provides an antibody-drug conjugate as described herein, its isomer, its pharma- ceutically acceptable salt, or a mixture thereof, or a pharmaceutical composition as described herein, for treating and / or preventing a Her3-mediated disease or condition, wherein preferably, the disease or condition is cancer.

[0236] In yet another aspect, the present invention provides a method for treating and / or preventing a Her3-mediated disease or condition, comprising administering to a subject in need thereof an antibody-drug conjugate as described herein, an isomer thereof, a pharma- ceutically acceptable salt thereof, or a mixture thereof, or a pharmaceutical composition as described herein, preferably, the disease or condition is cancer.

[0237] In some embodiments, the cancer is selected from lung cancer, kidney cancer, urinary tract cancer, colorectal cancer, prostate cancer, glioblastoma multiforme, ovarian cancer, pancreatic cancer, breast cancer, melanoma, liver cancer, bladder cancer, gastric cancer, and esophageal cancer.

[0238] In some embodiments, routes of administration of the present invention include, but are not limited to, oral administration, intravenous administration, subcutaneous administration, intramuscular administration, intra-arterial administration, intra-articular administration (e.g., in arthritic joints), inhalation, aerosol delivery, intratumoral administration, and the like.

[0239] In some embodiments, the present invention provides for co-administration to a subject of one or more therapies (e.g., therapeutic modalities and / or other therapeutic agents) in a therapeutically effective amount. In some embodiments, the therapy comprises surgical therapy and / or radiation therapy.

[0240] In some embodiments, the methods or uses provided herein further comprise administering to the individual one or more therapies (e.g., therapeutic modalities and / or other therapeutic agents). The antibody-drug conjugate of the present invention or a pharma- ceutically acceptable salt thereof can be used alone or in combination with other therapeutic agents in the treatment. For example, it can be co-administered with at least one additional therapeutic agent.

[0241] The drug conjugates described herein may have inhibitory activity against tumor cell proliferation in vitro. Inhibitory activity may be as follows: when the drug conjugates disclosed herein are added to a culture medium containing tumor cells, they may reduce the proliferation ability of tumor cells by 1% or more, 2% or more, 4% or more, 5% or more, 8% or more, 10% or more, 15% or more, 18% or more, 20% or more, 25% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, or 95% or more, compared to when a negative control or reference drug is added. For example, the inhibitory activity is as follows: IC50 value (nM) against tumor cells is 10000 or less, 5000 or less, 4000 or less, 3000 or less, 2000 or less, 1000 or less, 500 or less, 400 or less, 300 or less, 200 or less, 150 or less, 120 or less, 110 or less, 100 or less, 99 or less, 98 or less, 97 or less, 97 or less, 95 or less, 90 or less, 80 or less, 75 or less, 70 or less, 65 or less, 62 or less, 60 or less, 50 or less, 40 or less, 30 or less, 25 or less, 23 or less, 22 or less, 20 or less, 19 or less, 18 or less, 19 or less, 20 ... 8.5 or less, 17 or less, 15 or less, 12 or less, 10 or less, 9 or less, 8.5 or less, 7 or less, 6.7 or less, 6 or less, 5.9 or less, 5.5, 5.0 or less, 4.8 or less, 4.5 or less, 4.4 or less, 4 or less, 3.5 or less, 3 or less, 2.5 or less, 2 or less, 1.5 or less, 1.0, 0.5 or less, 0.3 or less, 0.29 or less, 0.25 or less, 0.21 or less, 0.20 or less, 0.18 or less, 0.17 or less, 0.15 or less, 0.12 or less, 0.10 or less, 0.09 or less, 0.08 or less, 0.07 or less, 0.06 or less, 0.05 or less, 0.04 or less, 0.03 or less, 0.02 or less, or 0.01 or less. For example, the tumor cells may include, but are not limited to, solid tumor cells; for example, the tumor cells may include, but are not limited to, gastric cancer cells or breast cancer cells; for example, the tumor cells may include, but are not limited to, HCC1569 cells or MDA-MB-453 cells.

[0242] The drug conjugates described herein may have targeting inhibition, such as: the proliferation ability of tumor cells with high expression of a specific target is reduced by 1% or more, 2% or more, 4% or more, 5% or more, 8% or more, 10% or more, 15% or more, 18% or more, 20% or more, 25% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, or 95% or more, compared to the culture medium of tumor cells with high expression of a specific target to which a negative control or reference drug is added. For example, target inhibition may be as follows: IC50 values ​​(nM) against tumor cells highly expressing a particular target are 10,000 or less, 5,000 or less, 4,000 or less, 3,000 or less, 2,000 or less, 1,000 or less, 500 or less, 400 or less, 300 or less, 200 or less, 185 or less, 150 or less, 120 or less, 110 or less, 100 or less, 99 or less, 98 or less, 97 or less, 95 or less, 91 or less, 80 or less, 74 or less, 70 or less, 65 or less, 62 or less, 60 or less, 50 or less, 40 or less, 30 or less, 25 or less, 23 or less, 22 or less, 20 or less, 19 or less, 18 or less, 18.5 or less. or less, 17 or less, 15 or less, 12 or less, 10 or less, 9 or less, 8.5 or less, 7 or less, 6.7 or less, 6 or less, 5.9 or less, 5.5, 5.0 or less, 4.8 or less, 4.5 or less, 4.4 or less, 4 or less, 3.5 or less, 3 or less, 2.5 or less, 2 or less, 1.5 or less, 1.0, 0.5 or less, 0.3 or less, 0.29 or less, 0.25 or less, 0.21 or less, 0.20 or less, 0.18 or less, 0.17 or less, 0.15 or less, 0.12 or less, 0.10 or less, 0.09 or less, 0.08 or less, 0.07 or less, 0.06 or less, 0.05 or less, 0.04 or less, 0.03 or less, 0.02 or less, or 0.01 or less. For example, tumor cells with high expression of specific targets include, but are not limited to, solid tumor cells; for example, tumor cells with high expression of specific targets include, but are not limited to, gastric cancer cells or breast cancer cells; for example, tumor cells with high expression of specific targets include, but are not limited to, HCC1569 cells or MDA-MB-453 cells. Specific targets include, but are not limited to, Her3.

[0243] The drug conjugates described herein may have plasma stability, where the drug conjugates disclosed herein provide no more than 50%, no more than 40%, no more than 30%, no more than 20%, no more than 10%, no more than 7%, no more than 5%, no more than 4%, no more than 3%, no more than 2%, no more than 1.9%, no more than 1.8%, no more than 1.7%, no more than 1.6%, no more than 1.5%, no more than 1.4%, no more than 1.3%, no more than 1.2%, no more than 1.1%, no more than 1.0%, no more than 0.9%, no more than 0.8%, no more than 0.7%, no more than 0.6%, no more than 0.5%, no more than 0.4%, no more than 0.3%, no more than 0.2%, or no more than 0.1% of the cytotoxic drug 1 day, 3 days, 5 days, 7 days, 14 days, 20 days, or 30 days after the drug conjugate is added to the plasma.

[0244] The drug conjugates described herein may have a tumor-inhibiting effect in vivo, such as a tumor-inhibiting effect in which the volume of the tumor in an animal is reduced by 1% or more, 2% or more, 4% or more, 5% or more, 8% or more, 10% or more, 15% or more, 18% or more, 20% or more, 25% or more, 40% or more, 50% or more, 55% or more, 60% or more, 70% or more, 73% or more, 75% or more, 80% or more, 90% or more, or 95% or more, as compared to when a negative control or reference drug is administered to the animal. One day, three days, five days, seven days, 14 days, 20 days, 21 days, or 30 days after administration of the drug conjugate disclosed herein, or the tumor volume of the animal is 1.1 times or less, 1.3 times or less, 1.5 times or less, 2 times or less, 3 times or less, 5 times or less, 10 times or less, 20 times or less, 22 times or less, 30 times or less, 50 times or less, 100 times or less, 500 times or less, 1000 times or less, or 1500 times or less. Animals include, but are not limited to, mammals. For example, animals include, but are not limited to, cats, dogs, horses, pigs, cows, sheep, rabbits, mice, rats, monkeys, or humans. Methods of administration include, but are not limited to, oral administration, intravenous injection, drip infusion, intraperitoneal injection, local administration, etc.

[0245] The drug conjugates described herein may have a bystander effect. The bystander effect may mean that the drug conjugates disclosed herein do not have obvious inhibitory effect on the cell proliferation of tumor cells with low expression of a specific target, but in co-culture with tumor cells with low expression of a specific target and tumor cells with high expression of a specific target, the drug conjugates disclosed herein may simultaneously inhibit the cell proliferation of tumor cells with low expression of a specific target and tumor cells with high expression of a specific target. For example, in a co-culture of tumor cells expressing a low level of a specific target with tumor cells expressing a high level of a specific target, the inhibitory activity may be as follows: IC50 values ​​(nM) for tumor cells expressing a low level of a specific target are 10,000 or less, 5,000 or less, 4,000 or less, 3,000 or less, 2,000 or less, 1,000 or less, 500 or less, 400 or less, 300 or less, 200 or less, 185 or less, 150 or less, 120 or less, 110 or less, 100 or less, 99 or less, 98 or less, 97 or less, 95 or less, 91 or less, 80 or less, 74 or less, 70 or less, 65 or less, 62 or less, 60 or less, 50 or less, 40 or less, 30 or less, 25 or less, 23 or less, 22 or less. or less, 20 or less, 19 or less, 18 or less, 18.5 or less, 17 or less, 15 or less, 12 or less, 10 or less, 9 or less, 8.5 or less, 7 or less, 6.7 or less, 6 or less, 5.9 or less, 5.5, 5.0 or less, 4.8 or less, 4.5 or less, 4.4 or less, 4 or less, 3.5 or less, 3 or less, 2.5 or less, 2 or less, 1.5 or less, 1.0, 0.5 or less, 0.3 or less, 0.29 or less, 0.25 or less, 0.21 or less, 0.20 or less, 0.18 or less, 0.17 or less, 0.15 or less, 0.12, 0.10 or less, 0.09 or less, 0.08 or less, 0.07 or less, 0.06 or less, 0.05 or less, 0.04 or less, 0.03 or less, 0.02 or less, or 0.01 or less. Compared to tumor cells with high expression of a specific target, expression of a specific target in tumor cells with low expression of a specific target may be reduced by 1% or more, 2% or more, 4% or more, 5% or more, 8% or more, 10% or more, 15% or more, 18% or more, 20% or more, 25% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, or 95% or more.For example, tumor cells with high expression of specific targets include, but are not limited to, solid tumor cells; for example, tumor cells with high expression of specific targets include, but are not limited to, gastric cancer cells or breast cancer cells; for example, tumor cells with high expression of specific targets include, but are not limited to, HCC1569 cells or MDA-MB-453 cells. For example, tumor cells with low expression of specific targets include, but are not limited to, solid tumor cells; for example, tumor cells with low expression of specific targets include, but are not limited to, breast cancer cells; for example, tumor cells with low expression of specific targets include, but are not limited to, HCC1187 cells.

[0246] The drug conjugates described herein may have the ability to inhibit transport by transporters. The ability to inhibit transport is that the efflux ratio of the drug conjugates described herein is reduced by 1% or more, 2% or more, 4% or more, 5% or more, 8% or more, 10% or more, 15% or more, 18% or more, 20% or more, 25% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, or 95% or more compared to a standard of the transport substrate. For example, the test of the efflux ratio may be a method commonly used by those skilled in the art or a method described in the examples of the present application.

