Antibody-drug conjugates, their preparation methods, and their antitumor uses

Abstract

The present invention relates to an anti-HER3 antibody or its antigen-binding fragment, and an antibody-drug conjugate linked to the antibody, a method for preparing the same, and its use in tumor resistance. The antibody-drug conjugate has a structure represented by Ab-(LD)n and is covalently linked to an effective cytotoxic load via a linker. The anti-HER3 antibody or its antigen-binding fragment and the antibody-drug conjugate are used in the preparation of therapeutic agents for the diagnosis, prevention, and treatment of tumor diseases.

Description

[Technical Field]

[0001] The present disclosure relates to anti-HER3 antibodies and antibody-drug conjugates that are linked to specifically targeted antibodies and anti-tumor drugs or toxins via hydrophobicity-improved linkers and exert anti-tumor effects by targeting HER3-expressing tumor cells. [Background technology]

[0002] Antibody-drug conjugates (ADCs) are vectorized chemotherapy agents that selectively deliver potent cytotoxic agents to tumor / cancer cells (Antibody-Drug Conjugates: The Last Decade, Nicolas Joubert et al., Pharmaceuticals (Basel) 2020 September 14; 13(9):245). The commercially available ADC drugs Enhertu and sacituzumab govitecan have excellent efficacy in treating tumors, particularly malignant tumors, and both incorporate DNA topoisomerase inhibitor camptothecin derivatives as cytotoxic agents, which are more hydrophobic than tubulin inhibitors (e.g., MMAE and MMAF). Sacituzumab govitecan incorporates an MCC-triazole spacer-PEG7-lysine-PABC linker, which degrades in the lysosomes and releases camptothecin SN38 (US 13 / 948,732). Enhertz, developed by AstraZeneca and Daiichi Sankyo, incorporates a cathepsin B-activating GGFG (an amino acid sequence consisting of glycine-glycine-phenylalanine-glycine linked by a peptide bond) tetrapeptide linker, which introduces a self-cleaving structure to release the exatecan derivative Dxd (Yusuke Ogitani et al., Clin Cancer Res (2016) 22(20):5097-5108). However, due to the physicochemical properties of DNA topoisomerase inhibitors, such as their rigid structure and poor hydrophilicity, the ADC molecules mentioned above tend to form numerous aggregates during preparation, which directly affects the safety and efficacy of ADC therapy.

[0003] HER3 (NCBI, Gene ID: 2065) is a member of the HER (EGFR / ErbB) receptor family, which consists of four closely related type 1 transmembrane receptors (EGFR, HER2, HER3, and HER4). Monoclonal antibodies and small molecule inhibitors targeting the tyrosine kinase activity of EGFR and HER2 have shown clinical benefit in the treatment of several types of cancer, but their clinical efficacy is limited by drug resistance. HER3 is unique among the EGFR family members. HER3 has almost no intracellular tyrosine kinase activity and is unable to form homodimers. As a result, HER3 cannot function alone. Its kinase activity depends on heterodimerization with other EGFR family members, such as EGFR or HER2. When the HER3 protein and HER2 heterodimerize, their downstream pathways are activated, and by inhibiting the activation of the HER3 downstream pathway, it can indirectly inhibit the growth, proliferation, and metastasis of cancer cells. Thus, HER3 plays an important role in drug resistance to EGFR- and HER2-targeted therapies (Clin Cancer Res; 20(6) March 15, 2014).

[0004] Currently, Daiichi Sankyo's patritumab deruxtecan (HER3-DXd, U3-1402) is the only HER3-targeted ADC drug in development. The ADC structure is formed by splicing the HER3 monoclonal antibody patritumab and the topoisomerase 1 inhibitor exatecan derivative (deruxtecan) via a cleavable linker, GGFG, and aims to fulfill the unmet clinical need of a broad group of patients with EGFR-TKI-resistant and chemotherapy-failed non-small cell lung cancer. Summary of the Invention [Problem to be solved by the invention]

[0005] Therefore, there remains a need for new antibodies and antibody-drug conjugates that target HER3. [Means for solving the problem]

[0006] The present disclosure provides an anti-HER3 antibody or antigen-binding fragment thereof comprising at least one VL and VH, wherein CDR1 of the VL comprises the amino acid sequence set forth in SEQ ID NO: 1, CDR2 of the VL comprises the amino acid sequence set forth in SEQ ID NO: 2, CDR3 of the VL comprises the amino acid sequence set forth in SEQ ID NO: 3, CDR1 of the VH comprises the amino acid sequence set forth in SEQ ID NO: 4, CDR2 of the VH comprises an amino acid sequence having 5, 4, 3, 2, 1 or less, or 0 mutations compared to the amino acid sequence set forth in SEQ ID NO: 5, and CDR3 of the VH comprises the amino acid sequence set forth in SEQ ID NO: 6.

[0007] In some embodiments, in the anti-HER3 antibody or antigen-binding fragment thereof, CDR2 of the VH comprises (a) and (b): (a) C52N, T62N, T66K, and G67S, (b) C52Y, T62N, T66K, and G67S The compound comprises one or more mutations selected from:

[0008] In some embodiments, in the anti-HER3 antibody or antigen-binding fragment thereof, the CDR2 of the VH is (a) or (b): (a) C52N, T62N, T66K, and G67S, (b) C52Y, T62N, T66K, and G67S Contains mutations of

[0009] The present disclosure provides nucleic acids encoding the above-described anti-HER3 antibodies or antigen-binding fragments thereof. The present disclosure provides a vector comprising a nucleic acid encoding the above-described anti-HER3 antibody or antigen-binding fragment thereof.

[0010] The present disclosure provides host cells comprising the above-described nucleic acids and / or vectors. The present disclosure provides a compound of formula I Ab-(LD)n (I) or an isomer, isotopic variant, pharmaceutically acceptable salt, prodrug, or solvate thereof, or a combination thereof. (wherein Ab is the anti-HER3 antibody or antigen-binding fragment thereof described above; L is a linker covalently linking Ab and D; D is the payload, (n is a number between 1 and 10) to provide.

[0011] The present disclosure provides a method for preparing an antibody-drug conjugate, comprising the steps of: reducing at least a portion of the interchain disulfide bonds of an antibody or antigen-binding fragment thereof by a reduction treatment; and reacting the reactive group of the linker in the linker-payload with the reactive group of the linker to form an antibody-drug conjugate of formula I. Ab-(LD)n (I) or an isomer, isotopic variant, pharmaceutically acceptable salt, prodrug, or solvate thereof, or a combination thereof. (wherein Ab is the anti-HER3 antibody or antigen-binding fragment thereof described above; L is a linker covalently linking Ab and D; D is the payload, (n is a number between 1 and 10) and obtaining:

[0012] The present disclosure provides a pharmaceutical composition comprising the above-mentioned anti-HER3 antibody or antigen-binding fragment thereof, or antibody-drug conjugate, or an isomer, isotopic variant, pharmaceutically acceptable salt, prodrug, or solvate thereof, or a combination thereof, and a pharmaceutically acceptable excipient.

[0013] The present disclosure provides a kit comprising the above-mentioned anti-HER3 antibody or antigen-binding fragment thereof, or antibody-drug conjugate, or an isomer, isotopic variant, pharmaceutically acceptable salt, prodrug, or solvate thereof, or a combination thereof.

[0014] The present disclosure provides use of the above-described anti-HER3 antibody or antigen-binding fragment thereof, antibody-drug conjugate, antibody-drug conjugate prepared by the above-described method, the above-described pharmaceutical composition, or kit in the preparation of a therapeutic agent for diagnosing, preventing, and treating tumor diseases.

[0015] In some embodiments, the use includes use in the preparation of a medicament that targets HER3. In some embodiments, the tumor comprises a HER3-expressing solid tumor. The present disclosure provides methods for diagnosing, preventing, and treating tumor diseases, comprising administering to a subject a therapeutic dose of a therapeutic agent comprising the above-described anti-HER3 antibody or antigen-binding fragment thereof, antibody-drug conjugate, antibody-drug conjugate prepared by the above-described method, and the above-described pharmaceutical composition or kit. [Brief explanation of the drawings]

[0016] [Figure 1] 1A and 1B show the size exclusion chromatography and hydrophobicity chromatography chromatograms, respectively, of the L1H5 antibody (naked antibody) prepared in Example 4. [Figure 2] 2A and 2B show chromatograms of size exclusion chromatography and hydrophobic chromatography, respectively, for the detection of aggregates in the antibody-drug conjugate L1H5-LP3 prepared in Example 6. FIG. [Figure 3] 3A and 3B show chromatograms of size exclusion chromatography and hydrophobic chromatography, respectively, detecting aggregates in the antibody-drug conjugate L1H5-LP1 prepared in Example 7. FIG. [Figure 4]4A and 4B show chromatograms of size exclusion chromatography and hydrophobic chromatography, respectively, detecting aggregates in the antibody-drug conjugate L1H5-LP2 prepared in Example 8. FIG. [Figure 5] FIG. 1 shows statistical plots of flow cytometry of the Mu4O3 antibody prepared in Example 4 in cells expressing different amounts of HER3. [Figure 6] FIG. 1 shows immunofluorescence images of the Mu4O3 antibody prepared in Example 4 in HER3-GFP Tag-overexpressing 293T cells. [Figure 7] FIG. 1 shows the cell survival curve of the Mu4O3 antibody prepared in Example 4 in an SW620 cell killing assay. [Figure 8] FIG. 1 shows the affinity curves of the L1H4, L1H5, L2H4, L2H5, L4H4, L4H5, and L4H7 antibodies prepared in Example 4 in the MDA-MB-453 cell line. [Figure 9] FIG. 1 shows the affinity curves of the L1H4, L1H5, L2H4, L2H5, L4H4, L4H5, and L4H7 antibodies prepared in Example 4 in the SW620 cell line. [Figure 10] FIG. 1 shows the cell growth inhibition rates of the L1H5 antibody prepared in Example 4, a reference antibody, and a blank control in the SK-BR-3 cell line. [Figure 11] FIG. 1 shows the curves of changes in tumor volume over time for the L1H5 antibody prepared in Example 4, the reference antibody, and the blank control PBS in a BT-474 breast cancer mouse model. [Figure 12] FIG. 1 shows cell viability curves of antibody-drug conjugates L1H4-LP1, L1H5-LP1, and L4H4-LP1 prepared in Example 7 and a reference ADC (positive control) prepared in Comparative Example 1 in the SW620 cell line. [Figure 13]FIG. 1 shows cell survival curves in a killing assay against the SK-BR-3 cell line using the L1H5 antibody prepared in Example 4, L1H5-LP3 prepared in Example 6, a reference antibody, and a reference ADC prepared in Comparative Example 1. [Figure 14] FIG. 1 shows cell survival curves in a killing assay against the BXPC-3 cell line using the L1H5 antibody prepared in Example 4, L1H5-LP3 prepared in Example 6, a reference antibody, and a reference ADC prepared in Comparative Example 1. [Figure 15] FIG. 1 shows cell viability curves of antibody-drug conjugates L1H5-LP1 prepared in Example 7, L1H5-LP2 prepared in Example 8, and the reference ADC prepared in Comparative Example 1 in the HCT-15 cell line. [Figure 16] FIG. 1 shows the curves of changes in tumor volume over time for the antibody-drug conjugate L1H5-LP prepared in Example 7, L1H5-LP2 prepared in Example 8, the reference ADC prepared in Comparative Example 1, and the blank control group in an HCT-15 colon cancer mouse model. [Figure 17] FIG. 1 shows curves of changes in tumor volume over time after treatment of a mouse colon cancer PDX model with a reference ADC prepared in Comparative Example 1, L1H5-LP3 prepared in Example 6, L1H5-LP1 prepared in Example 7, a Dxd isotype control ADC prepared in Comparative Example 4, and an LP3 isotype control ADC prepared in Comparative Example 5. [Figure 18] FIG. 1 shows the curves of changes in tumor volume over time after treating a mouse EGFR-TKI-resistant lung adenocarcinoma PDX model with the reference ADC prepared in Comparative Example 1, L1H5-LP3 prepared in Example 6, L1H5-LP1 prepared in Example 7, Dxd isotype control ADC prepared in Comparative Example 4, and LP3 isotype control ADC prepared in Comparative Example 5. [Figure 19]FIG. 1 shows the curves of changes in tumor volume over time after treating a mouse colon cancer CDX model with L1H5-DXd prepared in Comparative Example 3, the reference antibody-LP3 prepared in Comparative Example 2, L1H5-LP3 prepared in Example 6, and the reference ADC prepared in Comparative Example 1, and also setting up a negative control PBS group. [Figure 20] FIG. 20 is a partially enlarged view of FIG. 19 with the negative control PBS group removed. [Figure 21] FIG. 1 shows the curves of changes in tumor volume over time after treatment of a mouse pancreatic cancer CDX model with the reference ADC prepared in Comparative Example 1, L1H5-LP3 prepared in Example 6, L1H5-LP1 prepared in Example 7, Dxd isotype control ADC prepared in Comparative Example 4, and LP3 isotype control ADC prepared in Comparative Example 5. [Figure 22] FIG. 1 shows a comparison of hydrophobicity measurements of the L1H5 antibody prepared in Example 4, the antibody-drug conjugate L1H5-LP3 prepared in Example 6, a reference antibody, and a reference ADC prepared in Comparative Example 1. [Figure 23] Figures 23A and 23B show chromatograms of size exclusion chromatography and hydrophobic chromatography detection of aggregates in the reference ADC prepared in Comparative Example 1, respectively. [Figure 24] Figures 24A and 24B show chromatograms of size exclusion chromatography and hydrophobic chromatography, respectively, for the detection of aggregates in the antibody-drug conjugate Reference Antibody-LP3 prepared in Comparative Example 2. [Figure 25] 25A and 25B show chromatograms of size exclusion chromatography and hydrophobic chromatography, respectively, for the detection of aggregates in the antibody-drug conjugate L1H5-DXd prepared in Comparative Example 3. FIG. [Figure 26] Figures 26A and 26B show chromatograms of size exclusion chromatography and hydrophobic chromatography, respectively, detecting aggregates in the Dxd isotype control ADC prepared in Comparative Example 4. [Figure 27] Figures 27A and 27B show chromatograms of size exclusion chromatography and hydrophobic chromatography detection of aggregates in the LP3 isotype control ADC prepared in Comparative Example 5, respectively. DETAILED DESCRIPTION OF THE INVENTION

