Antibodies, conjugates and uses thereof directed against Her3

Abstract

The present invention relates to anti-Her3 antibodies, methods for preparing the same, conjugates thereof and uses thereof, in particular, antibodies and conjugates thereof that bind to Her3 and exhibit anti-tumor activity. The present invention further includes compositions of the antibodies and ADCs, and methods of using them.

Description

[Technical field]

[0001] The present disclosure is in the field of therapeutic biopharmaceuticals, and more specifically, the present disclosure relates to antibodies, conjugates against Her3, and further to the use of the antibodies or conjugates for the treatment of disease. [Background technology]

[0002] Her3 (ErbB3) is a transmembrane receptor tyrosine kinase molecule of the ERBB family, which includes four members: EGFR, Her2, Her3, and Her4. Activation of ERBB receptors requires the formation of homo- or heterodimers, and the family members can dimerize with each other. In the absence of ligand, Her3 is locked into a fixed conformation by interacting with cysteine-rich regions, and mutations in non-conserved amino acids in the intracellular protein kinase domain result in Her3 only weak kinase activity, and heterodimer formation with other EGFR family molecules is required to mediate signal transduction. Her3 plays an important role in embryonic development, and the absence of Her3 in mice can result in severe hypoplasia of the atrioventricular valves and cause embryonic death in mice. This effect is mainly determined by its NRG1 ligand. In addition, Her3 plays an important role in the differentiation of neural crest (e.g., Schwann cells) and the development of the sympathetic nervous system. Her3 is mutated or overexpressed in a variety of cancers, including several types of adenocarcinomas (breast, urethral, ​​gastric, colon, bile duct, bladder, endometrial, ovarian, uterine, prostate, and lung cancers), as well as melanomas and gliomas.

[0003] As antibody-drug conjugates (ADCs), biologically active small molecule compounds are conjugated with monoclonal antibodies or antibody fragments by chemical methods to take full advantage of the binding specificity of antibodies to surface antigens of normal and tumor cells, and the high antitumor biological activity of small molecules, while avoiding the drawbacks, such as the low specific efficacy of the former and the toxic side effects of the latter.Compared with traditional chemotherapy drugs or targeted drugs, antibody-drug conjugates can bind to tumor cells more precisely and reduce their effects on normal cells. Currently, patents have been reported on Her3 antibodies and ADC drugs, such as U3-1402 developed by Daiichi Sankyo and MP-Her3-ADC developed by MediaPharma. However, although antibody-drug conjugates (ADCs) combine the advantages of the target specificity and targeting properties of antibodies to target cells and the tumoricidal ability of administered drugs, the structural complexity of ADC drug molecules still causes the problem of insufficient safety margin in practical application.Current Her3 ADCs are still in early development stage, therefore, there is still an urgent need for safer and more effective Her3 antibodies or antibody-drug conjugates to meet unmet clinical needs. Summary of the Invention

[0004] In the present disclosure, a Her3 molecule binding domain comprising a light chain variable region (VL) and / or a heavy chain variable region (VH) was first developed, and a human antibody was further developed by using the above light chain variable region and / or heavy chain variable region. The antibody included in the present disclosure may be a fully human antibody and can be safely administered to human subjects without causing an immunogenic reaction. It has high binding ability to human and non-human Her3 molecules, including those that have cross-reactivity with Her3 from non-human primates (e.g. monkeys) and mammals (e.g. rats). The present disclosure also relates to antibody-drug conjugates (ADCs) that include Her3 antibodies. The antibodies or antibody-drug conjugates (ADCs) of the present disclosure have significant clinical value.

[0005] Antibodies of the present disclosure In a first aspect, the disclosure provides an antibody or antigen-binding fragment thereof that binds to Her3, the antibody or antigen-binding fragment thereof comprising the complementarity determining regions (CDRs) shown below: HCDR1 or a mutant form of its sequence, HCDR2 or a mutant form of its sequence, and HCDR3 or a mutant form of its sequence contained in the heavy chain variable region (VH) shown in SEQ ID NO: 23, 25, or 27, and / or LCDR1 or a mutant form of its sequence, LCDR2 or a mutant form of its sequence, and LCDR3 or a mutant form of its sequence contained in the light chain variable region (VL) shown in SEQ ID NO: 24, 26, or 28. Includes.

[0006] In some embodiments, the antibody or antigen-binding fragment thereof comprises a mutant form of HCDR1 or its sequence, a mutant form of HCDR2 or its sequence, and a mutant form of HCDR3 or its sequence contained in the VH shown in SEQ ID NO: 23, and / or a mutant form of LCDR1 or its sequence, a mutant form of LCDR2 or its sequence, and a mutant form of LCDR3 or its sequence contained in the VL shown in SEQ ID NO: 24. In some embodiments, the antibody or antigen-binding fragment thereof comprises a mutant form of HCDR1 or its sequence, a mutant form of HCDR2 or its sequence, and a mutant form of HCDR3 or its sequence contained in the VH shown in SEQ ID NO: 25, and / or a mutant form of LCDR1 or its sequence, a mutant form of LCDR2 or its sequence, and a mutant form of LCDR3 or its sequence contained in the VL shown in SEQ ID NO: 26. In some embodiments, the antibody or antigen-binding fragment thereof comprises a mutant form of HCDR1 or its sequence, a mutant form of HCDR2 or its sequence, and a mutant form of HCDR3 or its sequence contained in the VH shown in SEQ ID NO: 27, and / or a mutant form of LCDR1 or its sequence, a mutant form of LCDR2 or its sequence, and a mutant form of LCDR3 or its sequence contained in the VL shown in SEQ ID NO: 28.

[0007] In certain preferred embodiments, a sequence variant is a CDR that has one or several amino acid substitutions, deletions or additions (e.g. one, two or three amino acid substitutions, deletions or additions) compared to the CDR from which it is derived. In certain preferred embodiments, the substitutions are conservative substitutions. Preferably, the CDRs are defined according to the AbM, Chothia, Kabat, or IMGT numbering systems. In one aspect, the disclosure provides an antibody or antigen-binding fragment thereof capable of binding to Her3, the antibody or antigen-binding fragment thereof comprising a heavy chain variable region (VH) and / or a light chain variable region (VL).

[0008] In some embodiments, an antibody or antigen-binding fragment thereof of the disclosure comprises a heavy chain variable region (VH) and / or a light chain variable region (VL) as shown below, wherein the CDRs are aligned according to the Kabat numbering system: (a) a heavy chain variable region (VH) comprising the following three CDRs: HCDR1 consisting of the sequence of SEQ ID NO: 1 or a sequence having one or several amino acid substitutions, deletions or additions (e.g., one, two or three amino acid substitutions, deletions or additions) compared to SEQ ID NO: 1; HCDR2 consisting of the sequence of SEQ ID NO: 2 or a sequence having one or several amino acid substitutions, deletions or additions (e.g., one, two or three amino acid substitutions, deletions or additions) compared to SEQ ID NO: 2; HCDR3 consisting of the sequence of SEQ ID NO: 3 or a sequence having one or several amino acid substitutions, deletions or additions (e.g., one, two or three amino acid substitutions, deletions or additions) compared to SEQ ID NO: 3; and / or A light chain variable region (VL) comprising the following three CDRs: LCDR1 consisting of the sequence of SEQ ID NO: 7 or a sequence having one or several amino acid substitutions, deletions or additions (e.g., one, two or three amino acid substitutions, deletions or additions) compared to SEQ ID NO: 7; LCDR2 consisting of the sequence of SEQ ID NO: 8 or a sequence having one or several amino acid substitutions, deletions or additions (e.g., one, two or three amino acid substitutions, deletions or additions) compared to SEQ ID NO: 8; LCDR3 consisting of the sequence of SEQ ID NO: 9 or a sequence having one or several amino acid substitutions, deletions or additions (e.g., one, two or three amino acid substitutions, deletions or additions) compared to SEQ ID NO: 9; (b) a heavy chain variable region (VH) comprising the following three CDRs: HCDR1 consisting of the sequence of SEQ ID NO: 12 or a sequence having one or several amino acid substitutions, deletions or additions (e.g., one, two or three amino acid substitutions, deletions or additions) compared to SEQ ID NO: 12; HCDR2 consisting of the sequence of SEQ ID NO: 13 or a sequence having one or several amino acid substitutions, deletions or additions (e.g., one, two or three amino acid substitutions, deletions or additions) compared to SEQ ID NO: 13; HCDR3 consisting of the sequence of SEQ ID NO: 14 or a sequence having one or several amino acid substitutions, deletions or additions (e.g., one, two or three amino acid substitutions, deletions or additions) compared to SEQ ID NO: 14; and / or It is defined by a light chain variable region (VL) comprising the following three CDRs: LCDR1 consisting of the sequence of SEQ ID NO: 18 or a sequence having one or several amino acid substitutions, deletions or additions (e.g., one, two or three amino acid substitutions, deletions or additions) compared to SEQ ID NO: 18; LCDR2 consisting of the sequence of SEQ ID NO: 19 or a sequence having one or several amino acid substitutions, deletions or additions (e.g., one, two or three amino acid substitutions, deletions or additions) compared to SEQ ID NO: 19; and LCDR3 consisting of the sequence of SEQ ID NO: 20 or a sequence having one or several amino acid substitutions, deletions or additions (e.g., one, two or three amino acid substitutions, deletions or additions) compared to SEQ ID NO: 20.

[0009] In some embodiments, an antibody or antigen-binding fragment thereof of the disclosure comprises a heavy chain variable region (VH) and / or a light chain variable region (VL) as shown below, wherein the CDRs are aligned according to the Kabat numbering system: (a) a heavy chain variable region (VH) comprising the following three CDRs: HCDR1 consisting of the sequence of SEQ ID NO: 1, HCDR2 consisting of the sequence of SEQ ID NO: 2, and HCDR3 consisting of the sequence of SEQ ID NO: 3; and / or A light chain variable region (VL) comprising the following three CDRs: LCDR1 consisting of the sequence of SEQ ID NO: 7, LCDR2 consisting of the sequence of SEQ ID NO: 8, and LCDR3 consisting of the sequence of SEQ ID NO: 9; or (b) a heavy chain variable region (VH) comprising the following three CDRs: HCDR1 consisting of the sequence of SEQ ID NO: 12, HCDR2 consisting of the sequence of SEQ ID NO: 13, and HCDR3 consisting of the sequence of SEQ ID NO: 14; and / or It is defined by a light chain variable region (VL) comprising the following three CDRs: LCDR1 consisting of the sequence of SEQ ID NO: 18, LCDR2 consisting of the sequence of SEQ ID NO: 19, and LCDR3 consisting of the sequence of SEQ ID NO: 20.

[0010] In some embodiments, an antibody or antigen-binding fragment thereof of the disclosure comprises a heavy chain variable region (VH) and / or a light chain variable region (VL) as shown below, wherein the CDRs are represented by the IMGT numbering system: (a) a heavy chain variable region (VH) comprising the following three CDRs: HCDR1 consisting of the sequence of SEQ ID NO: 4 or a sequence having one or several amino acid substitutions, deletions or additions (e.g., one, two or three amino acid substitutions, deletions or additions) compared to SEQ ID NO: 4; HCDR2 consisting of the sequence of SEQ ID NO: 5 or a sequence having one or several amino acid substitutions, deletions or additions (e.g., one, two or three amino acid substitutions, deletions or additions) compared to SEQ ID NO: 5; HCDR3 consisting of the sequence of SEQ ID NO: 6 or a sequence having one or several amino acid substitutions, deletions or additions (e.g., one, two or three amino acid substitutions, deletions or additions) compared to SEQ ID NO: 6; and / or A light chain variable region (VL) comprising the following three CDRs: LCDR1 consisting of the sequence of SEQ ID NO: 10 or a sequence having one or several amino acid substitutions, deletions or additions (e.g., one, two or three amino acid substitutions, deletions or additions) compared to SEQ ID NO: 10; LCDR2 consisting of the sequence of AAS or a sequence having one or several amino acid substitutions, deletions or additions (e.g., one, two or three amino acid substitutions, deletions or additions) compared to AAS; LCDR3 consisting of the sequence of SEQ ID NO: 9 or a sequence having one or several amino acid substitutions, deletions or additions (e.g., one, two or three amino acid substitutions, deletions or additions) compared to SEQ ID NO: 9; or (b) a heavy chain variable region (VH) comprising the following three CDRs: HCDR1 consisting of the sequence of SEQ ID NO: 15 or a sequence having one or several amino acid substitutions, deletions or additions (e.g., one, two or three amino acid substitutions, deletions or additions) compared to SEQ ID NO: 15; HCDR2 consisting of the sequence of SEQ ID NO: 16 or a sequence having one or several amino acid substitutions, deletions or additions (e.g., one, two or three amino acid substitutions, deletions or additions) compared to SEQ ID NO: 16; HCDR3 consisting of the sequence of SEQ ID NO: 17 or a sequence having one or several amino acid substitutions, deletions or additions (e.g., one, two or three amino acid substitutions, deletions or additions) compared to SEQ ID NO: 17; and / or It is defined by a light chain variable region (VL) comprising the following three CDRs: LCDR1 consisting of the sequence of SEQ ID NO: 21 or a sequence having one or several amino acid substitutions, deletions or additions (e.g., one, two or three amino acid substitutions, deletions or additions) compared to SEQ ID NO: 21; LCDR2 consisting of the sequence of AAS or a sequence having one or several amino acid substitutions, deletions or additions (e.g., one, two or three amino acid substitutions, deletions or additions) compared to AAS; and LCDR3 consisting of the sequence of SEQ ID NO: 20 or a sequence having one or several amino acid substitutions, deletions or additions (e.g., one, two or three amino acid substitutions, deletions or additions) compared to SEQ ID NO: 20.

[0011] In some embodiments, an antibody or antigen-binding fragment thereof of the disclosure comprises a heavy chain variable region (VH) and / or a light chain variable region (VL) as shown below, wherein the CDRs are represented by the IMGT numbering system: (a) a heavy chain variable region (VH) comprising the following three CDRs: HCDR1 consisting of the sequence of SEQ ID NO: 4, HCDR2 consisting of the sequence of SEQ ID NO: 5, and HCDR3 consisting of the sequence of SEQ ID NO: 6; and / or A light chain variable region (VL) comprising the following three CDRs: LCDR1 consisting of the sequence of SEQ ID NO: 10, LCDR2 consisting of the sequence of AAS, and LCDR3 consisting of the sequence of SEQ ID NO: 9; (b) a heavy chain variable region (VH) comprising the following three CDRs: HCDR1 consisting of the sequence of SEQ ID NO: 15, HCDR2 consisting of the sequence of SEQ ID NO: 16, and HCDR3 consisting of the sequence of SEQ ID NO: 17; and / or It is defined by a light chain variable region (VL) comprising the following three CDRs: LCDR1 consisting of the sequence of SEQ ID NO:21, LCDR2 consisting of the sequence of AAS, and LCDR3 consisting of the sequence of SEQ ID NO:20.

[0012] In certain embodiments, an antibody or antigen-binding fragment thereof of the present disclosure comprises a heavy chain variable region (VH) and / or a light chain variable region (VL) as defined below, and at least one CDR of the heavy chain variable region (VH) and / or light chain variable region (VL) comprises a mutation compared to the CDR defined by the Kabat or IMGT numbering system as described above, which mutation is a substitution, deletion or addition of one or several amino acids or any combination thereof (substitution, deletion or addition of one, two or three amino acids or any combination thereof), and still has Her3 binding activity. Preferably, the substitutions described in this disclosure are conservative substitutions.

[0013] In certain embodiments, the antibody or antigen-binding fragment thereof binds to human Her3, monkey Her3, and / or rat Her3. In certain embodiments, the VH of the antibody or antigen-binding fragment thereof of the present disclosure comprises a framework region (FR) derived from a heavy chain variable region (VH) of a human immunoglobulin, and / or the VL of the antibody or antigen-binding fragment thereof comprises a framework region (FR) derived from a light chain variable region (VL) of a human immunoglobulin. Thus, in certain embodiments, the antibody or antigen-binding fragment thereof of the present disclosure is fully humanized. In certain embodiments, the antibody or antigen-binding fragment thereof of the present disclosure is humanized.

[0014] In certain embodiments, an antibody or antigen-binding fragment thereof of the present disclosure comprises: (a) a heavy chain framework region of a human immunoglobulin or variant thereof, wherein the variant has up to 20 conservative substitutions of amino acids (e.g. up to 20, up to 15, up to 10 or up to 5 conservative substitutions of amino acids, e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 conservative substitutions of amino acids) compared to the amino acid sequence encoded by the germline antibody gene from which it is derived, and / or (b) a light chain framework region of a human immunoglobulin or variant thereof, wherein the variant has up to 20 conservative substitutions of amino acids (e.g., up to 20, up to 15, up to 10 or up to 5 conservative substitutions of amino acids, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 conservative substitutions of amino acids) compared to the amino acid sequence encoded by the germline antibody gene from which it is derived.

[0015] In certain embodiments, the degree of humanization of an antibody or antigen-binding fragment thereof of the disclosure is at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%.

[0016] In certain embodiments, an antibody or antigen-binding fragment thereof of the present disclosure comprises: (a) a heavy chain variable region (VH) comprising an amino acid sequence selected from the following: (i) a sequence as set forth in SEQ ID NO: 23, 25, or 27; (ii) a sequence having one or several substitutions, deletions or additions, or any combination thereof, compared to the sequence shown in SEQ ID NO: 23, 25, or 27 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, deletions or additions, or any combination thereof); or (iii) a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the sequence set forth in SEQ ID NO: 23, 25, or 27; and / or (b) a light chain variable region (VL) comprising an amino acid sequence selected from the following: (iv) a sequence as set forth in SEQ ID NO: 24, 26, or 28; (v) a sequence having one or several substitutions, deletions or additions, or any combination thereof, compared to the sequence shown in SEQ ID NO: 24, 26, or 28 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, deletions or additions, or any combination thereof); or (vi) a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the sequence set forth in SEQ ID NO: 24, 26, or 28. Includes.

[0017] In certain embodiments, an antibody or antigen-binding fragment thereof of the disclosure comprises a VH set forth in SEQ ID NO:23 and / or a VL set forth in SEQ ID NO:24. In certain embodiments, an antibody or antigen-binding fragment thereof of the disclosure comprises a VH set forth in SEQ ID NO:25 and / or a VL set forth in SEQ ID NO:26. In certain embodiments, an antibody or antigen-binding fragment thereof of the disclosure comprises a VH set forth in SEQ ID NO:27 and / or a VL set forth in SEQ ID NO:28.

[0018] In certain embodiments, an antibody or antigen-binding fragment thereof of the present disclosure comprises: (a) a VH consisting of the sequence shown in SEQ ID NO: 23, and a VL consisting of the sequence shown in SEQ ID NO: 24; (b) a VH consisting of the sequence shown in SEQ ID NO: 25, and a VL consisting of the sequence shown in SEQ ID NO: 26; (c) a VH consisting of the sequence shown in SEQ ID NO: 27, and a VL consisting of the sequence shown in SEQ ID NO: 28; (d) a heavy chain variable region (VH) and a light chain variable region (VL) that have at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity, respectively, when compared independently with the VH and VL of any one of (a) to (c); or (e) a heavy chain variable region (VH) and a light chain variable region (VL) which independently have one or several amino acid substitutions, deletions or additions, or any combination thereof (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, deletions or additions, or any combination thereof), compared to the VH and VL of any one of (a) to (c), respectively; Preferably, the substitutions are conservative substitutions.

[0019] In certain embodiments, the present disclosure also provides an anti-Her3 antibody or antigen-binding fragment thereof that competes for binding with an antibody or antigen-binding fragment thereof defined above, such as antibody 202-2-1 or antigen-binding fragment thereof, characterized in that the antibody or antigen-binding fragment thereof is different from the antibody or antigen-binding fragment thereof with which it competes, e.g., it is not antibody 202-2-1 or antigen-binding fragment thereof.

[0020] In some embodiments, an anti-Her3 antibody or antigen-binding fragment thereof competes for binding with antibody 202-2-1, is not antibody 202-2-1 or an antigen-binding fragment thereof, and is characterized in that it has different CDRs than antibody 202-2-1 or an antigen-binding fragment thereof. In certain embodiments, an antibody of the disclosure binds to domain 3 of human Her3, e.g., to one or more amino acids in domain 3. Thus, in certain embodiments, the disclosure provides an antibody or antigen-binding fragment thereof that binds to HER3, where the antibody or antigen-binding fragment thereof binds to domain 3 of human Her3, preferably amino acids 328-499 of human Her3 as set forth in SEQ ID NO:31.

[0021] In some embodiments, the disclosure provides an antibody or antigen-binding fragment thereof that binds to HER3, where the antibody or antigen-binding fragment thereof binds to the following spatial epitopes: histidine at position 466, tryptophan at position 470, threonine at position 471, threonine at position 478, (aspartic acid-isoleucine-lysine-histidine-asparagine) at positions 483 to 487, isoleucine at position 484, arginine at position 490, and arginine at position 491 of human Her3 as set forth in SEQ ID NO:31. Anti-Her3 antibodies or antigen-binding fragments thereof that compete for binding with antibody 202-2-1 bind to the following spatial epitopes: histidine at position 466, tryptophan at position 470, threonine at position 471, threonine at position 478, (aspartic acid-isoleucine-lysine-histidine-asparagine) at positions 483 to 487, isoleucine at position 484, arginine at position 490, and arginine at position 491 of Her3 as shown in SEQ ID NO:31.

[0022] In certain embodiments, the antibodies or antigen-binding fragments thereof disclosed herein block ligand-dependent and / or independent signaling of Her3. In certain embodiments, the antibodies or antigen-binding fragments thereof disclosed herein inhibit ligand-dependent and / or independent AKT phosphorylation of Her3. In certain embodiments, the antibodies or antigen-binding fragments thereof disclosed herein inhibit ligand-dependent and / or independent Her3 / Her2 heterodimer formation.

[0023] In certain embodiments, the antibody of the present disclosure is a chimeric antibody, a humanized antibody, or a fully human antibody. In certain embodiments, the antibody of the present disclosure or an antigen-binding fragment thereof is selected from a Fab fragment, a Fab' fragment, a (Fab')2 fragment, an Fv fragment, such as an scFv or a disulfide-linked Fv (dsFv), a diabody, and a multispecific antibody (e.g., a bispecific antibody). In certain embodiments, the antibody of the present disclosure is an scFv.

[0024] In certain embodiments, an antibody or antigen-binding fragment thereof of the disclosure comprises a heavy chain constant region (CH) of a human immunoglobulin or variant thereof, wherein the variant has up to 50 amino acid conservative substitutions (e.g., up to 45, up to 40, up to 35, up to 30, up to 25, up to 20, up to 15, up to 10 or up to 5 amino acid conservative substitutions, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid conservative substitutions) compared to the wild-type sequence from which it is derived. In certain embodiments, an antibody or antigen-binding fragment thereof of the disclosure comprises a light chain constant region (CL) of a human immunoglobulin or variant thereof, where the variant has up to 50 amino acid conservative substitutions (e.g., up to 45, up to 40, up to 35, up to 30, up to 25, up to 20, up to 15, up to 10 or up to 5 amino acid conservative substitutions, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid conservative substitutions) compared to the wild-type sequence from which it is derived.

[0025] In some embodiments, the constant region is altered, e.g., mutated, to improve the properties of the anti-Her3 antibody molecule (e.g., to change one or more of the following properties: Fc receptor binding, antibody glycosylation, number of cysteine ​​residues, effector cell function, or complement function). The altered function can be achieved by substituting at least one amino acid residue in the constant region of the antibody with a different residue, e.g., altering the affinity of the antibody for an effector ligand (e.g., FcR or complement C1q), thereby altering (e.g., decreasing) the effector function. The Fc region of an antibody mediates several important effector functions, e.g., ADCC, phagocytosis (ADCP), CDC, etc. In certain embodiments, the antibody or antigen-binding fragment thereof of the present disclosure has a heavy chain constant region (CH) selected from, for example, IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD, and IgE heavy chain constant regions, particularly, for example, IgG1, IgG2, IgG3, and IgG4 heavy chain constant regions, and more particularly, IgG1 (e.g., human IgG1) heavy chain constant region. In some embodiments, the human IgG1 heavy chain constant region has the sequence set forth in SEQ ID NO: 29. In some embodiments, the antibody or antigen-binding fragment thereof of the present disclosure has a light chain constant region selected from, for example, a kappa or lambda light chain constant region, preferably a kappa light chain constant region (e.g., a human kappa light chain constant region). In some embodiments, the light chain constant region has the sequence set forth in SEQ ID NO: 30.

[0026] In some embodiments, the antibody or antigen-binding fragment thereof has a CH as set forth in SEQ ID NO:29 or a variant thereof, which variant has up to 20 conservative amino acid substitutions compared to SEQ ID NO:29 (e.g. up to 20, up to 15, up to 10 or up to 5 conservative amino acid substitutions, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 conservative amino acid substitutions), or at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity compared to SEQ ID NO:29. In some embodiments, the antibody or antigen-binding fragment thereof has a light chain constant region or variant thereof. In some embodiments, the light chain constant region comprises a kappa light chain constant region. In some embodiments, the light chain constant region comprises a light chain constant region (CL) as set forth in SEQ ID NO:30 or a variant thereof, the variant having up to 20 amino acid conservative substitutions (e.g. up to 20, up to 15, up to 10 or up to 5 amino acid conservative substitutions, e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid conservative substitutions) compared to SEQ ID NO:30, or at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity compared to SEQ ID NO:30.

[0027] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain constant region (CH) set forth in SEQ ID NO:29, and a light chain constant region (CL) set forth in SEQ ID NO:30. In some embodiments, an antibody or antigen-binding fragment thereof of the present disclosure comprises: (a) a heavy chain comprising an amino acid sequence selected from the following: (i) a sequence comprising the VH sequence shown in SEQ ID NO: 23 and the CH sequence shown in SEQ ID NO: 29; (ii) a sequence which has one or several amino acid substitutions, deletions or additions or any combination thereof compared to the sequence shown in (i) (e.g. up to 50, up to 45, up to 40, up to 35, up to 30, up to 25, up to 20, up to 15, up to 10 or up to 5 amino acid substitutions, deletions or additions or any combination thereof, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, deletions or additions or any combination thereof); or (iii) a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the sequence set forth in (i); and (b) a light chain comprising an amino acid sequence selected from the following: (iv) a sequence comprising the VL sequence shown in SEQ ID NO: 24 and the CL sequence shown in SEQ ID NO: 30; (v) a sequence which has one or several amino acid substitutions, deletions or additions or any combination thereof compared to the sequence shown in (iv) (e.g. up to 50, up to 45, up to 40, up to 35, up to 30, up to 25, up to 20, up to 15, up to 10 or up to 5 amino acid substitutions, deletions or additions or any combination thereof, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, deletions or additions or any combination thereof); or (vi) a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the sequence shown in (iv); Includes.

[0028] In certain embodiments, the substitutions described in (ii) or (v) are conservative substitutions. In certain embodiments, an antibody or antigen-binding fragment thereof of the present disclosure comprises: (a) a heavy chain comprising an amino acid sequence selected from the following: (i) a sequence comprising the VH sequence shown in SEQ ID NO: 25 and the CH sequence shown in SEQ ID NO: 29; (ii) a sequence which has one or several amino acid substitutions, deletions or additions or any combination thereof compared to the sequence shown in (i) (e.g. up to 50, up to 45, up to 40, up to 35, up to 30, up to 25, up to 20, up to 15, up to 10 or up to 5 amino acid substitutions, deletions or additions or any combination thereof, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, deletions or additions or any combination thereof); or (iii) a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the sequence set forth in (i); and (b) a light chain comprising an amino acid sequence selected from the following: (iv) a sequence comprising the VL sequence shown in SEQ ID NO: 26 and the CL sequence shown in SEQ ID NO: 30; (v) a sequence which has one or several amino acid substitutions, deletions or additions or any combination thereof compared to the sequence shown in (iv) (e.g. up to 50, up to 45, up to 40, up to 35, up to 30, up to 25, up to 20, up to 15, up to 10 or up to 5 amino acid substitutions, deletions or additions or any combination thereof, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, deletions or additions or any combination thereof); or (vi) a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the sequence shown in (iv); Includes. In certain embodiments, the substitutions described in (ii) or (v) are conservative substitutions.