[0247] The drug conjugates described herein may have in vivo tumor targeting ability. In vivo targeting ability may be as follows: when the drug conjugate labeled with a signal substance is administered to an animal, the distribution of the labeled drug conjugate in tumor tissues may be increased by 1% or more, 2% or more, 4% or more, 5% or less, 8% or less, 10% or less, 15% or less, 18% or less, 20% or less, 25% or less, 40% or less, 50% or less, 60% or less, 70% or less, 80% or less, 90% or less, or 95% or less, or 1.1 times or less, 1.3 times or less, 1.5 times or less, 2 times or less, 3 times or less, 5 times or less, 10 times or less, 20 times or less, 22 times or less, 30 times or less, 50 times or less, 100 times or less, 500 times or less, 1000 times or less, or 1500 times or less, when compared with other tissues and organs of the animal. The signal substance may be a radioactive substance. For example, the signal substance includes, but is not limited to, 125I. The animal includes, but is not limited to, a mammal. For example, it includes, but is not limited to, a cat, a dog, a horse, a pig, a cow, a sheep, a rabbit, a mouse, a rat, a monkey, a human, etc. The administration includes, but is not limited to, oral administration, intravenous injection, drip infusion, intraperitoneal injection, or local administration. The tissue or organ includes, but is not limited to, the heart, liver, spleen, lung, kidney, brain, or bone marrow.

[0248] The drug conjugates described herein may have good in vivo safety, where the in vivo free toxin release rate in an animal after the drug conjugates disclosed herein are administered to the animal is 50% or less, 40% or less, 30% or less, 20% or less, 10% or less, 7% or less, 5% or less, 4% or less, 3% or less, 2% or less, 1.9% or less, 1.8% or less, 1.7% or less, 1.6% or less, 1.5% or less, 1.4% or less, 1.3% or less, 1.2% or less, 1.1% or less, 1.0% or less, 0.9% or less, 0.8% or less, 0.7% or less, 0.6% or less, 0.5% or less, 0.4% or less, 0.3% or less, 0.2% or less, or 0.1% or less. For example, in vivo safety is as follows: the drug conjugates described herein can be administered to animals at concentrations of 0.5 mg / kg or more, 1 mg / kg or more, 2 mg / kg or more, 3 mg / kg or more, 4 mg / kg or more, 5 mg / kg or more, 10 mg / kg or more, 20 mg / kg or more, 30 mg / kg or more, 50 mg / kg or more, 70 mg / kg or more, 100 mg / kg or more, 200 mg / kg or more, 500 mg / kg or more, or 1000 mg / kg or more without causing toxicity. For example, animals include, but are not limited to, cats, dogs, horses, pigs, cows, sheep, rabbits, mice, rats, monkeys, or humans. Methods of administration include, but are not limited to, oral administration, intravenous injection, infusion, intraperitoneal injection, local administration, and the like.

[0249] Sample analysis 1.Analysis of DAR value of ADC - Hydrophobic interaction chromatography (HIC-HPLC) High-performance liquid chromatograph Waters E2965 high-performance liquid chromatography system. Chromatography column: MabPac™ HIC-Butyl 5 μm 4.6×100 mm (Manufacturer: Thermo). Mobile phase A: 1.5 M (NH4)2SO4 + 50 mM potassium phosphate (pH 7.0). Mobile phase B: 50 mM sodium phosphate (pH 7.0) / isopropanol (75:25 V / V).

[0250] [Table 2]

[0251] Elution was performed according to the following elution procedure.

[0252] Detection conditions: The flow rate of the mobile phase was set to 1 mL / min, the detection wavelength was set to 280 nm, and the column temperature was set to 30°C.

[0253] 2. SEC Purity Analysis - Size Exclusion Chromatography (SEC-HPLC) High performance liquid chromatograph Agilent 1260 liquid chromatograph. Chromatography column: Waters Xbridge BEH200 SEC (7.8 x 300 mm, 3.5 μm) Mobile phase: 50 mM potassium phosphate + 200 mM arginine (pH 6.80) + 10% isopropanol

[0254] [Table 3]

[0255] Detection conditions: The flow rate of the mobile phase was set to 0.5 mL / min, the detection wavelength was set to 280 nm, and the column temperature was set to 30°C.

[0256] The present invention includes any combination of the specific embodiments described. Further embodiments and the full scope of applicability of the present invention will become apparent from the detailed description provided below. However, it should be understood that the detailed description and specific examples, while showing preferred embodiments of the present invention, are provided for illustrative purposes only, since various changes and modifications within the spirit and scope of the present invention will become apparent to those skilled in the art from the detailed description. All publications, patents, and patent applications cited herein, including the cited references, are incorporated herein in their entirety by reference for all purposes. EXAMPLES

[0257] The following examples are provided to demonstrate and further illustrate certain preferred embodiments and aspects of the present invention and should not be construed as limiting the scope of the invention.

[0258] Example 1. Anti-Her3 antibody Materials and Methods: The following reagents were purchased from Southern Biotech and used at the indicated dilutions: goat anti-mouse IgG-HRP (1030-05, 1:5000 dilution), goat anti-human IgG-PE (2040-09, 1:1000 dilution), goat anti-human kappa IgG-HRP (2061-05, 1:20000 dilution), goat anti-rabbit IgG-HRP (4030-05, 1:5000 dilution), FITC-labeled streptavidin (7100-02, 1:500 dilution): mouse anti-human kappa-APC (9230-11, 1:500 dilution), and mouse IgG APC (0107-11, 0.1 mg / mL). NRG1 was obtained from Origene (TP723155). Cell culture media Roswell Park Memorial Institute (RPMI) 1640, Dulbecco's modified medium (DMEM), and fetal bovine serum were obtained from Hyclone. Recombinant patritumab was prepared in-house. HER3 antibody 3F8 was produced by hybridoma or recombinant. m3F8 represents murine 3F8 antibody, ch3F8 and hu3F8 represent chimeric and humanized 3F8 antibodies, respectively.

[0259] Cell culture: SP2 / 0, SP2 / 0-HER3, SP2 / 0-HER2, SP2 / O-EGFR, NCI-N87, MDA-MB-468, MDA-MB-453, 7901, HT29, MCF-7, and SK-BR-3 were purchased from ATCC and cultured in the appropriate media recommended by ATCC. SP2 / 0-EGFR, SP2 / 0-HER2, and SP2 / 0HER3 cells stably expressing human EGFR, HER2, and HER3 were generated in-house.

[0260] Preparation of anti-HER3 hybridomas: On the first day, BALB / c mice (female, 8-10 weeks old) were inoculated with 1-2 × 10 SP2 / 0-HER3 cells expressing human HER3 together with Freund's complete adjuvant. 6 Each mouse was intraperitoneally injected with 1000 cells / mL of the same amount of cells. On the 8th day, the mice were boosted with Freund's incomplete adjuvant and the same number of cells. From the 14th day, the mice were immunized with the above amount of cells every 3 days, and this procedure was repeated 3 times. Three days after the last immunization, B lymphocytes were isolated from the spleen and fused with immortal myeloma cells NS-1 to produce hybridoma cells.

[0261] Hybridoma cells were cultured in gradient dilution in 96-well plates, and the supernatants were harvested and screened for antibodies that recognized HER3 expressed on the surface of SP2 / 0 cells by flow cytometry or for recombinant HER3 antibodies by ELISA.

[0262] DNA cloning and sequencing of antibody variable regions: Total RNA extracted from hybridomas was reverse transcribed into the first strand of cDNA using Trizol (ThermoFisher), and then PCR was used to rapidly amplify 5' complementary DNA (5'RACE) according to the instructions of the 5'RACE kit (Invitrogen, 18374-058) to amplify the DNA sequences encoding the variable regions. The PCR products were cloned into pGM-T vector. Positive clones were subjected to DNA sequencing, from which the protein sequences were deduced. The amino acids of the variable regions were analyzed with the Kabat numbering scheme.

[0263] Antibody expression: DNA encoding the antibody heavy and light chains was cloned into the expression vector pCDNA3.1(+) (Invitrogen) and expressed in 293T cells. Antibodies were purified using protein A or G columns (GE).

[0264] Humanization: Humanization was performed by GenScript. First, a mouse-human chimeric antibody (ch3F8) was generated by replacing the constant region of the mouse antibody heavy chain with the constant region sequence of human IgG1, and the constant region of the mouse antibody light chain with the constant region sequence of human Igκ. The chimeric antibody was then humanized according to reference (Kuramochi et al.). The mouse framework was replaced with a human germline framework while retaining and maintaining the framework residues of the mouse antibody required for affinity and specificity to generate a humanized antibody.

[0265] Codon-optimized DNA sequences encoding the humanized antibodies were synthesized by GenScript.

[0266] Antibody expression and purification: Plasmids encoding the above antibody DNA sequences were transfected into ExpiCHO-S (Catl.# A2910001, Gibco) cells, after which the cells were grown in medium at 5% CO2 and 32°C and maintained for 12 days. After centrifugation at 4000g for 30 min, the supernatant was collected and filtered through a 0.22 μm membrane. Antibodies bound to Protein A (Catl.# 17508001, GE) were washed with 20 mM sodium phosphate (pH 7.0) and eluted with 0.1 M glycine (pH 3.0) as recommended in the manufacturer's manual. The eluted fraction was neutralized with 0.1 M Tris buffer (pH 9.0) and switched to PBS buffer by ultrafiltration centrifugation. Protein concentration was measured by BCA.

[0267] Affinity assay by surface plasmon resonance (SPR): Antibody kinetics and affinity were measured using a Biacore T200. Briefly, recombinant human HER3 antibody was immobilized on a Protein A chip (GE, Cat. 29-1275-5). Antigen concentrations were made in two-fold serial dilutions from 50 nM to a final concentration of 0.78125 nM, and affinity and kinetics were measured across the chip.

[0268] FACS assay: Cultured cells were digested with 0.25% trypsin-EDTA and centrifuged at 1500 rpm for 5 min. The cell pellet was diluted with PBS FACS solution containing 5% FBS and 0.2% ProClin300 to 5 × 10 6 The pellet was reconstituted with 100 μL of FACS solution containing goat anti-mouse IgG-PE (1:1000 dilution) and incubated on ice for 1 h in the dark.

[0269] Western Blot Assay: Proteins separated by SDS-PAGE were transferred to nitrocellulose membrane and subjected to Western blot analysis. Primary antibodies used were as follows: anti-HER2 (Cell Signaling, Catl#: 2165S), anti-HER3 (Cell Signaling, Catl#: 12708), anti-p-HER3 (Cell Signaling, Catl#: 4791), anti-β-actin (Cell Signaling, Catl#: 4967).

[0270] ELISA assay: Human HER2-ex-huFc, human HER3-huFc, human EGFR-his were diluted to 2 μg / mL, 50 μL / well in a 96-well plate and incubated overnight at 4 °C. The plate was washed with 0.5 × PBST, then incubated with 100 μL of blocking buffer (PBS + 3% BSA) for 2 h at 37 °C and washed with 0.5 × PBST. 3F8 antibody was diluted 1:3 in a serial gradient in blocking buffer, added at 50 μL / well, incubated at 37 °C for 40-50 min, then washed with 0.5 × PBST. Goat anti-mouse IgG-HRP (Southern Biotech, 1030-05) was diluted 1:20000 in blocking buffer, added at 50 μL / well, incubated in the dark for 30 min, then washed with 0.5 × PBST. Prior to the assay, 50 μL of Luminol Buffer A+B was added to each well in a 1:1 mix.

[0271] NRG1-induced HER3 phosphorylation: Cells were cultured in 6-well plates and used for the experiment when they reached 80% confluence. On the day of the experiment, cells were washed twice with PBS, cultured in serum-free medium for 6 hours, and then induced with 10 μg / mL of antibody overnight. To induce HER3 phosphorylation, NRG1 was added to a working concentration of 100 ng / mL 30 minutes before harvesting cells for Western blot.

[0272] Antibody stability studies: Purified antibodies at 5mg / mL were stored at 4℃ as normal, incubated at 40℃ for 7 and 14 days for thermal stability, regenerated in glycine solution at pH 3.5 and stored for 2, 4 and 6 hours for acid stability, and freeze-thawed for 4 and 6 cycles for freeze-thaw stability. Aggregation was measured by SEC-HPLC, and binding affinity was measured by ELISA.