[0017] Unless otherwise indicated, all numbers expressing content, concentration, ratio, mass, volume, time, temperature, thickness, technical effect, and the like used in the specification and claims should be understood to be modified in all instances by the term "about" or "approximately." Accordingly, unless indicated to the contrary, the numerical parameters set forth in the following specification and accompanying claims are approximations. It should be understood by those of ordinary skill in the art that the numerical parameters may vary depending upon the desired properties and effects sought and obtained by the present disclosure, and that each numerical parameter should be interpreted in accordance with the number of significant digits and conventional rounding techniques, or as understood by one of ordinary skill in the art.

[0018] Notwithstanding that the numerical ranges and parameters setting forth the broad scope of this disclosure are approximations, the numerical values ​​set forth in the specific examples are provided as precisely as possible. However, any numerical value inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing or measurements. Each numerical range given herein includes every narrower numerical range that falls within that broader numerical range, as if such narrower numerical ranges were all expressly written herein.

[0019] [antibody] The present disclosure provides an anti-HER3 antibody or antigen-binding fragment thereof comprising at least one light chain variable region (VL) and one heavy chain variable region (VH), wherein the VL and VH can be paired.

[0020] As used herein, the term "antibody" refers to any antigen-binding molecule or molecular complex containing at least one complementarity-determining region that specifically binds to or interacts with a specific antigen (e.g., HER3). The term "antibody" includes immunoglobulin molecules and multimers thereof (e.g., IgM), which contain four polypeptide chains, i.e., two heavy (H) chains and two light (L) chains, interconnected by disulfide bonds. Each heavy chain contains a heavy chain variable region and a heavy chain constant region. The heavy chain constant region contains three domains: CH1, CH2, and CH3. Each light chain contains a light chain variable region and a light chain constant region. The light chain constant region contains one domain (CL1). The VH and VL regions can be further subdivided into hypervariable regions called complementarity-determining regions (CDRs), separated by more conserved regions called framework regions (FRs). VH and VL are each composed of three CDRs and four FRs arranged from amino terminus to carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.

[0021] As used herein, the term "antibody" also encompasses antigen-binding fragments of intact antibody molecules. The term "antigen-binding fragment" encompasses any natural, synthetic, or genetically engineered polypeptide or glycoprotein that can be enzymatically obtained and specifically binds to an antigen to form a complex. Antigen-binding fragments of antibodies can be obtained, for example, from intact antibody molecules using any suitable standard technique. Non-limiting examples of antigen-binding fragments include Fab fragments, F(ab')2 fragments, Fd fragments, Fv fragments, single-chain Fv (scFv) molecules, dAb fragments, and minimal recognition units consisting of amino acid residues mimicking the hypervariable regions of an antibody (e.g., isolated complementarity-determining regions (CDRs), such as CDR3 peptides), or constrained FR3-CDR3-FR4 peptides. Other engineered molecules, such as domain-specific antibodies, single-domain antibodies, domain-deleted antibodies, chimeric antibodies, CDR-grafted antibodies, bifunctional antibodies, trifunctional antibodies, tetrafunctional antibodies, minibodies, and nanobodies (e.g., monovalent nanobodies, bivalent nanobodies, etc.), are also encompassed within the term "antigen-binding fragment" as used herein. Antigen-binding fragments of antibodies typically contain at least one variable domain. The variable domain may have any size or amino acid composition and typically contains at least one CDR adjacent to or in frame with one or more framework sequences. In antigen-binding fragments having a VH domain combined with a VL domain, the VH and VL domains can be arranged relative to each other in any suitable configuration. For example, the variable regions can be dimeric, containing VH-VH, VH-VL, or VL-VL dimers. Alternatively, antigen-binding fragments of antibodies can contain monomeric VH or VL domains.

[0022] In the anti-HER3 antibody or antigen-binding fragment thereof, CDR1 to 3 of the VL comprise the amino acid sequences set forth in SEQ ID NOs: 1 to 3, respectively, CDR1 of the VH comprises the amino acid sequence set forth in SEQ ID NO: 4, CDR2 of the VH comprises an amino acid sequence having 5, 4, 3, 2, 1 or less, or 0 mutations compared to the amino acid sequence set forth in SEQ ID NO: 5, and CDR3 of the VH comprises the amino acid sequence set forth in SEQ ID NO: 6. The CDRs of the anti-HER3 antibody or antigen-binding fragment thereof are defined and numbered using the Kabat system.

[0023] In some embodiments, the CDR2 of the VH comprises an amino acid sequence that has 4, 3, 2, 1 or less, or 0 mutations compared to the amino acid sequence set forth in SEQ ID NO: 5. In some embodiments, the CDR2 of the VH comprises an amino acid sequence that has 4 or less mutations compared to the amino acid sequence set forth in SEQ ID NO: 5. In some embodiments, the CDR2 of the VH comprises the amino acid sequence set forth in SEQ ID NO: 5.

[0024] CDR2 of VH contains mutations selected from any one or combination of C52N / Y, T62N, T66K, and G67S. C52N / Y indicates that the 52nd amino acid, "C," is mutated to "N" or "Y." As used herein, "C," "N," "Y," "T," "K," "G," and "S" are single-letter standard amino acids, representing cysteine, asparagine, tyrosine, threonine, lysine, glycine, and serine, respectively. For example, the first amino acid refers to the first amino acid from the amino terminus to the carboxyl terminus of VH.

[0025] In some embodiments, in the anti-HER3 antibody or antigen-binding fragment thereof, CDR2 of the VH comprises the mutations C52N, T62N, T66K, and G67S. In some embodiments, in the anti-HER3 antibody or antigen-binding fragment thereof, CDR2 of the VH comprises the mutations C52Y, T62N, T66K, and G67S.

[0026] Furthermore, the variable region of an anti-HER3 antibody or an antigen-binding fragment thereof includes framework regions (FRs), which are regions with relatively few changes in amino acid composition and sequence order other than the CDRs.

[0027] In some embodiments, there are no mutations in the framework region, or mutations that do not affect the binding of the antibody variable region to the antigen, which may increase the binding affinity of the antibody to the antigen, or may remain substantially unchanged. In some embodiments, the anti-HER3 antibody or its antigen-binding fragment further comprises conservatively modified variants, including single substitutions, deletions, or additions to the polypeptide sequence, resulting in the replacement of amino acids with chemically similar amino acids. Conservative substitution tables providing functionally similar amino acids are well known in the art. Such conservatively modified variants exist in addition to, and do not exclude, polymorphic variants, interspecies homologs, and alleles. The following eight groups contain amino acids that are conservatively substituted for one another: 1) alanine (A) and glycine (G), 2) aspartic acid (D) and glutamic acid (E), 3) asparagine (N) and glutamine (Q), 4) arginine (R) and lysine (K), 5) isoleucine (I), leucine (L), methionine (M) and valine (V); 6) phenylalanine (F), tyrosine (Y), and tryptophan (W), 7) serine (S) and threonine (T), and 8) cysteine ​​(C) and methionine (M) (see, e.g., Creighton, Proteins (1984)). In some aspects, the term "conservative sequence modification" is used to refer to amino acid alterations that do not significantly affect or alter the binding characteristics of the antibody containing the amino acid sequence.

[0028] In some embodiments, in the anti-HER3 antibody or antigen-binding fragment thereof, the VL comprises an amino acid sequence having at least 70%, 75%, 77%, 78%, 80%, 82%, 84%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the framework region sequences of the amino acid sequences set forth in SEQ ID NOs: 9, 11, 12, and 13, and the VH comprises an amino acid sequence having at least 70%, 75%, 77%, 78%, 80%, 82%, 84%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the framework region sequences of the amino acid sequences set forth in SEQ ID NOs: 10, 14, 15, and 16.

[0029] In some embodiments, in the anti-HER3 antibody or antigen-binding fragment thereof, the VL comprises an amino acid sequence having at least 70%, 75%, 77%, 78%, 80%, 82%, 84%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence set forth in SEQ ID NOs: 9, 11, 12, and 13, and the VH comprises an amino acid sequence having at least 70%, 75%, 77%, 78%, 80%, 82%, 84%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence set forth in SEQ ID NOs: 10, 14, 15, and 16.

[0030] In the present disclosure, the term "% identity" between two or more polypeptide sequences refers to the degree to which two or more sequences or subsequences are identical. Two sequences are "identical" if they have the same amino acid sequence over the region they are compared. Two sequences have a specified percentage of identical amino acid residues (e.g., 60% identity over a specified region, or, if not specified, over the entire sequence) when compared and aligned over a comparison window or specified region to maximize correspondence, or over a specified region as determined using one of the sequence comparison algorithms described below, or by manual alignment and visual inspection. Optionally, the identity exists over a region at least about 10 amino acids in length, and more preferably over a length of 20, 50, 200, or more amino acids. Two examples of algorithms that are suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms (Altschul et al., Nuc. Acids Res. 25:3389-3402, 1997, and Altschul et al., J. Mol. Biol. 215:403-410, 1990).

[0031] In addition to the percentage sequence identity discussed above, another meaning of two polypeptides being substantially identical refers to a polypeptide that is immunologically cross-reactive with a second polypeptide; thus, a polypeptide is typically substantially identical to a second polypeptide, for example, where the two peptides differ only by conservative substitutions.

[0032] In some aspects, in the anti-HER3 antibody or antigen-binding fragment thereof, the VL comprises the amino acid sequence set forth in SEQ ID NO: 9, 11, 12, or 13, and the VH comprises the amino acid sequence set forth in SEQ ID NO: 10, 14, 15, or 16.

[0033] In some embodiments, in the anti-HER3 antibody or antigen-binding fragment thereof, the VL and VH comprise an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence set forth in SEQ ID NO:9 and SEQ ID NO:10, respectively; the VL and VH comprise an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence set forth in SEQ ID NO:11 and SEQ ID NO:14, respectively; the VL and VH comprise an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence set forth in SEQ ID NO:11 and SEQ ID NO:15, respectively; and the VL and VH comprise an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence set forth in SEQ ID NO:12 and SEQ ID NO:14, respectively. wherein the VL and VH comprise an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequences set forth in SEQ ID NO:12 and SEQ ID NO:15, respectively; the VL and VH comprise an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequences set forth in SEQ ID NO:13 and SEQ ID NO:14, respectively; the VL and VH comprise an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequences set forth in SEQ ID NO:13 and SEQ ID NO:15, respectively; or the VL and VH comprise an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequences set forth in SEQ ID NO:13 and SEQ ID NO:16, respectively.