[0029] In certain embodiments, an antibody or antigen-binding fragment thereof of the present disclosure comprises: (a) a heavy chain comprising an amino acid sequence selected from the following: (i) a sequence comprising the VH sequence shown in SEQ ID NO: 27 and the CH sequence shown in SEQ ID NO: 29; (ii) a sequence which has one or several amino acid substitutions, deletions or additions or any combination thereof compared to the sequence shown in (i) (e.g. up to 50, up to 45, up to 40, up to 35, up to 30, up to 25, up to 20, up to 15, up to 10 or up to 5 amino acid substitutions, deletions or additions or any combination thereof, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, deletions or additions or any combination thereof); or (iii) a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the sequence set forth in (i); and (b) a light chain comprising an amino acid sequence selected from the following: (iv) a sequence comprising the VL sequence shown in SEQ ID NO: 28 and the CL sequence shown in SEQ ID NO: 30; (v) a sequence which has one or several amino acid substitutions, deletions or additions or any combination thereof compared to the sequence shown in (iv) (e.g. up to 50, up to 45, up to 40, up to 35, up to 30, up to 25, up to 20, up to 15, up to 10 or up to 5 amino acid substitutions, deletions or additions or any combination thereof, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, deletions or additions or any combination thereof); or (vi) a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the sequence shown in (iv); Includes.

[0030] In certain embodiments, the substitutions described in (ii) or (v) are conservative substitutions. In certain embodiments, an antibody or antigen-binding fragment thereof of the disclosure comprises a heavy chain and a light chain, wherein the heavy chain comprises: (i) the sequence shown in SEQ ID NO: 11; (ii) a sequence which has one or several amino acid substitutions, deletions or additions or any combination thereof compared to the sequence shown in (i) (e.g. up to 50, up to 45, up to 40, up to 35, up to 30, up to 25, up to 20, up to 15, up to 10 or up to 5 amino acid substitutions, deletions or additions or any combination thereof, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, deletions or additions or any combination thereof); or (iii) a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the sequence shown in (i). Including, The light chain, (iv) a sequence as set forth in SEQ ID NO: 22; (v) a sequence which has one or several amino acid substitutions, deletions or additions or any combination thereof compared to the sequence shown in (iv) (e.g. up to 50, up to 45, up to 40, up to 35, up to 30, up to 25, up to 20, up to 15, up to 10 or up to 5 amino acid substitutions, deletions or additions or any combination thereof, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, deletions or additions or any combination thereof); or (vi) a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the sequence shown in (iv); Including, Preferably, the substitutions described in (ii) or (v) are conservative substitutions.

[0031] In certain embodiments, the antibody or antigen-binding fragment thereof is an antibody, (a) a heavy chain comprising the VH sequence shown in SEQ ID NO: 23 and the CH sequence shown in SEQ ID NO: 29, and a light chain comprising the VL sequence shown in SEQ ID NO: 24 and the CL sequence shown in SEQ ID NO: 30, preferably a heavy chain shown in SEQ ID NO: 11, and a light chain shown in SEQ ID NO: 22; (b) a heavy chain comprising the VH sequence set forth in SEQ ID NO: 25 and the CH sequence set forth in SEQ ID NO: 29, and a light chain comprising the VL sequence set forth in SEQ ID NO: 26 and the CL sequence set forth in SEQ ID NO: 30; (c) a heavy chain comprising the VH sequence shown in SEQ ID NO: 27 and the CH sequence shown in SEQ ID NO: 29, and a light chain comprising the VL sequence shown in SEQ ID NO: 28 and the CL sequence shown in SEQ ID NO: 30. The compound is selected from the group consisting of:

[0032] In a second aspect, the antibody of the present disclosure is a multispecific antibody that binds to Her3 and one or more other antigens. Thus, the present disclosure provides a multispecific antibody. Preferably, the multispecific antibody is a bispecific antibody or a trispecific antibody or a tetraspecific antibody. In some embodiments, multispecific antibodies of the disclosure comprise antigen-binding fragments of the disclosure that bind to Her3 and other antibodies or antigen-binding fragments thereof or antibody mimetics.

[0033] Antibody Derivatives of the Present Disclosure The antibodies or antigen-binding fragments thereof of the present disclosure may be derivatized, e.g., conjugated to another molecule (e.g., another polypeptide or protein). In general, derivatization (e.g., labeling) of an antibody or antigen-binding fragment thereof does not adversely affect its binding to Her3 (e.g., human Her3). Thus, the antibodies or antigen-binding fragments thereof of the present disclosure are also intended to include such derivatized forms. For example, the antibodies or antigen-binding fragments thereof of the present disclosure may be conjugated (by chemical linkage, genetic fusion, non-covalent linkage, or otherwise) to one or more other molecular groups, e.g., another antibody (e.g., to form a bispecific antibody), a detection agent, a pharmaceutical agent, and / or a protein or polypeptide that can mediate binding of the antibody or antigen-binding fragment thereof to another molecule (e.g., avidin or a polyhistidine tag).

[0034] One type of derivatized antibody (e.g., bispecific antibody) is produced by crosslinking two or more antibodies (belonging to the same or different classes). Methods for obtaining bispecific antibodies are well known in the art and include, but are not limited to, chemical crosslinking, cell engineering (hybridoma) or genetic engineering methods. Another type of derivatized antibody is a labeled antibody. For example, the antibodies or antigen-binding fragments thereof of the present disclosure can be conjugated to a detectable marker. The detectable markers described in this disclosure can be any substance detectable by fluorescent, spectroscopic, photochemical, biochemical, immunological, electrical, optical, or chemical means. Such markers are well known in the art and examples include, but are not limited to, enzymes (e.g., horseradish peroxidase, alkaline phosphatase, β-galactosidase, urease, glucose oxidase, etc.), radionuclides (e.g., 3H, 125I, 35S, 14C, or 32P), fluorescent dyes (e.g., fluorescein isothiocyanate (FITC), fluorescein, tetramethylrhodamine isothiocyanate (TRITC), phycoerythrin (PE), Texas red, rhodamine, quantum dots, or cyanine dye derivatives (e.g., Cy7, Alexa 750)), acridine esters, magnetic beads (e.g., Dynabeads®), calorimetric markers, such as colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.) beads, and biotin for binding avidin (e.g., streptavidin) modified with the above markers. Patents that teach the use of markers include, but are not limited to, U.S. Patent Nos. 3,817,837, 3,850,752, 3,939,350, 3,996,345, 4,277,437, 4,275,149, and 4,366,241, all of which are hereby incorporated by reference in their entirety. The detectable markers described above can be detected by methods known in the art. For example, radioactive markers can be detected using photographic film or scintillation counters, and fluorescent markers can be detected using a photodetector to detect emitted light. Enzymatic markers are generally detected by providing the enzyme with a substrate and detecting the reaction product generated by the action of the enzyme on the substrate, and calorimetric markers are detected by simple visualization of the colored marker.In certain embodiments, such labeling may be suitable for immunological detection (e.g., enzyme-linked immunoassay, radioimmunoassay, fluorescent immunoassay, chemiluminescent immunoassay, etc.) In certain embodiments, the detectable markers described above may be attached to the antibodies or antigen-binding fragments of the present disclosure by linkers having various lengths to reduce potential steric hindrance.

[0035] Furthermore, the antibodies or antigen-binding fragments thereof of the present disclosure can also be derivatized with chemical groups, such as polyethylene glycol (PEG), methyl or ethyl, or glycosyl, which can be used to improve the biological properties of the antibody, such as increasing the serum half-life.

[0036] Preparation of antibodies The antibodies of the present disclosure can be prepared by obtaining them through various methods known in the art, such as recombinant genetic engineering techniques. For example, DNA molecules encoding the heavy and light chain genes of the antibodies of the present disclosure can be obtained by chemical synthesis or PCR amplification. The obtained DNA molecules are inserted into an expression vector and then transfected into a host cell. The transfected host cell is then cultured under specific conditions to express the antibodies of the present disclosure.

[0037] Antigen-binding fragments of the present disclosure can be obtained by hydrolysis of intact antibody molecules (see Morimoto et al., J. Biochem. Biophys. Methods 24: 107-117 (1992) and Brennan et al., Science 229: 81 (1985)). In addition, these antigen-binding fragments can also be produced directly by recombinant host cells (reviewed in Hudson, Curr. Opin. Immunol. 11: 548-557 (1999); Little et al., Immunol. Today, 21: 364-370 (2000)). For example, Fab' fragments can be obtained directly from host cells, or Fab' fragments can be chemically coupled to form F(ab')2 fragments (Carter et al., Bio / Technology, 10: 163-167 (1992)). Moreover, Fv, Fab, or F(ab')2 fragments can also be isolated directly from recombinant host cell culture. Other techniques for preparing these antigen-binding fragments are well known to those of skill in the art. Thus, in a third aspect, the present disclosure provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding an antibody or antigen-binding fragment thereof of the present disclosure, or a heavy chain variable region and / or a light chain variable region thereof, or one or more CDRs thereof, or a nucleotide sequence encoding a multispecific antibody of the present disclosure. In certain embodiments, the nucleotide sequence may be substituted by codon degeneracy. In certain embodiments, the nucleotide sequence is codon optimized.

[0038] In certain embodiments, an isolated nucleic acid molecule of the disclosure comprises (i) a first nucleic acid and a second nucleic acid encoding a heavy chain variable region and a light chain variable region, respectively, of an antibody or antigen-binding fragment thereof of the disclosure, or (ii) a first nucleic acid and a second nucleic acid encoding a heavy chain variable region and a heavy chain constant region and a light chain variable region and a light chain constant region, respectively, of an antibody or antigen-binding fragment thereof of the disclosure, or (iii) a first nucleic acid and a second nucleic acid encoding a heavy chain and a light chain, respectively, of an antibody or antigen-binding fragment thereof of the disclosure. In certain embodiments, the first nucleic acid and the second nucleic acid comprise a nucleic acid that is a degenerate sequence of or is substantially identical to the sequence of the first nucleic acid and the second nucleic acid of any of items (i)-(iii) above. In certain embodiments, a degenerate or substantially identical sequence refers to a sequence that has at least about 85%, 90%, 95%, or more sequence identity compared to a nucleic acid molecule set forth in items (i)-(iii), or has one or more nucleotide substitutions, or has no more than 3, no more than 6, no more than 15, no more than 30, or no more than 45 nucleotide differences.

[0039] In a fourth aspect, a vector (e.g., a cloning vector or an expression vector) is provided that comprises an isolated nucleic acid molecule of the present disclosure. In certain embodiments, the vector of the present disclosure is a cloning vector or an expression vector. In certain embodiments, the vector of the present disclosure is, for example, a plasmid, a cosmid, a bacteriophage, a lentivirus, etc. In certain embodiments, the vector is capable of expressing an antibody or an antigen-binding fragment thereof of the present disclosure in vivo in a subject (e.g., a mammal, e.g., a human). In a fifth aspect, a host cell is provided that comprises an isolated nucleic acid molecule of the present disclosure or a vector of the present disclosure. The host cell may be a eukaryotic cell (e.g., a mammalian cell, an insect cell, a yeast cell) or a prokaryotic cell (e.g., Escherichia coli). Suitable eukaryotic cells include, but are not limited to, NS0 cells, Vero cells, Hela cells, COS cells, CHO cells, HEK293 cells, BHK cells, and MDCKII cells. Suitable insect cells include, but are not limited to, Sf9 cells. In certain embodiments, the host cell of the present disclosure is a mammalian cell, such as a CHO (e.g., CHO-K1, CHO-S, CHO DXB11, CHO DG44). In a sixth aspect, there is provided a method for preparing an antibody or antigen-binding fragment thereof of the present disclosure, or a multispecific antibody of the present disclosure, comprising culturing a host cell of the present disclosure under conditions to express the antibody or antigen-binding fragment thereof, or multispecific antibody, and recovering the antibody or antigen-binding fragment thereof from the cultured host cell culture.

[0040] Antibody-drug conjugates In a seventh aspect, there is provided an antibody-drug conjugate, a stereoisomer thereof, a prodrug thereof, a pharma- ceutically acceptable salt thereof, or a pharma- ceutically acceptable solvate thereof, wherein the antibody is an antibody or antigen-binding fragment thereof as described above, which binds to Her3 and is attached to a coupling moiety via a connector. The coupling moiety is selected from detectable markers, radioisotopes, fluorescent agents, luminescent agents, colorants, enzymes, polyethylene glycol (PEG), nuclides, nucleic acids, small molecule toxins, polypeptides with binding activity, proteins, receptors, ligands, and other active agents that inhibit tumor cell proliferation and promote tumor cell apoptosis or necrosis.

[0041] In certain instances, the antibody-drug conjugate has the formula: Tb-(LD)q The present invention includes an anti-Her3 antibody-drug conjugate (Her3-ADC) of Tb is an antibody or antigen-binding fragment thereof that binds to Her3 as described above, D is a small molecule toxin drug moiety; L is a bond or linker covalently linking Tb and D; q is an integer between 1 and 16 and represents the number of LDs covalently bound to Tb. In some embodiments, the antibody-drug conjugate has a structure represented by formula I:

[0042] [ka] (I) L1 is

[0043] [ka] each Z is independently selected from a direct bond, a carbon-carbon triple bond, a carbon-carbon double bond, and an amide (preferably selected from a direct bond, a carbon-carbon triple bond, and a carbon-carbon double bond); Rx and Ry are each independently selected from H and C1-4 alkyl; each m is independently selected from 0, 1, 2, 3, 4, 5, and 6; y1 is selected from every integer between 1 and 6 (e.g., 4, 5, and 6); each y2 is independently selected from every integer between 0 and 15 (e.g., 6-15); each y3 is independently selected from 1, 2, and 3; and each y4 is independently selected from 0 and 1; position 1 is bonded to Tb via a S atom; position 2 is bonded to L2 or L3; L2 may be absent or present, and when L2 is present, L2 is

[0044] [ka] wherein y1 is selected from any integer between 1 and 6 (e.g., 4, 5, and 6), each y2 is independently selected from any integer between 0 and 10 (e.g., 6-10), each y3 is independently selected from 1 or 2, each y4 is independently selected from 0 and 1, position 1 is bonded to L1, position 2 is bonded to L3; L3 is selected from an amino acid residue or a short peptide consisting of 2 to 10 amino acid residues, and the amino acid residue is selected from a natural amino acid residue, a non-natural amino acid residue, or AA 1

[0045] [ka] or a stereoisomer thereof; 1 In the amino acid residue represented by a and R b One of the groups is H and the other is H,

[0046] [ka] or R a and R b form a 5- or 6-membered heterocyclic ring together with the carbon atom to which they are both attached, the 5- or 6-membered heterocyclic ring being a piperidine ring or a piperazine ring; r,r 1 , r 1a and r 1b are each independently 0, 1, 2, 3, 4, or 5; R m1 , R n1 , R m1a , R n1a , R m1b and R n1b are independently H, C 1-6 Alkyl, or -COOR x1 and R x1 is C 1-6 Is it an alkyl group? or R m1 and R n1 , Rm1a and R n1a , and R m1b and R n1b form a 5-6 membered heterocyclic ring together with the nitrogen atom to which they are both attached, the heteroatoms of the 5-6 membered heterocyclic ring being selected from one or two N atoms, and the 5-6 membered heterocyclic ring being selected from one or more R 0’ may be substituted with R z is C 1-6 alkyl, R 0 and R 0’ is C 1-6 Alkyl, -NR m2 R n2 , or C 1-6 each independently selected from a 5- to 6-membered heterocyclyl optionally substituted by alkyl, the heteroatoms of the 5- to 6-membered heterocyclyl being selected from 1 or 2 N atoms; R m2 and R n2 H and C 1-6 are each independently selected from alkyl, L4 may be absent or present, and when L4 is present, L4 is

[0047] [ka] wherein position 1 is linked to L3 and position 2 is linked to D; R1 and R2 are each independently selected from H, halogen, and C1-4 alkyl, or R1 and R2 together with the carbon atom to which they are both attached form a 5-6 membered heterocycle, the heterocycle containing 1, 2 or 3 O, S or N or any combination thereof; R3 is H and C 1-4 alkyl, or R and X together with the carbon atom to which they are both attached form a 5-6 membered carbocyclic ring; W may be absent or present, and when W is present, W is -O-, -S-, -NR4-,

[0048] [ka] wherein position 1 is bonded to X and position 2 is bonded to L4 or L3; X is an optionally substituted -(CH2) n1 -,

[0049] [ka] wherein position 1 is attached to the parent ring and position 2 is attached to W or L4, and the substituents are selected from one or two C1-4 alkyl; R4, R5 and R7 are each independently selected from H and C1-4 alkyl; n, n1, n2, and n3 are each independently selected from any integer between 0 and 6.

[0050] Furthermore, with regard to "position 1 of L1 binds to Tb via an S atom", it should also be noted that it can be understood by those skilled in the art that position 1 of L1 is linked to a sulfhydryl group contained in Tb (e.g., an antibody) after the disulfide bond is opened (e.g., reduction of the disulfide bond with a reducing agent TCEP can break the disulfide bond to generate a sulfhydryl-SH group). That is, the -S- between L1 and Tb is not an additional external sulfur atom. For example,

[0051] [ka] In the above, -S- is not an additional external sulfur atom, but rather a sulfhydryl group contained in Tb after opening the disulfide bond, which is attached to position 1 of L1, e.g.

[0052] [ka] is formed by linking to In some embodiments, L1 is

[0053] [ka] wherein each Z is independently selected from a direct bond, a carbon-carbon triple bond, a carbon-carbon double bond, a C6 aryl and an amide (preferably selected from a direct bond, a carbon-carbon triple bond, and a carbon-carbon double bond); Rx and Ry are independently selected from H and a C1-4 alkyl; each m is independently selected from 0, 1, 2, 3, 4, 5 and 6; y1 is selected from any integer between 1 and 6 (e.g., 4, 5 and 6); each y2 is independently selected from any integer between 0 and 15 (e.g., 6-15); each y3 is independently selected from 1, 2 and 3; and each y4 is independently selected from 0 and 1; the 1st position is bonded to Tb via a S atom, and the 2nd position is bonded to L2 or L3. In some embodiments, L1 is

[0054] [ka] wherein position 1 is bonded to Tb via an S atom and position 2 is bonded to L2 or L3. In some embodiments, L1 is

[0055] [ka] wherein position 1 is bonded to Tb via an S atom and position 2 is bonded to L2 or L3. In some embodiments, L2 may be absent or present, and when L2 is present, it is

[0056] [ka] wherein position 1 binds to L1 and position 2 binds to L3. In some embodiments, L2 is absent. In some embodiments, L3 is AA 1 , A.A. 1 -Gly, Val-Cit, Val-Ala, Val-AA1 , Val-AA 1 -Gly, AA 1 -Ala-Asn, Ala-Ala-Ala, Ala-Ala-Asn, and Gly-Gly-Phe-Gly.

[0057] In some embodiments, L3 is AA 1 , A.A. 1 -Gly, Val-Cit, Val-AA 1 -Gly, AA 1 -Ala-Asn, and Gly-Gly-Phe-Gly. In some embodiments, L3 is Val-AA 1 -Gly. In some embodiments, r, r 1 , r 1a and r 1b are each independently selected from 0 or 4, preferably r is 0 and r 1 , r 1a and r 1b is 4, or r is 4 and r 1 , r 1a and r 1b is 0. In some embodiments, r, r 1 , r 1a and r 1b is not all 0. In some embodiments, r is 0 and r 1 is 4.

[0058] In some embodiments, R m1 , R n1 , R m1a , R n1a , R m1b and R n1b are each independently H, methyl, ethyl, n-propyl, n-butyl, -COOCH3, -COOCH2CH3, -COOCH2CH2CH3, -COOCH(CH3)2, -COOC(CH3)3, or -COOCH2CH2CH2CH3. In some embodiments, R m1 , Rn1 , R m1a , R n1a , R m1b and R n1b are each independently H, methyl, ethyl, n-propyl, -COOCH, -COOCHCH, -COOCHCHCH, -COOCH(CH), -COOC(CH), or -COOCHCHCHCH. m1 and R n1 , R m1a and R n1a , and R m1b and R n1b form a piperidine or piperazine ring together with the nitrogen atom to which they are both attached, more preferably

[0059] [ka] The nitrogen atom shown by the number 1 is R m1 and R n1 are both nitrogen atoms bonded to. In some embodiments, R m1 and R n1 , R m1a and R n1a , and R m1b and R n1b form a piperidine ring together with the nitrogen atom to which they are both attached, more preferably

[0060] [ka] The nitrogen atom shown by the number 1 is R m1 and R n1 are both nitrogen atoms bonded to. In some embodiments, r and r 1 For r is 4 and r 1 If is 0, R m1 and R n1 are each independently selected from H, C alkyl (e.g., H, methyl), r is 0, and r 1 If is 4, R m1 and Rn1 are each independently selected from C1-6 alkyl (eg, methyl, ethyl, n-propyl), preferably selected from C2-6 alkyl (eg, ethyl, n-propyl). In some embodiments, r is 0 and r 1 If is 4, R m1 and R n1 are each independently selected from C1-6 alkyl (e.g., methyl, ethyl, and n-propyl). In some embodiments, R z is methyl. In some embodiments, R 0 is C 1-6 Alkyl or C 1-6 Alkyl-substituted piperidinyl or piperazinyl, preferably methyl, ethyl, or methyl-substituted piperidinyl, e.g., methyl or

[0061] [ka] It is. In some embodiments, R 01’ are each independently C 1-6 Alkyl or -NR m2 R n2 and more preferably methyl or -NR m2 R n2 and R m2 and R n2 are each independently preferably H or C 1-6 It is preferably an alkyl group, and more preferably a methyl group.

[0062] In some embodiments, AA 1 In the amino acid residue represented by a1 and R b together with the carbon atom to which they are both attached to a piperidine ring, e.g.

[0063] [ka] The carbon atom indicated by the number 1 is R a and R b are both attached to the carbon atom, e.g.

[0064] [ka] It is. In some embodiments, AA 1 The amino acid residue represented by

[0065] [ka] is selected from.

[0066] In some embodiments, AA 1 The amino acid residue represented by

[0067] [ka] is selected from. In some embodiments, AA 1 The amino acid residue represented by

[0068] [ka] In some embodiments, L3 is selected from:

[0069] [ka] wherein position 1 is attached to L1 or L2 and position 2 is attached to L4 or D. In some embodiments, L3 is

[0070] [ka] wherein position 1 is attached to L1 or L2 and position 2 is attached to L4 or D. In some embodiments, L3 is

[0071] [ka] wherein position 1 is attached to L1 or L2 and position 2 is attached to L4 or D. In some embodiments, L4 may be absent or present, and when L4 is present, L4 is selected from:

[0072] [ka] wherein position 1 binds to L3 and position 2 binds to D. In some embodiments, L4 is absent. In some embodiments, L4 is

[0073] [ka] wherein position 1 binds to L3 and position 2 binds to D. In some embodiments, L4 is

[0074] [ka] wherein position 1 binds to L3 and position 2 binds to D. In some embodiments, R1 is selected from H and halogen, and R2 is selected from H and C1-4 alkyl. In some embodiments, R1 and R2, together with the carbon to which they are attached, [ka] where the "dotted line" indicates where the heterocycle is fused to the benzene ring. In some embodiments, R1 is H or F and R2 is H or methyl. In some embodiments, R1 is F and R2 is methyl, or R1 and R2 together with the carbon atom to which they are attached are

[0075] [ka] Form. In some embodiments, R1 is F and R2 is methyl. In some embodiments, R1 and R2, together with the carbon atom to which they are attached,

[0076] [ka] Form. In some embodiments, R3 is H, or R3 and X together with the carbon atom to which they are attached are

[0077] [ka] where the dotted lines indicate where the carbocyclic ring is fused to the benzene and pyridine rings. In some embodiments, R3 is H. In some embodiments, each R4 is independently selected from H and C1-4 alkyl, and R5 is H. In some embodiments, each R4 is independently selected from H, methyl, ethyl, n-propyl, isopropyl, and tert-butyl; and R5 is H. In some embodiments, each R7 is independently selected from H and C1-4 alkyl. In some embodiments, R7 is H. In some embodiments, n is selected from 1, 2, and 3. In some embodiments, n is 1. In some embodiments, n1 is selected from 1, 2, 3, and 4. In some embodiments, n2 is 1. In some embodiments, n3 is 0. In some embodiments, W is -O-, -NR4-, and

[0078] [ka] wherein position 1 is bonded to X and position 2 is bonded to L4 or L3. In some embodiments, W is selected from -O- and -NR4-, position 1 is bonded to X and position 2 is bonded to L4 or L3. In some embodiments, X is optionally substituted. [ka] wherein position 1 is attached to the parent ring and position 2 is attached to W or L4, and the substituents are selected from one or two C1-4 alkyl groups (e.g., methyl), or two C1-4 alkyl groups (e.g., methyl). In some embodiments, X is

[0079] [ka] wherein position 1 is attached to the parent ring and position 2 is attached to W. In some embodiments, W is -O-, -NR4-, and

[0080] [ka] Position 1 is bonded to X, position 2 is bonded to L4 or L3, and X is selected from

[0081] [ka] wherein position 1 is attached to the parent ring and position 2 is attached to W. W may be absent or present, so that when W is absent, the 1-position of L4 is bonded to L3 and the 2-position is bonded to X, and when W is present, the 1-position of L4 is bonded to L3 and the 2-position is bonded to W. The following bond relationship of L4 can be understood with reference to the above. In some embodiments, the structure

[0082] [ka] But the following structural fragment [ka] Position 1 is selected from Tb, position 2 is selected from D, and 1 is defined above. In some embodiments, the structure

[0083] [ka] But the following structural fragment

[0084] [ka] [ka] [ka] wherein position 1 binds to Tb and position 2 binds to W. In some embodiments, the structure

[0085] [ka] But the following structural fragment

[0086] [ka] [ka] [ka] wherein position 1 binds to Tb and position 2 binds to W. In some embodiments, the structure

[0087] [ka] But the following structural fragment

[0088] [ka] wherein position 1 is bonded to Tb via an S atom and position 2 is bonded to W. In some embodiments,

[0089] [ka] The structural fragment represented by

[0090] [ka] and position 1 binds to L4. In some embodiments, the structure

[0091] [ka] But the following structural fragment

[0092] [ka] [ka] is selected from. In some embodiments, the structure

[0093] [ka] But the following structural fragment

[0094] [ka] is selected from. In some embodiments, the ligand-drug conjugate has the formula I-1

[0095] [ka] having a structure represented by Tb, L1, L2, L3, L4, X, R1, R2, R3, R4, and q are defined above and in any embodiment specifically described herein. In some embodiments, the ligand-drug conjugate has the formula I-1A or I-1B

[0096] [ka] having a structure represented by Tb, L2, L3, L4, X, R1, R2, R3, R4, and q are defined above and in any embodiment specifically described herein. In some embodiments, the ligand-drug conjugate has the formula I-2

[0097] [ka] having a structure represented by Tb, L1, L2, L3, L4, X, R1, R2, R3, and q are defined above and in any embodiment specifically described herein. In some embodiments, the ligand-drug conjugate has the formula I-2A or I-2B

[0098] [ka] having a structure represented by Tb, L2, L3, L4, X, R1, R2, R3, and q are defined above and in any embodiment specifically described herein. In some embodiments, the ligand-drug conjugate has the formula I-3

[0099] [ka] having a structure represented by Tb, L1, L2, L3, L4, X, R1, R2, R3, R4, R5, n, and q are defined above and in any embodiment specifically described herein. In some embodiments, the ligand-drug conjugate has the formula I-3A or I-3B

[0100] [ka] having a structure represented by Tb, L2, L3, L4, X, R1, R2, R3, R4, and q are defined above and in any embodiment specifically described herein. In some embodiments, the Ligand-Drug conjugate has the formula IA

[0101] [ka] having a structure represented by Tb, X, R1, R2, R3, R a , R b , and q are defined above and in any embodiment specifically described herein. In some embodiments, the Ligand-Drug conjugate has the formula IB

[0102] [ka] having a structure represented by Tb, X, R1, R2, R3, R a , R b , and q are defined above and in any embodiment specifically described herein. In some preferred embodiments, the Ligand-Drug conjugate is

[0103] [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] is selected from Tb is a Her3 antibody as described in any of the above embodiments, and q is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, and q is preferably 1, 2, 3, 4, 5, 6, 7, 8, The ligand-drug conjugate, e.g.

[0104] [ka] [ka] and Her3mAb is antibody 202-2-1. In some preferred embodiments, the Ligand-Drug conjugate is

[0105] [ka] is selected from Tb is the antibody Her3 as described in any of the above embodiments and q is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16, q is preferably 1, 2, 3, 4, 5, 6, 7 or 8. In some preferred embodiments, the Ligand-Drug conjugate is

[0106] [ka] is selected from Her3mAb is antibody 202-2-1.