[0273] antibody[ 89 [Zr]Zr labeling: DFO-NCS was conjugated to the antibody as described in (Zeglis and Lewis, 2015). DFO and antibody were mixed in a 5:1 molar ratio and incubated at 37°C for 1 h. DFO-conjugated antibody was purified by SEC-HPLC.

[0274] ZrOxalate 89 The solution (0.8 mCi) was mixed with DFO-labeled antibody (0.2 mg / mL) in HEPES / Na2CO2 buffer (pH 7.0-7.5) and incubated at room temperature for 30 min. Radiochemical purity was assessed by TLC. 89 The Rf of the Zr antibody is 0 to 0.3, and the free 89 The Rf of Zr was in the range of 0.6 to 1.0.

[0275] PET imaging: Each animal received approximately 100 μCi of 89 [Zr]Zr antibody was injected intravenously. At the indicated times after injection, images were collected and analyzed using a small animal PET scanner.

[0276] In vivo efficacy study: Animals were maintained and used in accordance with IACUC guidelines. BALC / b nude mice and NPG mice were purchased from Charles River and SPF Biotech, respectively, and were housed at 25°C with a 12-h light / dark cycle. Patient-derived xenograft (PDX) models were generated by subcutaneous implantation of cryopreserved tissue fragments. Tumor sizes of 100–200 mm were determined. 3 Drug treatment was started when the tumor size reached 1000 mm 3 Mice were euthanized when they reached a tumor size of 100 mg / kg / day. Health status was monitored daily. Tumor size and body weight were monitored and recorded every 3 days.

[0277] Software: Data were analyzed using Olinda, GraphPad Prism 6.0, or EXCEL.

[0278] Example 1.1 Mouse 3F8 cells were co-cultured with SP2 / 0 wild-type cells or cells overexpressing HER3, HER2 or EGFR. Binding intensity was measured on a FACS machine using a PE-anti-mouse secondary antibody. The results are shown in Figure 1. 3F8 bound specifically to SP2 / 0-HER3 cells, but did not bind to other cells.

[0279] Example 1.2 The binding affinity of murine 3F8 to human HER3, HER2 and EGFR was determined by ELISA. The results are shown in Figure 2. Mouse 3F8 recognized only HER3, but did not recognize HER2 or EGFR.

[0280] Example 1.3 The EC50 of murine 3F8 binding to human HER3, HER2, and EGFR was analyzed using GraphPad Prism 6.0. The results are shown in Table 1. Murine 3F8 showed strong binding affinity in the subnanomolar range.

[0281] [Table 4]

[0282] Example 1.4 The species selectivity of mouse 3F8 for human, monkey, rat and mouse HER3 was determined by ELISA, and the results are shown in Figure 3. Mouse 3F8 recognized human and monkey HER3 with similar titers, but did not recognize mouse or rat HER3.

[0283] Example 1.5 The EC50 of murine 3F8 binding to human, monkey, rat and mouse HER3 was analyzed using GraphPad Prism 6.0, and the results are shown in Table 2. Murine 3F8 showed the same binding affinity to human and cynomolgus HER3 in the subnanomolar range.

[0284] [Table 5]

[0285] Example 1.6 Mouse 3F8 inhibited phosphorylation of HER3 by NRG1. NCI-N87, MDA-MB-468 and MDA-MB-453 were treated with the HER3 ligand NRG1 to induce downstream phosphorylation of HER3. The effect of mouse 3F8 in inhibiting p-HER3 induced by NRG1 was determined by Western blot. 3D4 is an anti-HER3 antibody previously shown to compete with NRG1 in binding to HER3 and was applied here as a positive control.

[0286] The results are shown in Figure 4. The data show that murine 3F8 reduced the phosphorylation level of HER3 protein but did not affect total HER3 protein levels. Western blot of HER2 also shows that 3F8 did not affect HER2 protein levels.

[0287] Example 1.7 Murine 3F8 was rapidly internalized by cells with different HER3 surface levels. Cells with different HER3 surface levels were incubated with murine 3F8 on ice (control) or at 37° C. for 1 or 4 hours. Percent internalization was determined by subtracting the cell surface signal of the 37° C.-incubated cells from the ice-incubated control cells.

[0288] The results are shown in Figure 5. The data indicate that mouse 3F8 was rapidly internalized into cells: the majority was internalized within 1 h, and extending the incubation time to 4 h resulted in a slight increase in the number of intracellular fragments, indicating that 3F8 internalization is a rapid and continuous process.

[0289] Example 1.8 Anti-HER3 antibodies were effective in suppressing tumor growth in a BT474 subcutaneous xenograft model. Anti-HER3 antibodies 3F8, 3D4, or 3F8+3D4 combination therapy were administered intravenously at 25 mg / kg every 2 weeks for 3 weeks. Tumor size was monitored every 3-4 days. Both 3F8 and 3D4 are anti-HER3 antibodies. 3F8 is a murine antibody.

[0290] The results are shown in Figure 6. Both 3F8 and 3D4 significantly inhibited tumor growth (one-way ANOVA, p<0.05), with 3F8 showing superior efficacy. The combination of 3F8 and 3D4 showed efficacy equivalent to that of 3F8 alone.

[0291] Example 1.9 [ 89 Zr]Zr-ch3F8 was used to image the gastric PDX model GAS078. 89 [Zr]Zr-ch3F8 was injected intravenously into the gastric model GAS078. Images were collected 4, 24, 48, 72, 96, and 168 hours after injection. Radioactive uptake in each organ was analyzed by Olinda and expressed as %ID / g (injected dose / gram of tissue).

[0292] Figure 7 shows the [ 89Representative images of Zr]Zr-ch3F8 are shown. Ch3F8 represents the chimeric 3F8 antibody. The data show that the 89 Figure 1 shows that the uptake of [Zr]Zr-ch3F8 gradually increases over time. The tumor uptake remained stable at 96 hours post-injection and then decreased slightly at 168 hours post-injection.

[0293] The data are also shown in Table 3. The tumor and major organs of the heart, liver, kidney, and spleen 89 The uptake of Zr]Zr-ch3F8 was evaluated as %ID / g (% injected dose / g tissue).

[0294] [Table 6]

[0295] Example 1.10 [ 89 Zr]Zr-ch3F8 was used to image multiple PDX models. 89 [Zr]Zr-ch3F8 was intravenously injected. Images were collected 72 hours after injection. Figure 8 shows the effect of [ 89 Representative images of [Zr]Zr-ch3F8 imaging are shown. Table 4 shows HER3 expression levels in tumor tissues as determined by ELISA, and radioactive uptake in major organs and tumor tissues 72 hours after injection. Significant tumor uptake was observed in all six PDX models tested.

[0296] [Table 7]

[0297] Example 1.11 Binding affinity of humanized 3F8 (hu3F8) and chimeric 3F8. The binding kinetics of three humanized 3F8 clones and chimeric 3F8 were measured by Biacore. The three humanized 3F8 clones (clone 1, clone 2, and clone 3) had different heavy chains and one identical light chain. Clone 1, clone 2, and clone 3 corresponded to hu3F8-1, hu3F8-2, and hu3F8-3, respectively.

[0298] All data presented in Table 6 was processed using the Biacore T200 Evaluation Software version 3.1.

[0299] [Table 8]

[0300] Example 1.12 The three clones of hu3F8 were incubated at pH 3.5 for 0, 2, 4, and 6 hours, and then subjected to ELISA to measure the binding affinity. The results (Figure 9A) showed that acid treatment had little effect on the binding affinity.

[0301] Three clones of hu3F8 were incubated at pH 3.5 for 0, 2, 4, and 6 hours, and then EC50 assay was performed. EC50 was measured by ELISA. The results (Table 7) showed that acid treatment had little effect on binding affinity.

[0302] [Table 9]

[0303] The three clones of hu3F8 were incubated at 40°C for different times and subjected to ELISA to measure binding affinity. The chimeric antibody was also assayed in parallel. The results are shown in Figure 9B.

[0304] EC50 assay of three clones of hu3F8 incubated at 40℃ for different times. Three clones of humanized 3F8 antibody were incubated in saline at 40℃ for 7 or 14 days, and the binding affinity after heat stress test was measured by ELISA. Chimeric antibody 3F8 was also measured in parallel. The results (Table 8) showed that heat stress had little effect on the binding affinity.

[0305] [Table 10]

[0306] The three hu3F8 clones were freeze-thawed for 3 or 5 cycles and subjected to ELISA to measure the binding affinity. The chimeric antibody was also measured in parallel. The results (Figure 9C) showed that repeated freeze-thawing had little effect on the binding affinity. Table 9 also shows the EC50 of three clones of hu3F8 subjected to multiple freeze-thaw stress tests.

[0307] [Table 11]

[0308] In summary, hu3F8 was shown to maintain its binding affinity even after repeated heat, acid, and freeze-thaw cycles, demonstrating its high stability.

[0309] Example 1.13 Aggregation of three hu3F8 clones after stability testing and aggregation evaluation during storage at 4°C. Aggregation of three hu3F8 antibodies after acid treatment, repeated freeze-thawing, and incubation at 40°C was measured by SEC-HPLC and compared with each antibody stored at 4°C. The results are shown in Table 10. All clones retained 95% or more of the monomer even after the stress test, indicating little tendency to aggregate.

[0310] [Table 12]

[0311] Example 1.14 Post-translational modification (PTM) analysis of hu3F8. P1:hu3F8 was not stressed; P2:hu3F8 was stressed at 40°C for 2 weeks. hu3F8 that was not stressed or stressed at 40°C for 2 weeks was digested with trypsin and post-translational modifications were analyzed by mass spectrometry. The results showed a slight increase in deamidation at HC:372-393. In general, PTMs did not change much before and after stress, indicating good developability.

[0312] [Table 13]

[0313] The amino acid sequences of the antibodies disclosed herein are shown below. Amino acid sequence of mouse 3F8 light chain variable region: DIQMTQTTSSLSASLGDRVTISCRASQDISNYLNWYQQKPDGTFKLLIYYTSILHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGDTLPPTFGAGTKLELK (SEQ ID NO: 15)

[0314] Amino acid sequence of mouse 3F8 heavy chain variable region: DVQLQESGPGLVKPSQSLSLTCSVTGYSITSAYYWNWIRQFPGDKLEWMGYISYDGRNNFNPSLKNRISITRDTSKNQFFLKLNSVTSGDTATYYCARDGDYDYFDYWGQGTTLTVSS (SEQ ID NO: 16)

[0315] Amino acid sequence of humanized 3F8 heavy chain (clone 1 / hu3F8-1) QVQLQESGPGLVKPSETLSLTTCTVSGYSITSAYYWNWIRQPPGKGLEWIGYISYDGRNNFNPSLKNRVTISVDTSKNQFSLKLSSVTAADTAVYYCARDGDYDYFDYWGQGTTV TVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCP PCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 11)

[0316] Amino acid sequence of humanized 3F8 heavy chain (clone 2 / hu3F8-2) QVQLQESGPGLVKPSETLSLTTCTVSGYSITSAYYWNWIRQPFGKGLEWMGYISYDGRNNFNPSLKNRVTISRDTSKNQFSLKLSSVTAADTAVYYCARDGDYDYFDYWGQGTTV TVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCP PCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 13)

[0317] Amino acid sequence of humanized 3F8 heavy chain (clone 3 / hu3F8-3) QVQLQESGPGLVKPSETLSLTTCTVSGYSITSAYYWNWIRQPFGKGLEWMGYISYDGRNNFNPSLKNRVSISRDTSKNQFSLKLSSVTAADTATYYCARDGDYDYFDYWGQGTTV TVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCP PCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 14)

[0318] Amino acid sequence of humanized 3F8 light chain (clone 1-3 / hu3F8-1-3) DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKAPKLLIYYTSILHSGVPSRFSGSGSGTDYTFTISSLQPEDIATYFCQQGDTLPPTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 12)

[0319] Example 2. Preparation of anti-Her3 antibody-drug conjugates (ADCs) 1.1. Preparation of linker cytotoxin (linker payload) Linker Payload X1

[0320] [ka]

[0321] [ka]

[0322] Step 1 Benzyl bromide (11.0 g, 64.6 mmol) was added dropwise to a solution of 27a (5.00 g, 43.0 mmol) and NaHCO3 (10.9 g, 129 mmol) in DMF (50 mL) under nitrogen atmosphere, and the mixture was reacted at 25 °C for 17 h. After the reaction was completed as detected by TLC (PE / EA = 2 / 1), the reaction solution was added to water (500 mL) and extracted twice with EA (250 mL). The organic phase was separated, washed with saturated aqueous sodium chloride solution (500 mL), dried over anhydrous Na2SO4, concentrated, and purified by column chromatography (PE:EA = 3:2) to give a colorless liquid (5.1 g, 57.1% yield).