[0034] In some embodiments, the anti-HER3 antibody or antigen-binding fragment thereof comprises or has a VH and a VL that are amino acid sequences set forth in SEQ ID NOs: 9 and 10, 11 and 14, 11 and 15, 11 and 16, 12 and 14, 12 and 15, 12 and 16, 13 and 14, 13 and 15, or 13 and 16, respectively, or conservatively substituted variants thereof, and optionally the framework regions have conservative substitutions.

[0035] In some embodiments, the anti-HER3 antibody or antigen-binding fragment thereof comprises or has a VH and a VL that are amino acid sequences set forth in SEQ ID NOs: 9 and 10, 11 and 14, 11 and 15, 12 and 14, 12 and 15, 13 and 14, 13 and 15, or 13 and 16, respectively, or conservatively substituted variants thereof, and optionally the framework regions have conservative substitutions.

[0036] The HER3 antibody or antigen-binding fragment thereof provided by the present disclosure can bind to mammalian (e.g., human or mouse) HER3 protein. In some embodiments, the anti-HER3 antibody or antigen-binding fragment thereof specifically binds to human HER3. In some embodiments, the anti-HER3 antibody or antigen-binding fragment thereof specifically binds to mouse HER3.

[0037] The HER3 antibody or antigen-binding fragment thereof may have an IgM, IgG, IgA, IgD, or IgE class. In some embodiments, the HER3 antibody or antigen-binding fragment thereof is an IgG molecule. In some embodiments, the HER3 antibody or antigen-binding fragment thereof is an IgG1, IgG2, IgG3, or IgG4 subtype. In some embodiments, the HER3 antibody or antigen-binding fragment thereof is human IgG1. Antibody classes and subclasses are identified according to common methods in the art, for example, based on differences in the heavy chains α, δ, ε, γ, and μ, and the constructed antibodies are called IgA, IgD, IgE, IgG, and IgM. Among them, human γ can be further subdivided into γ1, γ2, γ3, and γ4, which correspond to the four subtypes of IgG1, IgG2, IgG3, and IgG4, respectively.

[0038] The HER3 antibody or antigen-binding fragment thereof may be a human antibody, a humanized antibody, or a chimeric antibody defined using means commonly understood in the art. For example, a human antibody refers to an antibody that is fully encoded by human antibody genes, a humanized antibody refers to an antibody in which at least a portion of the constant region is encoded by human genes, and a chimeric antibody refers to an antibody that is expressed by transforming mammalian cells using DNA recombinant technology to insert the light and heavy chain variable region genes of a heterologous monoclonal antibody into an expression vector containing a human antibody constant region.

[0039] The anti-HER3 antibodies or antigen-binding fragments thereof provided by the present disclosure have improved hydrophobicity, which is beneficial for improving stability in vitro and in vivo.

[0040] The anti-HER3 antibody or antigen-binding fragment thereof provided by the present disclosure has good targeting properties, can fix HER3-expressing tumor cells, and can play a role in tumor killing. Optionally, the anti-HER3 antibody or antigen-binding fragment thereof can play an anti-tumor role in vivo and in vitro.

[0041] The present disclosure also provides a nucleic acid encoding the above-mentioned anti-HER3 antibody or antigen-binding fragment thereof, a vector comprising the nucleic acid encoding the above-mentioned anti-HER3 antibody or antigen-binding fragment thereof, and a host cell comprising the nucleic acid and / or the vector.

[0042] Anti-HER3 antibodies or antigen-binding fragments thereof can be obtained by immunizing animals with HER3 or any polypeptide selected from the amino acid sequence of HER3, and then collecting and purifying the antibodies produced in vivo, according to methods commonly practiced in the art. In this case, antibodies applicable to human diseases can be selected by examining the cross-reactivity of the obtained antibodies binding to heterologous HER3 with human HER3. Alternatively, monoclonal antibodies can be obtained from hybridomas established by fusing antibody-producing cells that produce antibodies against HER3 with myeloma cells according to known methods (e.g., Kohler and Milstein, Nature (1975) 256, 495-497; Kennet, R, ed., Monoclonal Antibodies, 365-367, Plenum Press, NY (1980); Goding JW, Monoclonal Antibodies: Principles and Practice, 3rd Edition (1986), Academic Press, San Diego, CA). HER3 for use as an antigen can be obtained by expressing the HER3 gene in host cells using genetic engineering.

[0043] Chimeric antibodies can be obtained by engineering hybridomas, for example, chimeric antibodies in which antibody variable regions derived from a mouse or rat are linked to constant regions derived from a human (see Proc. Natl. Acad. Sci. USA, 81, pp. 6851-6855, (1984)).

[0044] Humanized antibodies can be produced by replacing most or all of the structural portions of a non-human monoclonal antibody with the corresponding human antibody sequences. For example, antibodies obtained by incorporating only the complementarity-determining regions (CDRs) into a human-derived antibody (see Nature (1986) 321, pp. 522-525) and antibodies obtained by grafting CDR sequences as well as some framework amino acid residues into a human antibody using the CDR grafting method (WO90 / 07861). Human antibodies can be obtained by using methods involving mice engineered to produce human antibodies that carry human chromosomal fragments containing human antibody heavy and light chain genes (Tomizuka, K. et al., Nature Genetics (1997) 16, pp. 133-143; Kuroiwa, Y. et al., Nucl. Acids Res. (1998) 26, pp. 3447-3448; Yoshida, H. et al., Animal Cell Technology: Basic and Applied Aspects, Vol. 10, pp. 69-73 (Kitagawa, Y., Matsuda, T., and Iijima, S., eds.), Kluwer Academic Publishers, 1999; Tomizuka, K., ed., Proc. Natl. Acad. Sci. USA (2000) 97, pp. 722-727, etc.). Methods for obtaining humanized antibodies include, for example, those described in Winter and Milstein, Nature, 1991, 349:293-299; Rader et al., Proc. Nat. Acad. Sci. USA, 1998, 95:8910-8915; Steinberger et al., J. Biol. Chem., 2000, 275:36073-36078; Queen et al., Proc. Natl. Acad. Sci. USA, 1989, 86:10029-10033.

[0045] [Antibody-drug conjugates] The present disclosure provides a compound of formula I Ab-(LD)n (I) or an isomer, isotopic variant, pharmaceutically acceptable salt, prodrug, or solvate thereof, or a combination thereof. (wherein Ab is the anti-HER3 antibody or antigen-binding fragment thereof described above; L is a linker covalently linking Ab and D; D is the payload, (n is a number between 1 and 10) to provide.

[0046] The term "antibody-drug conjugate" or "ADC" refers to a conjugate of an anti-HER3 antibody or antigen-binding fragment thereof described herein covalently linked to a payload. Generally, an antibody-drug conjugate may include an antibody, a payload, and optionally a linker between the antibody and the payload. An ADC can provide a therapeutic effect by delivering the payload to HER3 cells, particularly HER3 tumor cells, targeted by the antibody. Antibody-drug conjugates can be prepared by various methods known in the art for preparing antibody-drug conjugates.

[0047] In ADC molecules, the linker, which serves as the linking structure connecting the antibody and payload, is a key factor for successfully constructing an ADC molecule. The molecular design and properties of the linker are important determinants of the efficacy of the ADC in terms of pharmacokinetics (PK) / pharmacodynamics (PD) and therapeutic window. For optimal efficacy, an ideal linker should have the following properties: (1) The linker should be sufficiently stable in plasma to allow the ADC molecule to circulate in the bloodstream and localize at the tumor site without premature dissolution. An unstable linker can lead to premature release of the cytotoxic payload, damaging non-target healthy cells and potentially causing systemic toxicity and adverse reactions. (2) The linker should be capable of being rapidly cleaved upon internalization of the ADC by target tumor cells, rapidly releasing the free cytotoxic payload. (3) Linker design also requires consideration of hydrophobicity. Conjugation of a hydrophobic linker to a hydrophobic cytotoxic payload generally promotes aggregation of the ADC molecules, which is not only detrimental to ADC efficacy but may also lead to liver toxicity or induce unwanted immune responses (Kyoji Tsuchikama et al., Antibody-drug conjugates: recent advances in conjugation and linker chemistry, Protein Cell. 2018 January;9(1):33-46).

[0048] The term "isomer" refers to compounds with the same molecular formula but different structures, also known as isomers or structural isomers, and generally includes constitutional isomers and stereoisomers. Structural isomers refer to isomers caused by different bonding sequences or bonding characteristics of atoms in a molecule, and preferably include tautomers. Tautomers refer to functional isomers resulting from the rapid movement of atoms in a molecule between two positions. Stereoisomers refer to isomers caused by the same bonding sequence of atoms or groups of atoms and bonding substances in a molecule but different spatial arrangements, and preferably include optical isomers. Optical isomers refer to stereoisomers with different optical rotations due to the absence of axial symmetry in a molecule, such as enantiomers, diastereomers, racemates, and meso isomers.

[0049] The term "prodrug" refers to a compound that is chemically modified from an inactive or less active drug in vitro and is converted in vivo, either enzymatically or non-enzymatically, to release the active drug and become effective. In the present disclosure, the prodrug may be an ADC molecule or a payload.

[0050] In some aspects, the linkers are cleavable and non-cleavable linkers. In some embodiments, the linker comprises a cleavable peptide, and optionally the cleavable peptide is cleavable by an enzyme. In some embodiments, the enzyme comprises cathepsin B.

[0051] In some embodiments, the cleavable peptide or L comprises an amino acid unit that includes a dipeptide, tripeptide, tetrapeptide, or pentapeptide. In some embodiments, the amino acid units are selected from any one or combination of Val-Cit, Val-Ala, Glu-Val-Cit, Ala-Ala-Asn, Gly-Val-Cit, Gly-Gly-Gly, and Gly-Gly-Phe-Gly, where Val represents valine, Cit represents citrulline, Ala represents alanine, Glu represents glutamic acid, Asn represents asparagine, Gly represents glycine, and Phe represents phenylalanine.

[0052] In some embodiments, the linker comprises a self-immolative spacer, and optionally the self-immolative spacer comprises p-aminobenzyloxycarbonyl (PABC) or p-aminobenzyl (PAB).

[0053] In some aspects, the cleavable peptide is spliced ​​directly to the self-immolative spacer. In some embodiments, the linker is -L1-L2-L3-, where L1 is -(succinimid-3-yl-N)-(CH2)m 1 -C(=O)-, -CH2-C(=O)-NH-(CH2)m 2 -C(=O)-, or -C(=O)-(CH2)m 3 -C(=O)-(in the formula, m 1 represents an integer from 2 to 8, and m 2 represents an integer from 1 to 8, and m 3 represents an integer of 1 to 8), L2 represents an amino acid unit, and L3 represents a self-immolative spacer.

[0054] In some embodiments, m 1 represents 2, 3, 4, 5, 6, 7, or 8. In some embodiments, m 2 represents 1, 2, 3, 4, 5, 6, 7, or 8. In some embodiments, m 3 represents 1, 2, 3, 4, 5, 6, 7, or 8.