[0107] In some preferred embodiments, q in the Ligand-Drug conjugate is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16, with q preferably being 1, 2, 3, 4, 5, 6, 7, or 8. In an eighth aspect of the present disclosure, the present disclosure provides an antibody-drug conjugate comprising two or more of the above-mentioned antibody-drug conjugates, stereoisomers thereof, prodrugs thereof, pharma- ceutically acceptable salts thereof, or pharma-ceutically acceptable solvates thereof, wherein the antibody-drug conjugates in the antibody-drug conjugate have one, two or more q values. In some embodiments, the drug-to-antibody ratio (DAR) in the antibody-drug conjugate is selected from integers or decimals between 1-10. In some embodiments, the drug to antibody ratio (DAR) in the antibody-drug conjugate is selected from 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.2, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.7, 8.9 and 9.

[0108] In some embodiments, the DAR value of the antibody-drug conjugate is selected from 2±0.5, 4±0.5, 6±0.5, or 8±0.5. In some embodiments, the DAR value of the antibody-drug conjugate is selected from 2±0.5, 4±0.5, 5±0.5, 6±0.5, 7±0.5, or 8±0.5. In some embodiments, the antibody-drug conjugate has a DAR value of 8±0.5, 8±0.4, 8±0.3, 8±0.2, 8±0.1, or 8.0. In some embodiments, the Ligand-Drug conjugates include ADCs with a DAR distribution of 2-8, e.g., 1.5, 2, 4, 6, and 8 (i.e., 1.5, 2, 4, 6, and 8 payload species). It should be noted that degradants may be generated, so the Ligand-Drug conjugates may also include DARs of 1, 3, 5, and 7. Additionally, the ADCs of the mixture may also have a DAR greater than 8. The Ligand-Drug conjugates are generated by reduction of interchain disulfides followed by coupling. In some embodiments, the Ligand-Drug conjugates include both ADCs with a DAR of 4 or less (i.e., 4 or less payload species) and ADCs with a DAR of 6 or more (i.e., 6 or more payload species).

[0109] Uses, Treatment Methods, and Pharmaceutical Compositions In a ninth aspect of the present disclosure, the present disclosure provides a pharmaceutical composition comprising an antibody or antigen-binding fragment thereof according to the first aspect of the disclosure, a multispecific antibody according to the second aspect, a nucleic acid molecule according to the third aspect, a vector according to the fourth aspect, a host cell according to the fifth aspect, an antibody-drug conjugate according to the seventh aspect, a stereoisomer thereof, a prodrug thereof, a pharma- ceutically acceptable salt thereof or a pharma-ceutically acceptable solvate thereof, and / or an antibody-drug conjugate according to the eighth aspect, and a pharma- ceutically acceptable carrier and / or excipient. The antibody or antigen-binding fragment thereof described in the first aspect of the disclosure, the multispecific antibody described in the second aspect, the nucleic acid molecule described in the third aspect, the vector described in the fourth aspect, the host cell described in the fifth aspect, the antibody-drug conjugate described in the seventh aspect, a stereoisomer thereof, a prodrug thereof, a pharma- ceutically acceptable salt thereof or a pharma- ceutically acceptable solvate thereof, and / or the antibody-drug conjugate described in the eighth aspect may be in a therapeutically effective amount.

[0110] In certain embodiments, a pharmaceutical composition of the disclosure comprises an antibody or antigen-binding fragment thereof of the disclosure and a pharma- ceutically acceptable carrier and / or excipient. In certain embodiments, a pharmaceutical composition of the disclosure comprises a host cell of the disclosure and a pharma- ceutically acceptable carrier and / or excipient, wherein the host cell comprises an isolated nucleic acid molecule or vector as described above. In certain embodiments, a pharmaceutical composition of the present disclosure comprises a multispecific antibody of the present disclosure and a pharma- ceutically acceptable carrier and / or excipient. In certain embodiments, a pharmaceutical composition of the disclosure comprises an antibody-drug conjugate of the disclosure, a stereoisomer thereof, a prodrug thereof, a pharma- ceutically acceptable salt thereof, or a pharma- ceutically acceptable solvate thereof, and / or an antibody-drug conjugate and a pharma- ceutically acceptable carrier and / or excipient.

[0111] In another aspect of the disclosure, the disclosure provides the use of an antibody or antigen-binding fragment thereof, a nucleic acid, a vector, a host cell, a multispecific antibody, or an antibody-drug conjugate, a stereoisomer thereof, a prodrug thereof, a pharma- ceutically acceptable salt or a pharma- ceutically acceptable solvate thereof, an antibody-drug conjugate, or a pharmaceutical composition of the disclosure in the manufacture of a medicament for modulating (inhibiting or blocking) the activity of Her3. The antibody or antigen-binding fragment thereof, a nucleic acid molecule, a vector, a host cell, a multispecific antibody, or an antibody-drug conjugate, a stereoisomer thereof, a prodrug thereof, a pharma- ceutically acceptable salt or a pharma- ceutically acceptable solvate thereof of the disclosure may be in a therapeutically effective amount.

[0112] In some examples, the disclosure provides the use of an antibody or antigen-binding fragment thereof, a nucleic acid, a vector, a host cell, a multispecific antibody, an antibody-drug conjugate, a stereoisomer thereof, a prodrug thereof, a pharma- ceutically acceptable salt thereof, or a pharma-ceutically acceptable solvate thereof, an antibody-drug conjugate, or a pharmaceutical composition of the disclosure in the manufacture of a medicament for the treatment or prevention of a disease associated with Her3 activity. In some examples, the disclosure provides the use of an antibody or antigen-binding fragment thereof, a nucleic acid, a vector, a host cell, a multispecific antibody, an antibody-drug conjugate, a stereoisomer thereof, a prodrug thereof, a pharma- ceutically acceptable salt thereof, or a pharma-ceutically acceptable solvate thereof, an antibody-drug conjugate, or a pharmaceutical composition of the disclosure in the manufacture of a medicament for the treatment or prevention of a tumor associated with Her3 activity.

[0113] In some embodiments, the cancer disease is selected from esophageal cancer (e.g., esophageal adenocarcinoma and esophageal squamous cell carcinoma), brain cancer, lung cancer (e.g., small cell lung cancer and non-small cell lung cancer), squamous cell carcinoma, bladder cancer, gastric cancer, ovarian cancer, peritoneal cancer, pancreatic cancer, breast cancer, head and neck cancer, cervical cancer, endometrial cancer, colorectal cancer, liver cancer, renal cancer, urothelial cancer, solid tumors, non-Hodgkin's lymphoma, central nervous system tumors (e.g., glioma, glioblastoma multiforme, glioma, or sarcoma), prostate cancer, or thyroid cancer. In some embodiments, the cancer is a solid tumor. In some embodiments, the tumor is selected from colorectal cancer (eg, colon cancer), lung cancer (eg, non-small cell lung cancer), breast cancer, and prostate cancer. In some embodiments, the cancer is breast cancer or lung cancer (preferably non-small cell lung cancer).

[0114] In another aspect, the disclosure provides for the use of an antibody or antigen-binding fragment thereof, or a multispecific antibody of the disclosure in the manufacture of a kit for detecting the presence or level of Her3 in a sample. In another aspect, the disclosure provides a diagnostic or therapeutic kit comprising one or more of the following substances, an antibody or antigen-binding fragment thereof, a nucleic acid, a vector, a host cell, a multispecific antibody, an antibody-drug conjugate, or a pharmaceutical composition of the disclosure. The diagnostic or therapeutic kit may further comprise instructions for use.

[0115] definition In this disclosure, unless otherwise indicated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, cell culture, biochemistry, nucleic acid chemistry, immunology, and other laboratory procedures used herein are all routine procedures widely used in the corresponding fields. Meanwhile, for a better understanding of this disclosure, the definitions and explanations of relevant terms are provided below. As used herein, the term "HER3 ligand" refers to a polypeptide that binds to and activates HER3. Examples of HER3 ligands include, but are not limited to, neuregulin 1 (NRG) and neuregulin 2, betacellulin, heparin-binding epidermal growth factor (HB-EGF), and epiregulin. The present disclosure relates to an isolated antibody or fragment thereof that recognizes a conformational epitope of the HER3 receptor, the conformational epitope comprising amino acid residues in domain 3 of HER3, and the antibody or fragment thereof inhibits phosphorylation of HER3 as assessed by a ligand-independent phosphorylation assay of HER3. In one embodiment, the ligand-independent phosphorylation assay of HER3 uses HER2 amplified cells, and the HER2 amplified cells are SK-Br-3 cells.

[0116] The term "ligand-dependent signaling" as used herein refers to signaling that is dependent on activation of a ligand, for example activation of Erbb (e.g., HER3) by a ligand. Activation of HER3 can be demonstrated by increasing oligomerization (e.g., heterodimerization) and / or HER3 phosphorylation in downstream signaling pathways (e.g., PI3K). The antibody or fragment thereof can statistically significantly reduce the amount of phosphorylated HER3 in stimulated cells exposed to an antigen binding protein (e.g., antibody) compared to untreated (control) cells, as measured using the assays described in the Examples. The cells expressing HER3 can be naturally occurring cell lines (e.g., MCF7) or recombinant cells produced by introducing a nucleic acid encoding a HER3 protein into a host cell. Cell stimulation can be by exogenous addition of an activating HER3 ligand or by endogenous expression of an activating ligand. The term "ligand-independent signaling" as used herein refers to signaling that does not require ligand binding, such as activation (e.g., phosphorylation) of HER3 that is not dependent on ligand binding. For example, ligand-independent activation of HER3 can be the result of overexpression of HER2, or the result of activating mutations in HER3 heterodimer partners, such as EGFR and HER2. The antibody or fragment thereof can statistically significantly reduce the amount of phosphorylated HER3 in cells exposed to an antigen-binding protein (e.g., antibody) compared to untreated (control) cells. The HER3-expressing cell can be a naturally occurring cell line (e.g., SK Br-3) or a recombinant cell produced by introducing a nucleic acid encoding a HER3 protein into a host cell.

[0117] As used herein, examples of the term "pharmaceutical acceptable salt" are organic acid addition salts formed with organic acids that provide a pharmaceutically acceptable anion, including but not limited to formate, acetate, propionate, benzoate, maleate, fumarate, succinate, tartrate, citrate, ascorbate, α-ketoglutarate, α-glycerophosphate, alkylsulfonate or arylsulfonate, preferably alkylsulfonate being methylsulfonate or ethylsulfonate and arylsulfonate being benzenesulfonate or p-toluenesulfonate. Suitable inorganic salts may also be formed, including but not limited to hydrochloride, hydrobromide, hydroiodide, nitrate, bicarbonate and carbonate, sulfate or phosphate salts, and the like.

[0118] As used herein, the term "pharmacologically acceptable carriers and / or excipients" refers to carriers and / or excipients known in the art (see, e.g., Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995) that are pharmacologically and / or physiologically compatible with the subject and active ingredient, including, but not limited to, pH adjusters, surfactants, adjuvants, ionic strength enhancers, diluents, substances that maintain osmotic pressure, substances that delay absorption, and preservatives. Pharmaceutically acceptable salts can be obtained using standard procedures well known in the art, for example, by reacting a sufficient amount of a basic compound with a suitable acid to provide a pharma- ceutically acceptable anion.

[0119] In this disclosure, pharmaceutically acceptable adjuvants refer to excipients and additives used in the manufacture of drugs and preparations in addition to active ingredients, and refer to substances that are included in pharmaceutical preparations whose safety has been reasonably evaluated. In addition to forming, acting as a carrier and improving stability, pharmaceutically acceptable adjuvants also have important functions, such as solubilization, hydrotrope, sustained release and controlled release, and are important components that affect the quality, safety and efficacy of drugs. According to its source, it can be divided into natural, semi-synthetic and fully synthetic. According to its function and use, it can be divided into solvent, propellant, solubilizer, cosolvent, emulsifier, colorant, binder, disintegrant, filler, lubricant, wetting agent, osmotic pressure regulator, stabilizer, fluidizer, flavoring agent, preservative, suspending agent, coating material, flavor, anti-adhesive agent, antioxidant, chelating agent, penetration enhancer, pH adjuster, buffer, plasticizer, surfactant, foaming agent, defoaming agent, thickener, encapsulating agent, moisturizer, absorbent, diluent, flocculating and peptizing agent, filter aid, release blocker, etc. According to its administration route, it can be divided into oral administration, injection administration, mucosal administration, transdermal or topical administration, nasal or oral inhalation administration, and intraocular administration. The same pharmaceutically acceptable adjuvant can be used in pharmaceutical preparations of different administration routes and have different effects and uses.

[0120] The pharmaceutical composition may be prepared into various suitable dosage forms according to the route of administration. For example, tablets, capsules, granules, oral liquids, oral suspensions, oral emulsions, powders, tinctures, syrups, injections, suppositories, ointments, creams, pastes, ophthalmic preparations, pills, implants, aerosols, powder aerosols, sprays, etc., and the pharmaceutical composition or suitable dosage form may contain 0.01 mg to 1000 mg, suitably 0.1 mg to 800 mg, preferably 0.5 mg to 500 mg, preferably 0.5 mg to 350 mg, particularly preferably 1 mg to 250 mg of the antibody, antibody-drug conjugate, stereoisomer thereof, prodrug thereof, pharma- ceutically acceptable salt thereof, or pharma- ceutically acceptable solvate thereof of the present disclosure. The pharmaceutical composition can be administered in the form of injection, including injection solution, sterile powder for injection and concentrated solution for injection.Among them, useful vehicles and solvents include water, Ringer's solution and isotonic sodium chloride solution.In addition, sterile non-volatile oils such as monoglycerides or diglycerides can also be used as solvents or suspension media.

[0121] As used herein, the term "treatment" generally refers to obtaining a desired pharmacological and / or physiological effect. The effect may be prophylactic, in terms of complete or partial prevention of a disease or its symptoms, and / or therapeutic, in terms of partial or complete stabilization or cure of the disease and / or side effects caused by the disease. As used herein, "treatment" encompasses any treatment of a disease in a patient, including (a) prevention of a disease or condition in a patient susceptible to the disease or condition but not yet diagnosed as having the disease or condition, (b) suppression of a symptom of the disease, i.e., preventing its progression, or (c) alleviation of a symptom of the disease, i.e., causing the disease or condition to recede. In this disclosure, the term "individual" includes a human or a non-human animal. Exemplary human individuals include individuals (referred to as patients) with a human disease (e.g., a disease described herein) or normal individuals. In this disclosure, the term "non-human animal" includes all vertebrates, such as non-mammals (e.g., birds, amphibians, reptiles) and mammals, such as non-human primates, livestock and / or domesticated animals (e.g., sheep, dogs, cats, cows, pigs, etc.). In the present disclosure, the term "effective amount" refers to that amount of a compound which, upon administration, will relieve to some extent one or more of the symptoms of the disorder being treated.

[0122] In the present disclosure, the term "ligand drug conjugate" refers to a substance in which a bioactive molecule (drug molecule) is bound to a targeting moiety. In some embodiments of the present disclosure, the bioactive molecule is bound to the targeting moiety by a linker. The linker can be broken in a certain environment (e.g., the presence of hydrolytic enzymes in tumors and / or a low pH environment) or under a certain action (e.g., the action of lysosomal proteases), thereby separating the bioactive molecule from the targeting moiety. In some embodiments of the present disclosure, the linker comprises a cleavable or non-cleavable unit, such as a peptide or disulfide bond. In some embodiments of the present disclosure, the bioactive molecule is directly bound to the targeting moiety by a covalent bond that can be broken under a certain environment or action, thereby separating the bioactive molecule from the targeting moiety. In some embodiments of the present disclosure, the ligand drug conjugate comprises a targeting moiety, a linker, and a compound fragment of formula II of the present disclosure.

[0123] In the present disclosure, the term "ligand-drug conjugate" refers to a substance in which a biologically active molecule (drug molecule) is bound to an antibody or its antigen-binding fragment. In some embodiments of the present disclosure, the biologically active molecule is bound to the antibody or its antigen-binding fragment by a linker. The linker can be broken in a certain environment (e.g., the presence of hydrolases in tumors and / or a low pH environment) or under a certain action (e.g., the action of lysosomal proteases), thereby separating the biologically active molecule from the antibody or its antigen-binding fragment. In some embodiments of the present disclosure, the linker comprises a cleavable or non-cleavable unit, such as a peptide or disulfide bond. In some embodiments of the present disclosure, the biologically active molecule is directly bound to the antibody or its antigen-binding fragment by a covalent bond that can be broken under a certain environment or action, thereby separating the biologically active molecule from the antibody or its antigen-binding fragment. In some embodiments of the present disclosure, the ligand-drug conjugate comprises an anti-Her3 antibody or its antigen-binding fragment, a linker, and a compound fragment of formula II of the present disclosure.

[0124] In the present disclosure, the term "biologically active substance", "biologically active molecule" or "drug molecule" refers to a substance that inhibits or blocks the function of a cell and / or causes cell death or destruction. In some embodiments of the present disclosure, the biologically active substance, biologically active molecule or drug molecule in the complex is a molecule having antitumor biological activity. For example, radioisotopes such as At211, I131, I125, Y90, Re186, Re188, Sm153, Bi212, P32, Pb212 and Lu; metal complexes such as metal platinum complexes, metal gold complexes, oxaliplatin, etc.; glycopeptide antibiotics such as bleomycin, pingyangmycin; DNA topoisomerase inhibitors such as topoisomerase I inhibitors, camptothecin, hydroxycamptothecin, 9-aminocamptothecin, SN-38, iridium, erythritol ... Notecan, topotecan, belotecan, rubitecan, topoisomerase II inhibitors, actinomycin D, adriamycin, doxorubicin, duocarmycin, daunorubicin, mitoxantrone, podophyllotoxin, etoposide, etc.; drugs that interfere with DNA synthesis, e.g. methotrexate, 5-fluorouracil, cytarabine, gemcitabine, mercaptopurine, pentostatin, fludarabine, cladribine, nelarabine, etc.; drugs that act on structural proteins tubulin inhibitors, vinca alkaloids, vincristine, vinblastine, paclitaxel, docetaxel, cabazitaxel, etc.; tumor signaling pathway inhibitors, such as serine / threonine kinase inhibitors, tyrosine kinase inhibitors, aspartate kinase inhibitors or histidine kinase inhibitors; proteasome inhibitors, histone deacetylase inhibitors, tumor angiogenesis inhibitors, cyclin inhibitors, maytansine derivatives, calicheamicin derivatives, auristatin derivatives, pyrrolobenzodiazepine (PBD) derivatives, melphalan, mitomycin C, chlorambucil, or other active substances that inhibit tumor cell proliferation and promote tumor cell apoptosis and necrosis; enzymes and fragments thereof, such as nucleolysin; antibiotics; toxins, including small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, fragments and / or variants thereof; proliferation inhibitors; and also drug modules.The term "toxin" refers to a substance capable of causing adverse effects on cell growth or proliferation.

[0125] In this disclosure, the term "small molecule" refers to a biologically active small molecule drug. The term small molecule toxin has cytotoxic activity, i.e., the state of causing some form of cellular lesion, and cytotoxicity is not limited to direct injury, but includes any kind of damage to the structure and function of the cell, such as DNA breakage, base dimer formation, chromosome breakage, damage to the cell division mechanism, and reduction of various enzyme activities. In this disclosure, the term "linker" refers to a fragment that connects a bioactive molecule (a drug molecule) to a targeting moiety. In this disclosure, the term "targeting moiety" refers to a moiety in a conjugate that can specifically bind to a target (or a portion of a target) on a cell surface. The interaction of the targeting moiety with the target can allow the conjugate to be delivered to a specific population of cells. In this disclosure, when the targeting moiety in the conjugate is an antibody, the conjugate can be referred to as a "drug-antibody conjugate."

[0126] In the present disclosure, antibodies or antigen-binding fragments thereof include derivatized antibodies or antigen-binding fragments thereof, such as antibodies or antigen-binding fragments thereof bearing thiol groups, which allow the antibody to have a group or ability to react with a drug-linker conjugate. The thiol-SH can be derivatized by breaking a disulfide bond (e.g., by reduction with the reducing agent TCEP). As used in this disclosure and the appended claims, the singular forms, such as "a," "an," and "the," include plural references unless the context clearly dictates otherwise. Thus, the meaning of the singular word "a" includes "one or more."

[0127] As used herein, the terms "cancer" and "tumor" are used interchangeably. As used herein, the term "gene" includes not only DNA but also its mRNA, its cDNA and its cRNA. As used herein, the term "polynucleotide" is used interchangeably with nucleic acid and includes DNA, RNA, probes, oligonucleotides, and primers. As used herein, the term "Tb" is an abbreviation for "target binding" and includes an antibody or any molecule that binds to a target, and the term "Ab" is an abbreviation for "antibody" and is used interchangeably with "antibody." As used herein, the terms "polypeptide" and "protein" are used interchangeably. As used herein, the term "cell" also includes cells inside an individual animal and cells in culture.

[0128] As used in this disclosure, "an antibody or antigen-binding fragment thereof that binds to Her3" refers to an antibody or antigen-binding fragment thereof that specifically binds to Her3, for example, human Her3. As used in this disclosure, "Her3", also known as "ErbB3", is a transmembrane receptor tyrosine kinase molecule of the ERBB family. The HER3 gene is located on the long arm of chromosome 12 (12q13.2) and encodes a 180 kDa protein. The extracellular domain of HER3 is divided into four subdomains (domains 1-4), where domains 1 and 3 are leucine-rich beta-helical regions involved in ligand binding, and domains 2 and 4 are cysteine-rich regions. In addition, domain 2 also contains a dimerization arm required for interaction with other receptors. The transmembrane domain is followed by an intracellular domain, which contains a flexible juxtamembrane region, a kinase structure, and a C-terminal tail. In the absence of ligand, binding between domains 2 and 4 renders HER3 inactive. Upon ligand binding, the other half of the heterodimeric kinase structure phosphorylates tyrosine residues in the C-terminal tail of HER3.

[0129] The Her3 to which the present disclosure relates may be a conventional Her3 in the art (e.g., human Her3), such as soluble Her3, membrane-type Her3, etc., and also refers to Her3 variant 1 and / or Her3 variant 2. In some embodiments, the human Her3 is set forth in the accession number Uniport ID: P21860-1 or NCBI: NP_001973.2. In some embodiments, the human Her3 comprises the amino acid sequence set forth in SEQ ID NO: 31.

[0130] Her3(1-643) shown in SEQ ID NO:31 is derived from Uniport ID: P21860-1, and SEQ ID NO:31 is shown below. .

[0131] KD refers to the dissociation constant obtained from the ratio of Kd (dissociation rate of a specific binding molecule-target protein interaction) to Ka (onset rate of a specific binding molecule-target protein interaction) (or Kd / Ka, expressed as molar concentration (M)). The value of KD can be determined using methods well established in the art. A preferred method for determining the KD of a binding molecule is by surface plasmon resonance, for example, using a biosensor system, such as a Biacore TM (GE Healthcare Life Sciences) system.

[0132] As used herein, the term "antigen-binding fragment" refers to a partial fragment of an antibody having antigen-binding activity, which fragment has complete or partial function of an antibody, including, but not limited to, Fab, Fab', F(ab')2, Fv, such as single-chain Fv (scFv), disulfide-linked Fv (sdFv), or di-scFv. The term also includes Fab', which is a monovalent fragment of the variable region of an antibody obtained by treating F(ab')2 under reducing conditions. However, the term is not limited to these molecules, as long as the fragment has binding affinity to the antigen. Furthermore, these functional fragments include not only fragments obtained by treating the full-length molecule of an antibody protein with appropriate enzymes, but also proteins produced in appropriate host cells using genetically improved antibody genes.

[0133] As used herein, "antibody molecule" or "antibody" in its broadest sense refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen-binding site that allows immunospecific binding to an antigen. Thus, the term antibody broadly encompasses not only whole antibody molecules, but also fragments of the antibodies, and variants (including derivatives) of the antibodies and antibody fragments. When "antibody molecule" or "antibody" is used in the same context as an antigen-binding fragment, "antibody molecule" or "antibody" refers to a complete antibody molecule or a full-length antibody.

[0134] The term "single chain Fv" or "scFv" refers to a polypeptide comprising an antibody VL domain bound to an antibody VH domain. For example, an antibody that immunospecifically binds to Her3 may cross-react with other antigens, but preferably may not cross-react with other antigens. An antibody that immunospecifically binds to Her3 can be identified, for example, by immunoassays or other methods known to those of skill in the art. An "intact" or "full length" antibody refers to a protein comprising two heavy chains (H) and two light chains (L) bound together by disulfide bonds, which protein comprises: (1) for the heavy chains, a variable region (abbreviated herein as "VH") and a heavy chain constant region comprising three domains CH1, CH2, CH3; and (2) for the light chains, a light chain variable region (abbreviated herein as "VL") and a light chain constant region comprising a domain CL. Antibodies of the present disclosure include, but are not limited to, monoclonal antibodies, polyspecific antibodies, human or chimeric antibodies, single chain antibodies, Fab fragments, F(ab') fragments, anti-idiotypic (anti-Id) antibodies (including, for example, the anti-Id antibodies of the present disclosure), and epitope-binding fragments of any of the above-mentioned antibodies. Immunoglobulin molecules of the present disclosure may be of any immunoglobulin type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), any class (e.g., IgG1, IgG2, IgG3, IgG4, IgAl, and IgA2), and any subclass. Preferably, the antibodies of the present disclosure comprise or consist of a VH domain, VH-CDR, VL domain, or VL-CDR having any of the amino acid sequences set forth in Table 1, or fragments or variants thereof. As used herein, the term "Fab" refers to a monovalent fragment of the variable region of an antibody obtained by treating F(ab')2 under reducing conditions as described above. However, Fab' of the present disclosure also includes Fab' generated using genetically improved antibody genes.

[0135] As used herein, the term "scFv" refers to a single polypeptide chain comprising a VL domain and a VH domain, where the VL and VH are linked by a linker or directly (see, for example, Bird et al., Science 242: 423-426 (1988); Huston et al., Proc. Natl. Acad. Sci. USA 85: 5879-5883 (1988); and Pluckthun, The Pharmacology of Monoclonal Antibodies, Vol. 113, Eds. Roseburg and Moore, Springer-Verlag, New York, pp. 269-315 (1994)). Such scFv molecules can have the general structure NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH. Suitable linkers in the prior art consist of repeated GGGGS amino acid sequences or variants thereof. For example, a linker having the amino acid sequence (GGGGS)4 can be used, as can variants thereof (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90: 6444-6448). Other linkers that can be used in the present disclosure were described in Alfthan et al. (1995), Protein Eng. 8: 725-731, Choi et al. (2001), Eur. J. Immunol. 31: 94-106, Hu et al (1996), Cancer Res. 56: 3055-3061, Kipriyanov et al (1999), J. Mol. Biol. 293: 41-56, and Roovers et al (2001), Cancer Immunol. In some cases, a disulfide bond can also be present between the VH and VL of the scFv. As used herein, the term "di-scFv" refers to an antibody fragment formed by linking two scFvs.

[0136] The term "mutant of Her3" refers to a polypeptide that has a similar or identical function to a HER3 polypeptide, a HER3 fragment, an anti-HER3 antibody, or an antibody fragment thereof, but does not necessarily contain a similar or identical amino acid sequence to a HER3 polypeptide, a HER3 fragment, an anti-HER3 antibody, or a fragment thereof, or a similar or identical structure to a HER3 polypeptide, a HER3 fragment, an anti-Her3 antibody, or a fragment thereof. As used herein, even if an antibody or antigen-binding fragment thereof has mutations, and amino acid substitutions, deletions, or additions, it still has binding activity to Her3.

[0137] As used herein, the percentage of homology between two amino acid sequences is equal to the percentage of identity between the two sequences. The percentage of sequence identity between two sequences is a function of the number of identical positions shared by the sequences (i.e., % homology = number of identical positions / total number of positions x 100), taking into account the number of gaps and the length of each gap, which must be introduced for optimal alignment of the two sequences. Sequence comparison and determination of the percentage of identity between sequences can be performed by methods generally known in the art, and such sequence comparison and determination of the percentage of identity can be achieved by a mathematical algorithm. For example, the algorithm of Meyers and Miller, 1988, Comput. Appl. Biosci. 4: 11-17 (integrated into the ALIGN program (version 2.0)) can be used to determine the percentage of identity between amino acid sequences and / or between nucleotide sequences. In addition, the GAP program of the GCG package available online from Accelrys (using its default parameters) can be used to determine the percentage of identity between amino acid sequences or between nucleotide sequences. In one embodiment, the two sequences are equal in length.

[0138] The term "epitope" refers to a portion of Her3 that has antigenic or immunogenic activity in an animal, preferably a mammal. An epitope with immunogenic activity is a portion of Her3 that elicits an antibody response in an animal. An epitope with antigenic activity is a portion of Her3 to which an antibody immunospecifically binds and can be determined by methods known in the art. An antigenic epitope is not necessarily immunogenic. The epitope of the antibody or antigen-binding fragment thereof of the present disclosure can be determined by existing techniques, such as synthetic peptide methods, immunoinformatics prediction, polypeptide activity determination, epitope peptide scanning, phage display technology, X-ray diffraction and nuclear magnetic resonance analysis, alloantibody modeling, protein docking prediction methods. The antibody or antigen-binding fragment thereof of the present disclosure has an epitope that binds to domain 3 of Her3.