[0323] Step 2 A solution of 27b (4.50 g, 21.8 mmol) in THF (10 mL) was added dropwise to a solution of KI2 (4.00 g, 10.9 mmol) and TsOH (800 mg, 4.65 mmol) in THF (30 mL) at 0 °C under nitrogen atmosphere, and the mixture was reacted at 25 °C for 2 h. After the reaction was completed as detected by TLC (PE / EA = 1 / 2), the reaction solution was added to water (200 mL) and extracted twice with EA (200 mL). The organic phase was separated, dried over anhydrous Na2SO4, concentrated, and purified by column chromatography (PE / EA = 3 / 2) to give a white solid (1.56 g, 26% yield).

[0324] Step 3 Pd / C (80 mg) was added to a mixed solution of 27c (800 mg, 1.55 mmol) in EtOH (8 mL) and EA (8 mL) under hydrogen atmosphere at 0° C., and the mixture was stirred for 2.5 h at 0° C. The filtrate was concentrated, dissolved in THF (20 mL), and dried by rotary evaporation to give a white solid (600 mg, 91% yield).

[0325] Step 4 DIEA (152 mg, 1.18 mmol) was added to a solution of 27d (220 mg, 0.515 mmol), HY-13631A (250 mg, 0.47 mmol) and HATU (214 mg, 0.56 mmol) in DMF (6 mL) at 0 °C under nitrogen atmosphere and reacted at 0 °C for 2 h. After completion of the reaction detected by LCMS, the reaction solution was added to an aqueous citric acid solution (pH = 4) (150 mL) and filtered. The filter cake was washed with water (175 mL), filtered, dried and dried on an oil pump to give a brown solid (260 mg, 66% yield).

[0326] Step 5 Diethylamine (8 mL) was added dropwise to a solution of 27e (260 mg, 0.309 mmol) in DCM (30 mL) at 0° C. under nitrogen atmosphere, and the mixture was reacted at 0° C. for 3 h. After the reaction was completed as detected by LCMS, the reaction solution was added to a petroleum ether solution (600 mL) at 0° C. to precipitate a solid. The resulting mixture was left to stand until the solid was adsorbed to the bottom of the flask, and the solution was poured off and dried with an oil pump to give a brown solid (90 mg, 47.1% yield).

[0327] Step 6 HATU (74 mg, 0.19 mmol) was added to a solution of 27f (90 mg, 0.13 mmol), KI-1 (92 mg, 0.19 mmol) and DIEA (50 mg, 0.39 mmol) in DMF (2.5 mL) under nitrogen atmosphere at 0° C., and the mixture was reacted at 0° C. for 2 h. After the reaction was substantially complete as detected by LCMS, the reaction solution was added to an aqueous citric acid solution (30 mL) at pH 4 at 0° C. to precipitate a flocculent solid. The resulting mixture was filtered and purified by preparative plate (DCM / MecOH=10 / 1) to give X1 as a pale yellow solid (9.2 mg, 6% yield). MS m / z (ESI): 1074 [M+1] H-NMR (400 MHz, MeOD): 7.65 (d, 1H), 7.62 (s, 1H), 7.30-7.21(m, 5H), 6.79 (s, 2H), 5.69-5.65 (m, 1 H), 5.57 (d, 1H), 5.43-5.10 (m, 3H), 4.70 (d, 2H), 4.48-4.39 (m, 2H), 4.10-4.05 (m, 1H), 4.01-3.75 (m, 5H),3.46 (t, 2H), 3.22-3.15 (m, 2H), 3.07-3.00 (m, 1H), 2.75 (m, 1H), 2.62 (m, 1H), 2.45 (s, 3H), 2.37-2.20 (m, 6H), 2.10-2.02 (m, 2H), 2.00-1.92 (m, 2H) 1.68-1.57 (m, 6H), 1.01 (t, 3H)

[0328] Linker Payload X2

[0329] [ka]

[0330] [ka]

[0331] Step 1 34a (5 g, 48.0 mmol) and K2CO3 (19.9 g, 144.0 mmol) were dissolved in DMF (20 mL), followed by dropwise addition of benzyl bromide (12.3 g, 72.0 mmol). The mixture was reacted at 25 °C for 17 h. After the starting material was completely consumed as detected by TLC (PE / EA = 3 / 1), the reaction solution was added to water (200 mL) and extracted with EA (250 mL). The organic phase was separated, washed with saturated NaCl, dried over anhydrous Na2SO4, concentrated and purified by column chromatography (PE:EA = 2:1) to give 34b (8.7 g, 93% yield) as a colorless liquid. m / z 195.1 [M+H]+

[0332] Step 2 34c (7.3 g, 19.8 mmol) and TsOH (1.46 g, 8.5 mmol) were dissolved in THF (20 mL), and the mixture was cooled to 0 °C under nitrogen atmosphere. A solution of 43b (7.7 g, 39.6 mmol) in THF (10 mL) was added dropwise, and after the addition, the mixture was reacted at 0 °C for 2 h. After most of the starting material was consumed as detected by TLC (PE / EA = 2 / 1), the reaction solution was poured into water (100 mL) and extracted with DCM (100 mL). The organic phase was separated, washed with saturated NaCl, dried over anhydrous Na2SO4, and purified by column chromatography (PE / EA = 1 / 1) to give 34d as a colorless sticky material (3.9 g, 39% yield). MS-ESI: m / z 503.3 [M+H]+

[0333] Step 3 Pd / C (1 g, 10 wt.%) was added to a mixed solution of 34d (1.9 g, 3.78 mmol) in EtOH (100 mL) and EA (100 mL) under hydrogen atmosphere at 0 °C, and the mixture was reacted at 0 °C for 3 h. After the reaction was completed as detected by TLC (PE / EA = 2 / 1), the reaction solution was filtered through Celite, and the filter cake was washed with EA / EtOH (1:1, 100 mL x 3). The filtrate was concentrated, dissolved in THF (50 mL x 3), and dried by rotary evaporation. This procedure was repeated three times to give 34e as a grey solid (1 g, 64% yield). MS-ESI: m / z 435.2 [M+Na]+

[0334] Step 4 To a solution of 34e (426 mg, 1.03 mmol), KI4 (500 mg, 0.94 mmol) and HATU (429 mg, 1.13 mmol) in DMF (20 mL) was added dropwise at 0 °C under nitrogen atmosphere, and the mixture was reacted at 0 °C for 2 h after the addition was complete. After the reaction was completed as detected by LCMS, the reaction solution was added dropwise to water (300 mL). The resulting mixture was stirred, then left for 5 min, filtered, and the filter cake was dissolved in a DCM / MeOH (10:1, 100 mL) solution. The resulting solution was dried and concentrated to dryness by rotary evaporation, and the residue was mixed with silica gel and purified by column chromatography (EA:MeOH=30:1) to give 34f as a yellow solid (600 mg, 77% yield). MS-ESI: m / z 830.3 [M+H]+

[0335] Step 5 Diethylamine (5 mL) was added dropwise to a solution of 34f (150 mg, 0.18 mmol) in DCM (5 mL) at 0 °C under nitrogen atmosphere, and the mixture was reacted at 0 °C for 2 h. After the reaction was completed as detected by LCMS, petroleum ether solution (100 mL x 6) was added to the reaction solution, which precipitated a solid. The resulting mixture was left until the solid precipitated, and the solution was poured off. The residue was dried by oil pump to give 34f as a white powder (120 mg, 76% yield). MS-ESI: m / z 608.3 [M+H]+

[0336] Step 6 A solution of HATU (45 mg, 0.118 mmol) in DMF (1 mL) was added to a solution of 34g (60 mg, 0.099 mmol), 43h (51 mg, 0.108 mmol) and DIEA (32 mg, 0.25 mmol) in DMF (1 mL) at 0° C. under nitrogen atmosphere, and the mixture was reacted at 0° C. for 2 h. After the starting material was completely consumed as detected by LCMS, the reaction solution was directly purified by reverse phase column chromatography (eluent: (MeCN / MeOH=1 / 1):H2O=60%:40%) to give X2 as a yellow solid (14.8 mg, 14% yield). MS-ESI: m / z 1062.4 [M+H]+ 1H NMR (400 MHz, Methanol-d4) δ 7.69 - 7.61 (m, 2H), 7.22 - 7.16 (m, 2H), 7.16 - 7.09 (m, 3H), 6.76 (s, 2H), 5.70 - 5.64 (m, 1H), 5.60 (d, J = 16.4 Hz, 1H), 5.40 - 5.31 (m, 2H), 5.26 (d, J = 19.0 Hz, 1H), 4.65 - 4.50 (m, 7H), 4.25 - 4.16 (m, 1H), 3.87 (d, J = 16.7 Hz, 1H), 3.83 - 3.76 (m, 3H), 3.72 (d, J = 17.0 Hz, 2H), 3.44 (t, J = 7.1 Hz, 2H), 3.25 - 3.17 (m, 2H), 3.10 - 3.02 (m, 1H), 2.92 - 2.83 (m, 1H), 2.45 - 2.39 (m, 5H), 2.32 - 2.20 (m, 5H), 1.97 - 1.89 (m, 2H), 1.63 - 1.50 (m, 4H), 1.34 - 1.20 (m, 6H), 0.99 (t, J = 7.3 Hz, 3H).

[0337] Linker Payload X3

[0338] [ka]

[0339] [ka]

[0340] Step 1 Bromopropene (960 mg, 7.92 mmol) was added to 32a (2.00 g, 6.6 mmol) and K2CO3 (1.82 g, 13.2 mmol) in MeCN (20 mL), and the mixture was stirred at 20 °C for 5 h. After the reaction was completed as detected by TLC (PE / EA = 1 / 2), the reaction solution was poured into water (100 mL), adjusted to pH 5, and extracted three times with EA (100 mL). The organic phase was dried over anhydrous sodium sulfate and concentrated to dryness by rotary evaporation, and the residue was purified by column chromatography (PE / EA = 2 / 1) to give 32b as a white solid (1.83 g, 81% yield).

[0341] Step 2 TFA (10 mL) was added to 32b (1.38 g, 4.02 mmol) in DCM (10 mL), and the mixture was stirred for 17 h at 25° C. After the reaction was complete as detected by TLC (PE / EA=1 / 3), the reaction solution was concentrated to dryness by rotary evaporation to give 32c as a yellow sticky material (0.91 g, yield not calculated).

[0342] Step 3 41d (1.92 g, 4.87 mmol) was added to 32c (910 mg, 4.87 mmol) and NaHCO3 (613 mg, 7.3 mmol) in DME / H2O (20 mL / 10 mL), and the mixture was stirred at 25 °C for 3 h. After the reaction was complete as detected by TLC (DCM / MeOH = 1 / 1), the reaction solution was poured into water (100 mL), adjusted to pH 5 with aq. HCl (1 N), and extracted twice with EA (150 mL). The organic phase was dried over anhydrous sodium sulfate, concentrated to dryness by rotary evaporation, and the residue was purified by column chromatography (DCM / MeOH = 20 / 1) to give 32e as a white solid (1.53 g, 67% yield). MS-ESI: m / z 467.4 [M+H]+

[0343] Step 4 Pd / C (600 mg) was added to 32f (3 g, 5.83 mmol) in MeOH (50 mL), and the mixture was stirred under hydrogen atmosphere for 5 h at 25° C. After the reaction was complete as detected by TLC (EA), the reaction was filtered, and the filtrate was concentrated to dryness by rotary evaporation to give 32g as a white solid (1.9 g, 77% yield).