[0055] In some embodiments, L is the following group: -(succinimide-3-yl-N)-CH2CH2-C(=O)-GGFG-PABC-; -(succinimide-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-PABC-; -(succinimide-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-PABC-; -(succinimide-3-yl-N)-CH2CH2-C(=O)-NH-CH2CH2O-CH2CH2O-CH2CH2-C(=O)-GGFG-PABC-; -(succinimide-3-yl-N)-CH2CH2-C(=O)-NH-CH2CH2O-CH2CH2O-CH2CH2O-CH2CH2O-CH2CH2-C(=O)-GGFG-PABC-; -CH2-C(=O)-NH-CH2CH2-C(=O)-GGFG-PABC-; -C(=O)-CH2CH2CH2CH2CH2CH2-C(=O)-GGFG-PABC-; -(succinimide-3-yl-N)-CH2CH2-C(=O)-GGFG-NH-CH2CH2-C(=O)-; -(succinimide-3-yl-N)-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-; -(succinimide-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2-C(=O)-; -(succinimide-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-; -(succinimide-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2CH2CH2-C(=O)-; -(succinimide-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2-O-CH2-C(=O)-; -(succinimide-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2-O-CH2-C(=O)-; -(succinimide-3-yl-N)-CH2CH2-C(=O)-NH-CH2CH2O-CH2CH2O-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-; -(succinimide-3-yl-N)-CH2CH2-C(=O)-NH-CH2CH2O-CH2CH2O-CH2CH2-C(=O)-GGFG-NH-CH2CH2-C(=O)-; -(succinimide-3-yl-N)-CH2CH2-C(=O)-NH-CH2CH2O-CH2CH2O-CH2CH2O-CH2CH2O-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-; -(succinimide-3-yl-N)-CH2CH2-C(=O)-NH-CH2CH2O-CH2CH2O-CH2CH2O-CH2CH2O-CH2CH2-C(=O)-GGFG-NH-CH2CH2-C(=O)-; -CH2-C(=O)-NH-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-; -C(=O)-CH2CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-; -(succinimide-3-yl-N)-CH2CH2-C(=O)-VA-PABC-; -(succinimide-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-VA-PABC-; -(succinimide-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-VA-NH-PABC-; -(succinimide-3-yl-N)-CH2CH2-C(=O)-NH-CH2CH2O-CH2CH2O-CH2CH2-C(=O)-VA-PABC-; -(succinimide-3-yl-N)-CH2CH2-C(=O)-NH-CH2CH2O-CH2CH2O-CH2CH2O-CH2CH2O-CH2CH2-C(=O)-VA-PABC-; -CH2-C(=O)-NH-CH2CH2-C(=O)-VA-PABC-; -C(=O)-CH2CH2CH2CH2CH2CH2-C(=O)-VA-PABC-; -(succinimide-3-yl-N)-CH2CH2-C(=O)-VA-NH-CH2CH2-C(=O)-; -(succinimide-3-yl-N)-CH2CH2-C(=O)-VA-NH-CH2CH2CH2-C(=O)-; -(succinimide-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-VA-NH-CH2CH2-C(=O)-; -(succinimide-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-VA-NH-CH2CH2CH2-C(=O)-; -(succinimide-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-VA-NH-CH2CH2CH2CH2CH2-C(=O)-; -(succinimide-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-VA-NH-CH2-O-CH2-C(=O)-; -(succinimide-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-VA-NH-CH2CH2-O-CH2-C(=O)-; -(succinimide-3-yl-N)-CH2CH2-C(=O)-NH-CH2CH2O-CH2CH2O-CH2CH2-C(=O)-VA-NH-CH2CH2CH2-C(=O)-; -(succinimide-3-yl-N)-CH2CH2-C(=O)-NH-CH2CH2O-CH2CH2O-CH2CH2-C(=O)-VA-NH-CH2CH2-C(=O)-; -(succinimide-3-yl-N)-CH2CH2-C(=O)-NH-CH2CH2O-CH2CH2O-CH2CH2O-CH2CH2O-CH2CH2-C(=O)-VA-NH-CH2CH2CH2-C(=O)-; -(succinimide-3-yl-N)-CH2CH2-C(=O)-NH-CH2CH2O-CH2CH2O-CH2CH2O-CH2CH2O-CH2CH2-C(=O)-VA-NH-CH2CH2-C(=O)-; -CH2-C(=O)-NH-CH2CH2-C(=O)-VA-NH-CH2CH2CH2-C(=O)-; and -C(=O)-CH2CH2CH2CH2CH2CH2-C(=O)-VA-NH-CH2CH2CH2-C(=O)- is selected from one or more of: is selected from.

[0056] In some embodiments, p-aminobenzyloxycarbonyl (PABC) or p-aminobenzyl (PAB) is linked to a polysarcosine (poly-N-methylglycine) residue or a methylamino group.

[0057] In some embodiments, the antibody-drug conjugate comprises an antibody, a payload, and a compound of Formula II

[0058] [ka] Linker wherein the linker of formula II is covalently bonded via a succinimidyl group to a sulfhydryl moiety reduced from an interchain disulfide chain of an antibody by a thioether bond; The carbonyl in the ester group of the linker of formula II is linked to an amino group in the payload, In Formula II, R1 and R2 are independently selected from hydrogen, methyl, and isopropyl groups; R3 is -(CR5HCONH)n 1 -(CH2CONH)n 2 - or a single bond, R5 is hydrogen or benzyl, and n 1 represents an integer from 0 to 2, and n 2 represents an integer between 0 and 2, R4 is a methylamino group or -(NCH3COCH2)n 3 -NCH3COCH3, n 3 represents an integer between 1 and 20) Includes.

[0059] The term "payload" encompasses compounds that are cytotoxic or capable of killing cells upon release from the antibody-drug conjugate, compounds labeled with radioactive markers, fluorophores, chromophores, imaging agents, and / or metal ions for use as detectable markers or in cell killing applications, radionuclides or polypeptides, and compounds, nucleic acids, polypeptides or proteins, enzymes, hormones, or nucleic acids that can modulate (including activate or inhibit) the body's immune activity.

[0060] In some ideal cases, the conjugated payload of the antibody-drug conjugate exhibits little or no cytotoxicity to an effective therapeutic dose of the administered ADC without inducing a systemic toxic response in the subject due to the conjugated payload. The payload may be a drug that has been clinically validated for the treatment of a particular disease, or a compound, radionuclide, nucleic acid, protein, or polypeptide that has acceptable pharmacological activity under conditions of clinical use.

[0061] In the present disclosure, in Formula I, the linker is attached to the antibody by a thioether bond attached from a sulfhydryl reduced from the interchain disulfide chain of the antibody via a succinimidyl group at its terminus.

[0062] [ka] which forms a thioether bond with the carbon atom at position 3 and the sulfhydryl group reduced from the interchain disulfide chain of the antibody.

[0063] [ka] A bond with a represents a chemical bond that is attached to another group.

[0064] In the present disclosure, disulfide bonds of an antibody include interchain disulfide bonds and intrachain disulfide bonds, and preferably, the interchain disulfide bonds are treated, for example, activated (or reduced) to sulfhydryl groups, and then bound to a linker. Amino acids of an antibody that are chemically bound to the succinimidyl group of a linker include one or a combination of lysine, histidine, tyrosine, and cysteine, and preferably cysteine.

[0065] In some embodiments, the linker of Formula I can be linked to the hinge region, variable region, and / or constant region of an antibody. In some embodiments of the present disclosure, in the linker of Formula II, R4 is -(NCH3COCH2)n 3 -NCH3COCH3, n 3 represents an integer from 1 to 20. 3 may be selected from any integer of, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20.

[0066] In some embodiments, in the linker of Formula II, R4 is -(NCH3COCH2)n 3 -NCH3COCH3, n 3 represents an integer of 8 to 15. In some embodiments, in the antibody-drug conjugate, in the linker of Formula II, R4 is -(NCH3COCH2)n 3 -NCH3COCH3, n 3 represents an integer between 10 and 12.

[0067] In some embodiments, in the antibody-drug conjugate, in the linker of Formula II, R 4 represents a methylamino group. In the linker of Formula II of the present disclosure, R4 comprises a hydrophilic polysarcosine group or a methylamino group, which results in an increased hydrophilicity of the antibody-drug conjugate; particularly when a hydrophobic payload is conjugated in the antibody-drug conjugate, improving the hydrophilicity of the ADC molecule is beneficial in reducing aggregation of the ADC molecule during the preparation process, thereby improving the stability, homogeneity, and purity of the antibody-drug conjugate.

[0068] In some embodiments, in the antibody-drug conjugate, in the linker of Formula II, R3 represents a single bond. In some embodiments, in the antibody-drug conjugate, in the linker of Formula II, R3 is -(CR5HCONH)n 1 -(CH2CONH)n 2 -, R5 is benzyl, and n 1 represents an integer of 1 to 2, and n 2 represents an integer of 1 to 2.

[0069] In some embodiments, in the antibody-drug conjugate, in the linker of Formula II, R3 represents -CR5HCONH-, -CH2CONH-, or -CR5HCONH-CH2CONH-, -(CR5HCONH)2-CH2CONH-, -CR5HCONH-(CH2CONH)2-, or -(CR5HCONH)2-(CH2CONH)2-, and R5 is benzyl.

[0070] In some embodiments, in the antibody-drug conjugate, in the linker of Formula II, R is hydrogen. In some embodiments, in the linker of Formula II, R is isopropyl.

[0071] In some embodiments, in the antibody-drug conjugate, in the linker of Formula II, R2 is hydrogen. In some embodiments, in the linker of Formula II, R2 is methyl. In some embodiments, the linker in the antibody-drug conjugate comprises the group

[0072] [ka] is selected from one or more of:

[0073] In some embodiments, the payload in the antibody-drug conjugate is a marker containing a radioactive marker, fluorophore, chromophore, imaging agent, and / or metal ion as a detection marker, including, but not limited to, chemically synthesized organic compounds, radionuclides, metal complexes, or polypeptides. A radioactive marker refers to a labeled compound that can be identified and used as a tracer by replacing one or more atoms of the compound molecule with a radionuclide, including amino acids, polypeptides, proteins, sugars, nucleotides, nucleosides, purines, pyrimidines, steroids, lipids, tumor antigens, hormones, receptors, vitamins, and pharmaceuticals for medical research. Radionuclides are typically nuclear species that spontaneously emit radiation, including, but not limited to, tritium, iodine-125, iodine-131, sulfur-35, phosphorus-32, and carbon-14. A fluorophore is typically a group containing a conjugated double bond that emits fluorescence when the molecule returns from an excited state to a ground state. Chromophores refer to unsaturated groups and their associated chemical bonds contained in molecules that can absorb light radiation and undergo electronic transitions. Imaging agents generally refer to radiopharmaceuticals that, when introduced into the body, can be used to image organs, tissues, or molecules in nuclear medicine.

[0074] In some embodiments, the payload in the antibody-drug conjugate is a nucleic acid, which may be a ribonucleic acid and / or a deoxyribonucleic acid. In some embodiments, the payload in the antibody-drug conjugate is a hormone, a growth factor, a clotting factor, a plasminase (e.g., a prodrug converting enzyme capable of converting a prodrug into an active drug), and a ribonuclease.

[0075] In some embodiments, the payload in the antibody-drug conjugate is an immunomodulatory agent (including cytokines and chemokines that have immune-influencing effects), an agonist antibody with biological activity, or an antagonist antibody.

[0076] In some embodiments, the payload in the antibody-drug conjugate is a cytotoxic compound.In some embodiments, the payload in the antibody-drug conjugate has antitumor activity or is an antitumor drug, and the payload is selected from DNA topoisomerase inhibitors or selected from tubulin inhibitors.The DNA topoisomerase inhibitor can be a topoisomerase I inhibitor or a topoisomerase II inhibitor.

[0077] In this disclosure, the term "topoisomerase inhibitor" generally refers to a compound that inhibits the activity of a topoisomerase. Compounds that are topoisomerase I inhibitors are active against topoisomerase I, topoisomerase II inhibitors have activity against topoisomerase II, and some compounds are active against both topoisomerase I and topoisomerase II and are known as topoisomerase I / II inhibitors.

[0078] The term "tubulin inhibitor" generally refers to compounds that inhibit the microtubule system of eukaryotic cells, preventing cell division and inhibiting cell proliferation. In some embodiments, the payload is camptothecin or a derivative thereof having a topoisomerase inhibitory effect. The term "derivative": A compound formed by replacing an atom or atomic group in the molecule of a parent compound with another atom or atomic group is called a derivative of the parent compound. The term "camptothecin and its derivatives" generally encompasses camptothecin and camptothecin derivatives. Camptothecin exerts its pharmacological effect by irreversibly inhibiting topoisomerase I. Camptothecin derivatives include exatecan, irinotecan, topotecan, lurtotecan, siratecan, etirinotecan pegol, TAS 103, 9-aminocamptothecin, 7-ethylcamptothecin, 10-hydroxycamptothecin, 9-nitrocamptothecin, 10,11-methylenedioxycamptothecin, 9-amino-10,11-methylenedioxycamptothecin, 9-chloro-10,11-methylenedioxycamptothecin, (7-(4-methylpiperazinomethylene)-10,11-ethylenedioxy-20(S)-camptothecin), (7-(4-methylpiperazinomethylene)-10,11-methylenedioxy-20(S)-camptothecin), 7-(2-(N-isopropylamino)ethyl)-(20S)-camptothecin, and the like, as well as stereoisomers, salts, and esters thereof. Methods for synthesizing camptothecin and camptothecin analogs or derivatives are known and are summarized and described in US Pat. No. 5,244,903, which is incorporated herein by reference in its entirety.

[0079] In some embodiments, the payload is an auristatin or a derivative thereof, or maytansine or a derivative thereof, that has a tubulin inhibitory effect. The term "auristatin and its derivatives" generally encompasses auristatin F and derivatives of auristatin F, including monomethyl auristatin E (MMAE) and monomethyl auristatin F (MMAF). The term "maytansine and its derivatives" generally encompasses maytansine and derivatives of maytansine, including maytansine DM1, maytansine DM2, and maytansine DM4.