[0139] The antibody or antigen-binding fragment thereof of the present disclosure includes an antibody that binds to the same epitope. As used herein, the phrase "antibodies that bind to the same epitope" refers to different antibodies that bind to a common epitope. If the second antibody binds to a portion of the peptide or a portion of the tertiary structure that the first antibody binds to, it can be determined that the first antibody and the second antibody bind to the same epitope. "Epitope 202-2-1" or "202-2-1 epitope" is a region of the extracellular domain of HER3 that binds to antibody 202-2-1. This epitope is close to the transmembrane domain of HER3 and is within domain 3 of HER3. "Epitope 202-2-1" consists of histidine at position 466, tryptophan at position 470, threonine at position 471, threonine at position 478, (aspartic acid-isoleucine-lysine-histidine-asparagine) at positions 483 to 487, isoleucine at position 484, arginine at position 490, and arginine at position 491 of Her3 as shown in SEQ ID NO:31.

[0140] Here, the CDRs contained in the antibody or antigen-binding fragment thereof of the present disclosure can be identified by various numbering systems known in the art. In certain embodiments, the CDRs contained in the antibody or antigen-binding fragment thereof of the present disclosure are preferably determined by the Kabat, Chothia, AbM, or IMGT numbering system.

[0141] As used herein, the term "framework region" or "FR" residues refers to those amino acid residues of an antibody variable domain excluding the CDR residues defined above. As used herein, the term "germline antibody genes" refers to genes encoding immunoglobulins expressed by non-lymphocytes that have not undergone the maturation process that results in gene rearrangement and maturation of the particular immunoglobulin expressed. One advantage provided by various embodiments of the present disclosure resides in the recognition that amino acid sequences encoded by germline antibody genes retain more characteristic and important amino acid sequence structures of an individual of an animal species than those encoded by mature antibody genes, and are therefore less likely to be recognized as foreign by that species when applied therapeutically to that species.

[0142] Concerning amino acid substitution, conservative amino acid substitution is preferred herein. Conservative amino acid substitution refers to substitution occurring within a set of amino acids related to the side chain of the amino acid. Preferred sets of amino acids are as follows: acidic set (aspartic acid and glutamic acid), basic set (lysine, arginine and histidine), non-polar set (alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine and tryptophan), and uncharged polar family (glycine, asparagine, glutamine, cysteine, serine, threonine and tyrosine). More preferred sets of amino acids are as follows: aliphatic hydroxyl (serine and threonine), amide-containing set (asparagine and glutamine), aliphatic set (alanine, valine, leucine and isoleucine), and aromatic set (phenylalanine, tryptophan and tyrosine). Such amino acid substitution is preferably made within a set that does not impair the properties of the substance having the original amino acid sequence.

[0143] Furthermore, it is known that the lysine residue at the carboxyl terminus of the heavy chain of an antibody produced in cultured mammalian cells is deleted (Journal of Chromatography A, 705: 129-134 (1995)), and it is also known that two amino acid residues (glycine and lysine) at the carboxyl terminus of the heavy chain of an antibody produced in cultured mammalian cells are deleted and a new proline residue at the carboxyl terminus is amidated (Analytical Biochemistry, 360: 75-83 (2007)). However, such deletions and modifications of the heavy chain sequence do not affect the antigen-binding affinity and effector functions (complement activation, antibody-dependent cytotoxicity, etc.) of the antibody.

[0144] The IgG isotype control antibodies of the present disclosure are well known to those skilled in the art and can be purchased or prepared. For example, the sequence of human anti-hen egg lysozyme IgG (anti-HEL, e.g. human IgG1, abbreviated as hIgG1) is derived from the variable region of the Fab F10.6.6 sequence of Acierno et al., "Affinity maturation increases the stability and plasticity of the Fv domain of anti-protein antibodies" (Acierno et al. J Mol Biol. 2007; 374(1): 130-46). Anti-hel-hIgG1, a common hIgG1 antibody against hen egg lysozyme, can be obtained from Baiyin Biological, catalog number: B117901. It can also be prepared by oneself. The preparation method is as follows: Amino acid codon optimization and gene synthesis of the heavy and light chain (full sequence or variable region) genes of human IgG antibody are performed by Nanjing GenScript Biotech Corp. according to the standard techniques introduced in "Molecular Cloning, A Laboratory Manual (3rd Edition)". The heavy and light chain genes are subcloned into antibody heavy chain expression vectors and antibody light chain expression vectors of mammalian expression system, respectively, by standard molecular cloning techniques, such as PCR, enzyme digestion, DNA gel recovery, ligation transformation, colony PCR or enzyme digestion identification, and the heavy and light chain genes of the recombinant expression vectors are further sequenced. After the sequences are accurately confirmed by sequencing, a number of endotoxin-free grade expression plasmids are prepared, and then the heavy and light chain expression plasmids are transiently co-transfected into HEK293 cells for the expression of recombinant antibodies. After 7 days of culture, the cell culture is harvested and purified with rProtein A affinity column (GE). The quality of the collected antibody samples was determined by SDS-PAGE and SEC-HPLC standard analytical techniques.

[0145] The twenty conventional amino acids referred to herein are written according to conventional usage. See, for example, Immunology-A Synthesis (2nd Edition, ES Golub and DR Gren, Eds., Sinauer Associates, Sunderland, Mass. (1991)), which is incorporated herein by reference. As used herein, the terms "polypeptide" and "protein" have the same meaning and are used interchangeably. Also, in this disclosure, amino acids are generally represented by one-letter and three-letter abbreviations that are well known in the art. For example, alanine can be represented by A or Ala, arginine can be represented by R or Arg, glycine can be represented by G or Gly, and glutamine can be represented by Q or Gln.

[0146] As used herein, the term "pharmacologically acceptable carriers and / or excipients" refers to carriers and / or excipients known in the art (see, e.g., Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995) that are pharmacologically and / or physiologically compatible with the subject and active ingredient, including, but not limited to, pH adjusters, surfactants, adjuvants, ionic strength enhancers, diluents, substances that maintain osmotic pressure, substances that delay absorption, and preservatives.

[0147] As used herein, the term "prevention" refers to a method performed to prevent or delay the occurrence of a disease or disorder or condition (e.g., tumors and infectious diseases) in a subject. As used herein, the term "treatment" refers to a method performed to obtain beneficial or desired clinical results. For the purposes of this disclosure, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, whether detectable or undetectable, reduction in the extent of disease, stabilization of the disease state (i.e., not worsening), delay or slowing of disease progression, improvement or alleviation of the disease state, and elimination of symptoms (whether partial or complete). Furthermore, "treatment" can also mean prolonging survival compared to expected survival (in the absence of treatment). As used herein, the term "subject" refers to a mammal, such as a primate mammal, such as a non-human primate mammal, or a human. In certain embodiments, the subject (e.g., a human) has or is at risk of having a tumor or infection.

[0148] As used herein, the term "effective amount" refers to an amount sufficient to achieve or at least partially achieve a desired effect. For example, an effective amount for preventing a disease (e.g., tumors and infections) is an amount sufficient to prevent, stop, or delay the onset of the disease (e.g., tumors and infections), and an effective amount for treating a disease is an amount sufficient to treat or at least partially stop the disease and its complications in patients who already have them. Determining such an effective amount is entirely within the capabilities of a person skilled in the art. For example, the effective amount for therapeutic use depends on the severity of the disease to be treated, the general condition of the patient's own immune system, the general condition of the patient, such as age, weight, and sex, the method in which the drug is administered, and other treatments administered at the same time, etc.

[0149] As used herein, the term "effector functions" refers to those biological activities attributable to the Fc region of an antibody (either a native sequence Fc region or an amino acid sequence variant Fc region), which vary depending on the antibody isotype. The term "pharmaceutically acceptable" means that the molecule itself, molecular fragment, or composition does not produce adverse, allergic, or other side effects when properly administered to an animal or human. Specific examples of some substances that may be pharmaceutically acceptable carriers or components thereof include sugars (e.g., lactose), starch, cellulose and its derivatives, vegetable oils, gelatin, polyols (e.g., propylene glycol), alginic acid, and the like. The in vivo therapeutic efficacy of antibodies and antibody-drug conjugates against cancer can be determined using experimental animals, for example, by administering the antibodies to nude mice implanted with a tumor cell line expressing Her3 and measuring any changes in the cancer cells. Examples of cancer types include lung cancer, colon cancer, and the like. Here, the term "direct bond" means that the groups on both sides are directly bonded, for example, the formula II

[0150] [ka] When X is a direct bond in the compound represented by the formula [ka] Other direct bonds can be understood with reference to the above. As used herein, the term "absent" means that a group is not present, e.g., Formula II

[0151] [ka] When W is absent in a compound represented by the formula

[0152] [ka] It is. Formula II

[0153] [ka] In a compound represented by the formula:

[0154] [ka] The "dotted line" indicates where the heterocycle is fused to the benzene ring, e.g. [ka] Form. Here, X is, for example, "X is optionally substituted [ka] and the substituents are selected from one or two C1-4 alkyl groups (e.g., methyl) so that X can be, for example,

[0155] [ka] Other similar definitions of X can be understood with reference to the above. Here, X is, for example, "X is optionally substituted

[0156] [ka] and the substituents are selected from two C1-4 alkyl groups (e.g., methyl) which, together with the carbon atom to which they are both attached, form a C3-6 cycloalkyl (e.g., cyclopropyl) so that X is, for example,

[0157] [ka] Other similar definitions of X can be understood with reference to the above. AA 1

[0158] [ka] In the structure of an amino acid residue represented by the formula: 1 The structure of the amino acid residue represented by

[0159] [ka] It will be understood by those skilled in the art that AA 1

[0160] [ka] In the structure of an amino acid residue represented by a and R b When they both form a 4-10 membered heterocyclic ring together with the carbon atom to which they are attached, the 4-10 membered heterocyclic ring may be one or more R 0 The term "4-10 membered heterocycle" refers to one or more R 0 "optionally substituted with" means that the 4- to 10-membered heterocycle is optionally substituted with one or more R 0 means that it may be unsubstituted or substituted with more R 0 If there is, each R 0 The definitions of may be the same or different. Other similar definitions can be understood with reference to the above. Here, for example, when L3 is selected from Lys, Val-Cit, Ala-Ala-Asn, Ala-Ala-Asp, Gly-Gly-Phe-Gly, Val-Lys-Gly, Val-Ala, and Lys-Ala-Asn, "the terminal amino group of lysine (Lys) may be substituted with one, two, or three substituents selected from tert-butoxycarbonyl, C1-6 alkyl (preferably methyl), and O" means that the terminal amino group of Lys in each of the options for L3 may be substituted with one, two, or three substituents selected from tert-butoxycarbonyl, C1-6 alkyl (preferably methyl), and O. Here, "the terminal amino group of Lys" refers to a lysine residue.

[0161] [ka] "The terminal amino group of lysine (Lys) may be substituted with one, two or three substituents selected from tert-butoxycarbonyl, C1-6 alkyl (preferably methyl), and O" indicates that the terminal amino group of lysine (Lys) may be unsubstituted or substituted with one, two or three substituents selected from tert-butoxycarbonyl, C1-6 alkyl (preferably methyl), and O. For example, it may be substituted with one tert-butoxycarbonyl group, i.e.

[0162] [ka] or it can be substituted with two methyls, i.e.

[0163] [ka] or it can be substituted with two methyls and one O, i.e.

[0164] [ka] "The terminal amino group of lysine (Lys) is substituted with O" means that the terminal amino group of lysine (Lys) is substituted with oxo, that is,

[0165] [ka] When the terminal amino group is further substituted with two methyl groups,

[0166] [ka] It should be noted that this means that At various points in this specification, the substituents of the compounds of the present disclosure are disclosed according to group types or ranges. Specifically, the present disclosure includes each individual subcombination of each member of these group types or ranges. For example, the term "C1-6 alkyl" specifically refers to methyl, ethyl, C3 alkyl, C4 alkyl, C5 alkyl, and C6 alkyl, which are disclosed independently. As used herein, the term "C1-6 alkyl" refers to a straight or branched chain alkyl group containing 1 to 6 carbon atoms, including, for example, "C1-3 alkyl" or "C1-4 alkyl", methyl, ethyl, and the like, and specific examples include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, pentyl, and hexyl.

[0167] As used herein, the term "C 1-4 "Alkyl" refers to a straight or branched alkyl group containing 1 to 4 carbon atoms, e.g., "C 1-3 Alkyl includes methyl, ethyl, etc. Specific examples include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, and tert-butyl. As used herein, the term "C 2-6 "Alkenyl" refers to a straight-chain, branched-chain or cyclic alkenyl group containing at least one double bond and 2 to 6 carbon atoms, e.g., "C2-4 Alkenyl, etc. Examples include, but are not limited to, vinyl, 1-propenyl, 2-propenyl, 1-butenyl, 2-butenyl, 1,3-butadienyl, 1-pentenyl, 2-pentenyl, 3-pentenyl, 1,3-pentadienyl, 1,4-pentadienyl, 1-hexenyl, 2-hexenyl, 3-hexenyl, 1,4-hexadienyl, cyclopentenyl, 1,3-cyclopentadienyl, cyclohexenyl, 1,4-cyclohexadienyl, and the like.

[0168] As used herein, the term "C 2-6 "Alkynyl" refers to a straight or branched alkynyl group containing at least one triple bond and 2 to 6 carbon atoms, e.g., "C 2-4 Examples include, but are not limited to, ethynyl, propynyl, 2-butynyl, 2-pentynyl, 3-pentynyl, 4-methyl-2-pentynyl, 2-hexynyl, 3-hexynyl, 5-methyl-2-hexynyl, and the like. As used herein, the term "halogen" includes fluorine, chlorine, bromine, and iodine. As used herein, the term "3- to 6-membered cycloalkyl" or "C 3-6 "Cycloalkyl" refers to a saturated cyclic alkyl containing from three to six carbon atoms, including cyclopropanyl (ie, cyclopropyl), cyclobutanyl (ie, cyclobutyl), cyclopentanyl (ie, cyclopentyl), and cyclohexyl. As used herein, the term "3- to 7-membered carbocyclic alkyl" or "C 3-7 "Cycloalkyl" refers to a saturated cyclic alkyl containing 3 to 7 carbon atoms, including cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and cycloheptyl.

[0169] As used herein, the term "C 1-6"Alkoxy" refers to an alkyl group, as defined above, attached to the parent molecular moiety through an oxygen atom. Illustrative examples include, but are not limited to, methoxy, ethoxy, propoxy, isopropoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, tert-butoxy, pentoxy, hexyloxy, and the like. As used herein, the term "C 1-4 "Alkoxy" refers to an alkyl group, as defined above, attached to the parent molecular moiety through an oxygen atom. Illustrative examples include, but are not limited to, methoxy, ethoxy, propoxy, isopropoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, tert-butoxy, and the like.

[0170] As used herein, the term "4-10 membered heterocyclic group" refers to a cyclic group containing 4 to 10 ring atoms (at least one ring atom is a heteroatom, such as a nitrogen atom, an oxygen atom, or a sulfur atom). The term "4-6 membered heterocyclic group" refers to a cyclic group containing 4 to 10 ring atoms (at least one ring atom is a heteroatom, such as a nitrogen atom, an oxygen atom, or a sulfur atom). A ring atom (e.g., a carbon atom, a nitrogen atom, or a sulfur atom) of the ring structure may be substituted with oxygen. Examples of the "4- to 8-membered heterocyclic group" include a "4- to 8-membered nitrogen-containing heterocyclic group", a "4- to 8-membered oxygen-containing heterocyclic group", a "4- to 7-membered heterocyclic group", a "4- to 7-membered oxygen-containing heterocyclic group", a "4- to 7-membered heterocyclic group", a "4- to 6-membered heterocyclic group", a "5- to 7-membered heterocyclic group", a "5- to 6-membered heterocyclic group", and a "5- to 6-membered nitrogen-containing heterocyclic group", including, but not limited to, oxocyclobutanyl, pyrrolidinyl, tetrahydrofuryl, piperidinyl, piperazinyl, tetrahydropyranyl, homopiperazinyl, and the like.

[0171] As used herein, the term "4-10 membered heterocycle" refers to a ring containing 4-10 ring atoms (at least one ring atom is a heteroatom, such as a nitrogen atom, an oxygen atom, or a sulfur atom). The term "5-6 membered heterocycle" refers to a ring containing 5-6 ring atoms (at least one ring atom is a heteroatom, such as a nitrogen atom, an oxygen atom, or a sulfur atom), including, but not limited to, pyrrolidine, tetrahydrofuran, piperidine, piperazine, tetrahydropyran, and the like. As used herein, the term "aryl" refers to a monocyclic or polycyclic hydrocarbon group having aromaticity, such as 6-10 membered aryl, 5-8 membered aryl, etc. Specific examples include, but are not limited to, phenyl, naphthyl, anthracenyl, phenanthryl, etc. "6-10 membered aryl" refers to an aryl having 6-10 ring atoms. "C6-10 aryl" refers to an aryl group containing 6-10 carbon atoms.

[0172] As used herein, the term "heteroaryl" refers to a cyclic group having aromaticity, in which at least one ring atom is a heteroatom, such as a nitrogen atom, an oxygen atom, or a sulfur atom. The ring atoms (e.g., carbon atom, nitrogen atom, or sulfur atom) of the ring structure may be substituted with oxygen. Specific examples include 5-10 membered heteroaryl, 5-6 membered heteroaryl, 5-10 membered nitrogen-containing heteroaryl, 6-10 membered oxygen-containing heteroaryl, 6-8 membered nitrogen-containing heteroaryl, 5-8 membered oxygen-containing heteroaryl, etc., such as furyl, thienyl, pyrrolyl, thiazolyl, isothiazolyl, thiadiazolyl, oxazolyl, isoxazolyl, oxadiazolyl, imidazolyl, pyrazolyl, 1,2,3-triazolyl, 1,2,4-triazolyl, 1,2,3-oxadiazolyl, 1,2,4-oxadiazolyl, 1,2,5-oxadiazolyl, 1,2,5-oxadiazolyl, 1,2,6-oxadiazolyl, 1,2,7-oxadiazolyl, 1,2,8-oxadiazolyl, 1,2,9-oxadiazolyl, 1,3,4-oxadiazolyl, 1,4,5-oxadiazolyl, 1,5,6-oxadiazolyl, 1,6,7-oxadiazolyl, 1,7,8-oxadiazolyl, 1,8,9-oxadiazolyl, 1,9,10-oxadiazolyl, 1,9,10-oxadiazolyl, 1,10,11-oxadiazolyl, 1,11,12-oxadiazolyl, 1, ,3,4-oxadiazolyl, pyridyl, 2-pyridonyl, 4-pyridonyl, pyrimidinyl, 1,4-dioxinyl, 2H-1,2-oxazinyl, 4H-1,2-oxazinyl, 6H-1,2-oxazinyl, 4H-1,3-oxazinyl, 6H-1,3-oxazinyl, 4H-1,4-oxazinyl, pyridazinyl, pyrazinyl, 1,2,3-triazinyl, 1,3,5-triazinyl, 1,2,4,5-tetrazinyl, azacycloheptatrienyl, 1,3-diazacycloheptatrienyl, azacyclooctatetraenyl, and the like.

[0173] The bonds in structural formulas represented herein by a wavy line "~~" are intended to indicate that the structure represents either the cis or trans isomer, or a mixture of cis and trans isomers in any ratio. Here, "between" includes the values ​​at the ends of the interval, which can say 1 or 16, for example, between 1 and 16.

[0174] The term "drug-to-antibody ratio" or "DAR" refers to the amount of drug, e.g., the average ratio of small molecule toxins bound to the antibody of an ADC. The DAR of an ADC can range from 1 to 16, but depends on the number of binding sites on the antibody, i.e., higher loadings (e.g., 20) are also possible. The term DAR can be used to refer to the amount of drug loaded on a single antibody, or alternatively, to the average DAR of a group of ADCs. DAR can also be calculated as the average DAR of a population of molecules in a product, which is the overall ratio (molar ratio) of drug moieties (D) bound to the Ab moieties of the product, as measured by a detection method (e.g., conventional methods such as mass spectrometry, ELISA, electrophoresis, and / or HPLC). Based on the general knowledge of the art, the above preferred conditions may be combined to obtain the preferred embodiment of the present disclosure. The reagents and materials used in the present disclosure are commercially available.

[0175] The beneficial effects of the present disclosure are as follows: Compared with the prior art, the antibodies included in the present invention are fully human antibodies and can be safely administered to human subjects without causing immunogenic reactions. The antibodies included in the present invention have extremely high affinity and specificity, which can be beneficial for exerting anti-tumor effects. Unexpectedly, the antibody-drug conjugates of the present disclosure have significant in vivo anti-tumor activity.

[0176] Abbreviation [Table 2-1] [Table 2-2] [Brief description of the drawings]

[0177] [Figure 1A] FIG. 1A is a diagram of an assay for binding of anti-Her3 antibodies to human Her3-his protein by ELISA. [Figure 1B] FIG. 1B is a diagram of an assay for binding of anti-Her3 antibodies to monkey Her3-his protein by ELISA. [Figure 1C] FIG. 1C is a diagram of an assay for binding of anti-Her3 antibody to rat Her3-his protein by ELISA. [Figure 2A] FIG. 2A is an assay for binding of anti-Her3 antibodies to A549-human Her3 cells by flow cytometry. [Figure 2B] FIG. 2B is an assay for binding of anti-Her3 antibody 202-2-1 to T47D cells by flow cytometry. [Figure 2C] FIG. 2C is an assay for binding of anti-Her3 antibody 202-2-1 to MCF-7 cells by flow cytometry. [Diagram 3] FIG. 1 shows the inhibition rate of anti-Her3 antibody against phosphorylation of AKT by NRG1 in tumor cells. [Figure 4] FIG. 1 shows an assay of endocytosis of tumor cell A549-human Her3 cells for anti-Her3 antibodies. [Figure 5A] FIG. 5A is a diagram of an assay for anti-Her3 antibodies competing with biotinylated control antibody 2 for binding to human Her3 protein. [Figure 5B-1] FIG. 5B-1 is a diagram showing the formation of a complex by binding between Fab of antibody 202-2-1 and Her3. [Figure 5B-2] FIG. 5B-2 is a boundary diagram 1 of the complex between Fab of antibody 202-2-1 and Her3, with dotted lines and numbers indicating the amino acid side chains that interact with each other and their distance (Å). [Figure 5B-3] FIG. 5B-3 is a boundary diagram 2 of the complex between Fab of antibody 202-2-1 and Her3, where the dotted lines and numbers indicate the amino acid side chains that interact with each other and their distance (Å). [Figure 6A]FIG. 6A is a diagram of an assay for anti-Her3 antibody 202-2-1 ADC drug killing against PC-9 tumor cells. [Figure 6B] FIG. 6B is a diagram of anti-Her3 antibody 202-2-1 ADC drug killing assay against NCI-H358 tumor cells. [Figure 7] FIG. Pharmacodynamic study of anti-Her3 antibody 202-2-1 ADC drug on NCI-H358 tumor model. [Figure 8] FIG. 1. Pharmacodynamic study of anti-Her3 antibody 202-2-1 ADC drug on a SW480 tumor model. [Figure 9] FIG. 2: Pharmacodynamic study of anti-Her3 antibody 202-2-1 ADC drug on another SW480 tumor model. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0178] The present disclosure will be further illustrated by the following specific examples, which are not intended to limit the present disclosure. Based on the teachings of the present disclosure, those skilled in the art can make various improvements or modifications without departing from the basic spirit and scope of the present disclosure. Reagents or equipment used without manufacturer's instructions are commercially available conventional products. EXAMPLES

[0179] Example 1 Preparation of Her3 protein and control antibody The nucleic acid sequence of amino acids 1-643 of human Her3 (NCBI: NP_001973.2) was selected, and a 6×His detection tag was added to the C-terminus to name it human Her3-his, and the amino acid sequence of amino acids 20-641 of rat Her3 (Uniport ID: Q61526-1) was selected, and a 6×His purification tag was added to the C-terminus to name it rat Her3-his. Control antibody 1 was obtained from MediaPharma patent CN 103189392 B (heavy chain is SEQ ID NO: 10, light chain is SEQ ID NO: 14), and control antibody 2 was obtained from Daiichi Sankyo Company, referring to the IMGT database, IMGT / mAb DB ID: 964, INN No.: 11093. The gene of the above human Her3 antigen was synthesized, and at the same time, the genes of two control antibodies were synthesized by codon optimization (GenScript (Nanjing) Co., Ltd.), and then the genes were inserted into the PTT5 expression vector, and a number of plasmids were extracted. The extracted and prepared expression vector was transiently transfected into HEK293F cells by PEIMAX, expressed for 7 days, and the expression supernatant was purified and prepared by Ni column or ProA. The amino acid sequence of 1 to 643 of monkey Her3 (NCBI: XP_001113953.2) was selected, and a 6×His purification tag was added to the C-terminus to name monkey Her3-his.

[0180] Example 2 Screening of anti-Her3 antibodies from phage libraries Venous blood was collected from healthy individuals, PBMC cells were extracted by Ficol, and then mRNA was extracted for reverse transcription. Specific primers were designed to amplify the heavy chain variable region (VH) and light chain variable region (VL). ScFvs were integrated into phage vectors by homologous recombination and electrotransformed into TG1 together with auxiliary vectors, thereby generating a complete human ScFv phage library of 1.5×10 10A library volume of 10000 was obtained. After the first enrichment with human Her3-His, a second enrichment with monkey Her3-His (obtained from Sino Biological Inc.) was performed, and 96 phages were selected, and the optimal clones 202-2-1 and 202-3-1 were obtained by ELISA and T47D tumor cell flow cytometry. To improve the physicochemical properties of 202-3-1, site-directed mutagenesis was performed on its FR, resulting in a clone named 202-3-2.

[0181] Example 3 Expression of anti-Her3 antibodies The heavy chain variable regions of clone 202-2-1, clone 202-3-1, and clone 202-3-2 are each linked to an IgG1 constant region (SEQ ID NO: 29), while the light chain variable regions of clone 202-2-1, clone 202-3-1, and clone 202-3-2 are each linked to a kappa constant region (SEQ ID NO: 30), and then each is incorporated into a PTT5 vector by codon optimization and gene synthesis (General Biosystems (Anhui) Co., Ltd.). The expression plasmids of the heavy and light chains of the anti-Her3 antibody were co-transfected into HEK293F cells with PEI max reagent and expressed at 37°C in a shaker with 5% CO2 for 7 days. The supernatant was collected and purified by Protein A magnetic beads to obtain anti-Her3 antibodies, which were named antibody 202-2-1, antibody 202-3-1, and antibody 202-3-2, respectively. The heavy chain variable region, light chain variable region, and corresponding CDRs of antibodies 202-2-1, 202-3-1, and 202-3-2 are shown in Table 1.

[0182] [Table 3-1] [Table 3-2]

[0183] Example 4 Affinity assay of anti-Her3 antibodies to Her3 protein The affinity of the anti-Her3 antibodies to the Her3 protein was tested by ELISA. Specific experimental procedure: Human Her3-his, monkey Her3-his protein, and rat Her3-his were diluted with pH 9.6 carbonate buffer to a final concentration of 1 μg / mL, added to a 96-well microplate at 100 μL / well, coated overnight at 4°C, discarded the coating solution, washed the plate once with PBST, then added 100 μL of PBS (containing 2% BSA) to each well, blocked at 37°C for 1 hour, then added anti-Her3 antibody (starting at 1 μg, serially diluted 3-fold in duplicate wells) diluted with 100 μL of PBS (containing 2% BSA) to each well, then incubated at 37°C for 2 hours, washed the plate three times with PBST, added horseradish peroxidase-labeled anti-human Fc antibody (diluted 1:10000 in PBS) at 100 μL / well, and reacted at 37°C for 1 hour. The plate was washed 5 times with PBST. TMB development solution was added at 100 μL / well and incubated at 37° C. for 7 min for color development, then stop solution was added at 50 μL / well to stop the reaction. Absorbance was measured at 450 nm and EC 50 The values ​​were calculated and imported into Graph-prism. The results are shown in Figure 1A-1C, and the detailed results are listed in Table 2. 202-2-1 had high affinity to human Her3 protein and monkey Her3 protein, comparable to Control Antibody 1, but had no affinity to rat Her3 protein, and 202-3-1 and 202-3-2 had high affinity to human Her3 protein and monkey Her3 protein, but lower than the affinity of Control Antibody 1, and both of them bound to rat Her3 protein.