[0344] Step 5 HATU (707 mg, 1.86 mmol) was added to 32g (789 mg, 1.86 mmol), KI4 (900 mg, 1.69 mmol) and triethylamine (342 mg, 3.38 mmol) in DMF (10 mL) and the mixture was stirred at 0 °C for 3.5 h. After the reaction was complete as detected by TLC (EA), the reaction solution was poured into H2O (80 mL) and extracted twice with EA (100 mL). The organic phase was dried over anhydrous sodium sulfate and concentrated to dryness by rotary evaporation, and the residue was purified by column chromatography (EA) to give 32h as a white solid (1.186 g, 83% yield). MS-ESI: m / z 842.3 [M+H]+

[0345] Step 6 32h (1.186 g, 1.41 mmol) was stirred in DCM / diethylamine (20 mL, 20 / 1) at 25° C. for 17 h. After the reaction was complete as detected by TLC (DCM / MeOH=10 / 1), the reaction solution was poured into petroleum ether (200 mL) and the resulting mixture was filtered to give 32i as a white solid (768 mg, 88% yield). MS-ESI: m / z 620.3 [M+H]+

[0346] Step 7 HATU (414 mg, 1.09 mmol) was added to 32i (676 mg, 1.09 mmol), 32e (508 mg, 1.09 mmol) and DIEA (423 mg, 3.27 mmol) in DMF (10 mL), and the mixture was stirred at 20 °C for 17 h. After the reaction was completed as detected by TLC (PE / EA = 1 / 5), the reaction solution was poured into water (30 mL). The resulting mixture was filtered, and the filter cake was purified by column chromatography (DCM / MeOH = 50 / 1) to give 32j as a white solid (511 mg, 44% yield). MS-ESI: m / z 1068.3 [M+H]+

[0347] Step 8 A solution of 32j (482 mg, 0.451 mmol) in diethylamine / DCM (10 mL, 1 / 5) was stirred for 17 h at 10° C. After the reaction was complete as detected by TLC (EA), the reaction solution was poured into PE (300 mL) and the resulting mixture was filtered to give 32k as a white solid (301 mg, yield not calculated).

[0348] Step 9 Morpholine (93 mg, 1.07 mmol) was added to 32k (301 mg, 0.356 mmol) and Pd(PPh3)4 (82 mg, 0.071 mmol) in THF (5 mL), and the mixture was stirred at 25° C. for 5 h. After the reaction was complete as detected by LCMS, the reaction was purified by preparative chromatography to give 32l as a white solid (108 mg, 38% yield). MS-ESI: m / z 806.3 [M+H]+

[0349] Step 10 Bromoacetyl bromide (27 mg, 0.134 mmol) was added to 32l (108 mg, 0.134 mmol) and triethylamine (41 mg, 0.402 mmol) in THF (2 mL) and DMF (2 mL), and the mixture was stirred at 0° C. for 1 h. After the reaction was complete as detected by TLC (DCM / MeOH=10 / 1), the reaction solution was directly purified by preparative chromatography to give X3 as a white solid (15 mg, 12% yield).

[0350] MS-ESI: m / z 926.3 [M+H]+ 1H NMR (400 MHz, DMSO-d6) δ 12.11 (s, 1H), 8.54 - 8.42 (m, 3H), 8.27 - 8.16 (m, 2H), 7.78 (d, J = 11.0 Hz, 1H), 7.30 (s, 1H), 6.53 (s, 1H), 5.61 - 5.51 (m, 1H), 5.42 (s, 2H), 5.20 - 5.05 (m, 2H), 4.56 - 4.42 (m, 2H), 4.32 - 4.22 (m, 1H), 3.96 - 3.87 (m, 3H), 3.79 (d, J = 5.6 Hz, 2H), 3.70 (d, J = 5.9 Hz, 2H), 3.25 - 3.08 (m, 2H), 2.61 - 2.53 (m, 2H), 2.45 - 2.36 (m, 4H), 2.36 - 2.22 (m, 3H), 2.20 - 2.03 (m, 4H), 1.99 - 1.68 (m, 4H), 0.87 (t, J = 7.3 Hz, 3H).

[0351] Linker Payload X4

[0352] [ka]

[0353] [ka]

[0354] Step 1 To 33a (2.00 g, 2.58 mmol) in MeOH (20 mL) was added Pd / C (400 mg, 10 wt.%), and the mixture was stirred for 5 h at 20° C. After the reaction was completed as detected by TLC (EA), the reaction solution was filtered, and the filtrate was concentrated to dryness by rotary evaporation to give 33b as a white solid (1.3 g, 74% yield).

[0355] Step 2 HATU (305 mg, 0.802 mmol) was added to 33b (0.55 g, 0.802 mmol), KI4 (427 mg, 0.802 mmol) and DIPEA (310 mg, 2.40 mmol) in DMF (5 mL), and the mixture was stirred at 0 °C for 2 h. After the reaction was completed as detected by TLC (DCM / MeOH = 1 / 10), the reaction solution was poured into water (40 mL). The resulting mixture was filtered to obtain the crude product, which was purified by column chromatography (DCM / MeOH = 20 / 1) to give 33c as a yellow solid (360 mg, 41% yield).

[0356] Step 3 Diethylamine (2 mL) was added to 33c (360 mg, 0.326 mmol) in DCM (10 mL), and the mixture was stirred at 25° C. for 17 h. After the reaction was complete as detected by TLC (DCM / MeOH=5 / 1), the reaction solution was poured into PE (100 mL), and the resulting mixture was filtered to give 33d as a white solid (205 mg, 71% yield). MS-ESI: m / z 881.3 [M+H]+

[0357] Step 4 A solution of bromoacetyl bromide (94 mg, 0.446 mmol) in THF (2 mL) was added to 33d (205 mg, 0.233 mmol) and triethylamine (118 mg, 1.17 mmol) in DMF (1 mL) and water (1 mL) and the mixture was stirred at 0° C. for 1 h.

[0358] MS-ESI: m / z 1001.2 [M+H]+ 1H NMR (400 MHz, DMSO-d6) δ 8.57 - 8.50 (m, 1H), 8.50 - 8.43 (m, 2H), 8.35 - 8.29 (m, 1H), 8.19 - 8.12 (m, 2H), 7.80 (d, J = 10.8 Hz, 1H), 7.27 - 7.14 (m, 7H), 6.53 (s, 1H), 5.59 - 5.51 (m, 1H), 5.44 - 5.39 (m, 2H), 5.20 - 5.07 (m, 2H), 4.56 - 4.44 (m, 3H), 3.92 (s, 3H), 3.80 - 3.68 (m, 5H), 3.41 (s, 1H), 3.21 - 3.12 (m, 2H), 2.83 - 2.74 (m, 1H), 2.58 - 2.55 (m, 3H), 2.39 (s, 4H), 2.18 - 2.03 (m, 4H), 1.93 - 1.78 (m, 2H), 0.87 (t, J = 7.3 Hz, 3H).

[0359] 2.2 Modulation of anti-Her3 antibody-drug conjugates Modulation of antibody conjugate ADC-1-X1 (hu3F8-2-X1)

[0360]

change

[0361] The reducing agent and protecting agent were prepared by mixing 2 mg / mL TCEP (tris-2-carboxyethyl-phosphine, manufacturer: Thermo) and 100 mmol / L ethylenediaminetetraacetic acid sodium salt (manufacturer: Sigma) in ultrapure water. 150 mg of 7.4 mg / mL anti-Her3 antibody (hu3F8-2) was placed in a 50 mL centrifuge tube and diluted to an antibody reaction concentration of 5 mg / mL by adding 50 mM sodium phosphate buffer (pH 7.5). 100 mmol / L ethylenediaminetetraacetic acid sodium salt was added in an amount of 5% of the total reaction solution, and the resulting mixture was shaken to mix well. A 10 mg / mL solution of linker payload X1 in dimethyl sulfoxide (manufacturer: Sinopharm) was prepared and slowly added according to a molar ratio of drug to antibody of 18.0:1. The resulting mixture was shaken to mix well and reacted on a thermomixer at 4 °C for 1 h. The sample was exchanged into a storage buffer using an ultrafiltration tube (MWCO 30KD, manufacturer: Millipore). First, ultrafiltration was performed three times using 30 mM histidine acetate buffer (pH 5.5) containing 10% dimethyl sulfoxide, then ultrafiltration was performed three times using 30 mM histidine acetate buffer (pH 5.5) without DMSO, and concentrated by ultrafiltration to obtain ADC-1-X1 (concentration 26.8 mg / mL, yield 73%). Purity and DAR value were measured using size exclusion chromatography and hydrophobic chromatography.

[0362] Testing revealed that the purity of the antibody-drug conjugate ADC-1-X1 was 98.68% and the DAR value p was 7.12.

[0363] Preparation of antibody-drug conjugate ADC-2-X1 (hu3F8-3-X1)

[0364] [ka]

[0365] The reducing agent and protecting agent were prepared by mixing 2 mg / mL TCEP (tris-2-carboxyethyl-phosphine, manufacturer: Thermo) and 100 mmol / L ethylenediaminetetraacetic acid sodium salt (manufacturer: Sigma) in ultrapure water. 150 mg of 7.5 mg / mL anti-Her3 antibody (hu3F8-3) was placed in a 50 mL centrifuge tube and diluted to an antibody reaction concentration of 5 mg / mL by adding 50 mM sodium phosphate buffer (pH 7.5). 100 mmol / L ethylenediaminetetraacetic acid sodium salt was added in an amount of 5% of the total reaction solution, and the resulting mixture was shaken to mix well. A 10 mg / mL solution of linker payload X1 in dimethyl sulfoxide (manufacturer: Sinopharm) was prepared and slowly added according to a drug-to-antibody molar ratio of 18.0:1. The resulting mixture was shaken to mix well and reacted on a thermomixer at 4 °C for 1 h. The sample was exchanged into a storage buffer using an ultrafiltration tube (MWCO 30KD, manufacturer: Millipore). First, ultrafiltration was performed three times using 30 mM histidine acetate buffer (pH 5.5) containing 10% dimethyl sulfoxide, then ultrafiltration was performed three times using 30 mM histidine acetate buffer (pH 5.5) without DMSO, and then ultrafiltration concentration was performed to obtain the antibody-drug conjugate ADC-2-X1 (concentration 21.15 mg / mL, yield 67.3%). Purity and DAR value were measured using size exclusion chromatography and hydrophobic chromatography.

[0366] Testing revealed that the purity of the antibody-drug conjugate ADC-2-X1 was 99.08% and the DAR value p was 7.01.

[0367] Preparation of antibody drug conjugate ADC-1-X2 (hu3F8-2-X2)

[0368] [ka]

[0369] The reducing agent and protecting agent were prepared by mixing 2 mg / mL TCEP (tris-2-carboxyethyl-phosphine, manufacturer: Thermo) and 100 mmol / L ethylenediaminetetraacetic acid sodium salt (manufacturer: Sigma) in ultrapure water. 150 mg of 7.4 mg / mL anti-Her3 antibody (hu3F8-2) was placed in a 50 mL centrifuge tube and diluted to an antibody reaction concentration of 5 mg / mL by adding 50 mM sodium phosphate buffer (pH 7.5). 100 mmol / L ethylenediaminetetraacetic acid sodium salt was added in an amount of 5% of the total reaction solution, and the resulting mixture was shaken to mix well. A 10 mg / mL solution of linker payload X2 in dimethyl sulfoxide (manufacturer: Sinopharm) was prepared and slowly added according to a molar ratio of drug to antibody of 21:1. The resulting mixture was shaken to mix well and reacted on a thermomixer at 4 °C for 1 h. The sample was exchanged into a storage buffer using an ultrafiltration tube (MWCO 30KD, manufacturer: Millipore). First, ultrafiltration was performed three times using 30 mM histidine acetate buffer (pH 5.5) containing 10% dimethyl sulfoxide, then ultrafiltration was performed three times using 30 mM histidine acetate buffer (pH 5.5) without DMSO, and then ultrafiltration concentration was performed to obtain the antibody-drug conjugate ADC-1-X2 (concentration 20.56 mg / mL, yield 64%). Purity and DAR value were measured using size exclusion chromatography and hydrophobic chromatography.