[0080] In some embodiments, the payload is the camptothecin derivative exatecan, which is a topoisomerase inhibitor that acts throughout the cell cycle with strong penetration and good therapeutic effect on slow-growing solid tumors.In addition, the number of targets in cells is much smaller than that of tubulin inhibitors, and ADC molecules can carry the same number of payloads and enter cells to exert a better killing effect.The exatecan molecule is not a substrate of P-gp, which is beneficial for reducing or alleviating the problem of drug resistance.

[0081] In some embodiments, the payload has Formula III

[0082] [ka] of exatecan, which is connected to the linker by the nitrogen atom of the amino group on its cyclohexane ring.

[0083] The structure of the exatecan molecule is rigid and poorly hydrophilic. When conjugated with the GGFG tetrapeptide linker, which is commonly used in the prior art to prepare ADCs, the exatecan molecule tends to cause aggregation between ADC molecules, which does not meet the requirements for developing ADC drugs (Bioorg.Med.Chem.Lett.26(2016) pp.1542-1545). Therefore, the selection and compatibility of the linker and payload affect the safety and stability of ADC drugs.

[0084] Without being bound by any theory, the antibody-drug conjugates provided in the present disclosure improve the hydrophilicity of the linker-payload structure due to the presence of multiple hydrophilic groups in the linker, which can to some extent reduce phenomena such as aggregation and precipitation of ADC molecules caused by hydrophobic payloads.

[0085] After the ADC molecule is endocytosed into a cell, the payload or a compound of the linker (or part of the linker)-payload structure is released, depending on whether the linker is degraded. In some embodiments, the amino group on the cyclohexane ring of the exatecan of Formula III and the carbonyl group in the ester group of the linker of Formula II form a carbamate-containing linker-payload structure. Without being bound by any theory, in the linker-payload structure provided in the present disclosure, after the ADC molecule is endocytosed into a cell, the linker is enzymatically cleaved by a cathepsin (e.g., cathepsin B), releasing a compound of Formula V

[0086] [ka] (R4 is a methylamino group or -(NCH3COCH2)n 3 -NCH3COCH3, n 3 represents an integer between 1 and 20) forming intermediates or active metabolites of

[0087] Next, the PABC group in the intermediate or active metabolite of Formula V undergoes 1,6-elimination to release exatecan. The mechanism of 1,6-elimination of PABC is described in detail in Angew. Chem. Int. Ed. 2015, 54, 7492-7509. Therefore, the linker-payload structure in the ADC molecule provided by the present disclosure has good in vivo stability and biological activity.

[0088] Without being bound by any theory, the enzyme cleavage site in the linker-payload structure may be an amide bond in the linker, for example, an amide bond between the carbon atom on which the substituent represented by R2 is located and the group represented by R3, or an amide bond in the group represented by R3.

[0089] In some embodiments, in the antibody-drug conjugate, n is the ratio of the number of conjugated payload molecules to the number of antibody molecules (drug-to-antibody ratio, DAR). In some embodiments, in the antibody-drug conjugate, n is 1 to 10, 1 to 2, 2 to 4, 4 to 6, 2 to 8, 4 to 8, 4 to 10, 6 to 10, 7 to 10, or 8 to 10, with exemplary DAR values ​​being 4, 6, 7.78, 8.038, or 9.92.

[0090] DAR represents the average number of conjugated payload or drug molecules per antibody molecule, i.e., the average number of conjugated drug molecules. In antibody-drug conjugates, the number of conjugated payload molecules per antibody molecule is an important factor affecting its efficacy and safety. The production of antibody-drug conjugates is carried out by specifying reaction conditions, such as the amount of starting materials and reagents used in the reaction, to achieve a certain number of conjugated payload molecules. Usually, the preparation of antibody-drug conjugates produces mixtures containing various numbers of conjugated payload molecules. Unless otherwise specified, the number of conjugated payload or drug molecules per antibody molecule is defined as an average value, i.e., the average number of conjugated payload or drug molecules, in this disclosure.

[0091] In some embodiments, the antibody-drug conjugate has the following structure:

[0092] [ka]

[0093] [ka] (Ab represents an anti-HER3 antibody or antigen-binding fragment, and n is 1 to 10 or 4 to 10.) This includes any one of the following:

[0094] The antibody in the antibody-drug conjugate is a HER3 target-specific antibody or its antigen-binding fragment, which is linked to a linker after forming an active sulfhydryl through a disulfide bond. In some embodiments, the disulfide bond in the hinge region of the antibody forms an active sulfhydryl, which is then linked to a linker.

[0095] In some embodiments, the antibody in the antibody-drug conjugate is an anti-HER3 antibody or antigen-binding fragment thereof provided in the "Antibodies" section above of this disclosure. In some embodiments, the anti-HER3 antibody or antigen-binding fragment thereof in the antibody-drug conjugate comprises the VL sequence set forth in SEQ ID NO: 11 and the VH sequence set forth in SEQ ID NO: 15.

[0096] In the antibody-drug conjugates provided by the present disclosure, due to the use of an anti-HER3 antibody or antigen-binding fragment thereof and a linker with improved hydrophobicity, both the hydrophilicity and homogeneity of the prepared product are significantly improved, which is beneficial for improving the target cell killing effect, and improving or maintaining pharmaceutical properties such as biological activity and safety, for example, improving in vitro / in vivo stability and in vivo drug metabolism properties (longer half-life, fewer free small molecule toxins, etc.).

[0097] The antibody drugs provided by the present disclosure have excellent in vivo antibody-tumor effects, particularly HER3-dependent cell-killing or anti-tumor activity, which have varying degrees of tumor cell-killing effect depending on the tumor cell HER3 expression level. In some embodiments, the antibody-drug conjugates provided by the present disclosure have extremely high cell-killing activity against HER3-high expressing tumor cells, and the level of HER3 expression can be determined according to methods known in the art, such as H-Score scoring: low expression (H-Score = 10-99), medium expression (H-Score = 100-199), and high expression (H-Score = 200-300).

[0098] It should be noted that the antibody-drug conjugates of the present disclosure may absorb water, retain adsorbed water, or become hydrated when left in the atmosphere or recrystallized, and such hydrated compounds and salts are also included in the present disclosure. Furthermore, isotopically variant compounds labeled with various radioactive or non-radioactive isotopes are also included in the present disclosure. The antibody-drug conjugates of the present disclosure may contain one or more atoms with unnatural ratios of isotopes. Examples of atomic isotopes include deuterium (H), tritium (H), iodine-125 (I), and carbon-14 (C). Furthermore, compounds of the present disclosure may be radiolabeled with radioactive isotopes such as tritium (H), iodine-125 (I), or carbon-14 (C). Radiolabeled compounds are useful as therapeutic or preventative agents, research reagents such as laboratory reagents, and diagnostic agents such as in vivo imaging agents. All isotopic variations of the antibody-drug conjugates of the present disclosure, whether radioactive or not, are intended to be encompassed within the scope of the present disclosure.

[0099] [Method for preparing antibody-drug conjugates] The present disclosure provides a method for preparing an antibody-drug conjugate, comprising the steps of: reducing at least a portion of the interchain disulfide bonds of an antibody or antigen-binding fragment thereof by a reduction treatment; and reacting the reactive group of the linker in the linker-payload with the reactive group of the linker to form an antibody-drug conjugate of formula I. Ab-(LD)n (I) or an isomer, isotopic variant, pharmaceutically acceptable salt, prodrug, or solvate thereof, or a combination thereof. (wherein Ab is the anti-HER3 antibody or antigen-binding fragment thereof described above; L is a linker covalently linking Ab and D; D is the payload, (n is a number between 1 and 10) and obtaining:

[0100] In some embodiments, the carbon atom at the 3-position of the maleimido-N-yl in LD is reacted with a reduced antibody to covalently link the antibody to provide an ADC. In some aspects, the preparation method comprises the steps of: reducing at least a portion of the interchain disulfide bonds of the antibody by reduction treatment; and conjugating with a linker-payload, wherein the sulfhydryls after disulfide bond reduction are represented by Formula IV in the linker-payload.

[0101] [ka] Linker wherein the carbonyl in the ester group of the linker of formula IV is linked to an amino group in the payload of the antibody-drug conjugate; In Formula IV, R1 and R2 are independently selected from hydrogen, methyl, and isopropyl groups; R3 is -(CR5HCONH)n 1 -(CH2CONH)n 2 - or a single bond, R5 is selected from hydrogen and benzyl, n 1 represents an integer from 0 to 2, and n 2 represents an integer between 0 and 2, R4 is a methylamino group or -(NCH3COCH2)n 3 -NCH3COCH3, n 3 represents an integer between 1 and 20) It reacts with the carbon atom at the 3-position of the maleimide-N-yl group.

[0102] In many practical cases, in antibody-drug conjugates, a linker bearing a payload of the structure of Formula IV above is linked to the same antibody molecule bearing a reactive sulfhydryl. In some embodiments, the antibody is reacted with a reducing agent such as dithiothreitol (DTT), 2-mercaptoethanol, or tris(2-carboxyethyl)phosphine hydrochloride (TCEP) to form reactive sulfhydryls from disulfide bonds on the antibody chain. The amount of reducing agent can be 0.3 to 10 times the molar equivalent of the antibody, for example, 1 to 10, 3 to 10, 5 to 10, and 7 to 10 times the molar equivalent.

[0103] In some embodiments, the method further comprises reacting the antibody with a reducing agent in a buffer solution containing a chelator, followed by adding a linker-payload solution and carrying out the reaction. The linker-payload is specifically a compound formed by combining a linker of formula IV and a payload, wherein an amino group (primary amino group) in the payload is linked to a carbonyl group in the ester group of the linker of formula IV. The payload is selected from the payloads described in [Antibody-Drug Conjugates]. In some embodiments, the payload is exatecan. The term "chelator" refers to a compound capable of binding to a metal atom or ion through a coordinate bond and forming a complex having a cyclic structure.

[0104] In some embodiments, a reducing agent is reacted with an antibody in a buffer solution containing a chelating agent to yield an antibody in which interchain disulfide bonds have been partially or completely reduced. Chelating agents include, but are not limited to, ethylenediaminetetraacetic acid (EDTA) and diethylenetriaminepentaacetic acid (DTPA). The chelating agent is used at a concentration of 1 mM to 20 mM, e.g., 2 mM to 20 mM, 5 mM to 20 mM, 8 mM to 20 mM, 1 mM to 15 mM, or 1 mM to 10 mM. Components of the buffer solution may be buffer salts commonly used in the art, such as sodium phosphate, sodium borate, sodium acetate, or similar buffer salts.

[0105] The reaction of the antibody with the reducing agent is carried out under controlled pH conditions. In some embodiments, the antibody is reacted with the reducing agent at a pH of 5-9, optionally at a pH of 6-8, 6-7, 6.5-7.5, or 7-8, for example, at about pH 7. The pH of the solution may be adjusted using a chemical having acidic or basic properties, and exemplary chemicals having acidic or basic properties include acetic acid, hydrochloric acid, phosphoric acid, sulfuric acid, sodium bicarbonate, sodium carbonate, sodium hydroxide, and triethylamine.

[0106] The reaction of the antibody with the reducing agent is carried out at a controlled temperature, for example, -10 to 40°C, -5 to 40°C, 0 to 40°C, 5 to 40°C, 10 to 40°C, 15 to 40°C, 20 to 40°C, 25 to 40°C, 30 to 40°C, and 35 to 38°C, for example, about 37°C.

[0107] The linker-payload may be dissolved in an organic solvent selected from any one or combination of dimethyl sulfoxide (DMSO), dimethylformamide (DMF), dimethylacetamide (DMA), and N-methyl-2-pyrrolidone (NMP).

[0108] In some embodiments, the linker-payload solution is added to a buffer solution containing an antibody that has been subjected to a reduction treatment or has active sulfhydryls in an amount of 1-20% by volume based on the volume of the antibody buffer solution, hi some embodiments, the linker-payload solution is added at a volume ratio of 1-20%, 2-20%, 5-20%, 10-20%, 15-20%, 1-18%, 1-15%, 1-13%, 1-10%, or 5-15% based on the volume of the antibody buffer solution.

[0109] In some embodiments, the molar ratio of linker-payload to antibody is between 4 and 20, optionally between 8 and 20. In some embodiments, the molar ratio of linker-payload to antibody is between 10 and 20, 14 and 20, 16 and 20, or 18 and 20.