[0184] [Table 4]

[0185] Example 5 Dynamic affinity assay of anti-Her3 antibodies The dynamic affinity of anti-Her3 antibody to Her3 protein was detected by Fortibio, a commonly used dynamic affinity assay device. The method is briefly described as follows: serial dilutions of human Her3-His were performed with PBST to obtain 400nM, 200nM, 100nM, 50nM, 25nM, 12.5nM, 6.25nM, 3.125nM, 1.5625nM and 0nM. ProteinA biosensor (Pall Life Sciences) was pre-humidified with PBST buffer before use. Anti-Her3 antibody was diluted to 5μg / mL in PBST and immobilized on ProA sensor. The sensor with immobilized antibody was then equilibrated in PBST buffer for 60s to obtain a baseline, and then transferred to antigen diluent for 60s for binding, followed by dissociation in PBST for 180s. After one analysis cycle, the sensor was regenerated with 10mM Gly (pH 1.5). The association (Ka) and dissociation (Kd) rate constants were determined in a 1:1 model using Date analysis and used to calculate the dissociation equilibrium constant (KD). As shown in Table 3, the kinetic affinity of 202-2-1, 202-3-1, and 202-3-2 for human Her3 was higher and comparable to that of the control antibody 1.

[0186] [Table 5]

[0187] Example 6 Assay of affinity of anti-Her3 antibodies to cells Her3 protein was highly expressed in various solid tumor cells. The affinity of Her3 antibody to tumor cells was detected by FACS assay. Specific experimental procedure: MCF-7 (breast cancer cells, ATCC, product number HTB-22), A549-human Her3 (human non-small cell lung cancer, ATCC, product number CRM-CCL-185), and T47D (human breast ductal carcinoma, ATCC) cancer cells grown to logarithmic growth phase were digested with trypsin and diluted at 5 × 10 cells / mL in PBS buffer containing 2% BSA. 5The antibodies were resuspended at 150 μg / mL cells / ml. 100 μL of the cell suspension was added to a 96-well plate. Control antibody 1 or control antibody 2, 202-2-1, 202-3-1, and 202-3-2 were diluted in PBS buffer containing 2% BSA by 3-fold serial dilutions at a starting concentration of 150 μg / mL or 50 μg / mL to obtain approximately 10 concentrations. 100 μL of the antibody suspension was then added to the 96-well plate and mixed, the plate was incubated at 4° C. for 1 hour, washed twice with pre-chilled PBS, 300 μL / well each time, then centrifuged at 500 g for 5 minutes, and 100 μL / well of 1:50 fluorescent secondary antibody APC anti-human IgG (obtained from Biolegend) was added and mixed. The plates were incubated at 4°C for 0.5 hours, washed twice with pre-cooled PBS, 300 μL / well each time, centrifuged at 500g for 5 minutes, and then resuspended in 500 μL PBS. The mean fluorescent signal values ​​were measured by Beckman flow cytometry. The results are shown in Figures 2A-2C and Table 4. The affinity of 202-2-1 to cells was comparable to that of control antibody 1 and better than that of control antibody 2, but the maximum MFI was higher than that of control antibody 1. The cell affinity of 202-3-1 and 202-3-2 was slightly weaker than that of control antibody 1.

[0188] [Table 6]

[0189] Example 7 Assay of the inhibition rate of anti-Her3 antibody against NRG1-induced AKT phosphorylation After binding with high affinity to Her3 / Her2 heterodimer, NRG1 induced phosphorylation of AKT in the downstream signaling pathway and stimulated the proliferation of tumor cells. The inhibition rate of anti-Her3 antibody against the NRG1-induced AKT phosphorylation pathway was detected by cell blotting. Specific experimental procedure: On a certain day, MCF-7 tumor cells (human breast cancer cells) were cultured in a 96-well plate at 50000 / well in advance, left overnight, and then starved with serum-free RPMI1640 medium for about 24 hours. Antibodies 202-2-1 and 202-3-1 were diluted by 3-fold serial dilution at a starting concentration of 100nM, then added to the cells, followed by incubation at 37℃ for 30 minutes, and then NRG1 (Sino Biological, Part No.: 11609 HNCH) was added at a final concentration of 10ng / ml. The plate was incubated at 37℃ for 30 minutes, and then the reaction was stopped and the supernatant was discarded. The plate was washed once with PBS, then formaldehyde was added at 100 μL / well for 30 minutes to fix at room temperature. The plate was washed twice with PBS, pre-cooled methanol was added at 100 μL / well for 30 minutes for membrane permeation, and then washed three times with PBS. PBS (containing 2% BSA) was added to each well, and the plate was blocked for 2 hours. 100 μL of 1:500 anti-Phospho Akt (Ser473) antibody (CST, part number 9271-S) was added, and the plate was incubated overnight at 4°C. The plate was then washed three times with PBST, and 1:1000 HRP alpaca anti-rabbit IgG antibody (NB-biolab, Sichuan) was added, and then incubated at 37°C for 1 hour. The plate was washed five times with PBST. 100 μL of ECL developer was added (Vazyme, Part No. E412-01) and the luminescence value was read using a microplate reader (MD, Model: i3x). IC50 values ​​were calculated using Graph-prism as shown in Figure 3. Both anti-Her3 antibodies 202-2-1 and 202-3-1 significantly inhibited NRG1-induced AKT phosphorylation, but antibody 202-2-1 was significantly more potent than antibody 202-3-1, with IC50 values ​​of 0.2434 nM and 1.037 nM, respectively.

[0190] Example 8 Assay of anti-Her3 antibody specificity The specificity of the Her3 antibody, which binds to the Her3 protein but not to its cognate proteins EGFR, Her2 and Her4, was determined by ELISA. Specific experimental procedure: Human EGFR-His, human Her2-His, human Her4-His (obtained from Sino Biological Inc.), and human Her3-his proteins were diluted with carbonate buffer at pH 9.6 to a final concentration of 1 μg / mL, respectively, and added to a 96-well microplate at 100 μL / well, coated overnight at 4°C, discarded the coating solution, washed the plate once with PBST, then added 100 μL of PBS (containing 2% BSA) to each well, blocked the plate at 37°C for 1 hour, then added Her3 antibody diluted with 100 μL of PBS (containing 2% BSA) at 1 μg / mL, 200 ng / mL, and 40 ng / mL to each well, incubated the plate at 37°C for 2 hours, washed the plate three times with PBST, and added horseradish peroxidase-labeled anti-human Fc antibody (1:10000 100μL / well of 202-2-1, 202-3-1, and 202-3-2 were added to the plate at 37℃ for 1 hour, and the plate was washed 5 times with PBST. 100μL / well of TMB development solution was added to the plate at 37℃ for 7 minutes, and the reaction was stopped by adding 50μL / well of stop solution, and the plate was read at 450nm in a microplate reader. The results are shown in Table 5. 202-2-1, 202-3-1, and 202-3-2 specifically bound to human Her3 protein, but did not bind to human EGFR, human Her2, and human Her4 proteins.

[0191] [Table 7]

[0192] Example 9 Assay of endocytosis of anti-Her3 antibody Endocytosis of anti-Her3 antibodies by A549-human Her3 (human non-small cell lung cancer cells) was detected by FACS. A549-human Her3 cells grown to logarithmic growth phase were digested with trypsin, then the cells were collected by centrifugation and washed three times with cold PBS, the cells were resuspended in PBS (containing 1% BSA), and the anti-Her3 antibodies to be tested were added at a concentration of 10 μg / mL, followed by incubation at 4°C for 1 h, the cells were collected by centrifugation, washed three times with PBS, resuspended in DMEM+10% FBS, and incubated in four portions at 37°C for 0, 1, 2, and 4 h, respectively, for the incubation phase. After completion of the above, the cells were collected by centrifugation, washed three times with cold PBS, and resuspended in 50 μL of 1% BSA (in PBS), then 1 μL of fluorescent secondary antibody APC anti-human IgG (Biolegend) was added to each well, the mixture was mixed thoroughly, the plate was incubated at 4°C for 0.5 hours, the cells were collected by centrifugation, washed three times with cold PBS, resuspended in 200 μL of PBS, and loaded into a flow cytometer (Beckman, Model: CytoFlex) for detection. According to the formula: endocytosis rate (%) = [1-(mean fluorescence value of test sample at this time point-mean fluorescence value of negative control sample at this time point) / (mean fluorescence value of test sample at 0 hours-mean fluorescence value of negative control sample at 0 hours)] x 100. The results are shown in Figure 4. Anti-Her3 antibody 202-2-1 had strong endocytosis activity against A549-human Her3 tumor cells, reaching about 26.9% at 4 hours, which was stronger than the endocytosis activity of control antibody 2 at 16.6% and comparable to that of control antibody 1. However, anti-Her3 antibody 202-2-1 reached its maximum endocytosis rate before the control antibody and had a faster endocytosis rate than the control antibody. The endocytosis activity of antibody 202-3-2 was stronger than that of control antibody 1 and control antibody 2.

[0193] Example 10 Antigen epitope assay 10.1 Assay for epitope competition of anti-Her3 antibodies A competitive ELISA was used to determine whether anti-Her3 antibody 202-2-1 competes with control antibodies 1 and 2 for epitope binding. The specific steps were as follows: human Her3-his antigen was coated with CBS coating solution at 100ng / well, then the plate was incubated overnight at 4°C, the coating solution was removed, the wells were washed once with 300μL of PBS, then each well was blocked with PBS (containing 2% BSA) solution at 100μL / well, then incubated at 37°C for 2 hours, the blocking solution was discarded, and antibody 202-2-1, control antibody 1, and control antibody 2 were diluted by 3-fold dilutions starting from 15μg / mL to a total of 11 dilution points, and the 12th point was replaced with diluent. Then, 50μL of antibody was added to each well in duplicate, and 50μL of control antibody 1, labeled with biotin, diluted to 10ng / mL, and control antibody 2, diluted to 88ng / mL, were added to each well in duplicate, and the plate was then incubated at 25℃ for 2 hours, and the wells were washed three times with 300μL of PBST, and then 100μL of HRP anti-biotin secondary antibody, diluted to a ratio of 1:5000 in PBS (containing 2%BSA), was added, and then incubated at 25℃ for 1 hour, and the wells were washed five times with 300μL of PBST, and 100μL of TMB color development solution (Huzhou InnoReagents, Anhui) was added to each well, and after 5 minutes of color development at room temperature, 50μL of H2SO4 was added to each well to stop the reaction, and the plate was immediately read in a microplate reader at OD450nm. The raw data was imported into Graph-prism to calculate the EC50 values, and the results are shown in Figure 5 A. The results showed that the anti-Her3 antibody 202-2-1 did not compete with the control antibody 12 for epitope binding.

[0194] 10.2 Determination of the antigenic epitope of anti-Her3 antibody To further investigate the specific mode of action of the Her3 antibody and the epitope recognized by it, a complex of the 202-2-1 Fab fragment with the Her3 protein was prepared, and then the structure of the antibody-antigen complex was analyzed by cryo-electron microscopy.

[0195] (1) Antigen preparation: DNA encoding the amino acid sequence of the extracellular domain of HER3 (20-643) obtained from the uniprot database was synthesized after sequence optimization (293F as host), and a 6xHis tag was added to the C-terminus of the protein. The gene was incorporated into pLVX (MIAO LING BIO, product number P0249) expression vector and transfected into 293F cells (ATCC; PTA-5077), and then screening was performed to obtain a stable cell pool. The culture supernatant containing the antigen was obtained by fermentation and cultivation of the cells. The antigen was recovered by Ni affinity column chromatography and further purified by molecular sieve. The final antigen was dissolved in Tris-NaCl solution at pH 7.5 (0.7mg / ml). Antigen quality characterization: 1. SDS-PAGE experiment showed that the band was sharp under both non-reducing and reducing conditions. 2. The purity was 95.6% by SEC-HPLC. 3. The purity was 97.44% by reverse phase chromatography HPLC. 4. The affinity of the antigen and antibody tested by ELISA showed that the affinity of this batch of antigen and antibody 202-2-1 was less than 0.1nM, and the next step of the experiment could be carried out.

[0196] (2) Preparation of Fab-antigen complex: Antibody 202-2-1 was dissolved in 20 mM acetic acid + 150 mM sodium chloride to a concentration of 20.7 mg / ml. Pepsin and antibody were incubated at 37°C for 240 minutes, the digestion products were analyzed by SDS-PAGE and separated by SEC (Superdex 200 Increase 10 / 300 GL column) column, and the main peak was collected. The above product was incubated with papain for 120 minutes at 37°C, the digestion products were analyzed by SDS-PAGE and separated by SEC (Superdex 200 Increase 10 / 300 GL column) column, and the main peak was collected to obtain the Fab of antibody 202-2-1. The antigen prepared in step (1) was mixed with the Fab and then incubated overnight at 4°C. The incubated products were concentrated by centrifugation and analyzed by SDS-PAGE, then separated using a SEC (Superdex 200 Increase 10 / 300 GL column) column, and the major peak was collected to obtain the Fab-antigen complex. Before preparing the samples for cryo-EM, the quality of the complexes was examined by a negative staining method (Vitrobot™ MarkIV, easiGlow™ Glow Discharge, 200kV TF20, CCD camera 895). The negative staining results showed that many of the complexes had consistent morphology. Eighty pictures were selected for the negative staining reconstruction, and the PDB model (4LEO) was used for the simulation of the density map. The simulation results showed that the complexes could overlap with most of the PDB model (including the Fab binding domain), indicating that the quality of the complexes met the requirements for cryo-EM observation.

[0197] (3) Preparation of samples for cryo-microscopy Glacios / cryo-microscopy in data collection / screening: The complex prepared in (2) was diluted to 0.2mg / ml with 25mM Tris-HCl (pH7.5) and 150mM NaCl, centrifuged at 12000g for 15min, all related equipment and reagents were prepared, 3μL of sample was dropped onto a 300 mesh R1.2 / 1.3 holey carbon film (Quantifoil) grid, and the complex pictures were observed and screened using a 200KV Glacios electron microscope. High resolution cryo-electron micrographs were collected by a 300kV Titan Krios electron microscope (equipped with a K3 direct electron detector, Gatan brand), and a total of 3272 pictures were collected for further analysis. Parameter settings: magnification 105K, physical pixel 0.819A, total of 56.3e- / A divided into 40 frames. 2 electron dose, and the underfocus range set to -1.0 µM to -2.4 µM.

[0198] (4) Photo processing / atomic model construction and epitope analysis: All 3272 photo data were processed using CryoSPARC v3.3.1, including contrast transfer function evaluation and correction, 2D classification, heterogeneous refinement, and homogeneous refinement. To construct a structural model of the Her3-Fab complex, a Fab structure with high homology to the Her3 structure (PDB 1M6B) and antibody 202-2-1 (light chain: PDB 7CDJ, heavy chain: PDB 6D01) was fitted to the cryo-EM map using UCSF Chimera software. Guided by the cryo-EM map, a model was manually constructed in Coot (Emsley P et al., Acta Crystallogr D Biol Crystallogr. 2010 Apr;66(Pt 4):486-501) in combination with real-space refinement using CCP-EM. The results are shown in Figures 5B-1, 5B-2, and 5B-3.

[0199] The results of the structural analysis showed that the antigen epitope to which the 202-2-1 antibody Fab binds is located in domain 3 of human Her3, amino acids 328 to 499 of SEQ ID NO: 31, and if the Her3 antigen is numbered according to the amino acid sequence of SEQ ID NO: 31, it is particularly located at histidine at position 466, (tryptophan-threonine) at positions 470 to 471, threonine at position 478, and (aspartic acid-isoleucine-lysine-histidine-asparagine) at positions 483 to 487. ), isoleucine at position 484, and (arginine-arginine) at positions 490-491 of the Fab heavy chain, and based on the amino acid numbering of SEQ ID NO:23, (serine) at position 30, (tyrosine-aspartic acid) at positions 53-54, (serine-histidine) at positions 56-57, tyrosine at position 59, and tryptophan at position 100 of the Fab heavy chain are involved in antigen binding, and asparagine at position 92 of the Fab light chain numbered according to the amino acid sequence of SEQ ID NO:24 is involved in antigen binding.

[0200] Example 11 Synthesis of intermediates used in the synthesis of biologically active molecules and "drug-linker compounds" Example 11.1: Synthesis of (S)-7-ethyl-7-hydroxy-14-(3-hydroxypropyl)-10,13-dihydro-11H-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-8,11(7H)-dione (A1)

[0201] [ka] Step 1: Synthesis of (S)-14-(3-chloropropyl)-7-ethyl-7-hydroxyl-10,13-dihydro-11H-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-8,11(7H)-dione (A1B)

[0202] [ka] In an ice bath, to a solution of compound (S) 7-ethyl-7-hydroxy-10,13-dihydro-11H-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indeno[1,2-b]quinoline-8,11(7H)-dione (A1.5-A, 500 mg) in 75% sulfuric acid solution (5 mL), iron sulfate heptahydrate (570 mg of iron sulfate heptahydrate was dissolved in 1 mL of water) and 4-dimethoxychlorobutane (3.89 g) were added, and the reaction solution was stirred for 3 minutes, followed by dropwise addition of hydrogen peroxide (29%, 2.5 mL). The reaction solution was stirred at 0°C for 5 minutes, warmed to room temperature, and then reacted under stirring for 3 hours. The reaction solution was diluted with water (50 mL) and extracted with ethyl acetate (80 mL x 2). The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated to give the crude product, which was further purified by C18 column (acetonitrile / 0.05% formic acid water: 5%-60%) to give the target compound A1B (yellow solid, 400 mg, yield: 67%). LCMS (ESI) [M+H] + : 468.9; 1H NMR (400 MHz, DMSO-d6) δ 7.65 (s, 1H), 7.51 (s, 1H), 7.24 (s, 1H), 6.50 (s, 1H), 6.30 (s, 2H), 5.42 (s, 2H), 5.26 (s, 2H), 3.81 (d, J = 5.9 Hz, 2H), 3.22 (s, 2H), 1.98 (d, J = 6.7 Hz, 4H), 0.88 (t, J = 7.2 Hz, 3H).

[0203] Step 2: Synthesis of (S)-7-ethyl-7-hydroxyl-14-(3-hydroxypropyl)-10,13-dihydro-11H-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-8,11(7H)-dione (A1)

[0204] [ka] The compound (S)-7-ethyl-7-hydroxyl-14-(3-chloropropyl)-10,13-dihydro-11H-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-8,11(7H)-dione (100 mg, 0.213 mmol) was dissolved in 10% sulfuric acid (5 mL) and reacted at 110°C for 48 hours. Saturated sodium bicarbonate (30 mL) solution was added to the reaction solution, and then the reaction solution was extracted with dichloromethane (10 mL x 5), dried over anhydrous sodium sulfate, suction filtered, and concentrated under reduced pressure to obtain the crude product, which was purified by preparative HPLC (acetonitrile / water containing 0.05% formic acid) to obtain (S)-7-ethyl-7-hydroxyl-14-(3-hydroxypropyl)-10,13-dihydro-11H-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-8,11(7H)-dione (A1, 1.78 mg). LCMS (ESI) [M+H] + : 451.0; 1 H NMR (400 MHz, DMSO-d6) δ 7.63 (s, 1H), 7.50 (s, 1H), 7.24 (s, 1H), 6.48 (s, 1H), 6.28 (s, 2H), 5.47 - 5.37 (m, 2H), 5.32 - 5.19 (m, 2H), 3.51 - 3.46 (m, 2H), 3.17 - 3.13 (m, 2H), 1.92 - 1.76 (m, 4H), 0.90 - 0.84 (m, 3H).

[0205] Example 11.2: Synthesis of (S)-4-ethyl-8-fluoro-4-hydroxyl-11-(3-hydroxypropyl)-9-methyl-1,12-dihydro-14H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione (A2)

[0206] [ka] Step 1: At 0°C, 1 mol / L boron trichloride (96 mL) and 4-chlorobutyronitrile (9.9 g) were added dropwise successively to a solution of compound A2A (10 g) in 1,2-dichloroethane (200 mL). The reaction was stirred at 80°C for 2 hours. The reaction solution was cooled to room temperature, then 2 mol / L hydrochloric acid (90 mL) was added, and the mixture was stirred at 80°C under reflux for 0.5 hours. The reaction solution was cooled to room temperature, diluted with a small amount of water, and extracted with dichloromethane (200 mL x 3). The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the crude product, which was separated and purified by column chromatography (petroleum ether:ethyl acetate = 10 / 1) to obtain the target compound A2B (4 g). LCMS(ESI)[M+H] + :230.0

[0207] Step 2: Compound A2B (50 mg) was added with (S)-4-ethyl-4-hydroxy-7,8-dihydro-1H-pyrano[3,4-f]indolizine-3,6,10(4H)-trione (35 mg) and p-toluenesulfonic acid monohydrate (41.4 mg) successively, and then the mixture was dissolved in dichloromethane (30 mL). The solution was mixed until clear, concentrated under reduced pressure, and oil pump vacuum was applied, and the reaction was carried out under vacuum at 120° C. for 3 hours. LCMS showed that the reaction was complete. The reaction mixture was cooled to room temperature, and then water (20 mL) was added. The resulting mixture was extracted with dichloromethane (20 mL×3). The combined organic phase was subsequently dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain the crude product. The crude product was separated and purified by column chromatography (dichloromethane:methanol=20:1) to obtain the target compound A2C (80 mg) as a white solid. LCMS(ESI)[M+H] + :457.0

[0208] Step 3: Compound A2C (75 mg) was dissolved in hexamethylphosphoric triamide, pure water (0.8 mL) was added, and the reaction solution was then stirred at 100° C. for 72 hours. LCMS test showed that the reaction was complete. Target compound A2 (10 mg) was purified by preparative chromatography (0.01% TFA water, MeCN). LCMS (ESI) [M+H] + : 439.2; 1 H NMR (400 MHz, DMSO-d6) δ 8.22 (d, J = 8.4 Hz, 1H), 7.87 (d, J = 10.9 Hz, 1H), 7.31 (s, 1H), 6.50 (s, 1H), 5.43 (s, 2H), 5.30 (s, 2H), 4.67 (t, J = 4.9 Hz, 1H), 3.55 - 3.47 (m, 2H), 3.28 - 3.20 (m, 2H), 2.51 (s, 3H), 1.93 - 1.81 (m, 4H), 0.88 (t, J = 7.3 Hz, 3H).

[0209] Example 11.3: Synthesis of (S,E)-14-(3-amino-1-propylene-1-yl)-7-ethyl-7-hydroxyl-10,13-dihydro-11H-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-8,11(7H)-dione (A3)

[0210] [ka]

[0211] Step 1: Compound A3A (200 mg, 0.39 mmol), compound A3B (112 mg, 0.39 mmol), cesium fluoride (152 mg, 0.975 mmol) and tetrakis(triphenylphosphine)palladium (45 mg, 0.039 mmol) were added to 1,4-dioxane (8 mL). The reaction was sonicated under nitrogen atmosphere at 120° C. for 0.5 hours. LCMS showed the reaction was complete. A mixed solvent of dichloromethane (20 mL) and methanol (10 mL) was added to the reaction, and then the resulting solution was filtered. The filtrate was concentrated, and the crude product was purified by preparative TLC (dichloromethane:methanol=30:1) to obtain the target compound (S,E)-14-(3-((tert-butoxycarbonyl)amino)-1-propylene-1-yl)-7-ethyl-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-7-yl acetate (A3C, 60 mg, yield: 26%) as a brown solid. LCMS (ESI) [M+H] + = 590.3; 1 H NMR (400 MHz, DMSO-d6) δ 7.63 (s, 1H), 7.49 (s, 1H), 7.35 (s, 1H), 7.09 (d, J = 16.7 Hz, 1H), 6.93 (s, 1H), 6.45 (d, J = 16.6 Hz, 1H), 6.30 (s, 2H), 5.47 (s, 2H), 5.34 - 5.24 (m, 2H), 3.94 (s, 2H), 2.21 (br s, 3H), 2.03 - 1.96 (m, 2H), 1.45 (s, 9H), 0.91 (t, J = 6.7Hz, 3H).

[0212] Step 2: To a solution of compound (S,E)-14-(3-((tert-butoxycarbonyl)amino)-1-propylene-1-yl)-7-ethyl-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-7-yl acetate (A3C, 50 mg, 0.085 mmol) in methanol (15 mL) was added sodium methoxide (9.2 mg, 0.17 mmol) and the reaction was then stirred at 50° C. for 2 hours. LCMS indicated the reaction was complete. The reaction solution was concentrated to obtain the crude target compound (S,E)-14-(3-((tert-butoxycarbonyl)amino)-1-propylene-1-yl)-7-ethyl-7-hydroxy-10,13-dihydro-11H-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-8,11(7H)-dione (A3D, 50 mg) as a brown solid. LCMS (ESI) [M+H] + =548

[0213] Step 3: To a solution of compound (S,E)-14-(3-((tert-butoxycarbonyl)amino)-1-propylene-1-yl)-7-ethyl-7-hydroxy-10,13-dihydro-11H-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-8,11(7H)-dione (A3D, 50 mg, 0.091 mmol) in dichloromethane (2 mL) was added trifluoroacetic acid (1 mL) and the reaction was then stirred at room temperature for 30 minutes. LCMS indicated the reaction was complete. The reaction solution was concentrated, and the crude product was purified by preparative HPLC (acetonitrile / 0.05% formic acid water) to obtain the target compound (S,E)-14-(3-amino-1-propylene-1-yl)-7-ethyl-7-hydroxy-10,13-dihydro-11H-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-8,11(7H)-dione (A3) (8.1 mg, 21% yield) as a brown solid. LCMS (ESI) [M+H] += 448.3; 1 H NMR (400 MHz, DMSO-d6) δ 8.20 (s, 2H), 7.72 (s, 1H), 7.54 (s, 1H), 7.40 (d, J = 16.5 Hz, 1H), 7.27 (s, 1H), 6.52 - 6.46 (m, 2H), 6.31 (s, 2H), 5.42 (s, 2H), 5.27 (s, 2H), 3.86 (br s, 2H), 1.90 - 1.83 (m, 2H), 0.88 (t, J = 7.1 Hz, 3H).

[0214] Example 11.4: Synthesis of (S)-2-amino-N-((4-(4-ethyl-8-fluoro-4-hydroxyl-9-methyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-11-yl)propoxy)methyl)acetamide (B1)

[0215] [ka] Step 1: Compound A2 (160 mg, 0.365 mmol) was dissolved in N,N-dimethylformamide (3 mL), (2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetamido)methyl acetate ((B1A, 672 mg, 1.83 mmol) was added, and then hydrochloric acid in ethyl acetate (0.073 mL, 3 M) was added to the reaction solution. The reaction solution was stirred at room temperature overnight. LCMS detection showed that the reaction was complete. The reaction solution was directly purified by reverse phase chromatography (acetonitrile / 0.05% FA water: 5%-50%) to obtain the target compound B1B (80 mg, 29.0% yield) as a white solid. LCMS (ESI) [M+H] + = 747.4; 1H NMR (400 MHz, DMSO-d6) δ 8.74 (t, J = 6.4 Hz, 1H), 8.28 - 8.17 (m, 1H), 7.99 - 7.88 (m, 3H), 7.73 (d, J = 7.3 Hz, 2H), 7.63 (t, J = 5.7 Hz, 1H), 7.44 (t, J = 7.4 Hz, 2H), 7.35 (t, J = 7.2 Hz, 3H), 6.58 (s, 1H), 5.48 (s, 2H), 5.29 (s, 2H), 4.66 (d, J = 6.3 Hz, 2H), 4.32 (d, J = 6.9 Hz, 2H), 4.26 (d, J = 6.1 Hz, 1H), 3.71 (d, J = 5.8 Hz, 2H), 3.57 (t, J = 5.7 Hz, 2H), 3.29 - 3.20 (m, 2H), 2.55 (s, 3H), 2.00 - 1.86 (m, 4H), 0.93 (t, J = 7.2 Hz, 3H).

[0216] Step 2: B1B (240 mg) was dissolved in DMF (5 ml), piperidine (1 ml) was added, and the mixture was stirred for 20 min. The solution was evaporated under reduced pressure to remove low boiling components, and the residue was used directly in the next step. ESI-MS(m / z):525.2[M+H] + A small amount of the crude product was purified by reverse phase chromatography (acetonitrile / 0.05% FA water: 5% to 50%) to obtain the target compound. ESI-MS (m / z): 525.1 [M+H] + ; 1H NMR (400 MHz, DMSO) δ 9.13 (t, J = 6.6 Hz, 1H), 8.21 (d, J = 8.1 Hz, 1H), 8.02 (brs, 2H), 7.89 (d, J = 10.8 Hz, 1H), 7.32 (s, 1H), 6.54 (s, 1H), 5.44 (s, 2H), 5.28 (s, 2H), 4.66 (d, J = 6.5 Hz, 2H), 3.64 (s, 2H), 3.53 (t, J = 6.1 Hz, 2H), 3.25-3.18 (m, 2H), 2.52 (s, 3H), 1.98-1.84 (m, 4H), 0.88 (t, J = 7.3 Hz, 3H).