[0370] As a result of the test, the purity of the antibody-drug conjugate ADC-1-X2 was 99.02% and the DAR value p was 7.53.

[0371] Preparation of antibody-drug conjugate ADC-2-X2 (hu3F8-3-X2)

[0372] [ka]

[0373] The reducing agent and protecting agent were prepared by mixing 2 mg / mL TCEP (tris-2-carboxyethyl-phosphine, manufacturer: Thermo) and 100 mmol / L ethylenediaminetetraacetic acid sodium salt (manufacturer: Sigma) in ultrapure water. 150 mg of 7.5 mg / mL anti-Her3 antibody (hu3F8-3) was placed in a 50 mL centrifuge tube and diluted to an antibody reaction concentration of 5 mg / mL by adding 50 mM sodium phosphate buffer (pH 7.5). 100 mmol / L ethylenediaminetetraacetic acid sodium salt was added in an amount of 5% of the total reaction solution, and the resulting mixture was shaken to mix well. A 10 mg / mL solution of linker payload X2 in dimethyl sulfoxide (manufacturer: Sinopharm) was prepared and slowly added according to a molar ratio of drug to antibody of 18.0:1. The resulting mixture was shaken to mix well and reacted on a thermomixer at 4 °C for 1 h. The sample was exchanged into a storage buffer using an ultrafiltration tube (MWCO 30KD, manufacturer: Millipore). First, ultrafiltration was performed three times using 30 mM histidine acetate buffer (pH 5.5) containing 10% dimethyl sulfoxide, then ultrafiltration was performed three times using 30 mM histidine acetate buffer (pH 5.5) without DMSO, and then ultrafiltration concentration was performed to obtain the antibody-drug conjugate ADC-2-X2 (concentration 21.63 mg / mL, yield 67.3%). Purity and DAR value were measured using size exclusion chromatography and hydrophobic chromatography.

[0374] Testing revealed that the purity of the antibody-drug conjugate ADC-2-X2 was 99.24% and the DAR value p was 7.11.

[0375] Preparation of reference ADC-1 (U3-1402) The reducing agent and protecting agent were mixed with ultrapure water: 2 mg / mL TCEP (tris-2-carboxyethyl-phosphine, manufacturer: Thermo) and 100 mmol / L ethylenediaminetetraacetic acid sodium salt (manufacturer: Sigma). 40 mg of 6.9 mg / mL Patritumab monoclonal antibody (antibody sequence consists of heavy chain described in SEQ ID NO: 70 and light chain described in SEQ ID NO: 72 of patent "WO2007077028"; this antibody was produced by transfection of CHO cells followed by routine antibody expression and purification, and the purity is 95% or more) was placed in a 50 mL centrifuge tube and diluted to an antibody reaction concentration of 5 mg / mL by adding 50 mM sodium phosphate buffer (pH 7.5). 100 mmol / L ethylenediaminetetraacetic acid sodium salt was added in an amount of 5% of the total reaction solution, and the resulting mixture was shaken to mix well. Then, 2mg / mL TCEP was added at a molar ratio of TCEP to antibody of 6.0:1 to reduce the antibody, and the resulting mixture was shaken to mix well and reacted on a thermomixer at 37℃ for 2 hours. A 10mg / mL solution of Deruxtecan (CAS 1599440-13-7, purchased from Shanghai Haoyuan Chemexpress Co.Ltd.) in dimethylsulfoxide (manufacturer: Sinopharm) was prepared and slowly added according to a molar ratio of drug to antibody of 18.0:1. The resulting mixture was shaken to mix well and reacted on a thermomixer at 4℃ for 1 hour. The sample was exchanged into storage buffer using ultrafiltration tubes (MWCO 30KD, manufacturer: Millipore). First, ultrafiltration was performed three times using 30 mM histidine acetate buffer (pH 5.5) containing 10% dimethyl sulfoxide, and then ultrafiltration was performed three times using 30 mM histidine acetate buffer (pH 5.5) without DMSO to obtain reference ADC-1 (concentration 8.77 mg / mL, yield 65%). Purity and DAR value were measured using size exclusion chromatography and hydrophobic chromatography.

[0376] As a result of the testing, the purity of the reference ADC-1 was 99.42% and the DAR value p was 7.58.

[0377] Example 3. Internalization activity of antibody-drug conjugates the purpose The uptake of the antibody-drug conjugate of the present invention targeting HER3 was detected in HER3-expressing MDA-MB-453 cells. Cells, a constant concentration of antibody-drug conjugate (ADC), and an internalization indicator reagent pHrodo were co-cultured, and the endocytosis ability of the antibody-drug conjugate was evaluated by observing the number of fluorescent signal foci generated by pHrodo that entered the cells together with the antibody-drug conjugate at different time points.

[0378] method 1. Cell culture: MDA-MB-453 cells were cultured in 10% FBS Leibovitz's L-15 medium. 2. Preparation of cells: MDA-MB-453 cells in log phase were harvested, washed once with PBS, and then digested with 2-3 mL of Versene for 2-3 min. After the cells were completely digested, 10-15 mL of cell culture medium was added to elute the digested cells. The eluate was centrifuged at 1000 rpm for 5 min, and the supernatant was discarded. The obtained cells were resuspended in cell culture medium to obtain a single cell suspension. This suspension was diluted with 3 × 10 5 Adjusted to viable cell density in cells / mL. 3. Cell plating: The cell suspension was added to a 96-well cell culture plate at 50 μL / well. The culture plate was cultured in an incubator (37° C., 5% CO2) for 18 hours. 4. Sample addition: The test antibody-drug conjugate was co-incubated with Fab-pHrodo to form a complex, adjusted to a concentration of 10 nM, and added to the cells at 50 μL / well. 5. Cell culture: The culture plates were incubated in an incubator (37°C, 5% CO2) for 2, 4, 6, 24, or 48 hours. 6. Plate reading: When the corresponding time point was reached, the 96-well cell culture plate was taken out, Hoechst33342 was added to perform cell nucleus staining, and the plate was placed in operatta CLS for photography to read the cell number and the number of fluorescent dots of each cell. The average number of cell fluorescent dots was calculated using Harmony® imaging and analysis software.

[0379] [Table 14]

[0380] Conclusion: According to the results in Figure 10 and Table 12, the antibody-drug conjugate of the present invention exhibited uptake in MDA-MB-453 cells highly expressing HER3, and its endocytosis ability was stronger than that of the reference ADC-1.

[0381] Example 4. Inhibition of tumor cell proliferation by HER3-targeting antibody-drug conjugates in vitro the purpose The growth inhibitory activity of the antibody-drug conjugate of the present invention targeting HER3 against HCC1569 and MDA-MB-453 cells highly expressing HER3, and against MDA-MB-231 tumor cells not expressing HER3, was tested in vitro. The cells were treated in vitro with different concentrations of the antibody-drug conjugate, and after culturing for 6 days, the cell growth was detected using CTG reagent, and the in vitro activity of the compound was evaluated as the 50% growth inhibitory concentration (IC 50 ) values ​​were used to evaluate the

[0382] method 1. Cell culture: HCC1569, MDA-MB-453, or MDA-MB-231 cells were cultured in RPMI-1640 medium containing 10% FBS. 2. Preparation of cells: Log-phase HCC1569, MDA-MB-453, or MDA-MB-231 cells were harvested and washed once with PBS, and then digested with 2-3 mL of trypsin for 2-3 min. After the cells were completely digested, 10-15 mL of cell culture medium was added to elute the digested cells. The eluate was centrifuged at 1000 rpm for 5 min, and the supernatant was discarded. The obtained cells were resuspended in 10-20 mL of cell culture medium to obtain a single cell suspension. The cells were stained with trypan blue, and viable cells were counted. 3. Cell plating: HCC1569, MDA-MB-453, or MDA-MB-231 single cell suspensions were mixed thoroughly and cultured to a viable cell density of 6 × 10 4The cell suspension with the adjusted density was mixed well and added to a 96-well cell culture plate at 50 μL / well. The culture plate was cultured in an incubator (37° C., 5% CO2) for 18 hours. 4. Antibody-drug conjugate concentration: The antibody-drug conjugate was diluted 3-fold from 100 nM to 0.0152 nM, for a total of 9 concentrations. 5. Addition of samples: The samples prepared for detection at different concentrations were added to the culture plate, and two duplicate wells were set up for each sample. The culture plate was cultured in an incubator (37°C, 5% CO2) for 6 days. 6. Color development: The 96-well cell culture plate was removed and left to equilibrate to room temperature for 30 minutes. 50 μL / well of CTG reagent was added, the plate was shaken on an orbital shaker for 2 minutes, and incubated in the dark at room temperature for 10 minutes. 7. Plate reading: The 96-well cell culture plate was taken out and placed in a 2104 EnVision microplate reader, and the chemiluminescence was measured using the microplate reader.

[0383] Data analysis The inhibition rate (IR) of the detected compounds was calculated according to the following formula: IR(%)=(1-(RLU compound-RLU blank control) / (RLU vehicle control-RLU blank control))×100%. The inhibition rates of different concentrations of compounds were calculated using Microsoft Excel, and the data was processed and analyzed using Graphpad Prism 9.

[0384] [Table 15]

[0385] Conclusion: According to the results shown in Table 13 and Figure 11 (11A / 11B / 11C), the antibody-drug conjugate of the present invention has significant inhibitory activity against the proliferation of Her3-positive highly expressing cells HCC1569 and MDA-MB-453, which is stronger than that of the reference ADC-1, while it has no inhibitory activity against the proliferation of MDA-MB-231 cells that do not express HER3, and therefore has good selectivity.

[0386] Example 5. Evaluation of the efficacy of antibody drug conjugates in HCC1569 tumor-bearing mice the purpose The antitumor effect of the antibody-drug conjugate of the present invention is evaluated using a female NOD / SCID mouse model with subcutaneous xenografts of HCC1569 breast cancer cells. 1. Test Compounds and Materials Blank control (negative control group): PBS Reference ADC-1 (positive control group): 1 mg / kg ADC-1-X1 (treatment group): 1mg / kg ADC-1-X2 (treatment group): 1mg / kg 2. Preparation: All samples were diluted with PBS. 3. Experimental Animals NOD / SCID mice aged 8-10 weeks were purchased from Jiangsu GemPharmatech Co, Ltd. 4. Experimental Process 2 × 10 cells suspended in PBS / Matrigel (1:1) mixture (0.1 mL / mouse) 6 HCC1569 cells were inoculated subcutaneously into the right anterior scapula. Tumor growth was monitored. Tumors had an average volume of 100–150 mm. 3 When the tumors had grown to 100 mg / kg, the mice were randomly divided into groups based on tumor size and mouse weight, with six mice per group receiving treatment. The day of treatment for each group was designated day 0.

[0387] The administration was performed once a week via tail vein injection, and the experiment was terminated 21 days after administration. Tumor volume and weight were measured twice a week, and the data were recorded. After the experiment was completed, the mice were euthanized, and the tumor growth inhibition rate TGI% was calculated as follows: (TGI=(1-(Ti-T0) / (Vi-V0)). Ti: average tumor volume of the treatment group and positive control group on day i of administration; T0: average tumor volume of the treatment group and positive control group on day 0 of administration; Vi: average tumor volume of the negative control group on day i of administration; V0: average tumor volume of the negative control group on day 0 of administration.

[0388] Data analysis The data was analyzed using Excel 2016 statistical software, and the mean value was calculated as avg, the SD value as STDEV, and the P value for the difference between groups as TTEST.