[0110] In some embodiments, the temperature at which the antibody and linker-payload are reacted is 0 to 37° C. In some embodiments, the reaction temperature is −10 to 40° C., −5 to 40° C., 0 to 40° C., 5 to 40° C., 5 to 37° C., 10 to 37° C., 10 to 25° C., or 15 to 30° C.

[0111] In some embodiments, the antibody is reacted with the linker-payload for 0.5 to 2 hours, hi some embodiments, the antibody is reacted with the linker-payload for 0.5 to 1.75 hours, 0.5 to 1.5 hours, 0.5 to 1.25 hours, 0.75 to 2 hours, or 1 to 2 hours.

[0112] The reaction can be terminated by inactivating the reactivity of unreacted linker-payload using a thiol-containing reagent. Examples of thiol-containing reagents include, but are not limited to, cysteine ​​or N-acetyl-(L)-cysteine ​​(NAC). More specifically, 1 to 2 molar equivalents of the thiol-containing reagent with the linker-payload are added to the reaction solution, and the reaction solution is incubated at room temperature (10 to 25°C) for 10 to 30 minutes to terminate the reaction.

[0113] In the case of an antibody having a sulfhydryl, an antibody-drug conjugate can also be obtained by reacting the compound using a known method (for example, it can be obtained by the method described in Patent Publication US2016 / 297890 (for example, it can be obtained by the method described in paragraphs

[0336] to

[0374] ). Antibodies having a sulfhydryl can be obtained by methods well known to those skilled in the art (Hermanson, GT, Bioconjugate Techniques, pp. 56-136, pp. 456-493, Academic Press (1996)).

[0114] In some embodiments, the antibody in the antibody-drug conjugate is selected from the anti-HER3 antibodies or antigen-binding fragments thereof disclosed under "Antibodies." In some embodiments, the anti-HER3 antibody or antigen-binding fragment thereof in the antibody-drug conjugate comprises the VL sequence set forth in SEQ ID NO: 11 and the VH sequence set forth in SEQ ID NO: 15.

[0115] The antibody-drug conjugates provided by the present disclosure can be obtained by the above-mentioned preparation methods. In some embodiments, the prepared antibody-drug conjugates are subjected to a purification process, including, but not limited to, gel filtration, for example, purification using a gel column.

[0116] In the antibody-drug conjugates prepared by the method disclosed herein, the linker and the payload exatecan are conjugated to the HER3-targeting antibody by a simple chemical method. Compared with traditional random conjugation methods, the anti-HER3 antibody-drug conjugates obtained by applying the linker have higher DAR values ​​(e.g., DAR 8).

[0117] [Pharmaceutical composition] The present disclosure provides a pharmaceutical composition comprising the above-mentioned anti-HER3 antibody or antigen-binding fragment thereof, or antibody-drug conjugate, or an isomer, isotopic variant, pharmaceutically acceptable salt, prodrug, or solvate thereof, or a combination thereof, and a pharmaceutically acceptable excipient.

[0118] An appropriate mode of administration can be selected for the pharmaceutical compositions of the present disclosure depending on the specific application form, physicochemical properties, etc. of the pharmaceutically acceptable excipients. In some embodiments, the pharmaceutical compositions may be formulated in the form of a lyophilized or liquid preparation, which may contain appropriate formulation additives known in the art. For example, the above-mentioned pharmaceutical compositions typically contain one or more pharmaceutical carriers, such as sterile liquids, such as water and oils (including those of petroleum, animal, vegetable, or synthetic origin (e.g., peanut oil, soybean oil, mineral oil, sesame oil, etc.)). For intravenous administration of the above-mentioned pharmaceutical compositions, water is a more typical carrier. In addition, aqueous solutions of saline, glucose, and glycerol can also be used as liquid carriers, particularly for injections. Suitable pharmaceutical excipients are known in the art. The above-mentioned compositions may further contain minor amounts of wetting agents, emulsifiers, or pH buffering agents, if desired. Pharmaceutical compositions are typically administered parenterally and may be administered by intradermal, intramuscular, intraperitoneal, intravenous, or subcutaneous injection, but are not limited thereto, and may be administered by, for example, infusion or bolus injection. In general, the active ingredient, e.g., an anti-HER3 antibody or antigen-binding fragment thereof, or an antibody-drug conjugate, can be prepared as a pharmaceutical composition using techniques and methods well known in the art (see, e.g., Ansel Introduction to Pharmaceutical Dosage Forms, 7th ed. 1999).

[0119] The pharmaceutical composition of the present disclosure may be a pharmaceutical composition containing only an anti-HER3 antibody or antigen-binding fragment thereof, or an antibody-drug conjugate of the present disclosure, or may be a pharmaceutical composition containing an anti-HER3 antibody or antigen-binding fragment thereof, or an antibody-drug conjugate, and at least one second therapeutic agent (e.g., a cancer therapeutic agent). In some embodiments, the antibody-drug conjugate of the present disclosure may also be administered together with other cancer therapeutic agents to enhance the anti-cancer effect. The other anti-cancer agents used for this purpose may be administered to an individual simultaneously with the antibody-drug conjugate, separately, or sequentially, or may be administered at various administration intervals. Exemplary other cancer therapeutic agents include paclitaxel, cisplatin, and vinblastine, but are not limited thereto, as long as they have anti-tumor activity.

[0120] [kit] The present disclosure also provides a kit comprising the above-described anti-HER3 antibody or antigen-binding fragment thereof, or antibody-drug conjugate. The kit may further comprise, if desired, a container, a buffer, and controls such as a positive control and a negative control. Those skilled in the art may make corresponding selections as desired. Therefore, the kit may further comprise instructions for use to facilitate the operation and use of the kit by those skilled in the art.

[0121] [use] The present disclosure provides the use of the above-described anti-HER3 antibodies or antigen-binding fragments thereof, antibody-drug conjugates, pharmaceutical compositions, antibody-drug conjugates prepared by the above-described methods, and kits in the preparation of therapeutic agents for diagnosing, preventing, and treating tumor diseases.

[0122] In some aspects, there is provided the use of a HER3 antibody or antigen-binding fragment thereof, an antibody-drug conjugate, a pharmaceutical composition, and an antibody-drug conjugate prepared by the above-described methods, and a kit in the preparation of an anti-tumor drug or in the preparation of a medicament that targets HER3.

[0123] The anti-HER3 antibodies or antigen-binding fragments thereof and antibody-drug conjugates provided by the present disclosure have the function of killing cells that express HER3, and can be used to kill cells that express HER3 in vivo or in vitro.

[0124] Such tumor diseases include benign tumors and malignant tumors (e.g., cancers), and the antibody-drug conjugates are particularly suitable for use in tumors or cancers in which HER3 expression is observed. In some embodiments, the tumor or cancer is a solid tumor that expresses HER3. In some embodiments, the tumor or cancer includes, but is not limited to, lung cancer, kidney cancer, urothelial carcinoma, colorectal cancer, prostate cancer, glioblastoma multiforme, ovarian cancer, pancreatic cancer, breast cancer, metastatic breast cancer, luminal breast cancer, melanoma, liver cancer, bladder cancer, gastric cancer (cancer of the stomach), gastrointestinal stromal tumor, cervical cancer, head and neck cancer, esophageal cancer, epidermoid carcinoma, peritoneal cancer, adult glioblastoma multiforme, liver cancer, hepatocellular carcinoma, colon cancer, rectal cancer, colorectal cancer, endometrial cancer, uterine cancer, salivary gland cancer, renal cancer, vulvar cancer, thyroid cancer, hepatocarcinoma, anal cancer, penile cancer, and lung adenocarcinoma (including EGFR-TKI-resistant lung adenocarcinoma).

[0125] In some embodiments, the cancer classes are breast cancer, lung adenocarcinoma, pancreatic cancer, and colon cancer. In some embodiments, the cancer classes are breast cancer, EGFR-TKI-resistant lung adenocarcinoma, pancreatic cancer, and colon cancer.

[0126] When a subject has or is suspected of having a solid tumor that expresses HER3, a therapeutic agent comprising a therapeutic dose of the above-described anti-HER3 antibody or antigen-binding fragment thereof, antibody-drug conjugate, antibody-drug conjugate prepared by the above-described method, and the above-described pharmaceutical composition is administered to the subject, which can be used to diagnose, prevent, and treat tumor diseases.

[0127] The present disclosure also provides methods for diagnosing, preventing, and treating tumor diseases, comprising administering to a subject in need thereof a therapeutic dose of a therapeutic agent comprising the above-described anti-HER3 antibody or antigen-binding fragment thereof, antibody-drug conjugate, antibody-drug conjugate prepared by the above-described method, and the above-described pharmaceutical composition or kit.

[0128] The subject is a mammal, including humans, non-human primates, dogs, pigs, and mice; preferably, the subject is a human, eg, a patient with a HER3-expressing tumor. The therapeutic dosage of a therapeutic agent will vary depending on factors such as the particular condition being treated, the severity of the condition, individual patient parameters (including age, physical condition, size, sex, and weight), the duration of treatment, the nature of concurrent therapy (if any), the particular route of administration, and the knowledge of the medical professional. In some embodiments, the dosage of an active ingredient, such as an anti-HER3 antibody or antigen-binding fragment thereof, or an antibody-drug conjugate, can be empirically determined in an individual who has received one or more administrations of the antibody.

[0129] In some embodiments, the therapeutically acceptable dose of the anti-HER3 antibody or antigen-binding fragment thereof, or antibody-drug conjugate is 0.1 to 30 mg / kg, 0.5 to 30 mg / kg, 1 to 30 mg / kg, 1 to 25 mg / kg, 0.1 to 25 mg / kg, 0.1 to 20 mg / kg, 1 to 20 mg / kg, or 0.5 to 20 mg / kg. In some embodiments, the frequency of administration is every 12 hours, daily, weekly, every two weeks, every four weeks, every five weeks, every six weeks, every seven weeks, every eight weeks, every nine weeks, or every ten weeks, or once monthly, every two months, or every three months or more. The therapeutic dose and frequency of administration can vary depending on the treatment regimen.

[0130] The anti-HER3 antibodies or antigen-binding fragments thereof, antibody-drug conjugates, or pharmaceutical compositions of the present disclosure may also be used in combination with one or more second therapeutic agents, which, when combined, can be administered simultaneously or sequentially by the same or different routes, which can vary depending on the metabolic properties of the therapeutic agents themselves and the disease being treated.

[0131] The various aspects and preferences of the present disclosure may be combined with one another (provided they are not essentially contradictory to one another), and the various aspects formed thereby are considered to be part of the present disclosure.

[0132] The technical solutions of the present disclosure are more clearly and explicitly illustrated in the following examples. It should be understood that the examples are for illustrative purposes only and are not intended to limit the scope of the present disclosure in any way. The scope of the present disclosure is limited only by the claims. [Example]

[0133] The present disclosure is specifically illustrated by the following examples, but the present disclosure is not limited thereto. Furthermore, the examples should not be construed as limiting in any way. Furthermore, reagents, solvents, and starting materials not specifically described herein can be easily obtained from commercially available sources.

[0134] Example 1: Preparation of compound LP-1

[0135] [ka] Step 1: Synthesis of intermediate 11-1 A mixture of dichloromethane (DCM) and methanol (MeOH) (v:v = 2:1, 90 mL) and 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline (EEDQ, 1.86 g, 7.55 mmol) was added to a mixture of compound 11-1A (Mc-Val-Ala-OH, purchased from Shanghai Haoyuan Chemexpress Co., Ltd., 2.4 g, 6.29 mmol) and 11-1B (3.18 g, 6.29 mmol) at room temperature (25 °C-30 °C). The mixture was reacted at room temperature for 24 h, and the solvent was removed under vacuum. The crude residue was purified by flash chromatography to give compound 11-1 (3.9 g, 71%). LC-MS (ESI, m / z): 868.49 (M+H).

[0136] Step 2: Synthesis of intermediate 11-2 Compound 11-1 (2 g, 2.3 mmol) was dissolved in anhydrous tetrahydrofuran (THF, 50 mL). Hydrogen fluoride-pyridine (4.6 g, 46 mmol) was added under argon atmosphere at 0 ° C. The mixture was stirred for 2 hours, and water was added to quench the reaction. The mixture was extracted with DCM, and the organic phase was dried and concentrated. The residue was purified by silica gel chromatography to give compound 11-2 (1.1 g, 76%). LC-MS (ESI, m / z): 630.31 (M+H).