[0217] Example 11.5: Synthesis of (S)-2-amino-N-((3-(7-ethyl-7-hydroxyl-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-14-yl)propoxy)methyl)acetamide (B2)

[0218] [ka] Step 1: Compound (B1A) (368 mg), compound (A1) (440 mg) and pyridinium p-toluenesulfonate (PPTS) (25 mg) were refluxed in dichloromethane (20 ml) for 20 hours, and then the reaction solution was washed with an aqueous sodium bicarbonate solution and an aqueous hydrochloric acid solution, respectively. The organic solvent was removed under reduced pressure to obtain a crude product, which was separated and purified by column chromatography (dichloromethane:methanol = 10 / 1) to obtain the target compound B2A (240 mg). LCMS(ESI)[M+H] + :759 Step 2: B2A (240 mg) was dissolved in DMF (5 ml), piperidine (1 ml) was added, and the compound was then stirred for 20 minutes. Low boiling components were removed under reduced pressure, and the residue was used directly in the next synthesis step. A small amount of the crude product was purified by reverse phase chromatography (acetonitrile / 0.05% FA water: 5% to 50%) to obtain the target compound B2. ESI-MS (m / z): 537 [M+H] + ; 1 H NMR (400 MHz, DMSO-d6) δ 9.13 (t, 1H), 8.04 (br, 2H), 7.58 (s, 1H), 7.51 (s, 1H), 7.25 (s, 1H), 6.29 (s, 2H), 5.43 (S, 2H), 5.21 (s, 2H), 4.65 (d, 2H), 3.63 (m, 2H), 3.53 (m, 2H), 3.11 (m, 2H), 1.87 (m, 4H), 0.88 (t, 3H).

[0219] Example 11.6: (S,E)-2-amino-N-(3-(7-ethyl-7-hydroxyl-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-14-yl)allyl)acetamide (B3)

[0220] [ka]

[0221] Step 1: At room temperature, triethylamine (135 mg, 1.341 mmol) and N-hydroxy-2,5-dioxopyrrolidin-1-yl(tert-butoxycarbonyl)glycinate (183 mg, 0.671 mmol) were added to a solution of compound A3 (200 mg, 0.447 mmol) in N,N-dimethylformamide (5 mL), and then the mixture was reacted at room temperature for 1 hour. LCMS showed that the reaction was complete. Water (20 mL) was added to the reaction solution, and the resulting mixture was extracted with ethyl acetate (20 mL x 3). The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated to obtain the target compound B3A (130 mg, yield: 48%) as a brown solid. LCMS(ESI)[M+H] + =605.6

[0222] Step 2: At room temperature, compound B3A (130 mg, 0.215 mmol) was dissolved in a solution of HCl in 1,4-dioxane (5 mL) and then reacted at room temperature for 30 minutes. LCMS showed that the reaction was complete. The reaction solution was purified by reverse phase column chromatography to obtain the target compound B3 (60 mg, 52% yield) as a brown solid. LCMS (ESI) [M+H] + = 505.2; 1 H NMR (400 MHz, DMSO-d6) δ 8.85 (s, 1H), 8.15 (s, 2H), 7.70 (s, 1H), 7.54 (s, 1H), 7.26 (s, 1H), 7.21 (d, J = 16.2 Hz, 1H), 6.51 (d, J = 16.2 Hz, 1H), 6.31 (s, 2H), 5.42 (s, 2H), 5.28 (s, 2H), 4.19 (s, 2H), 1.86 - 1.82 (m, 4H), 0.87 (t, J = 7.3 Hz, 3H).

[0223] Example 11.7: N 6 ,N 6 -Dimethyl-N 2-((6-(2-(methanesulfonyl)pyrimidin-5-yl)hexan-5-ynoyl)-L-valinyl)-L-lysine (C1) [ka] 6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynoic acid (268 mg), compound C1A (328 mg), and triethylamine (322 mg) were dissolved in N,N-dimethylformamide (5 mL). Then, 1-hydroxybenzotriazole (HOBT, 162 mg) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI, 229 mg) were added, and the reaction solution was stirred at room temperature for 16 hours. The reaction solution was directly purified with a reversed-phase C18 column (acetonitrile and 0.05% formic acid aqueous solution system) to obtain the target compound N. 6 ,N 6 -Dimethyl-N 2 -((6-(2-(methanesulfonyl)pyrimidin-5-yl)hexan-5-ynoyl)-L-valinyl)-L-lysine (C1, white solid, 327 mg) was obtained. LCMS (ESI) [M+H] + : 524.4. 1 H NMR (400 MHz) δ 9.13 (s, 2H), 7.95 (t, J = 8.8 Hz, 2H), 4.21 (dd, J = 8.8, 6.9 Hz, 1H), 4.08 - 4.03 (m, 1H), 3.41 (s, 3H), 2.55 (t, J = 7.0 Hz, 2H), 2.42 - 2.32 (m, 4H), 2.27 (s, 6H), 1.98 (dd, J = 13.6, 6.8 Hz, 1H), 1.86 - 1.77 (m, 2H), 1.74 - 1.55 (m, 2H), 1.47 - 1.37 (m, 2H), 1.31 - 1.23 (m, 2H), 0.85 (dd, J = 12.8, 6.8 Hz, 6H).

[0224] Example 11.8: N 6 ,N 6 -Dimethyl-N2 -((6-(4-(2-(methanesulfonyl)pyrimidin-5-yl)-1H-1,2,3-triazol-1-yl)hexanoyl)-L-valinyl)-L-lysine (C2)

[0225] [ka] Step 1: Compound 6-(4-(2-(methylthio)pyrimidin-5-yl)-1H-1,2,3-triazol-1-yl)hexanoic acid (C2A, 1.0 g, 3.3 mmol) was dissolved in a mixed solvent of tetrahydrofuran and water (40 mL, 3:1) and potassium peroxymonosulfonate (10.0 g, 16.3 mmol) was added. The reaction was stirred at room temperature for 3 h and the reaction was completed as shown by LCMS detection. The reaction solution was filtered. The cake was washed with DMSO and the combined filtrate was subjected to reverse phase purification (C18, acetonitrile:0.1% formic acid = 5%-55%) to obtain 6-(4-(2-(methanesulfonyl)pyrimidin-5-yl)-1H-1,2,3-triazol-1-yl)hexanoic acid (C2B, 750 mg, 67.9% yield) as a white solid. LCMS (ESI) [M+H] + = 340.1; 1 H NMR (400 MHz, DMSO-d6) δ 9.48 (s, 2H), 8.95 (s, 1H), 4.49 (t, J = 7.0 Hz, 2H), 3.45 (s, 3H), 2.22 (t, J = 7.3 Hz, 2H), 1.95 - 1.84 (m, 2H), 1.61 - 1.50 (m, 2H), 1.36 - 1.26 (m, 2H).

[0226] Step 2: Compound 6-(4-(2-(methanesulfonyl)pyrimidin-5-yl)-1H-1,2,3-triazol-1-yl)hexanoic acid (C2B, 300 mg, 0.88 mmol) was dissolved in N,N-dimethylformamide (6 mL), HATU (337 mg, 0.88 mmol) and DIPEA (286 mg, 2.21 mmol) were added, and the reaction was stirred at room temperature for 30 min. Then, dipeptide C1.16-D (243 mg, 0.88 mmol) was added, and the reaction was stirred at room temperature for 2 h. LCMS showed that the reaction was complete, and the reaction solution was separated and purified on a C18 column (acetonitrile / 0.01% FA aqueous solution: 5%-50%) to obtain the target compound N. 6 ,N 6 -Dimethyl-N 2 -((6-(4-(2-(methanesulfonyl)pyrimidin-5-yl)-1H-1,2,3-triazol-1-yl)hexanoyl)-L-valinyl)-L-lysine (C2, 300 mg, 57% yield) was obtained as a white solid. LCMS (ESI) [M+H] + = 595.5, t R = 2.024 minutes. 1 H NMR (400 MHz, DMSO-d6) δ 9.49 (s, 2H), 9.01 (s, 1H), 7.85 (br s, 1H), 7.84 (d, J = 8.9 Hz, 1H), 4.48 (t, J = 6.8 Hz, 2H), 4.17 - 4.15 (m, 1H), 4.02 (br s, 1H), 3.44 (s, 3H), 2.42 (br s, 2H), 2.30 (s, 6H), 2.18 - 2.14 (m, 2H), 1.99 - 1.96 (m, 1H), 1.88 (dd, J = 14.6, 7.1 Hz, 2H), 1.67 (br s, 1H), 1.59 - 1.53 (m, 2H), 1.43 (br s, 2H), 1.28 - 1.26 (m, 5H), 0.83 - 0.89 (m, 6H).

[0227] Example 11.9: N 6 ,N 6-Diethyl-N 2 -((6-(2-(methanesulfonyl)pyrimidin-5-yl)hexan-5-ynoyl)-L-valinyl)-L-lysine (C3) [ka]

[0228] Step 1: Compound C3A (5.0 g, 12.05 mmol) was dissolved in dichloromethane (100 mL), acetaldehyde (3.2 g, 72.3 mmol) was added, and the reaction was stirred at room temperature for 10 min. Sodium triacetoxyborohydride (12.8 g, 60.25 mmol) was then added to the reaction solution, and the reaction was stirred at room temperature for 1 h. LCMS showed that the reaction was complete. A saturated aqueous solution of ammonium chloride was added to the reaction solution, which was then stirred for 1 h. The reaction solution was evaporated to dryness under rotary evaporation. The crude product was separated and purified on a reversed-phase C18 column (acetonitrile to 0.05% formic acid aqueous solution: 5% to 55%) to obtain the target compound C3B (4.57 g, 82.0% yield) as a white solid. LCMS (ESI) [M+H] + = 436.4; 1 H NMR (400 MHz, DMSO-d6) δ 7.70 (d, J = 7.0 Hz, 1H), 7.41 (d, J = 9.0 Hz, 1H), 7.38 - 7.26 (m, 5H), 5.08 - 4.99 (m, 2H), 4.00 (dd, J = 12.6, 6.5 Hz, 1H), 3.86 (dd, J = 8.6, 6.8 Hz, 1H), 2.74 (dd, J = 14.0, 6.9 Hz, 4H), 2.64 - 2.54 (m, 2H), 2.05 - 1.94 (m, 1H), 1.72 - 1.52 (m, 2H), 1.52 - 1.38 (m, 2H), 1.38 - 1.18 (m, 2H), 1.04 (t, J = 7.1 Hz, 6H), 0.87 - 0.81 (m, 6H).

[0229] Step 2: At room temperature, compound C3B (1.6 g, 3.68 mmol) was dissolved in methanol (80 mL), then Pd / C (0.16 g) was added, and the reaction was stirred at room temperature under hydrogen atmosphere for 12 hours. LCMS showed the reaction was complete. The reaction solution was filtered, and the filtrate was concentrated under reduced pressure to give the target compound C3C (900 mg, 82% yield) as an off-white solid. 1 H NMR (400 MHz, DMSO-d6) δ 8.04 (br s, 1H), 4.02 - 3.99 (m, 1H), 3.10 (d, J = 4.5 Hz, 1H), 2.65 (q, J = 7.1 Hz, 4H), 2.55 - 2.51 (m, 2H), 2.06 - 1.93 (m, 1H), 1.73 - 1.54 (m, 2H), 1.47 - 1.38 (m, 2H), 1.30 - 1.21 (m, 2H), 1.01 (t, J = 7.1 Hz, 6H), 0.89 (d, J = 6.9 Hz, 3H), 0.79 (d, J = 6.8 Hz, 3H).

[0230] Step 3: Compound 6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynoic acid (268 mg, 1 mmol) was dissolved in DMF (8 mL), and HATU (380 mg, 1 mmol) and triethylamine (322 mg, 2.5 mmol) were added successively, and then stirred at room temperature for 20 minutes, after which compound C3C (301 mg, 1 mmol) was added. The reaction solution was further stirred at room temperature for 30 minutes. After the reaction was completed by LCMS detection, the reaction solution was directly purified on a reversed-phase C18 column (acetonitrile and 0.05% formic acid aqueous system) to obtain the target compound C3 (280 mg, 51% yield) as a white solid. LCMS (ESI) [M+H] + = 552.3; 1H NMR (400 MHz, DMSO-d6) δ 9.13 (s, 2H), 7.95 (d, J = 8.9 Hz, 1H), 7.86 (d, J = 7.2 Hz, 1H), 4.18 (dd, J = 8.8, 6.8 Hz, 1H), 4.02 (dd, J = 12.8, 7.2 Hz, 1H), 3.41 (s, 3H), 2.74 - 2.69 (m, 4H), 2.62 - 2.52 (m, 4H), 2.44 - 2.29 (m, 2H), 2.04 - 1.94 (m, 1H), 1.86 - 1.77 (m, 2H), 1.72 - 1.54 (m, 2H), 1.51 - 1.39 (m, 2H), 1.33 - 1.23 (m, 2H), 1.02 (t, J = 7.2 Hz, 6H), 0.87 - 0.82 (m, 6H).

[0231] Example 11.10: N 6 ,N 6 -Dipropyl-N 2 -((6-(2-(methanesulfonyl)pyrimidin-5-yl)hexan-5-ynoyl)-L-valinyl)-L-lysine (C4)

[0232] [ka] Step 1: Compound C3A (5.0 g, 12 mmol) was dissolved in dichloromethane (100 mL), n-propionaldehyde (4.2 g, 72.3 mmol) was added, and the reaction was then stirred at room temperature for 10 minutes, after which sodium triacetoxyborohydride (12.8 g, 60.25 mmol) was added. The reaction was stirred at room temperature for 1 hour. LCMS showed that the reaction was complete. A saturated aqueous solution of ammonium chloride was added to the reaction solution, and the solution was then stirred for 1 hour and evaporated to dryness under rotary evaporation. After filtration, the filtrate was separated and purified on a reversed-phase C18 column (acetonitrile to 0.05% formic acid aqueous solution: 5% to 55%) to obtain the target compound C4A (4.57 g, 82.0% yield) as a white solid. LCMS (ESI) [M+H]+ = 464.0; 1 H NMR (400 MHz, DMSO-d6) δ 7.81 (d, J = 7.3 Hz, 1H), 7.38 - 7.28 (m, 5H), 5.04 (d, J = 1.7 Hz, 2H), 4.12 - 4.02 (m, 1H), 3.94 - 3.82 (m, 1H), 2.65 - 2.52 (m, 6H), 2.06 - 1.94 (m, 1H), 1.76 - 1.64 (m, 1H), 1.64 - 1.53 (m, 1H), 1.52 - 1.40 (m, 6H), 1.34 - 1.18 (m, 2H), 0.92 - 0.80 (m, 12H).

[0233] Step 2: At room temperature, compound C4A (2.0 g, 4.32 mmol) was dissolved in methanol (80 mL), then Pd / C (0.16 g) was added, and the reaction was stirred at room temperature under hydrogen atmosphere for 12 hours. LCMS showed the reaction was complete. The reaction solution was filtered, and the filtrate was concentrated under reduced pressure to give the target compound C4B (1.2 g, 85.5% yield) as a white solid.

[0234] Step 3: Compound 6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynoic acid (100 mg, 0.373 mmol) was dissolved in N,N-dimethylformamide (1 mL), then HATU (142 mg, 0.373 mmol) and N,N-diisopropylethylamine (120 mg, 0.93 mmol) were added, and the reaction was stirred for 30 minutes. Compound C4B (122 mg, 0.371 mmol) was then added, and the reaction solution was stirred at room temperature for 1 hour. After LCMS showed that the reaction was complete, the reaction solution was directly purified on a reverse phase C18 column (acetonitrile and 0.05% formic acid aqueous system) to obtain the target compound N. 6 ,N 6 -Dipropyl-N 2-((6-(2-(methanesulfonyl)pyrimidin-5-yl)hexan-5-ynoyl)-L-valinyl)-L-lysine (C4, 50 mg, 28% yield) was obtained as a pale yellow solid. LCMS (ESI) [M+H] + = 580.0; 1 H NMR (400 MHz, DMSO-d6) δ 8.24 (s, 2H), 7.98 - 7.93 (m, 2H), 4.24 - 4.16 (m, 1H), 4.10 (d, J = 5.2 Hz, 1H), 3.41 (s, 3H), 2.79 - 2.64 (m, 6H), 2.55 (t, J = 7.1 Hz, 2H), 2.45 - 2.26 (m, 2H), 2.06 - 1.91 (m, 1H), 1.89 - 1.78 (m, 2H), 1.76 - 1.66 (m, 1H), 1.64 - 1.57 (m, 1H), 1.57 - 1.42 (m, 6H), 1.37 - 1.24 (m, 2H), 0.93 - 0.78 (m, 12H).

[0235] Example 12 Synthesis of drug-linker compounds Example 12.1: N-((11S,14S)-11-(4-(dimethylamino)butyl)-1-((S)-4-ethyl-8-fluoro-4-hydroxyl-9-methyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-11-yl)-15-methyl-7,10,13-trioxo-4-oxa-6,9,12-triazahexadecan-14-yl)-6-(2-(methanesulfonyl)pyrimidin-5-yl)hexan-5-ynamide (DL-01)

[0236] [ka] Compound N 6 ,N 6 -Dimethyl-N 2-((6-(2-(methanesulfonyl)pyrimidin-5-yl)hexan-5-ynoyl)-L-valinyl)-L-lysine (126 mg) (C1), B1 (150 mg), 1-hydroxybenzotriazole (49 mg), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (69 mg) and diisopropylethylamine (156 mg, 1.21 mmol) were dissolved in N,N-dimethylformamide (10 mL) and the reaction was then stirred at 40° C. for 12 hours. LCMS showed the reaction was complete. The reaction solution was filtered and purified by preparative HPLC (acetonitrile / water containing 0.05% formic acid) to obtain the compound N-((11S,14S)-11-(4-(dimethylamino)butyl)-1-((S)-4-ethyl-8-fluoro-4-hydroxy-9-methyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-11-yl)-15-methyl-7,10,13-trioxo-4-oxa-6,9,12-triazahexadecan-14-yl)-6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynamide (DL-01) (12 mg, yield 6.0%) as a yellow solid. LCMS (ESI) [M+H] + = 1030.6; 1H NMR (400 MHz, DMSO-d6) δ 9.10 (s, 2H), 8.62 (t, J = 6.4 Hz, 1H), 8.23 ​​- 8.13 (m, 2H), 8.07 (d, J = 7.2 Hz, 1H), 7.95 - 7.83 (m, 2H), 7.31 (s, 1H), 6.53 (s, 1H), 5.44 (s, 2H), 5.29 (s, 2H), 4.64 - 4.53 (m, 2H), 4.28 - 4.20 (m, 1H), 4.19 - 4.11 (m, 1H), 3.76 - 3.69 (m, 2H), 3.50 (t, J = 5.7 Hz, 2H), 3.41 (s, 3H), 3.23 - 3.19 (m, 2H), 2.67 - 2.62 (m, 2H), 2.57 - 2.54 (m, 2H), 2.54 (s, 3H), 2.42 - 2.26 (m, 3H), 2.02 - 1.77 (m, 6H), 1.68 - 1.66 (m, 1H), 1.58 - 1.45 (m, 3H), 1.36 - 1.25 (m, 2H), 0.88 (t, J = 7.4 Hz, 3H), 0.82 (t, J = 6.3 Hz, 6H).

[0237] Example 12.2: N-((S)-1-(((S)-6-(diethylamino)-1-((2-(((E)-3-((S)-7-ethyl-7-hydroxyl-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-14-yl)allyl)amino)-2-oxoethyl)amino)-1-oxohexan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)-6-(2-(methanesulfonyl)pyrimidin-5-yl)hexan-5-ynamide (DL-02)

[0238] [ka] To a solution of compound B3 (40 mg, 0.074 mmol) and compound C3 (40 mg, 0.072 mmol) in N,N-dimethylformamide (4 mL) was added HBTU (30 mg, 0.079 mmol) and DIPEA (26 mg, 0.2 mmol) successively. The reaction solution was stirred at room temperature for 1 hour. LCMS showed that the reaction was complete. The reaction solution was purified by preparative HPLC (acetonitrile / 0.05% trifluoroacetic acid water) to obtain the compound N-((S)-1-(((S)-6-(diethylamino)-1-((2-(((E)-3-((S)-7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-14-yl)allyl)amino)-2-oxoethyl)amino)-1-oxohexan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)-6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynamide (DL-02, 11.1 mg, yield 14%).

[0239] LCMS (ESI) [M+H] + =1038.6; 1H NMR (400 MHz, DMSO-d6) δ 9.10 (s, 2H), 9.02 (s, 1H, TFA), 8.33 - 8.25 (m, 2H), 8.12 (d, J = 7.3 Hz, 1H), 7.92 (d, J = 8.4 Hz, 1H), 7.68 (d, J = 4.1 Hz, 1H), 7.52 (d, J = 2.8 Hz, 1H), 7.26 (s, 1H), 7.15 (d, J = 16.2 Hz, 1H), 6.52 - 6.50 (m, 2H), 6.30 (s, 2H), 5.43 (s, 2H), 5.28 - 5.24 (m, 2H), 4.27 (d, J = 6.2 Hz, 2H), 4.12 (br s, 2H), 3.81 (br s, 2H), 3.41 (s, 3H), 3.11 - 3.07 (m, 6H), 2.97 (br s, 2H), 2.53 - 2.50 (m, 1H), 2.39 - 2.24 (m, 4H), 1.85 - 1.79 (m, 8H), 1.16 (t, J = 6.4 Hz, 6H), 0.88 (t, J = 6.4 Hz, 3H), 0.78 (d, J = 6.4 Hz, 6H).

[0240] Example 12.3: (S)-6-(dimethylamino)-N-(2-(((3-((S)-4-ethyl-8-fluoro-4-hydroxyl-9-methyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-11-yl)propyloxy)methyl)amino)-2-oxoethyl)-2-((S)-3-methyl-2-(6-(4-(2-(methanesulfonyl)pyrimidin-5-yl)-1H-1,2,3-triazol-1-yl)hexanamide)butanamide)hexanamide (DL-03)

[0241] [ka] Compound C2 (113 mg, 0.19 mmol) and compound B1 (100 mg, 0.19 mmol) were dissolved in DMF (1 mL), and then HBTU (72 mg, 0.19 mmol) and N,N-diisopropylethylamine (61 mg, 0.48 mmol) were added. After the addition, the reaction solution was stirred at room temperature for 1 hour. LCMS showed that the reaction was complete. The reaction solution was purified by preparative chromatography (0.01% TFA water, MeCN) to obtain the target compound (S)-6-(dimethylamino)-N-(2-(((3-((S)-4-ethyl-8-fluoro-4-hydroxy-9-methyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-11-yl)propyloxy)methyl)amino)-2-oxoethyl)-2-((S)-3-methyl-2-(6-(4-(2-(methanesulfonyl)pyrimidin-5-yl)-1H-1,2,3-triazol-1-yl)hexanamide)butanamide)hexanamide (DL-03, 16 mg, yield 8%) as a white solid.

[0242] LCMS (ESI) [M+H] + = 1101.6; 1H NMR (400 MHz, DMSO) δ 9.46 (s, 2H), 8.93 (s, 1H), 8.63 (t, J = 6.3 Hz, 1H), 8.20 - 8.28 (m, 2H), 8.04 (d, J = 7.2 Hz, 1H), 7.88 (d, J = 10.8 Hz, 1H), 7.81 (d, J = 8.3 Hz, 1H), 7.31 (s, 1H), 6.53 (s, 1H), 5.44 (s, 2H), 5.29 (s, 2H), 4.63 - 4.54 (m, 2H), 4.46 (t, J = 7.0 Hz, 2H), 4.29 - 4.19 (m, 1H), 4.15 - 4.08 (m, 1H), 3.73 (d, J = 6.9 Hz, 2H), 3.50 (t, J = 5.8 Hz, 2H), 3.44 (s, 3H), 3.24 - 3.14 (m, 2H), 3.01 - 2.91 (m, 2H), 2.72 (s, 6H), 2.53 (s, 3H), 2.23 - 2.07 (m, 2H), 1.93 - 1.84 (m, 6H), 1.72 - 1.64 (m, 1H), 1.61 - 1.45 (m, 6H), 1.34 - 1.22 (m, 4H), 0.87 (t, J = 7.4 Hz, 3H), 0.82 - 0.76 (m, 6H).

[0243] Example 12.4: N-((11S,14S)-11-(4-(di-n-propylamino)butyl)-1-((S)-7-ethyl-7-hydroxyl-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-14-yl)-15-methyl-7,10,13-trioxo-4-oxa-6,9,12-triazahexadecan-14-yl)-6-(2-(methanesulfonyl)pyrimidin-5-yl)hexan-5-ynamide (DL-04)

[0244] [ka] Compound C4 (43 mg, 0.074 mmol) and compound B2 (40 mg, 0.075 mmol) were dissolved in N,N-dimethylformamide (1 mL), and then HBTU (28 mg, 0.075 mmol) and N,N-diisopropylethylamine (24 mg, 0.187 mmol) were added. The reaction solution was stirred at room temperature for 1 h. LCMS detection showed that the reaction was complete. The reaction solution was directly purified by preparative chromatography (0.01% trifluoroacetic acid water, acetonitrile) to obtain the target compound (DL-04, 8.5 mg, 10% yield) as a yellow solid. LCMS (ESI) [M+H] + = 1098.6; 1 H NMR (400 MHz, DMSO-d6) δ 9.10 (s, 2H), 9.03 (s, 1H), 8.64 (t, J = 6.4 Hz, 1H), 8.19 (t, J = 5.9 Hz, 1H), 8.08 (d, J = 7.4 Hz, 1H), 7.92 (d, J = 8.5 Hz, 1H), 7.59 (s, 1H), 7.51 (s, 1H), 7.24 (s, 1H), 6.49 (s, 1H), 6.29 (s, 2H), 5.42 (s, 2H), 5.24 (s, 2H), 4.66 - 4.52 (m, 2H), 4.31 - 4.21 (m, 1H), 4.19 - 4.10 (m, 1H), 3.74 (d, J = 5.5 Hz, 2H), 3.49 - 3.48 (m, 2H), 3.40 (s, 3H), 3.15 - 3.06 (m, 2H), 3.02 - 2.95 (m, 6H), 2.59 - 2.52 (m, 3H), 2.41 - 2.29 (m, 2H), 2.05 - 1.90 (m, 2H), 1.91 - 1.77 (m, 6H), 1.63 - 1.57 (m, 6H), 1.31 - 1.29 (m, 2H), 0.92 - 0.80 (m, 15H).

[0245] Example 12.5: (S)-6-(dimethylamino)-N-(2-(((3-((S)-7-ethyl-7-hydroxyl-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-14-yl)propyloxy)methyl)amino)-2-oxoethyl)-2-((S)-3-methyl-2-(6-(4-(2-(methanesulfonyl)pyrimidin-5-yl)-1H-1,2,3-triazol-1-yl)hexanamide)butanamide)hexanamide (DL-05)

[0246] [ka] Compound C2 (40 mg, 0.06 mmol) was dissolved in N,N-dimethylformamide (1.5 mL), HBTU (26 mg, 0.06 mmol), B2 (36 mg, 0.06 mmol) and DIPEA (66 mg, 0.17 mmol) were added successively, and then the reaction was stirred at room temperature for 1 h. LCMS detection showed that the reaction was complete. The reaction solution was separated and purified by preparative chromatography (acetonitrile / 0.05% trifluoroacetic acid water) to obtain the target compound DL-05 (13.88 mg, TFA salt, 16% yield) as a light yellow solid. LCMS (ESI) [M+H] + = 1113.7; 1H NMR (400 MHz, DMSO-d6) δ 9.46 (s, 2H), 9.29 (s, 1H, TFA salt), 8.92 (s, 1H), 8.63 (t, J = 6.6 Hz, 1H), 8.18 (br s, 1H), 8.03 (d, J = 7.6 Hz, 1H), 7.81 (d, J = 8.3 Hz, 1H), 7.59 (s, 1H), 7.50 (s, 1H), 7.24 (s, 1H), 6.48 (s, 1H), 6.28 (s, 2H), 5.42 (s, 2H), 5.24 (s, 2H), 4.64 - 4.55 (m, 2H), 4.46 (t, J = 7.0 Hz, 2H), 4.28 - 4.20 (m, 1H), 4.15 - 4.09 (m, 1H), 3.76 - 3.71 (m, 4H), 3.53 - 3.48 (m, 4H), 3.44 (s, 3H), 3.10 - 3.08 (m, 2H), 2.97 - 2.95 (m, 2H), 2.75 (d, J = 4.9 Hz, 6H), 2.20 - 2.09 (m, 2H), 1.92 - 1.83 (m, 5H), 1.68 - 1.65 (m, 1H), 1.63 - 1.50 (m, 5H), 0.90 - 0.74 (m, 9H).