[0389] The experimental results are shown in Table 14 and Figure 12. At the end of the experiment, the average tumor volume in the blank control group was 1078.32 mm 3 It was.

[0390] [Table 16]

[0391] Example 6. Evaluation of the efficacy of antibody-drug conjugates in Colo205 tumor-bearing mice the purpose To evaluate the antitumor effect of the antibody-drug conjugate of the present invention using BALB / c nude mice as test animals; To evaluate the efficacy of ADC-III-28 and reference ADC-2 in nude mice bearing Colo205 xenograft tumors after intraperitoneal injection. 1. Test Compounds and Materials Blank control (negative control group): PBS Reference ADC-1 (positive control group): 1 mg / kg ADC-1-X1 (treatment group): 1mg / kg ADC-1-X2 (treatment group): 1mg / kg 2. Preparation method: All were diluted with PBS. 3. Experimental Animals BALB / c nude mice aged 8–10 weeks (purchased from Jiangsu GemPharmatech Co, Ltd.). 4. Experimental Process Colo205 cells were inoculated into the right flank of mice at 1 × 10 7 (100 μL / mouse) was inoculated subcutaneously. 3 When the animals had grown to 100 mg / kg, they were randomly divided into groups, with 5 animals per group. The day of administration for each group was designated as day 0. Administration was performed via tail vein injection once every 2 weeks, and the experiment was terminated 22 days after administration. Tumor volume and weight were measured twice a week, and the data were recorded. After the experiment was completed, the mice were euthanized, and the tumor growth inhibition rate TGI% was calculated as follows: (TGI=(1-(Ti-T0) / (Vi-V0)). Ti: average tumor volume of the treatment group and positive control group on day i of administration; T0: average tumor volume of the treatment group and positive control group on day 0 of administration; Vi: average tumor volume of the negative control group on day i of administration; V0: average tumor volume of the negative control group on day 0 of administration.

[0392] The data was analyzed using Excel 2016 statistical software, and the mean value was calculated as avg, the SD value as STDEV, and the P value for the difference between groups as TTEST.

[0393] The experimental results are shown in Table 15 and Figure 13. At the end of the experiment, the mean tumor volume in the blank control group was 515.92 mm 3 It was.

[0394] [Table 17]

[0395] Conclusion: The antibody-drug conjugate of the present invention can significantly reduce tumor volume. Moreover, the antibody-drug conjugate of the present invention exhibited tumor suppression effects at lower doses than the reference ADC-1.

[0396] Example 7. Evaluation of the efficacy of antibody drug conjugates in NCI-H441 lung cancer cell tumor-bearing mice the purpose The antitumor effect of the antibody-drug conjugate of the present invention is evaluated in NCI-H441 tumor-bearing mice using male NU / NU nude mice as test animals. 1. Test Compounds and Materials Blank control (negative control group): saline Reference ADC-1 (positive control group): 0.3 mg / kg or 1 mg / kg ADC-1-X2 (treatment group): 0.3 mg / kg or 1 mg / kg 2. Preparation method: All were diluted with saline. 3. Test animals: 6-8 week old NU / NU nude mice (purchased from Vital River Laboratory Animal Technology Co.) 4. Experimental Process Test mice were 5 × 10 6 NCI-H441 cells were inoculated subcutaneously in the right dorsum and suspended in 0.1 mL of PBS and Matrigel (1:1). Tumor growth was monitored periodically. Tumors reached a mean volume of 115.24 mm. 3 When the mice had grown to 1000 mg / kg, they were randomly divided into groups according to tumor size and mouse weight, and six mice were administered per group. The administration day for each group was designated as day 0. Administration was performed once via tail vein injection, and the experiment was terminated 28 days after administration. Tumor volume and weight were measured twice a week, and the data were recorded. After the experiment was completed, the mice were euthanized, and the tumor growth inhibition rate TGI% was calculated as follows: (TGI=(1-(Ti-T0) / (Vi-V0)). Ti: average tumor volume of the treatment group and positive control group on day i of administration; T0: average tumor volume of the treatment group and positive control group on day 0 of administration; Vi: average tumor volume of the negative control group on day i of administration; V0: average tumor volume of the negative control group on day 0 of administration.

[0397] The data was analyzed using Excel 2016 statistical software, and the mean value was calculated as avg, the SD value as STDEV, and the P value for the difference between groups as TTEST.

[0398] The experimental results are shown in Table 16 and Figure 14. At the end of the experiment, the average tumor volume in the blank control group was 1138.85 mm 3 It was.

[0399] [Table 18]

[0400] Conclusion: The antibody-drug conjugate of the present invention can significantly reduce tumor volume, and the antibody-drug conjugate of the present invention has a better tumor-inhibiting effect than the reference ADC-1 under the same concentration.

[0401] Example 8. Evaluation of the efficacy of antibody-drug conjugates in mice bearing human lung cancer LU1542 subcutaneous xenograft tumors the purpose Female BALB / c nude mice are used as test animals to evaluate the antitumor effect of the antibody-drug conjugate of the present invention in mice bearing human lung cancer LU1542 subcutaneous xenograft tumors. 1. Test Compounds and Materials Blank control (negative control group): PBS Reference ADC-1 (positive control group): 3 mg / kg or 10 mg / kg ADC-1-X2 (treatment group): 3 mg / kg or 10 mg / kg 2. Preparation method: All were diluted with PBS. 3. Test animals: 6-8 week-old BALB / c nude mice (purchased from Beijing AniKeeper Biotech Co., Ltd.). 4. Experimental Process Tumor tissues were collected from mice bearing the HuPrime® lung cancer xenograft model LU1542, cut into tumor masses with a diameter of 2 to 3 mm, and then subcutaneously inoculated into the right anterior scapula of Balb / c nude mice. The average tumor volume of the tumor-bearing mice was 136.8 mm. 3When the mice reached the age of 18 days, they were randomly divided into groups, with 6 mice per group. The administration day for each group was designated as day 0. Administration was performed once via tail vein injection, and the experiment was terminated 28 days after administration. Tumor volume and weight were measured twice a week, and the data were recorded. After the experiment was completed, the mice were euthanized, and the tumor growth inhibition rate TGI% was calculated as follows: (TGI=(1-(Ti-T0) / (Vi-V0))). Ti: average tumor volume of the treatment group and positive control group on day i of administration; T0: average tumor volume of the treatment group and positive control group on day 0 of administration; Vi: average tumor volume of the negative control group on day i of administration; V0: average tumor volume of the negative control group on day 0 of administration.

[0402] The data was analyzed using Excel 2016 statistical software, and the mean value was calculated as avg, the SD value as STDEV, and the P value for the difference between groups as TTEST.

[0403] The experimental results are shown in Table 17 and Figure 15. At the end of the experiment, the average tumor volume in the blank control group was 1580.09 mm 3 It was.

[0404] [Table 19]

[0405] Conclusion: The antibody-drug conjugate of the present invention can significantly reduce tumor volume. Furthermore, the antibody-drug conjugate of the present invention exhibits tumor-inhibitory effect at a lower administration concentration than the reference ADC, and the antibody-drug conjugate of the present invention has a superior tumor-inhibitory effect at the same concentration.

[0406] [Table 20-1]

[0407] [Table 20-2]

Claims

1. An anti-Her3 antibody-drug conjugate, an isomer thereof, a pharma- ceutically acceptable salt thereof, or a mixture thereof, wherein the anti-Her3 antibody-drug conjugate has the formula (I): Ab-(L-M-D) ｐ (I) wherein: L and M are linker units; D is a cytotoxic drug; p represents the average number of connections, where p is selected from an integer or decimal number between 1 and 10, preferably an integer or decimal number between 3 and 8; Ab is an anti-Her3 antibody or antigen-binding fragment thereof, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 of the amino acid sequences shown in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, respectively, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3 of the amino acid sequences shown in SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, respectively, an anti-Her3 antibody-drug conjugate, an isomer thereof, a pharma- ceutically acceptable salt thereof, or a mixture thereof.

2. The anti-Her3 antibody-drug conjugate has the formula (I-1): 【Chemistry 1】 [During the ceremony, L and M are linker units; p represents the average number of connections, where p is selected from an integer or decimal number between 1 and 10, preferably an integer or decimal number between 3 and 8; Ab is an anti-Her3 antibody or antigen-binding fragment thereof, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 of the amino acid sequences shown in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, respectively, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3 of the amino acid sequences shown in SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, respectively. The anti-Her3 antibody-drug conjugate of claim 1, having the structure:

3. The anti-Her3 antibody or antigen-binding fragment thereof is (I) a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO: 7, or a heavy chain variable region having at least 95%, 96%, 97%, 98% or 99% identity thereto, and a light chain variable region having an amino acid sequence as set forth in SEQ ID NO: 8, or a light chain variable region having at least 95%, 96%, 97%, 98% or 99% identity thereto; or (II) a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO: 9, or a heavy chain variable region having at least 95%, 96%, 97%, 98%, or 99% identity thereto, and a light chain variable region having an amino acid sequence as set forth in SEQ ID NO: 8, or a light chain variable region having at least 95%, 96%, 97%, 98%, or 99% identity thereto; or (III) A heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO: 10, or a heavy chain variable region having at least 95%, 96%, 97%, 98%, or 99% identity thereto, and a light chain variable region having an amino acid sequence as set forth in SEQ ID NO: 8, or a light chain variable region having at least 95%, 96%, 97%, 98%, or 99% identity thereto. The anti-Her3 antibody-drug conjugate of claim 1 or 2, an isomer thereof, a pharma- ceutically acceptable salt thereof, or a mixture thereof.

4. The anti-Her3 antibody-drug conjugate, its isomer, its pharma- ceutically acceptable salt, or a mixture thereof according to claim 1 or 2, wherein the anti-Her3 antibody or its antigen-binding fragment is a murine antibody or a fragment thereof, a chimeric antibody or a fragment thereof, or a humanized antibody or a fragment thereof.

5. The anti-Her3 antibody or antigen-binding fragment thereof is selected from the group consisting of Fab, Fab', Fab'-SH, Fv, scFv, F(ab') ２ 3. The anti-Her3 antibody-drug conjugate of claim 1 or 2, which is selected from a sdAb, a bispecific antibody, or a linear antibody, its isomer, its pharma- ceutically acceptable salt, or a mixture thereof.

6. The anti-Her3 antibody-drug conjugate of claim 1 or 2, wherein the antibody is of type IgG1, IgG2, IgG3 or IgG4, or an isomer thereof, a pharma- ceutically acceptable salt thereof or a mixture thereof.

7. The anti-Her3 antibody or antigen-binding fragment thereof is (1) a heavy chain having an amino acid sequence as set forth in SEQ ID NO: 11, or a heavy chain having at least 95%, 96%, 97%, 98% or 99% identity thereto, and a light chain having an amino acid sequence as set forth in SEQ ID NO: 12, or a light chain having at least 95%, 96%, 97%, 98% or 99% identity thereto; or (2) a heavy chain having an amino acid sequence as set forth in SEQ ID NO: 13, or a heavy chain having at least 95%, 96%, 97%, 98% or 99% identity thereto, and a light chain having an amino acid sequence as set forth in SEQ ID NO: 12, or a light chain having at least 95%, 96%, 97%, 98% or 99% identity thereto; or (3) A heavy chain having the amino acid sequence shown in SEQ ID NO: 14, or a heavy chain having at least 95%, 96%, 97%, 98% or 99% identity thereto, and a light chain having the amino acid sequence shown in SEQ ID NO: 12, or a light chain having at least 95%, 96%, 97%, 98% or 99% identity thereto. The anti-Her3 antibody-drug conjugate according to any one of claims 1 to 6, an isomer thereof, a pharma- ceutically acceptable salt thereof, or a mixture thereof, comprising:

8. M is -L ２ -L ３ -XL １ - and L ２ is —O— or —S—; L ３ But -(C(R １ａ ) (R １ｂ )) ｍ -, m is selected from 0, 1, 2 or 3, where L ３ When contains a methylene unit, L ３ may be replaced by -C(O)- or -C(S)-; L １ But -(C(R ２ａ ) (R ２ｂ )) ｎ -, n is selected from 1, 2 or 3, where L １ When L may contain methylene units, １ may be replaced by -C(O)- or -C(S)-; X is selected from 3- to 6-membered saturated carbocyclyl, 3- to 6-membered saturated heterocyclyl, or a single bond, where 3- to 6-membered saturated carbocyclyl and 3- to 6-membered saturated heterocyclyl are selected from 0, 1, 2, or 3 R ３ａ optionally replaced by Here, each R １ａ , each R １ｂ , each R ２ａ , each R ２ｂ , and each R ３ａ is independently hydrogen, halogen, or C optionally substituted with R １－６ may be an aliphatic group, The anti-Her3 antibody-drug conjugate of any one of claims 1 to 7, wherein each R can independently be hydrogen or halogen, an isomer thereof, a pharma- ceutically acceptable salt thereof, or a mixture thereof.