[0137] Step 3: Synthesis of intermediate 11-3 Compound 11-2 (700 mg, 1.11 mmol) was dissolved in anhydrous N,N-dimethylformamide (DMF, 4 mL), and N,N-diisopropylethylamine (DIPEA, 0.39 mL, 2.23 mmol) and 4,4'-dinitrodiphenyl carbonate (406 mg, 1.33 mmol) were added at room temperature under an argon atmosphere. The reaction mixture was stirred overnight at ambient temperature. The mixture was concentrated to remove the solvent, and methyl tert-butyl ether (MTBE) was added to precipitate the product. The mixture was filtered to collect the yellow solid, which was washed with diethyl ether and dried to give compound 11-3. LC-MS (ESI, m / z): 795.41 (M+H).

[0138] Step 4: Synthesis of intermediate 11-4 Compound 11-3 (300 mg, 0.44 mmol) was dissolved in 4 mL of anhydrous DMF, and 1 mL of dry pyridine was added, followed by the addition of exatecan mesylate (purchased from Shanghai Haoyuan Chemexpress Co., Ltd., 234 mg, 0.44 mmol) and 1-hydroxybenzotriazole (HOBt, 60 mg, 0.44 mmol). The reaction mixture was stirred overnight at room temperature under an argon atmosphere. The product was purified by preparative high-performance liquid chromatography (pre-HPLC) to give intermediate 11-4 (230 mg, 48%). LC-MS (ESI, m / z): 1091.53 (M+H).

[0139] Step 5: Synthesis of intermediate 11-5 Compound 11-4 (200 mg, 0.183 mmol) was dissolved in 1 mL of anhydrous DCM, and 300 μL of TFA was added at 0 ° C. The mixture was stirred at room temperature for 30 minutes and concentrated to remove the solvent, giving the TFA salt of intermediate 11-5. LC-MS (ESI, m / z): 991.47 (M+H).

[0140] Step 6: Synthesis of compound LP-1 Compound 11-5 (120 mg, 0.109 mmol) was dissolved in 1 mL of anhydrous DMF, and N-acetyldecasarcosine (Ac-Sar10-COOH, 84 mg, 0.109 mmol) was added, followed by 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU, 50 mg, 0.130 mmol) and DIPEA (38 μL, 0.22 mmol). The reaction mixture was stirred overnight at room temperature and concentrated to remove the solvent. The crude product was purified by pre-HPLC to give compound LP-1 (74 mg, 38%). LC-MS (ESI, m / z): 1743.85 (M+H).

[0141] Example 2: Preparation of compound LP-2

[0142] [ka] Compound LP2 was synthesized according to the procedure for compound LP-1, where starting material 11-1A was replaced with Mc-GGFG-OH (purchased from Shanghai Haoyuan Chemexpress Co. Ltd., where GGFG represents the amino acid sequence consisting of glycine-glycine-phenylalanine-glycine linked by a peptide bond), to obtain compound LP-2 as a beige amorphous solid. LC-MS (ESI, m / z): 1891.90 (M+H).

[0143] Example 3: Preparation of compound LP-3

[0144] [ka] Compound LP-3 is an intermediate of LP-1. Step 6 was removed, and intermediate 11-5 was LP-3.

[0145] Example 4: Preparation of monoclonal antibodies Balb / c mice (8-12 weeks old) were immunized with a human HER3 protein extracellular domain fragment antigen (NCBI reference sequence: NM_001982.4, amino acid sequence: positions 20-643), and their serum titers were monitored to determine the number of immunizations. After the first immunization, three to four booster immunizations were administered. Mouse sera were collected, and their titers were determined according to routine methods in the art. After a single central immunization of mice qualified by titer detection, the entire spleen and half of the lymph nodes were harvested and PEG-fused with the myeloma SP2 / 0 cell line. The fused cells were plated and cultured, and supernatants were collected from all wells and the antigen was screened by ELISA. Positive wells containing cells identified microscopically were transferred to a 96-well plate for further culture. After 7 days of growth, supernatants were collected from all wells and tested for reactivity with the antigen using ELISA. Positive wells were further examined with various dilutions of antigen binding for affinity ranking. Twenty parent clones with the highest immunogenic affinity were selected for subcloning. Subcloning was performed by limiting dilution and ELISA screening to obtain hybridoma cell lines. The hybridoma cell line was used to prepare mouse ascites. After collection and purification, a monoclonal antibody named Mu4O3 was obtained, and the amino acid sequences of its CDRs and variable regions were determined as shown in Table 1 below, where the CDRs (Kabat system definition and numbering) are underlined.

[0146] [Table 1] The hybridoma sequences were subjected to humanization modification to obtain humanized 4O3 antibodies (abbreviated as "Hu4O3") L1H4, L1H5, L2H4, L2H5, L4H4, L4H5, and L4H7. L1, L2, and L4 represent the numbers of the antibody light chain variable region sequences, and H4, H5, and H7 represent the numbers of the antibody heavy chain variable region sequences; that is, the L1H4 antibody contains a light chain variable region numbered L1 and a heavy chain variable region numbered H4, the L1H5 antibody contains a light chain variable region numbered L1 and a heavy chain variable region numbered H5, and the L2H4 antibody contains a light chain variable region numbered L2 and a heavy chain variable region numbered H4. The L2H5 antibody comprises a light chain variable region numbered L2 and a heavy chain variable region numbered H5, the L4H4 antibody comprises a light chain variable region numbered L4 and a heavy chain variable region numbered H4, the L4H5 antibody comprises a light chain variable region numbered L4 and a heavy chain variable region numbered H5, and the L4H7 antibody comprises a light chain variable region numbered L4 and a heavy chain variable region numbered H7.

[0147] The CDR sequences (underlined) and variable region sequences of Hu4O3 are shown below in Table 2, where mutated amino acids in CDR2 of VH are shown in bold italics. CDRs are defined and numbered using the Kabat system.

[0148] [Table 2] For exemplary preparation and / or detection purposes, the constant regions of the above antibodies are selected from the constant regions of human IgG1, wherein the heavy chain constant region sequence is selected from the amino acid sequence set forth in SEQ ID NO: 17 and the light chain constant region sequence is selected from the amino acid sequence set forth in SEQ ID NO: 18.

[0149] Example 5: Methods for detecting antibody-drug conjugates The antibody-drug conjugates were identified by concentration, medium exchange, purification, measurement of antibody concentration, and calculation of the average number of drug molecules carried by each antibody according to the methods described below.

[0150] Procedure A: Concentration of antibodies or antibody-drug conjugates The solution of antibody or antibody-drug conjugate to be concentrated was added to an ultrafiltration tube (Amicon Ultra, 50000 MWCO, Millipore), centrifuged to the desired volume, and removed.

[0151] Procedure B: Measurement of antibody concentration The absorbance of the antibody was measured using a microplate reader (Multiskan GO, Thermo Fisher) according to the method specified by the manufacturer, and the ratio of the absorbance of the antibody at that wavelength to the extinction coefficient was the concentration of the antibody.

[0152] Procedure C: Antibody medium exchange Zeba desalting columns (5 mL, 40K MWCO) were pre-equilibrated with phosphate buffer containing sodium chloride (50 mM) and EDTA (2 mM) (abbreviated as "PBS 7.0 / EDTA", 50 mM, pH 7.0) according to the instructions provided by the manufacturer (Thermo Fisher). 2 mL of sample was loaded onto each zeba desalting column and centrifuged (1000 g, 4 min). The flow-through fraction was collected and concentrated by procedure A. The antibody concentration was determined in procedure B and adjusted with PBS 7.0 / EDTA.

[0153] Procedure D: Purification of antibody-drug conjugates A Zeba desalting column (5 mL, 40K MWCO) was pre-equilibrated with storage buffer according to the manufacturer's instructions. Histidine-acetate buffer (20 mM histidine, pH 5.5) containing 150 mM sodium chloride or phosphate buffer (50 mM, pH 7.0) containing 50 mM sodium chloride was used as the storage buffer. The reaction solution (approximately 2 mL) containing the antibody-drug conjugate was added to the Zeba desalting column and centrifuged (1000 g, 4 min). The flow-through fraction (approximately 2 mL) was collected. The elution process was repeated twice to remove low molecular weight compounds, including unbound linker-payload and reducing agent.

[0154] Procedure E: Determination of antibody concentration and average number of drug molecules linked per antibody (DAR value) in antibody-drug conjugates The concentration of the conjugated drug in the antibody-drug conjugate can be obtained by measuring the ultraviolet absorbance at 280 nm and 370 nm of an aqueous solution of the antibody-drug conjugate and calculating using the following formula:

[0155] At any given wavelength, the total absorbance of a system is equal to the sum of the absorbances of all light-absorbing chemicals present in the system (additivity of absorbance). Therefore, assuming that the molar extinction coefficients of the antibody and drug do not change before and after the antibody and drug are conjugated, the antibody and drug concentrations in the antibody-drug conjugate can be expressed as follows:

[0156] A 280 =A D,280 +A A,280 =ε D,280 C D +ε A,280 C A Formula (1) A 370 =A D,370 +A A,370 =ε D,370 C D +ε A,370 C A Formula (2) A 280 represents the total absorbance at 280 nm of the aqueous antibody-drug conjugate solution, and A 370 represents the total absorbance at 370 nm of an aqueous solution of the antibody-drug conjugate. A,280 represents the absorbance of the antibody at 280 nm, and A A,370 represents the absorbance of the antibody at 370 nm, and A D,280 represents the absorbance of the drug molecule at 280 nm, and A D,370 represents the absorbance of the drug molecule at 370 nm, and ε A,280 represents the molar extinction coefficient of the antibody at 280 nm, and ε A,370 represents the molar extinction coefficient of the antibody at 370 nm, and εD,280 represents the molar extinction coefficient of the drug molecule at 280 nm, and ε D,370 represents the molar extinction coefficient of the drug molecule at 370 nm, and C A is the concentration of antibody in the antibody-drug conjugate, C D represents the concentration of drug molecules in the antibody-drug conjugate.

[0157] ε A,280 , ε A,370 , ε D,280 , and ε D,370 is a known value (calculated from the sequence of the antibody or measured by UV absorption of the compound). For example, ε A,280 can be calculated from the amino acid sequence of the antibody by known methods (Protein Science, 1995, Vol. 4, pp. 2411-2423). Antibodies usually do not absorb at 370 nm, and therefore, ε A,370 is usually 0. D,280 and εD,370 The value of C can be calculated by measuring the change in absorbance of the drug molecule at 280 nm and 370 nm, respectively, using the Lambert-Beer law (absorbance = molar concentration × molar extinction coefficient × path length). A and C D is the absorbance A of the antibody-drug conjugate at 280 nm and 370 nm 280 and A 370 and then solving the simultaneous linear equations of two variables in equations (1) and (2). Furthermore, the number of drug molecules linked to each antibody (DAR value) can be obtained by measuring C D C A can be obtained by dividing by

[0158] Procedure F: Measurement of Aggregation in Antibody-Drug Conjugates Aggregates in antibody-drug conjugates were detected using high performance liquid chromatography size exclusion chromatography as follows: High-performance liquid chromatography system: Agilent 1260 Infinity II HPLC system Detector: UV absorption spectrometer (detection wavelength: 280 nm) Chromatography column model: TOSOH TSKgel G3000SWXL (7.8 x 300 mm, 5 μm) Mobile phase: 200 mmol / L KHPO4, 150 mmol / L NaCl, 15% (v / v) isopropanol, pH 7.0 Flow rate: 0.75mL / min Analysis time: 18 minutes Column temperature: room temperature Sample injection amount: 50 μg Data Analysis: The size-exclusion chromatogram of the quality control (QC, L1H5 naked antibody, i.e., the antibody not conjugated with a linker-payload and numbered L1H5) is shown in Figure 1A. The retention time of the major peak (single peak) of the 150 kDa quality control is 9.5-10.5 min. The retention time of the aggregates should be earlier than that of the monomers.

[0159] Procedure G: Measuring the hydrophobicity of antibody-drug conjugates The hydrophobicity of the antibody-drug conjugates was analyzed using high performance liquid chromatography hydrophobic interaction chromatography (HIC) as follows: High-performance liquid chromatography system: Agilent 1260 Infinity II HPLC system Detector: UV absorption spectrometer (detection wavelength: 280 nm) Chromatography column model: TOSOH TSKgel Butyl-NPR (inner diameter 4.6 mm x 3.5 cm, 2.5 μm) Mobile phase A: (NH4)2SO4, 50 mmol / L KHPO4, pH 7.0 Mobile phase B: 50 mmol / L KHPO4, 25% (v / v) isopropanol, pH 7.0 Analysis time: 25 minutes Column temperature: room temperature Elution procedure (B%): 0% to 25% (0 to 1 min), 25% (1 to 3 min), 25% to 80% (3 to 13 min), 80% (13 to 17 min), 80% to 0% (17 to 17.10 min), 0% (17.10 to 25 min) Sample injection volume: 10 μL Data Analysis: The hydrophobicity chromatograms of the quality control (QC, L1H5 naked antibody) are shown in Figure 1B and Figure 22, and the hydrophobicity chromatogram of the quality control reference antibody is shown in Figure 22. Generally, samples with shorter retention times are less hydrophobic, and antibody-drug conjugates are more hydrophobic than naked antibodies without conjugated drug molecules and should therefore have longer retention times.