[0247] Example 13 Preparation of anti-Her3 antibody-drug conjugate Example 13.1: Preparation of Her3-ADC-07

[0248] [ka] q can be estimated from the data given in determining the DAR.

[0249] 13.1.1 Her3-ADC-07(DAR8) Sample Preparation 30mg of anti-Her3 antibody 202-2-1 was diluted with diluent (20mM PB+105mM NaCl, pH7.7), sodium edetate solution was added to a final concentration of 5mM, the solution was then mixed uniformly, 20mmol / L TCEP solution was added at 4.5 times the molar equivalent of the antibody, the solution was then mixed uniformly and left at room temperature for 30 minutes, DL-04 dissolved in 10mmol / L dimethylsulfoxide was added to the above solution at 10 times the molar equivalent (relative to the antibody), the solution was then mixed uniformly and left at room temperature for 2 hours to obtain a conjugate sample. After the reaction was completed, the sample was transferred to 10mM pH5.5 histidine buffer using a 30KDa ultrafiltration tube to remove low molecular weight substances, and finally the sample was concentrated to obtain a solution containing antibody 202-2-1 ADC composition Her3-ADC-07(DAR8). The average DAR value determined by mass spectrometry for Example 14 was 8.0. According to the mass spectrometry results of Her3-ADC-07(DAR8), the light chain of the antibody was complexed with 0-1 toxin molecules (the percentages of LC, DAR1 were 0% and 100%, respectively), and the heavy chain was complexed with 0-3 toxin molecules (the percentages of mAb, DAR1, DAR2, DAR3 were 0%, 0%, 0%, and 100%, respectively). It can be inferred that the q value is 8.

[0250] 13.1.2 Her3-ADC-07(DAR4) Sample Preparation 30mg of anti-Her3 antibody 202-2-1 was diluted with diluent (20mM PB+105mM NaCl, pH7.7), sodium edetate solution was added to a final concentration of 5mM, the solution was then mixed uniformly, 20mmol / L TCEP solution was added at 4.5 times the molar equivalent of the antibody, the solution was then mixed uniformly and left at room temperature for 30 minutes, DL-04 dissolved in 10mmol / L dimethylsulfoxide was added at 4.8 times the molar equivalent (relative to the antibody) to the above solution, the solution was then mixed uniformly and left at room temperature for 2 hours to obtain a conjugate sample. After the reaction was completed, the sample was transferred to 10mM pH5.5 histidine buffer using a 30KDa ultrafiltration tube to remove low molecular weight substances, and finally the sample was concentrated to obtain a solution containing antibody 202-2-1 ADC composition Her3-ADC-07(DAR4). The average DAR value, determined by mass spectrometry, was 4.5.

[0251] According to the mass spectrometry results of Her3-ADC-07(DAR4), the light chain of the antibody was complexed with 0-1 toxin molecules (LC, DAR1 percentages were 93.67%, 6.33%, respectively), and the heavy chain was complexed with 0-3 toxin molecules (mAb, DAR1, DAR2, DAR3 percentages were 19.23%, 7.01%, 10.66%, 63.1%, respectively). The q value can be estimated to be 1, 2, 3, 4, 5, 6, 7, or 8.

[0252] 13.1.3 Her3-ADC-07(DAR6) Sample Preparation 30mg of anti-Her3 antibody 202-2-1 was diluted with diluent (20mM PB+105mM NaCl, pH7.7), sodium edetate solution was added to a final concentration of 5mM, the solution was then mixed uniformly, 20mmol / L TCEP solution was added at 4.5 times the molar equivalent of the antibody, the solution was then mixed uniformly and left at room temperature for 30 minutes, DL-04 dissolved in 10mmol / L dimethylsulfoxide was added at 7.2 times the molar equivalent (relative to the antibody) to the above solution, the solution was then mixed uniformly and left at room temperature for 2 hours to obtain a conjugate sample. After the reaction was completed, the sample was transferred to 10mM pH5.5 histidine buffer using a 30KDa ultrafiltration tube to remove low molecular weight substances, and finally the sample was concentrated to obtain a solution containing antibody 202-2-1 ADC composition Her3-ADC-07(DAR6). The average DAR value, determined by mass spectrometry, was 5.8. According to the mass spectrometry results of Her3-ADC-07(DAR6), the light chain of the antibody was complexed with 0-1 toxin molecules (LC, DAR1 percentages were 81.31%, 18.69%, respectively), and the heavy chain was complexed with 0-3 toxin molecules (mAb, DAR1, DAR2, DAR3 percentages were 7.17%, 2.36%, 4.44%, 86.03%, respectively). The q value can be estimated to be 1, 2, 3, 4, 5, 6, 7, or 8.

[0253] Example 13.2: Preparation of Her3-ADC-05 (DAR4) Compound DL-01 was substituted for compound DL-04 in Example 13.1.2 to obtain Her3-ADC-05(DAR4), the coupling product of DL-01 and antibody 202-2-1, with an average DAR value of 4.0 as determined by mass spectrometry. According to the mass spectrometry results of Her3-ADC-05(DAR4), the light chain of the antibody was complexed with 0-1 toxin molecules (LC, DAR1 percentages were 90.89%, 9.11%, respectively), and the heavy chain was complexed with 0-3 toxin molecules (mAb, DAR1, DAR2, DAR3 percentages were 25.1%, 7.17%, 18.19%, 49.53%, respectively). The q value can be estimated to be 1, 2, 3, 4, 5, 6, 7, or 8.

[0254] [ka] Example 13.3: Her3-C3-ADC-05(DAR4)(DAR4) Sample Preparation Compound DL-04 of Example 13.1.2 was replaced with compound DL-01, and anti-Her3 antibody 202-2-1 of Example 13.1.2 was replaced with control antibody 2 to obtain Her3-C3-ADC-05 (DAR4), the coupling product of DL-01 and control antibody 2. The average DAR value was determined by mass spectrometry to be 4.7.

[0255] [ka] Example 13.4: Preparation of Her3-ADC-15

[0256] [ka] q can be estimated from the data given in determining the DAR.

[0257] 13.4.1 Her3-ADC-15(DAR8) Sample Preparation Compound DL-04 from Example 13.1.1 was replaced with DL-02 to give the coupling product of DL-02 and antibody 202-2-1, Her3-ADC-15 (DAR8), with an average DAR value of 7.9 as determined by mass spectrometry. According to the mass spectrometry results of Her3-ADC-15(DAR8), the light chain of the antibody was complexed with 0-1 toxin molecules (the percentages of LC and DAR1 were 0% and 100%, respectively), and the heavy chain was complexed with 0-3 toxin molecules (the percentages of mAb, DAR1, DAR2, and DAR3 were 0%, 0%, 6.31%, and 93.69%, respectively). The q value can be estimated to be 3, 4, 5, 6, 7, or 8.

[0258] 13.4.2 Her3-ADC-15(DAR2) Sample Preparation Compound DL-04 from Example 13.1.1 was replaced with 2 molar equivalents of DL-02 to give the coupling product of DL-02 and antibody 202-2-1, Her3-ADC-15(DAR2), with an average DAR value of 2.0 as determined by mass spectrometry. According to the mass spectrometry results of Her3-ADC-15(DAR2), the light chain of the antibody was complexed with 0-1 toxin molecules (LC, DAR1 percentages were 93.82%, 6.18%, respectively), and the heavy chain was complexed with 0-3 toxin molecules (mAb, DAR1, DAR2, DAR3 percentages were 61.16%, 5.77%, 11.02%, 22.05%, respectively). The q value can be estimated to be 1, 2, 3, 4, 5, 6, 7, or 8.

[0259] Example 13.5: Preparation of Her3-ADC-23 (DAR4) Compound DL-04 in Example 13.1.2 was replaced with DL-03 to give the coupling product of DL-03 and antibody 202-2-1, Her3-ADC-23(DAR4), with an average DAR value of 4.2 as determined by mass spectrometry. According to the mass spectrometry results of Her3-ADC-23(DAR4), the light chain of the antibody was complexed with 0-1 toxin molecules (LC, DAR1 percentages were 91.28% and 8.72%, respectively), and the heavy chain was complexed with 0-3 toxin molecules (mAb, DAR1, DAR2, DAR3 percentages were 19.24%, 8.67%, 21.97%, and 50.13%, respectively). The q value can be estimated to be 1, 2, 3, 4, 5, 6, 7, or 8.

[0260] [ka] Example 13.6: Her3-C3-ADC-23(DAR4) Sample Preparation Compound DL-04 from Example 13.1.2 was replaced with DL-03, and Her3 antibody 202-2-1 from 13.1.2 was replaced with control antibody 2 to obtain Her3-C3-ADC-23 (DAR4), the coupling product of DL-03 and control antibody 2. The average DAR value was determined by mass spectrometry to be 4.9.

[0261] [ka] Example 13.7: Her3-C3-ADC-21(DAR4) Sample Preparation With reference to CN104755494B, the following drug-linker compounds were prepared:

[0262] [ka] Compound DL-04 from Example 13.1.2 was replaced with the above drug-linker compound, and anti-Her3 antibody 202-2-1 from Example 13.1.2 was replaced with control antibody 2 to yield Her3-C3-ADC-21 (DAR4). The average DAR value was determined by mass spectrometry to be 4.4.

[0263] [ka] Example 13.8: Her3-C3-ADC-21(DAR8) Sample Preparation Compound DL-04 from Example 13.1.1 was replaced with the drug-linker compound from Example 13.7, and anti-Her3 antibody 202-2-1 from Example 13.1.1 was replaced with control antibody 2 to yield Her3-C3-ADC-21 (DAR8). The average DAR value was determined by mass spectrometry to be 9.5.

[0264] [ka] Example 13.9: Her3-ADC-21(DAR4) Sample Preparation Compound DL-04 from Example 13.1.2 was replaced with the drug-linker compound from Example 13.7 to obtain Her3-ADC-21(DAR4), the coupling product of the drug-linker compound and the 202-2-1 antibody, with an average DAR value of 4.1 as determined by mass spectrometry. According to the mass spectrometry results of Her3-ADC-21(DAR4), the light chain of the antibody was complexed with 0-1 toxin molecules (LC, DAR1 percentages were 100%, 0%, respectively), and the heavy chain was complexed with 0-3 toxin molecules (mAb, DAR1, DAR2, DAR3 percentages were 20.57%, 8.01%, 17.3%, 54.12%, respectively). It can be inferred that the q value is 1, 2, 3, 4, 5, or 6.

[0265] [ka] Example 13.10: Preparation of Her3-ADC-33 The following drug-linker compounds were prepared with reference to Example 4-1 of WO2019195665.

[0266] [ka]

[0267] Compound DL-04 from Example 13.1.2 was replaced with the above drug-linker compound to obtain Her3-ADC-33(DAR4), the coupling product of the above drug-linker compound and antibody 202-2-1, with an average DAR value of 5.6 as determined by mass spectrometry. According to the mass spectrometry results of Her3-ADC-33(DAR4), the light chain of the antibody was complexed with 0-1 toxin molecules (LC, DAR1 ratios were 49% and 51%, respectively), and the heavy chain was complexed with 0-3 toxin molecules (mAb, DAR1, DAR2, DAR3 ratios were 18.01%, 3.25%, 9.13%, and 69.62%, respectively). The q value can be estimated to be 1, 2, 3, 4, 5, 6, 7, or 8.

[0268] [ka] Example 13.11: Preparation of IgG-ADC-07(DAR8) The antibody 202-2-1 in Example 13.1.1 was replaced with an IgG1 isotype antibody (anti-hen egg lysozyme antibody from MediLink Therapeutics (Suzhou) Co., Ltd.) to obtain IgG1-ADC-07, a coupling product of DL-04 and antibody IgG1. The average DAR value was determined by mass spectrometry to be 8.0.

[0269] [ka] Note: Her3mAb and Her3Ab both refer to anti-Her3 antibodies or antigen-binding fragments thereof, specifically, in the above examples, Her3mAb is antibody 202-2-1 and Her3Ab is control antibody 2.

[0270] Example 13.12: Preparation of Her3-ADC-08 [ka] q can be estimated from the data given in determining the DAR.

[0271] (1) Preparation of Her3-ADC-08(DAR6) samples Compound DL-04 in Example 13.1.3 "Preparation of Her3-ADC-07(DAR6) sample" was replaced with DL-05 to obtain Her3-ADC-08(DAR6), which is the coupling product of DL-05 and antibody 202-2-1. The average DAR value was measured by mass spectrometry and was 4.9. According to the mass spectrometry results of Her3-ADC-08(DAR6), the light chain of the antibody was complexed with 0-1 toxin molecules (LC, DAR1 percentages were 66.58%, 33.42%, respectively), and the heavy chain was complexed with 0-3 toxin molecules (mAb, DAR1, DAR2, DAR3 percentages were 16.51%, 11.72%, 16.63%, 55.59%, respectively). It can be inferred that the q value is 1, 2, 3, 4, 5, 6, 7, or 8.

[0272] (2) Preparation of Her3-ADC-08(DAR2) samples Compound DL-04 of Example 13.1.1 was replaced with 2 molar equivalents of DL-05 to obtain Her3-ADC-08(DAR2), which is the coupling product of DL-05 and antibody 202-2-1. The average DAR value was measured by mass spectrometry to be 1.9. The mass spectrometry results of Her3-ADC-08(DAR2) showed that the light chain of the antibody was complexed with 0-1 toxin molecules (LC, DAR1 percentages were 96.61%, 3.39%, respectively), and the heavy chain was complexed with 0-3 toxin molecules (mAb, DAR1, DAR2, DAR3 percentages were 58.72%, 9.05%, 15.41%, 16.83%, respectively). It can be inferred that the q value is 1, 2, 3, 4, 5, 6, 7, or 8.

[0273] Example 14 Determination of DAR values ​​of complex samples by mass spectrometry Detection: The molecular weight and DAR value of Her3-ADC-07 (DAR8) were analyzed by LC-MS using the following conditions. Chromatographic conditions: Column: PLRP-S, 2.1 x 50 mm, 5 μm Mobile phase A: 0.1% FA / H2O; Mobile phase B: 0.1% FA / ACN Column temperature: 30°C; Sample chamber temperature: 8°C; Flow rate: 0.6 ml / min; Injection volume: 2 μl

[0274] [Table 8] Sample treatment: 1M DTT (2 μl) was added to 50 μg of sample, and ultrapure water was added up to 50 μl to dilute the solution to a concentration of approximately 1.0 mg / ml, then mixed uniformly, and then reduced at room temperature for 30 min. LC / MS model: Agilent 1290-6545XT Q-TOF MS conditions: Gas temperature: 320°C, Drying gas: nebulizer: 35 psi; Sheath gas temperature: 350°C; Sheath gas flow rate: 11 l / min: m / z 500-3000 The results are shown below:

[0275] [Table 9] In Table 6, mAb represents the unconjugated antibody, LC represents the light chain of the antibody, HC represents the heavy chain of the antibody, DAR1 represents a conjugate comprising a light chain or a heavy chain conjugated to one toxin molecule, DAR2 represents a conjugate comprising a light chain or a heavy chain conjugated to two toxin molecules, and DAR3 represents a conjugate comprising a light chain or a heavy chain conjugated to three toxin molecules, and the theoretical molecular weight of the monoclonal antibody is calculated as the G0F glycotype. Hereinafter, mAb, LC, HC, DAR1, DAR2, and DAR3 are defined as above.

[0276] In the detection results shown, the antibody light chain in Her3-ADC-07(DAR8) was conjugated with 0-1 toxin molecules (the percentages of LC, DAR1 were 0%, 100%, respectively), and the heavy chain was conjugated with 0-3 toxin molecules (the percentages of mAb, DAR1, DAR2, DAR3 were 0%, 0%, 0%, 100%, respectively), and therefore the antibody-drug coupling ratio (DAR value) of Her3-ADC-07(DAR8) was calculated to be 8.0.

[0277] Example 15 Assay of in vitro killing activity of anti-Her3 ADC drugs against tumor cell lines A. Determination of the inhibitory activity of Her3 ADC against PC-9 and NCI-H358 tumors PC-9 tumor cells (human lung adenocarcinoma, Nanjing COBIOER Bioscience Co., Ltd.) and NCI-H358 tumor cells (human non-small cell lung carcinoma, ATCC, CRL-5807) were selected to detect the killing activity of Her3-ADC-07 against tumor cell lines with endogenous expression of human Her3. The specific experimental steps were as follows: PC-9 cells and NCI-H358 cells were digested, collected by centrifugation, and resuspended in 1640+4%FBS medium to 50000 cells / mL, then plated in 96-well plates at 100μL / well, ADC molecules were gradient-diluted with 1640 basal medium in 3-fold dilutions starting from a final concentration of 150μM to a total of 11 dilution points, and added to the corresponding wells at 100μL / well to a final serum concentration of 2%, the plates were incubated for 120 hours in a 37℃, 5%CO2 incubator, CCK8 (Rhinogen) was added to each well at 20μL / well, and the plates were then incubated for 0.5-2.5 hours in a 37℃, 5%CO2 incubator. The plates were read in a microplate reader (MD, model ABS Plus) at 450nm every 30 minutes, and the data was then imported into Graphpad Prism for curve fitting. The experimental results are shown in Figures 6A and 6B. Her3-ADC-07 had killing activity against PC-9 tumor cells with an EC50 value of 49.26 nM, as well as against NCI-H358 tumor cells with an EC50 value of 183.3 nM.

[0278] B. Determining the inhibitory activity of Her3 ADC against KYSE520 tumors KYSE520 (esophageal squamous cell carcinoma tumor cells, ATCC) were digested by conventional trypsin-based method, cells were collected and counted in tubes, then resuspended in corresponding assay medium (containing 2% FBS) and added to 96-well plates at 2000-5000 cells / well. ADC (shown in Table 7) diluted with 100 μL of medium containing 2% FBS was added to the 96-well plate to give an initial concentration of 50 μg / ml, followed by 3-fold dilutions (11 concentration gradients). The plate was incubated at 37°C under 5% CO2 for 5 days. Then, 20 μL of CCK8 reagent was added to each well, and the reaction was carried out for 1-4 hours. The plate was read by a microplate reader (detection wavelength was 450 nm). The cells used in the experiment and the experimental results are shown in Table 7.

[0279] [Table 10] The experimental results showed that both Her3-ADC-05 and Her3-C3-ADC-05 had strong killing effects on tumor cells.

[0280] C. Determination of the inhibitory activity of Her3 ADC against NCI-H358 tumors In vitro cell activity assay: NCI-H358 tumor cells were digested by conventional trypsin-based method, cells were collected and counted in tubes, then resuspended in corresponding assay medium (containing 2% FBS) and added to 96-well plates at 2000-5000 cells / well. ADC diluted in 100 μL of medium containing 2% FBS was added to 96-well plates to give an initial concentration of 150 μg / ml, followed by 3-fold dilutions (11 concentration gradients). The plates were incubated at 37°C and 5% CO2 for 7 days. Then, 20 μL of CCK8 reagent was added to each well, and the reaction was carried out for 2-6 hours. The plates were read by a microplate reader (detection wavelength was 450 nm). The samples and experimental results of each group are shown in Table 8.

[0281] [Table 11] The experimental results of this example demonstrate that the ADCs with various DAR values ​​disclosed herein all had killing effects on tumor cells.

[0282] (Example 16) Assay of in vivo activity of anti-Her3 antibody drug conjugates and IgG-ADC 16.1 Efficacy Assay of Anti-Her3 Antibody Drug Conjugates Against NCI-H358 Xenograft Tumors In vivo efficacy was demonstrated by evaluating the antitumor effect of the anti-Her3 antibody ADC drug in a subcutaneously implanted tumor model of human non-small cell lung cancer cell line NCI-H358 cells (available from ATCC, approximately 10% Her3 expression positive rate) in Balb / c nude mice.

[0283] The specific steps are as follows: Balb / c nu nude mice, female, 5-6 weeks old, were obtained from Vital River Laboratory Animal Co., Ltd. NCI-H358 cells were cultured in 1640 medium (containing 10% FBS) in a 15 cm diameter Petri dish, and when the confluence reached about 80-90%, they were digested by trypsin-EDTA, washed twice with PBS, and then centrifuged and resuspended in pre-cooled PBS. The cells were counted by a cell counter, and then the cells were diluted to a cell concentration of 5 × 10 7 After 7 days of laboratory adaptation, the inoculum was diluted to 5 × 10 6 Balb / c nu mice were inoculated subcutaneously with NCI-H358 cells at the right rib cage with 0.2 ml of cells / mouse and 50% Matrigel inoculation volume. Tumors grew to approximately 300 mm 3When tumors grew to 100x, mice were randomly divided into groups according to tumor size, with 5 mice per group, which were anti-hen egg lysozyme human IgG1 isotype antibody (negative control, from MediLink Therapeutics (Suzhou) Co., Ltd.) group, IgG1-ADC-07 (DAR8) dose group, Her3-C3-ADC-21 (DAR4) dose group, Her3-ADC-21 (DAR4) dose group and Her3-ADC-07 (DAR8) dose group, respectively. All samples were injected via tail vein once a week for a total of 3 weeks. Tumor volume and body weight of mice were observed and measured periodically after administration. Relative tumor growth rate T / C (%) is the ratio of relative tumor volume or tumor weight of the treatment group to the control group at a specific time point. The relative tumor inhibition rate TGI (%) was calculated as the formula: TGI%=(1-T / C)×100%, where T and C are the relative tumor volume (RTV) or tumor weight (TW) of the treatment and control groups, respectively, at a particular time point. The results are shown in Table 9 and FIG.

[0284] [Table 12]

[0285] Based on the experimental results, compared with the IgG1 group, both 5mg / kg and 1mg / kg of Her3-ADC-07(DAR8) significantly inhibited tumor growth in the NCI-H358 human non-small cell lung cancer xenograft model, and showed a tendency of dose gradient. The TGI value of the Her3-ADC-07 1mg / kg group was 76.26%, which was slightly higher than the TGI value of the Her3-ADC-21 5mg / kg group (75.74%). The TGI value of the Her3-ADC-21 1mg / kg group was 29.92%, which was slightly higher than the TGI value of the Her3-C3-ADC-21 1mg / kg group (24.11%). Furthermore, animal weight results indicated that the dosing and recovery periods were well tolerated by animals in each test group.

[0286] 16.2. Efficacy Assay I of Anti-Her3 Antibody Drug Conjugates Against SW480 Xenograft Tumors The present disclosure demonstrated the in vivo efficacy by evaluating the anti-tumor effect of anti-Her3 antibody ADC drugs in a subcutaneously implanted tumor model of human colon cancer cell line SW480 cells (available from ATCC, approximately 30% Her3 expression positive rate) in Balb / c nude mice. The specific steps are as follows: 36 Balb / c nu nude mice, female, 5-6 weeks old, were obtained from Vital River Laboratory Animal Technology Co., Ltd. SW480 cells were cultured in 1640 medium (containing 10% FBS) in 15 cm diameter Petri dishes, and when the confluence reached approximately 80-90%, they were digested by trypsin-EDTA, washed twice with PBS, and then centrifuged and resuspended in pre-cooled PBS. The cells were counted by a cell counter, and then the cells were diluted to a concentration of 5 × 10 7 After 7 days of laboratory adaptation, the inoculum was diluted to 5 × 10 6 SW480 cells were inoculated subcutaneously into the right rib of Balb / c nu mice with an inoculation volume of 0.2 ml (containing 50% Matrigel) per cell / mouse. Tumors grew to approximately 250 mm 3 When the tumors grew to 100x, the mice were randomly divided into groups according to tumor size, with 5 mice per group, which were anti-chicken lysozyme human IgG1 isotype control antibody (negative control, from MediLink Therapeutics (Suzhou) Co., Ltd.) group, IgG1-ADC-07 dose group, and Her3-ADC-07 dose group, respectively. All samples were injected via tail vein once a week for a total of 3 weeks. The tumor volumes and body weights of the mice were observed and measured periodically after administration.

[0287] 1) Relative tumor growth rate T / C (%) is the ratio of tumor volume or tumor weight in a treatment group to that in a control group at a particular time point. 2) Relative tumor inhibition rate TGI (%) was calculated using the formula: TGI% = (1-T / C) x 100%, where T and C are the relative tumor volume (RTV) or tumor weight (TW) of the treatment and control groups, respectively, at a particular time point. The specific results are shown in Table 10 and Figure 8. Based on the experimental results, both 10 mg / kg and 3 mg / kg doses of Her3-ADC-07 had significant inhibitory effects on tumor growth in SW480 human colon cancer xenograft model, and showed a trend of dose gradient.

[0288] [Table 13]

[0289] The antitumor effects of IgG1, IgG1-ADC-07(DAR8), Her3-ADC-07(DAR8) and Her3-C3-ADC-21(DAR8) in a transplanted tumor model of human colon cancer cell line SW480 (available from ATCC, approximately 30% Her3 expression positive rate) were determined by referring to the same model and method as in Example 16.2. Specific results are shown in Table 11 and FIG.

[0290] [Table 14] Based on the experimental results, both 10 mg / kg and 3 mg / kg doses of Her3-ADC-07 had significant inhibitory effects on tumor growth in SW480 human colon cancer xenograft model, and showed a trend of dose gradient.

[0291] Unexpectedly, the tumor inhibitory effect of the Her3-ADC-07(DAR8) 3 mg / kg group was significantly more potent than that of the Her3-C3-ADC-21(DAR8) 3 mg / kg group, and the tumor inhibitory effect of the Her3-ADC-07(DAR8) 10 mg / kg group was significantly more potent than that of the Her3-C3-ADC-21(DAR8) 10 mg / kg group. Furthermore, animal weight results indicated that the dosing and recovery periods were well tolerated by animals in each test group.

[0292] (Example 17) Plasma stability studies of Her3 antibody drug conjugates The stability of Her3-ADC-07(DAR8) in plasma was evaluated by measuring the release of the bioactive molecule A1 in human plasma incubated with Her3-ADC-07(DAR8).

[0293] Her3-ADC-07(DAR8) was incubated with filter-sterilized plasma from mouse, rat, dog, monkey, and human in triplicate wells. Incubation was performed at 37°C, 5% CO2, with a final concentration of Her3-ADC-07(DAR8) of 50μg / mL and a total incubation time of 504 hours. During the incubation process, samples were removed at 0, 24, 72, 168, 336, and 504 hours and the reaction was stopped by adding a methanol solution containing an internal standard (0.5μM tolbutamide) and 0.1% formic acid. The test samples were analyzed using UPLC-MS / MS under the following conditions:

[0294] [Table 15] The concentration and release rate of bioactive molecule A1 were calculated according to the following formulas: Release rate = A1 concentration at time of measurement / theoretical maximum release concentration Theoretical maximum release concentration = ADC incubation concentration / ADC drug molecular weight × DAR × molecular weight of bioactive molecule A1 The stability results of Her3-ADC-07(DAR8) in mouse, rat, dog, monkey, and human plasma at a test concentration of 50 μg / mL are shown in Table 12.

[0295] [Table 16] The experimental results showed that at a test concentration of 50 μg / mL, Her3-ADC-07(DAR8) was incubated in the plasma of mice, rats, dogs, monkeys, and humans for 504 hours, and the release rates of the bioactive molecule A1 were 0.726%, 0.497%, 0.521%, 0.474%, and 0.372%, respectively. The stability of Her3-ADC-07(DAR8) in the plasma of mice, rats, dogs, monkeys, and humans was good. After 504 hours (21 days) of incubation in vitro, the release of A1 was less than 1% of the theoretical maximum release concentration.

[0296] (Example 18) Safety evaluation of Her3 antibody-drug conjugates The therapeutic window was evaluated according to the highest non-serious toxicity dose (HNSTD) in toxicity studies and the lowest effective dose (MED) in pharmacodynamic studies. The therapeutic window of Her3-ADC-07 (DAR8) was calculated according to the ratio of in vivo exposure of the HNSTD dose and the MED dose.