9. L ３ is a single bond, -C(R １ａ ) (R １ｂ ) - or -C(R １ａ ) (R １ｂ ) C(R １ａ ) (R １ｂ ) - is selected from Here, each R １ａ And each R １ｂ is independently hydrogen, halogen, or C optionally substituted with R １－６ may be an aliphatic group, 9. The anti-Her3 antibody-drug conjugate of claim 8, wherein each R can independently be hydrogen or halogen, an isomer thereof, a pharma- ceutically acceptable salt thereof, or a mixture thereof.

10. L ３ is a single bond, -CH ２ -, -CH(CH ３ ) -, -C(CH ３ ) ２ --, --CH ２ CH ２ -, -CH(CH ３ ) CH ２ - or -C(CH ３ ) ２ 10. The anti-Her3 antibody-drug conjugate of claim 9, wherein the conjugate is selected from the group consisting of: CH-, an isomer thereof, a pharma- ceutically acceptable salt thereof, or a mixture thereof.

11. L １ But -C(R ２ａ ) (R ２ｂ ) -, -C(R ２ａ ) (R ２ｂ )C(O)—, or —C(O)—; Here, each R ２ａ And each R ２ｂ is independently hydrogen, halogen, or C optionally substituted with R １－６ may be an aliphatic group, 9. The anti-Her3 antibody-drug conjugate of claim 8, wherein each R can be independently hydrogen or halogen, an isomer thereof, a pharma- ceutically acceptable salt thereof, or a mixture thereof.

12. L １ But -CH ２ --, --CH ２ C(O)-, -CH(CH ３ 12. The anti-Her3 antibody-drug conjugate of claim 11, wherein the moiety is selected from -C(O)-, -C(O)-, -C(O)-, an isomer thereof, a pharma- ceutically acceptable salt thereof, or a mixture thereof.

13. X is 0, 1, 2, or 3 R ３ａ or a 3- to 6-membered saturated carbocyclyl optionally substituted with a single bond; Here, each R ３ａ is independently hydrogen, halogen, or C optionally substituted with R １－６ may be an aliphatic group, 9. The anti-Her3 antibody-drug conjugate of claim 8, wherein each R can be independently hydrogen or halogen, an isomer thereof, a pharma- ceutically acceptable salt thereof, or a mixture thereof.

14. The anti-Her3 antibody-drug conjugate, its isomer, its pharma- ceutically acceptable salt or a mixture thereof according to claim 13, wherein X is a 3- to 6-membered saturated carbocyclyl or a single bond.

15. -M-, 【Chemistry 2】 [During the ceremony, L ２ is —O— or —S—; X is 0, 1, 2 or 3 R ３ａ selected from 3-6 membered saturated carbocyclyl optionally substituted with L １ But -C(R ２ａ ) (R ２ｂ ) -, -C(R ２ａ ) (R ２ｂ )C(O)—, or —C(O)—; Here, each R ２ａ , each R ２ｂ , or each R ３ａ is independently hydrogen, halogen, or C optionally substituted with R １－６ may be an aliphatic group, Each R can be independently hydrogen or halogen. The anti-Her3 antibody-drug conjugate according to any one of claims 1 to 14, an isomer thereof, a pharma- ceutically acceptable salt thereof, or a mixture thereof, selected from the group consisting of:

16. -M-, 【Chemistry 3】 [During the ceremony, L ２ is —O— or —S—; X is selected from 3- to 6-membered saturated carbocyclyl. The anti-Her3 antibody-drug conjugate of claim 15, an isomer thereof, a pharma- ceutically acceptable salt thereof, or a mixture thereof, selected from the group consisting of

17. -M-, 【Chemistry 4】 [During the ceremony, L ２ is —O— or —S—; L ３ But -C(R １ａ ) (R １ｂ )-or-C(R １ａ ) (R １ｂ ) C(R １ａ ) (R １ｂ )-selected from; L １ But -C(R ２ａ ) (R ２ｂ ) -, -C(R ２ａ ) (R ２ｂ )C(O)—, or —C(O)—; Here, each R １ａ , each R １ｂ , each R ２ａ , or each R ２ｂ is independently hydrogen, halogen, or C optionally substituted with R １－６ may be an aliphatic group, The anti-Her3 antibody-drug conjugate of any one of claims 1 to 14, its isomer, its pharma- ceutically acceptable salt, or mixtures thereof, wherein each R can be independently hydrogen or halogen.

18. -M-, 【Chemistry 5】 [During the ceremony, L ２ is —O— or —S—; L ３ But -CH ２ -, -CH(CH ３ ), -C(CH ３ ) ２ --, --CH ２ CH ２ -, -CH(CH ３ ) CH ２ - or -C(CH ３ ) ２ CH ２ -, its isomer, its pharma- ceutically acceptable salt, or a mixture thereof, of claim 17.

19. -M-, 【Chemistry 6】 The anti-Her3 antibody-drug conjugate according to any one of claims 1 to 18, an isomer thereof, a pharma- ceutically acceptable salt thereof, or a mixture thereof, selected from the group consisting of:

20. L is -L ａ -L ｂ -L ｃ - and -L ａ -teeth, 【Chemistry 7】 [During the ceremony, W is -(C(R ｗａ ) (R ｗｂ )) ｗｎ - and Y is -(OCH ２ CH ２ ) ｙｎ -O ｙｐ - and Z is -(C(R ｚａ ) (R ｚｂ )) ｚｎ and where wn is 1, 2, 3, or 6; Each methylene unit of W is independently -Cyr-, -N(R ｗｘ )C(O)-, -C(O)N(R ｗｘ )- or -C(O)-; where yn is 0, 4 or 8 and yp is 0 or 1; where zn is 1, 2 or 3; Each methylene unit of Z is independently -Cyr-, -N(R ｚｘ )C(O)-, -C(O)N(R ｚｘ )- or -C(O)-; -Cyr- is a 3- to 10-membered saturated carbocyclylene, where -Cyr- is unsubstituted or independently represents 1 to 3 substituents R ｃｘ is replaced by; Here, each R ｗａ , each R ｗｂ , each R ｚａ , each R ｚｂ , each R ｗｘ , each R ｚｘ And each R ｃｘ are independently hydrogen, halogen, -OR ｒ , or R ｒ C optionally substituted with １－６ is an aliphatic group, Here, each R ｒ are independently hydrogen, halogen or C １－６ is an aliphatic group. and -L ｂ -teeth, 【Chemistry 8】 Selected from: -L ｃ -teeth, 【Chemistry 9】 [During the ceremony, R Ｌ１ and R Ｌ２ are each independently hydrogen, halogen, —OH, or C １－６ aliphatic groups] The anti-Her3 antibody-drug conjugate according to any one of claims 1 to 19, an isomer thereof, a pharma- ceutically acceptable salt thereof, or a mixture thereof, wherein

21. -L ａ -teeth, 【Chemistry 10】 and -L ｂ -teeth, 【Chemistry 11】 and -L ｃ -teeth, 【Chemistry 12】 The anti-Her3 antibody-drug conjugate of claim 20, wherein

22. L is, 【Chemistry 13】 The anti-Her3 antibody-drug conjugate according to any one of claims 1 to 21, an isomer thereof, a pharma- ceutically acceptable salt thereof, or a mixture thereof,

23. L is, 【Chemistry 14】 The anti-Her3 antibody-drug conjugate according to any one of claims 1 to 21, an isomer thereof, a pharma- ceutically acceptable salt thereof, or a mixture thereof,

24. The anti-Her3 antibody-drug conjugate has the structure of formula (II-1) or (II-2): 【Chemistry 15】 [During the ceremony, p is as defined in claim 1; Ab is as defined in any one of claims 1 to 7; L ２ is —O— or —S—; X is 0, 1, 2 or 3 R ３ａ selected from 3- to 6-membered saturated carbocyclyl optionally substituted with L ３ is -C(R １ａ ) (R １ｂ )-or-C(R １ａ ) (R １ｂ ) C(R １ａ ) (R １ｂ ) - is selected from Here, each R １ａ , each R １ｂ , or each R ３ａ is independently hydrogen, halogen, or C optionally substituted with R １－６ may be an aliphatic group, where each R can independently be hydrogen or halogen. The anti-Her3 antibody-drug conjugate according to any one of claims 1 to 23, having the structure:

25. The anti-Her3 antibody-drug conjugate has the following structural formula: 【Chemistry 16】 【Chemistry 17】 【Chemistry 18】 【Chemistry 19】 【Chemistry 20】 【Chemistry 21】 [During the ceremony, p is as defined in claim 1; Ab is as defined in any one of claims 1 to 7. The anti-Her3 antibody-drug conjugate according to any one of claims 1 to 24, an isomer thereof, a pharma- ceutically acceptable salt thereof, or a mixture thereof, selected from the group consisting of:

26. An anti-Her3 antibody-drug conjugate, an isomer thereof, a pharma- ceutically acceptable salt thereof, or a mixture thereof, wherein the anti-Her3 antibody-drug conjugate is 【Chemical 22】 【Chemistry 23】 【Chemistry 24】 【Chemistry 25】 【Chemistry 26】 【Chemical 27】 【Chemistry 28】 [During the ceremony, p represents the average number of connections, and p is selected from an integer or decimal number of 1 to 10, preferably an integer or decimal number of 3 to 8. an anti-Her3 antibody-drug conjugate selected from the group consisting of an isomer thereof, a pharma- ceutically acceptable salt thereof, or a mixture thereof.

27. A pharmaceutical composition comprising the anti-Her3 antibody-drug conjugate according to any one of claims 1 to 26, an isomer thereof, a pharma- ceutically acceptable salt thereof, or a mixture thereof, and a pharma- ceutically acceptable carrier or excipient.

28. Use of an anti-Her3 antibody-drug conjugate according to any one of claims 1 to 26, its isomer, its pharma- ceutically acceptable salt, or a mixture thereof, or a pharmaceutical composition according to claim 27, in the preparation of a medicament for treating and / or preventing a disease or condition mediated by Her3, wherein preferably the disease or condition is cancer; more preferably, the cancer is selected from lung cancer, renal cancer, urinary tract cancer, colorectal cancer, prostate cancer, glioblastoma multiforme, ovarian cancer, pancreatic cancer, breast cancer, melanoma, liver cancer, bladder cancer, gastric cancer, and esophageal cancer.

29. A method for treating and / or preventing a Her3-mediated disease or condition, comprising administering to a subject in need thereof an anti-Her3 antibody-drug conjugate, its isomer, its pharma- ceutically acceptable salt, or a mixture thereof, according to any one of claims 1 to 26, or a pharmaceutical composition according to claim 27, wherein preferably the disease or condition is cancer; more preferably, the cancer is selected from lung cancer, renal cancer, urinary tract cancer, colorectal cancer, prostate cancer, glioblastoma multiforme, ovarian cancer, pancreatic cancer, breast cancer, melanoma, liver cancer, bladder cancer, gastric cancer, and esophageal cancer.

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