[0297] 18.1 GLP Toxicology Studies In a GLP toxicology study consisting of six consecutive weeks of Her3-ADC-07(DAR8) followed by four weeks of recovery, each dose group contained 10 animals, half male and half female, and Her3-ADC-07(DAR8) was repeatedly administered to cynomolgus monkeys at doses of 0, 3, 10, and 20 mg / kg by intravenous injection once every three weeks for three consecutive doses. The animals were then allowed to recover for four weeks after administration. The toxicity of Her3-ADC-07(DAR8) was evaluated in cynomolgus monkeys, and the concentration of the ADC in serum samples was detected in a toxicokinetics (TK) study. The results of GLP toxicology studies showed that the maximum dose without severe toxicity was 10 mg / kg, and at this dose there were no significant abnormalities in the clinical observations, body weight, blood biochemistry, coagulation, etc. of the animals.

[0298] Compared with similar ADC products of Her3-ADC-07 (DAR8), patritumab deruxtecan is Daiichi Sankyo's ADC targeting HER3, and the doses of toxicology experiments in cynomolgus monkeys were 0 mg / kg, 3 mg / kg, 10 mg / kg, and 30 mg / kg, which were administered once every three weeks for five consecutive doses. The results showed that at doses of 3 mg / kg and above, reductions in reticulocytes, lymphocytes, and platelets were observed, and transferase was elevated at doses of 10 mg / kg and above, with no significant abnormalities in body weight at all doses, and the toxic target organs were the thymus, bone marrow, and skin (Hashimoto Y, et al. Clin Cancer Res. 2019 Dec 1; 25 (23): 7151-7161).

[0299] It should be noted that patritumab deruxtecan showed thrombocytopenia at doses of 3 mg / kg or more, and in its clinical studies, thrombocytopenia was one of the dose-limiting toxicities in dose-escalation studies (Janne PA, et al. Cancer Discov. 2022 Jan; 12(1): 74-89) and was also one of the most common (30%) treatment-emergent adverse events (TEAEs) in multicenter clinical studies (Yu-H, et al., Abstract # MA21.06, WCLC 2019). In comparison, it was found that Her3-ADC-07(DAR8) did not show significant platelet count abnormalities at the maximum dose of 20 mg / kg, and no toxic reactions were observed in important organs, such as the liver, kidneys, and lungs, at the maximum dose of 20 mg / kg.

[0300] 18.2 Nude Mouse PK / PD Experiments Nude mice bearing NCI-H358 tumors were given 0.3 mg / kg or 3 mg / kg Her3-ADC-07(DAR8) via a single tail vein injection, and serum and tumors were collected at 0, 3, 6, 24, 48, 96, 168, and 336 hours (five animals at each time point). Tumor volume and tumor weight were analyzed, and ADC concentrations in serum were measured. Following a single intravenous injection of 0.3 mg / kg or 3 mg / kg of Her3-ADC-07(DAR8) into mice bearing NCI-H358 tumors, tumor growth was inhibited at doses of 0.3 mg / kg or higher, indicating that the minimal effective dose of Her3-ADC-07(DAR8) in this study was 0.3 mg / kg.

[0301] AUC at maximum non-severely toxic dose in cynomolgus monkeys 0-336h Or C max AUC at the minimum effective dose vs. CDX mice 0-336h Or C max The ratio of these parameters was defined as the therapeutic index (TI), and the therapeutic indices for each parameter were as follows:

[0302] [Table 17] In summary, Her3-ADC-07(DAR8) was well tolerated at a dose of 10 mg / kg (HNSTD) in cynomolgus monkeys, with a therapeutic index close to 100-fold. Furthermore, thrombocytopenia did not occur at this dose, and no toxicity was observed in vital organs, such as lungs, liver, or kidneys. The above results indicated that Her3-ADC-07(DAR8) has a high safety profile in long-term use.

[0303] Although specific embodiments of the present disclosure have been described in detail, those skilled in the art will recognize in light of all the teachings published herein that various minor improvements and modifications may be made, all of which are within the scope of the present disclosure. The claimed scope of the present disclosure is defined by the appended claims and any equivalents thereof.

Claims

1. 1. An antibody or antigen-binding fragment thereof that binds to Her3, wherein the antibody or antigen-binding fragment has a complementarity determining region CDR as shown below: (a) HCDR1 or a mutant form of its sequence, HCDR2 or a mutant form of its sequence, and HCDR3 or a mutant form of its sequence contained in the heavy chain variable region VH shown in SEQ ID NO: 23, 25, or 27, and / or (b) LCDR1 or a mutant form of its sequence, LCDR2 or a mutant form of its sequence, and LCDR3 or a mutant form of its sequence contained in the light chain variable region VL shown in SEQ ID NO: 24, 26, or 28. Including, Preferably, the variant of the sequence is a CDR having one or several amino acid substitutions, deletions or additions compared to the CDR from which it is derived, for example one, two or three amino acid substitutions, deletions or additions, more preferably the substitutions are conservative substitutions.

2. The antibody or antigen-binding fragment thereof, (1) VH and / or VL as defined by the Kabat numbering system, (a) the VH comprises an HCDR1 consisting of the sequence of SEQ ID NO: 1, an HCDR2 consisting of the sequence of SEQ ID NO: 2, and an HCDR3 consisting of the sequence of SEQ ID NO: 3; and / or VL comprises an LCDR1 consisting of the sequence of SEQ ID NO: 7, an LCDR2 consisting of the sequence of SEQ ID NO: 8, and an LCDR3 consisting of the sequence of SEQ ID NO: 9; (b) the VH comprises an HCDR1 consisting of the sequence of SEQ ID NO: 12, an HCDR2 consisting of the sequence of SEQ ID NO: 13, and an HCDR3 consisting of the sequence of SEQ ID NO: 14; and / or VH and / or VL, wherein VL comprises an LCDR1 consisting of the sequence of SEQ ID NO: 18, an LCDR2 consisting of the sequence of SEQ ID NO: 19, and an LCDR3 consisting of the sequence of SEQ ID NO: 20; or (2) VH and / or VL as defined by the IMGT numbering system, (a) the VH comprises an HCDR1 consisting of the sequence of SEQ ID NO: 4, an HCDR2 consisting of the sequence of SEQ ID NO: 5, and an HCDR3 consisting of the sequence of SEQ ID NO: 6; and / or VL comprises LCDR1 consisting of the sequence of SEQ ID NO: 10, LCDR2 consisting of the sequence of AAS, and LCDR3 consisting of the sequence of SEQ ID NO: 9; (b) the VH comprises an HCDR1 consisting of the sequence of SEQ ID NO: 15, an HCDR2 consisting of the sequence of SEQ ID NO: 16, and an HCDR3 consisting of the sequence of SEQ ID NO: 17; and / or VH and / or VL comprising LCDR1 consisting of the sequence of SEQ ID NO: 21, LCDR2 consisting of the sequence of AAS, and LCDR3 consisting of the sequence of SEQ ID NO:

20. Including, The antibody or antigen-binding fragment thereof may comprise the following heavy chain variable region VH and / or light chain variable region VL, wherein at least one CDR of the heavy chain variable region VH and / or the light chain variable region VL comprises a mutation compared to the CDR defined by the Kabat or IMGT numbering system, said mutation being a substitution, deletion or addition of one or several amino acids or any combination thereof, such as a substitution, deletion or addition of one, two or three amino acids or any combination thereof, preferably wherein the substitution is a conservative substitution: More preferably, the antibody or antigen-binding fragment thereof of claim 1 binds to human Her3, monkey Her3, and / or rat Her3.

3. The antibody or antigen-binding fragment thereof, (a) a VH as set forth in any one of SEQ ID NOs: 23, 25, or 27, and / or a VL as set forth in any one of SEQ ID NOs: 24, 26, or 28; (b) a VH having at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any one of the VHs of (a), and / or a VL having at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any one of the VLs of (a); or (c) a VH having one or several amino acid substitutions, deletions or additions or any combination thereof, such as one, two, three, four, five, six, seven, eight, nine or ten amino acid substitutions, deletions or additions or any combination thereof, compared to any one of the VHs in (a); and / or a VL having one or several amino acid substitutions, deletions or additions or any combination thereof, such as one, two, three, four, five, six, seven, eight, nine or ten amino acid substitutions, deletions or additions or any combination thereof, compared to any one of the VLs in (a).

3. The antibody or antigen-binding fragment thereof of claim 1 or 2, wherein the substitution is preferably a conservative substitution.

4. The antibody or antigen-binding fragment thereof, (a) a VH consisting of the sequence shown in SEQ ID NO: 23, and a VL consisting of the sequence shown in SEQ ID NO: 24; (b) a VH consisting of the sequence shown in SEQ ID NO: 25, and a VL consisting of the sequence shown in SEQ ID NO: 26; (c) a VH consisting of the sequence shown in SEQ ID NO: 27, and a VL consisting of the sequence shown in SEQ ID NO: 28; (d) a VH and a VL, wherein the VH has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity and / or the VL has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity compared to the VH and VL of any one of the pairs (a)-(c); or (e) VH and VL in which, compared to the VH and VL of any one of the pairs (a) to (c), the VH has one or several amino acid substitutions, deletions or additions, or any combination thereof, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, deletions or additions, or any combination thereof, and / or the VL has one or several amino acid substitutions, deletions or additions, or any combination thereof, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, deletions or additions, or any combination thereof. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, wherein the substitution is preferably a conservative substitution.

5. An anti-Her3 antibody or antigen-binding fragment thereof that competes for binding with the antibody or antigen-binding fragment thereof according to any one of claims 1 to 4 and is different from the antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, preferably an anti-Her3 antibody or antigen-binding fragment thereof that competes for binding with antibody 202-2-1 and is not antibody 202-2-1 or an antigen-binding fragment thereof and has different CDRs from antibody 202-2-1 or an antigen-binding fragment thereof; The anti-Her3 antibody or antigen-binding fragment thereof may competitively bind to domain 3 of human Her3 with antibody 202-2-1, and preferably may bind to amino acids 328 to 499 of human Her3 as set forth in SEQ ID NO:31, and preferably the anti-Her3 antibody or antigen-binding fragment thereof may competitively bind to the following spatial epitopes with antibody 202-2-1: histidine at position 466, tryptophan at position 470, threonine at position 471, threonine at position 478, (aspartic acid-isoleucine-lysine-histidine-asparagine) at positions 483 to 487, isoleucine at position 484, arginine at position 490, and arginine at position 491 of human Her3 as set forth in SEQ ID NO:31; Preferably, the anti-Her3 antibody or antigen-binding fragment thereof blocks Her3 ligand-dependent signaling and Her3 ligand-independent signaling; Preferably, the anti-Her3 antibody or antigen-binding fragment thereof inhibits Her3 ligand-dependent AKT phosphorylation and Her3 ligand-independent AKT phosphorylation.

6. the antibody or antigen-binding fragment thereof is a chimeric antibody, a humanized antibody, or a fully human antibody; The antibody or antigen-binding fragment thereof may be a Fab fragment, a Fab' fragment, a (Fab') 2 The antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, which may be selected from fragments, Fv fragments such as scFv or disulfide-linked Fv (dsFv), diabodies, and multispecific antibodies.

7. The antibody or antigen-binding fragment thereof, (a) a heavy chain constant region CH of a human immunoglobulin or a variant thereof, and / or (b) a human immunoglobulin light chain constant region CL or a variant thereof Further comprising: the variant has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity compared to the wild-type sequence from which it is derived, or the variant has one or more amino acid substitutions, deletions or additions or any combination thereof compared to the wild-type sequence from which it is derived, such as up to 50, up to 45, up to 40, up to 35, up to 30, up to 25, up to 20, up to 15, up to 10 or up to 5 amino acid substitutions, deletions or additions or any combination thereof, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, deletions or additions or any combination thereof, preferably wherein the substitutions are conservative substitutions, Preferably, the heavy chain constant region is an IgG heavy chain constant region, e.g. an IgG1, IgG2, IgG3 or IgG4 heavy chain constant region, and / or the light chain constant region is a κ or λ light chain constant region; More preferably, the antibody or antigen-binding fragment thereof comprises a human IgG1 heavy chain constant region and / or the antibody or antigen-binding fragment thereof comprises a human κ light chain constant region.

8. the heavy chain constant region comprises a CH as set forth in SEQ ID NO:29 or a variant thereof, which variant has up to 20 conservative amino acid substitutions compared to SEQ ID NO:29, such as up to 20, up to 15, up to 10 or up to 5 conservative amino acid substitutions, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 conservative amino acid substitutions, or has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity compared to SEQ ID NO:29; and / or the light chain constant region comprises CL as set forth in SEQ ID NO: 30 or a variant thereof, wherein the variant has up to 20 amino acid conservative substitutions compared to SEQ ID NO: 30, such as up to 20, up to 15, up to 10 or up to 5 amino acid conservative substitutions, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid conservative substitutions, or has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity compared to SEQ ID NO: 30; Preferably, the antibody or antigen-binding fragment thereof comprises a heavy chain constant region CH as set forth in SEQ ID NO:29, and a light chain constant region CL as set forth in SEQ ID NO:

30.

9. The antibody, (a) a heavy chain comprising a VH as set forth in SEQ ID NO: 23 and a CH as set forth in SEQ ID NO: 29, and a light chain comprising a VL as set forth in SEQ ID NO: 24 and a CL as set forth in SEQ ID NO: 30, preferably a heavy chain as set forth in SEQ ID NO: 11, and a light chain as set forth in SEQ ID NO: 22; (b) a heavy chain comprising a VH set forth in SEQ ID NO: 25 and a CH set forth in SEQ ID NO: 29, and a light chain comprising a VL set forth in SEQ ID NO: 26 and a CL set forth in SEQ ID NO: 30; or (c) a heavy chain comprising a VH set forth in SEQ ID NO: 27 and a CH set forth in SEQ ID NO: 29, and a light chain comprising a VL set forth in SEQ ID NO: 28 and a CL set forth in SEQ ID NO: 30 The antibody or antigen-binding fragment thereof according to any one of claims 1 to 8, selected from the group comprising:

10. the antibody is a multispecific antibody, The antibody according to any one of claims 1 to 9, wherein the multispecific antibody is preferably a bispecific antibody, a trispecific antibody or a tetraspecific antibody.

11. An isolated nucleic acid molecule encoding the antibody or antigen-binding fragment thereof of any one of claims 1 to 10.

12. A vector comprising the isolated nucleic acid molecule of claim 11, which is preferably a cloning vector or an expression vector.

13. 13. A host cell comprising the isolated nucleic acid molecule of claim 11 or the vector of claim 12.

14. A method for preparing the antibody or antigen-binding fragment thereof of any one of claims 1 to 10, comprising culturing a host cell of claim 13 under conditions for expression of the antibody or antigen-binding fragment thereof, and recovering the antibody or antigen-binding fragment thereof from the cultured host cell culture.

15. The antibody-drug conjugate, its stereoisomer, its prodrug, its pharma- ceutically acceptable salt or its pharma-ceutically acceptable solvate, wherein the antibody or antigen-binding fragment thereof according to any one of claims 1 to 10 is attached to a coupling moiety via a connector; The antibody-drug conjugate, its stereoisomer, its prodrug, its pharmaceutically acceptable salt or its pharmaceutically acceptable solvate, wherein the coupling moiety can be selected from a detectable marker, a radioisotope, a fluorescent agent, a luminescent agent, a colorant, an enzyme, a polyethylene glycol, a nuclide, a nucleic acid, a small molecule toxin, a polypeptide having binding activity, a protein, a receptor, a ligand and other active agents that inhibit tumor cell proliferation and promote tumor cell apoptosis or necrosis.

16. The antibody-drug conjugate has the formula: Tb-L-D) q is shown in Tb is an antibody or an antigen-binding fragment thereof according to any one of claims 1 to 10, D is a small molecule toxin drug moiety; L is a bond or a linker covalently linking Tb and D; q is an integer between 1 and 16 and represents the number of L-D covalently bonded to Tb. The antibody-drug conjugate according to claim 15, a stereoisomer thereof, a prodrug thereof, a pharma- ceutically acceptable salt thereof or a pharma- ceutically acceptable solvate thereof.

17. The antibody-drug conjugate has a structure represented by Formula I: 【Chemistry 1】 (I) L 1 teeth, 【Chemistry 2】 each Z is independently selected from a direct bond, a carbon-carbon triple bond, a carbon-carbon double bond, and an amide; each Rx and Ry are independently selected from H and C1-4 alkyl; each m is independently selected from 0, 1, 2, 3, 4, 5, and 6; y1 is independently selected from any integer from 1 to 6; each y2 is independently selected from any integer from 0 to 15; each y3 is independently selected from 1, 2, and 3; and each y4 is independently selected from 0 and 1; position 1 is bonded to Tb via a S atom; position 2 is bonded to L 2 Or L 3 Binds to L 2 is absent or present, L 2 If there is L 2 teeth, 【Chemistry 3】 wherein y1 is selected from any integer from 1 to 6, each y2 is independently selected from any integer from 0 to 10, each y3 is independently selected from 1 or 2, each y4 is independently selected from 0 or 1, and the first position is L 1 and position 2 is L 3 Binds to L 3 is selected from an amino acid residue or a short peptide consisting of 2 to 10 amino acid residues, the amino acid residue being selected from a natural amino acid residue, a non-natural amino acid residue, or AA 1 【Chemistry 4】 or a stereoisomer thereof; 1 In the amino acid residue represented by a and R b One of the groups is H, and the other is H, 【Chemistry 5】 or R a and R b form a 5- to 6-membered heterocyclic ring together with the carbon atom to which they are both attached, said 5- to 6-membered heterocyclic ring being a piperidine ring or a piperazine ring; r,r 1 , r 1a and r 1b are each independently selected from 0, 1, 2, 3, 4, or 5; R m1 , R n1 , R m1a , R n1a , R m1b and R n1b are each independently H, C 1-6 Alkyl, or -COOR x1 and R x1 is C 1-6 Is it an alkyl group? Or R m1 and R n1 , R m1a and R n1a , and R m1b and R n1b form a 5-6 membered heterocyclic ring together with the nitrogen atom to which they are both attached, the heteroatoms of said 5-6 membered heterocyclic ring being selected from one or two N atoms, and said 5-6 membered heterocyclic ring being selected from one or more R 0’ may be substituted with R z is C 1-6 alkyl, R 0 and R 0’ is C 1-6 Alkyl, -NR m2 R n2 , or C 1-6 each independently selected from a 5- to 6-membered heterocyclyl optionally substituted by alkyl, wherein the heteroatoms of said 5- to 6-membered heterocyclyl are selected from 1 or 2 N atoms; R m2 and R n2 is H and C 1-6 are each independently selected from alkyl, L 4 is absent or present, L 4 If there is L 4 teeth, 【Chemistry 6】 The first position is selected from L 3 and position 2 is bonded to D. R 1 and R 2 are each independently selected from H, halogen, and C1-4 alkyl, or R 1 and R 2 form a 5-6 membered heterocyclic ring together with the carbon atom to which they are both attached, the heterocyclic ring containing 1, 2 or 3 O, S or N or any combination thereof; R 3 is H and C 1-4 alkyl, or R 3 and X together with the carbon atom to which they are both attached form a 5- to 6-membered carbocyclic ring; W is absent or present, and when W is present, W is -O-, -S-, -NR 4 --, 【Chemistry 7】 Position 1 is bonded to X and position 2 is selected from L 4 Or L 3 Binds to X is an optionally substituted -(CH 2 ) n1 --, 【Chemistry 8】 Position 1 is attached to the parent ring and position 2 is selected from W or L 4 and the substituents are selected from one or two C1-4 alkyl; R 4 , R 5 and R 7 are each independently selected from H and C1-4 alkyl; n, n1, n2, and n3 are each independently selected from any integer from 0 to 6. The antibody-drug conjugate according to claim 16, a stereoisomer thereof, a prodrug thereof, a pharma- ceutically acceptable salt thereof or a pharma- ceutically acceptable solvate thereof.

18. The following conditions (1) L 1 teeth, 【Chemistry 9】 and wherein position 1 is bonded to Tb via an S atom and position 2 is selected from L 2 Or L 3 Binds to (2) L 2 is absent or present, L 2 If there is L 2 teeth, 【Chemistry 10】 The first position is selected from L 1 and position 2 is L 3 Binds to (3) L 3 AA 1 , A.A. 1 -Gly, Val-Cit, Val-AA 1 -Gly, AA 1 - selected from Ala-Asn, and Gly-Gly-Phe-Gly; (4) A.A. 1 The amino acid residue represented by 【Chemistry 11】 Selected from: (5) L 4 is absent or present, L 4 If there is L 4 teeth, 【Chemistry 12】 The first position is selected from L 3 and position 2 is bonded to D. (6) R 1 is selected from H and halogen; R 2 is selected from H and C1-4 alkyl, or R 1 and R 2 along with the carbon atoms to which they are attached. 【Chemistry 13】 and the dotted line indicates where the heterocycle is fused to the benzene ring. (7) R 3 is H or R 3 and X together with the carbon atom to which they are attached 【Chemistry 14】 where the dotted lines indicate where the carbocyclic ring is fused to the benzene and pyridine rings. (8) Each R 4 is independently selected from H, methyl, ethyl, n-propyl, isopropyl, and t-butyl; R 5 is H, (9) Each R 7 is independently selected from H and C1-4 alkyl; (10) n is selected from 1, 2 and 3; (11) n1 is selected from 1, 2, 3 and 4; (12) n2 is 1. (13) n3 is 0. (14) W is -O-, -NR 4 - and 【Chemistry 15】 Position 1 is bonded to X and position 2 is selected from L 4 Or L 3 Binds to (15) X is optionally substituted 【Chemistry 16】 Position 1 is attached to the parent ring and position 2 is selected from W or L 4 and the substituents are selected from one or two C1-4 alkyl groups, or two C1-4 alkyl groups; The antibody-drug conjugate, its stereoisomer, its prodrug, its pharma- ceutically acceptable salt or its pharma- ceutically acceptable solvate according to claim 17, characterized in that one or more of the following conditions are satisfied:

19. The following conditions (1) L 1 teeth, 【Chemistry 17】 wherein position 1 is bonded to Tb via an S atom and position 2 is bonded to L2 or L3; (2) L 2 does not exist, (3) L 3 teeth, 【Chemistry 18】 The first position is selected from L 1 Or L 2 and position 2 is L 4 or attached to D; (4) L 4 teeth, 【Chemistry 19】 The first position is selected from L 3 and position 2 is bonded to D. (5) R 1 is H or F, R 2 is H or methyl, or R 1 and R 2 along with the carbon atoms to which they are attached. 【Chemistry 20】 Forming (6) R 3 is H, (7) W is -O-, -NR 4 - and 【Chemistry 21】 Position 1 is bonded to X and position 2 is selected from L 4 Or L 3 Binds to (8) X is ​ wherein position 1 is attached to the parent ring and position 2 is attached to W; The antibody-drug conjugate, its stereoisomer, its prodrug, its pharma- ceutically acceptable salt or its pharma- ceutically acceptable solvate according to claim 18, characterized in that one or more of the following conditions are satisfied:

20. The following conditions (1) Structure 【Chemistry 23】 But the following structural fragment 【Chemistry 24】 is selected from Position 1 binds to Tb, position 2 binds to D, For example, the structure 【Chemistry 25】 But the following structural fragment 【Chemistry 26-1】 【Chemistry 26-2】 【Chemistry 26-3】 is selected from Position 1 binds to Tb and position 2 binds to W; （２） 【Chemical 27】 The structural fragment represented by 【Chemistry 28】 And the first place is L 4 Binds to For example, the structure 【Chemical 29】 But the following structural fragment 【Chemistry 30-1】 【Chemistry 30-2】 Selected from: (3) Structure 【Chemistry 31】 But the following structural fragment 【Chemistry 32】 is selected from Position 1 is bonded to Tb via an S atom, and position 2 is bonded to W. (4) Structure 【Chemical 33】 But the following structural fragment 【Chemical Formula 34】 is selected from Position 1 is attached to L4, The antibody-drug conjugate, its stereoisomer, its prodrug, its pharma- ceutically acceptable salt or its pharma- ceutically acceptable solvate according to claim 17 or 18, characterized in that one or more of the following conditions are satisfied:

21. The following conditions (1) The antibody-drug conjugate is represented by the formula I-1, I-2, or I-3 【Chemistry 35】 having a structure represented by (2) The antibody-drug conjugate is represented by the formula I-1A, I-1B, I-2A, I-2B, I-3A, or I-3B 【Hua 36-1】 【Chemistry 36-2】 having a structure represented by (3) The antibody-drug conjugate is represented by the formula IA or IB 【Chemical 37】 having a structure represented by one or more of Tb, L 1 , L 2 , L 3 , L 4 , X, R 1 , R 2 , R 3 , R 4 , R 5 , R a , R b The antibody-drug conjugate, its stereoisomer, its prodrug, its pharma- ceutically acceptable salt or its pharma- ceutically acceptable solvate according to claim 17 or 18, characterized in that n and q are defined in any one of claims 16 to 20.

22. The antibody-drug conjugate 【Table 1-1】 【Table 1-2】 【Table 1-3】 【Table 1-4】 is selected from Tb is an anti-Her3 antibody according to any one of claims 1 to 21, q is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, and q is preferably 1, 2, 3, 4, 5, 6, 7, 8; The antibody-drug conjugate, e.g. 【Hua 38-1】 【Chemistry 38-2】 and The antibody-drug conjugate, its stereoisomer, its prodrug, its pharmaceutically acceptable salt or its pharmaceutically acceptable solvate according to any one of claims 15 to 21, wherein Her3mAb is antibody 202-2-1.

23. The antibody-drug conjugate 【Chemical Formula 39】 is selected from Tb is an anti-Her3 antibody according to any one of claims 1 to 22, q is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, and q is preferably 1, 2, 3, 4, 5, 6, 7, 8; The antibody-drug conjugate may be, for example, 【Chemistry 40】 and The antibody-drug conjugate, its stereoisomer, its prodrug, its pharmaceutically acceptable salt or its pharmaceutically acceptable solvate according to any one of claims 15 to 21, wherein Her3mAb is antibody 202-2-1.

24. An antibody-drug conjugate comprising two or more antibody-drug conjugates according to any one of claims 16 to 23, a stereoisomer thereof, a prodrug thereof, a pharma- ceutically acceptable salt thereof, or a pharma- ceutically acceptable solvate thereof, wherein the antibody-drug conjugate in the antibody-drug conjugate has a q value of 1, 2 or greater.

25. the drug-to-antibody ratio (DAR) in the antibody-drug conjugate is selected from an integer or decimal number between 1 and 10; For example, the antibody-drug conjugate of claim 24, characterized in that the DAR is selected from 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.2, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.7, 8.9 and 9.

26. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 10, the nucleic acid molecule according to claim 11, the vector according to claim 12, the host cell according to claim 13, the antibody-drug conjugate according to claims 15 to 23, a stereoisomer thereof, a prodrug thereof, a pharma- ceutically acceptable salt thereof or a pharma-ceutically acceptable solvate thereof, and / or the antibody-drug conjugate according to claims 24 to 25, and a pharma-ceutically acceptable carrier and / or excipient.

27. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 10, the nucleic acid molecule according to claim 11, the vector according to claim 12, the host cell according to claim 13, the antibody-drug conjugate according to claims 15 to 23, a stereoisomer thereof, a prodrug thereof, a pharma- ceutically acceptable salt thereof or a pharma-ceutically acceptable solvate thereof, the antibody-drug conjugate according to claims 24 to 25, and / or the pharmaceutical composition according to claim 26 in the manufacture of a medicament for the prevention and / or treatment of a tumor and / or an infectious disease, The tumor may be a solid tumor; the tumor may be selected from esophageal cancer (e.g. esophageal adenocarcinoma and esophageal squamous cell carcinoma), brain tumor, lung cancer (e.g. small cell lung cancer and non-small cell lung cancer), squamous cell carcinoma, bladder cancer, gastric cancer, ovarian cancer, peritoneal cancer, pancreatic cancer, breast cancer, head and neck cancer, cervical cancer, endometrial cancer, colorectal cancer, liver cancer, renal cancer, urothelial cancer, solid tumors, non-Hodgkin's lymphoma, central nervous system tumors (e.g. glioma, glioblastoma multiforme, glioma or sarcoma), prostate cancer, or thyroid cancer; The use, wherein the tumor is selected from colorectal cancer (e.g., colon cancer), lung cancer (e.g., non-small cell lung cancer), breast cancer, and prostate cancer.

28. A method for preventing and / or treating a tumor and / or an infectious disease in a subject, the method comprising the step of administering an effective amount of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 10, the nucleic acid molecule according to claim 11, the vector according to claim 12, the host cell according to claim 13, the antibody-drug conjugate according to claims 15 to 23, a stereoisomer thereof, a prodrug thereof, a pharma-ceutically acceptable salt thereof, or a pharma-ceutically acceptable solvate thereof, the antibody-drug conjugate according to claims 24 to 25, and / or the pharmaceutical composition according to claim 26 to a subject in need thereof, wherein the subject is a mammal, Preferably, the subject is a human.

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