[0160] Example 6: Preparation of antibody-drug conjugate L1H5-LP3 Antibody reduction: Antibody L1H5 prepared in Example 4 was reduced to 1.49 mL mg as the extinction coefficient of the antibody at 280 nm using procedure B of Example 5. -1 cm -1 The medium was then exchanged using PBS 7.0 / EDTA according to procedure C, and the antibody concentration after the medium exchange was 20.76 mg / mL. To 481.70 μL of the L1H5 antibody aqueous solution, 93.33 μL of 5 mM TCEP solution (equivalent to 7 times the antibody content) was added, followed by 200 μL of 50 mM phosphate buffer (pH 7.0, PBS 7.0) and 224.97 μL of ultrapure water. The mixture was incubated at 37°C for 2 hours.

[0161] Conjugation of antibody to linker-payload: The above mixture was incubated for 10 minutes at 4° C. The linker-payload LP3 prepared in Example 3 was dissolved in N,N-dimethylacetamide (DMA) and added to the mixture in a volume of 93.33 μL (equivalent to 14 times the antibody content), and the mixture was allowed to react for another 30 minutes at 22° C.

[0162] Purification of antibody-drug conjugate: The above reaction solution was purified by the method of operation D in Example 5 to obtain antibody-drug conjugate L1H5-LP3. Characterization of antibody-drug conjugates: The resulting antibody-drug conjugates were analyzed by procedure E(ε) of Example 5. D,280 =5186 and ε D,370 =13688), procedure F, and procedure G.

[0163] The concentration of the antibody-drug conjugate was calculated to be 5.73 mg / mL by procedure E, and the average number of conjugated payloads per antibody was calculated to be 7.78 by procedure E. Figure 2A shows the aggregate detection profile, with an aggregate content of 2.20% for antibody-drug conjugate L1H5-LP3 as measured by procedure F. Figure 2B shows the hydrophobic chromatography detection profile of antibody-drug conjugate L1H5-LP3, with a retention time of 6.169 minutes for the antibody-drug conjugate as measured by procedure G.

[0164] Example 7: Preparation of antibody-drug conjugate L1H5-LP1 Antibody reduction: The antibody was reduced to 1.49 mL mg as in procedure B of Example 5 (the extinction coefficient of the antibody at 280 nm). -1 cm -1 The cells were then subjected to a medium exchange using PBS 7.0 / EDTA according to Procedure C, and the antibody concentration after the medium exchange was 11.44 mg / mL. 160 μL of a 5 mM TCEP aqueous solution (equivalent to 10 times the antibody content) was added to 1048.95 μL of the aqueous solution of L1H5 antibody prepared in Example 4, and 320 μL of 50 mM PBS 7.0 and 71.05 μL of ultrapure water were added thereto simultaneously. The mixture was reacted at 37°C for 2 hours.

[0165] Conjugation of antibody to linker-payload: The above mixture was incubated for 10 minutes at 4° C. The linker-payload LP1 prepared in Example 1 was dissolved in DMA and added to the mixture in a volume of 120 μL (equivalent to 15 times the antibody content), and the mixture was reacted for another 30 minutes at 22° C.

[0166] Purification of antibody-drug conjugate: The above reaction solution was purified by the method of operation D in Example 5 to obtain antibody-drug conjugate L1H5-LP1. Characterization of antibody-drug conjugates: The resulting antibody-drug conjugates were analyzed by procedure E(ε) of Example 5. D,280 = 6384 and ε D,370 =16180), procedure F, and procedure G.

[0167] The concentration of the antibody-drug conjugate was calculated to be 6.12 mg / mL by procedure E, and the average number of conjugated payloads per antibody was calculated to be 8.03 by procedure E. Figure 3A shows the aggregate detection profile, with an aggregate content of 1.60% for antibody-drug conjugate L1H5-LP1 as measured by procedure F. Figure 3B shows the hydrophobic chromatography detection profile of antibody-drug conjugate L1H5-LP1, with a retention time of 6.234 minutes for antibody-drug conjugate L1H5-LP1 as measured by procedure G.

[0168] Drug conjugates of other antibodies, such as L1H4 and L4H4, to LP1 were prepared as described in Example 7. Example 8: Preparation of antibody-drug conjugate L1H5-LP2 Antibody reduction: The antibody was reduced to 1.49 mL mg as in procedure B of Example 5 (the extinction coefficient of the antibody at 280 nm). -1 cm -1 The cells were then subjected to a medium exchange using PBS 7.0 / EDTA according to Procedure C, and the antibody concentration after the medium exchange was 11.44 mg / mL. 160 μL of a 5 mM TCEP aqueous solution (equivalent to 10 times the antibody content) was added to 1048.95 μL of the aqueous solution of L1H5 antibody prepared in Example 4, and 320 μL of 50 mM PBS 7.0 and 71.05 μL of ultrapure water were added thereto simultaneously. The mixture was reacted at 37°C for 2 hours.

[0169] Conjugation of antibody to linker-payload: The above mixture was incubated for 10 minutes at 4° C. The linker-payload LP2 prepared in Example 2 was dissolved in DMA and added to the mixture in a volume of 120 μL (equivalent to 15 times the antibody content), and the mixture was reacted for another 30 minutes at 22° C.

[0170] Purification of antibody-drug conjugate: The above reaction solution was purified by the method of operation D in Example 5 to obtain antibody-drug conjugate L1H5-LP2. Characterization of antibody-drug conjugates: The resulting antibody-drug conjugates were analyzed by procedure E(ε) of Example 5. D,280 =5814 and ε D,370 =14742), procedure F, and procedure G.

[0171] The concentration of the antibody-drug conjugate was calculated to be 6.28 mg / mL by procedure E, and the average number of conjugated payloads per antibody was calculated to be 8.89 by procedure E. Figure 4A shows the aggregate detection profile, with an aggregate content of 1.89% for antibody-drug conjugate L1H5-LP2 as measured by procedure F. Figure 4B shows the hydrophobic chromatography detection profile of antibody-drug conjugate L1H5-LP2, with a retention time of 6.599 minutes for antibody-drug conjugate L1H5-LP2 as measured by procedure G.

[0172] Comparative Example 1: Preparation of antibody-drug conjugate reference ADC Antibody reduction: The antibody was reduced to 1.77 mL mg as in procedure B of Example 5 (the extinction coefficient of the antibody at 280 nm). -1 cm -1The cells were then subjected to a medium exchange using PBS 7.0 / EDTA according to procedure C, and the antibody concentration after the medium exchange was 9.44 mg / mL. To 0.037 mL of an aqueous solution of a reference antibody (see SEQ ID NOS: 69-72, U1-59 in Patent US20190151328A1 for the antibody sequence), 3.27 μL of 5 mM TCEP solution (equivalent to 7 times the antibody content) was added, and 14 μL of 50 mM PBS 7.0 and 15.66 μL of deionized water were simultaneously added. The mixture was allowed to react at 37°C for 2 hours.

[0173] Conjugation of antibody to linker-payload: The above mixture was incubated for 10 minutes at 4° C. Linker-payload GGFG-DXd (purchased from DC Chemicals, DC50025) was dissolved in DMA and added to the mixture in a volume of 3.27 μL (equivalent to 14 times the antibody content), and the mixture was allowed to react for another 30 minutes at 22° C.

[0174] Purification of antibody-drug conjugate: The above reaction solution was purified by the method of operation D in Example 5 to obtain the antibody-drug conjugate reference ADC. Characterization of antibody-drug conjugates: The resulting antibody-drug conjugates were analyzed by procedure E(ε) of Example 5. D,280 =5178 and ε D,370 =20217), procedure F, and procedure G were used to characterize the samples.

[0175] The antibody-drug conjugate concentration was calculated to be 3.10 mg / mL as measured by Procedure B, and the average number of conjugated payloads per antibody was calculated to be 6.63 as measured by Procedure E. Figure 23A shows the aggregate detection profile, with an aggregate content of 2.04% for the antibody-drug conjugate reference ADC as measured by Procedure F. Figure 23B shows the hydrophobic chromatography detection profile of the antibody-drug conjugate reference ADC, with a retention time of 8.215 minutes for the antibody-drug conjugate reference Antibody-DXd as measured by Procedure G.

[0176] Figure 22 also shows a comparison of hydrophobicity measurements for the L1H5 antibody prepared in Example 4, the antibody-drug conjugate L1H5-LP3 prepared in Example 6, the reference antibody, and the antibody-drug conjugate reference ADC, and Table 3 shows the retention time for each test sample. The results show that the L1H5 antibody and L1H5-LP3 exhibited shorter retention times than the positive control reference antibody and the drug-antibody conjugate reference ADC, indicating that both the reference antibody and the drug-antibody conjugate reference ADC are more hydrophobic than the L1H5 antibody and the antibody-drug conjugate L1H5-LP3. The L1H5 antibody and L1H5-LP3 are predicted to have relatively high in vivo stability due to their weak hydrophobicity.

[0177] [Table 3] Comparative Example 2: Preparation of antibody-drug conjugate Reference Antibody-LP3 Antibody reduction: The antibody was reduced to 1.77 mL mg as in procedure B of Example 5 (the extinction coefficient of the antibody at 280 nm). -1 cm -1 The cells were then subjected to a medium exchange using PBS 7.0 / EDTA according to procedure C, and the antibody concentration after the medium exchange was 15.5 mg / mL. 268.80 μL of 5 mM TCEP solution (equivalent to 7 times the antibody content) was added to 1.858 mL of an aqueous solution of a reference antibody (the sequence of which was the same as in Comparative Example 1), and 360 μL of 100 mM PBS 7.0 and 1113.14 μL of deionized water were added thereto simultaneously. The mixture was reacted at 37°C for 2 hours.

[0178] Conjugation of antibody to linker-payload: The above mixture was incubated for 10 minutes at 4° C. Linker-payload LP3 was dissolved in DMA and added to the mixture in a volume of 268.80 μL (equivalent to 14 times the antibody content), and the mixture was allowed to react for another 30 minutes at 22° C.

[0179] Purification of antibody-drug conjugate: The above reaction solution was purified by the method of operation D in Example 5 to obtain antibody-drug conjugate Reference antibody-LP3. Characterization of antibody-drug conjugates: The resulting antibody-drug conjugates were analyzed by procedure E(ε) of Example 5. D,280 =5186 and ε D,370 =13688), procedure F, and procedure G.

[0180] The concentration of the antibody-drug conjugate was calculated to be 8.45 mg / mL by procedure E, and the average number of conjugated payloads per antibody was calculated to be 7.60 by procedure E. Figure 24A shows the aggregate detection profile, with an aggregate content of 1.92% for the antibody-drug conjugate Reference Antibody-LP3 as measured by procedure F. Figure 24B shows the hydrophobic chromatography detection profile of the antibody-drug conjugate Reference Antibody-LP3, with a retention time of 6.262 minutes for the antibody-drug conjugate Reference Antibody-MCE8 as measured by procedure G.

[0181] Comparative Example 3: Preparation of antibody-drug conjugate L1H5-DXd Antibody reduction: The antibody was reduced to 1.49 mL mg as in procedure B of Example 5 (the extinction coefficient of the antibody at 280 nm). -1 cm -1 The medium was then exchanged using PBS 7.0 / EDTA according to procedure C, and the antibody concentration after the medium exchange was 11.44 mg / mL. To 0.59 mL of the aqueous solution of L1H5 antibody, 90 μL of 5 mM TCEP solution (equivalent to 10 times the antibody content) was added, and 180 μL of 50 mM PBS 7.0 and 39.97 μL of deionized water were simultaneously added thereto. The mixture was reacted at 37°C for 2 hours.

[0182] Conjugation of antibody to linker-payload: The above mixture was incubated for 10 minutes at 4° C. Linker-payload GGFG-DXd (purchased from DC Chemicals, DC50025) was dissolved in DMA and added to the mixture in a volume of 67.50 μL (equivalent to 15 times the antibody content), and the mixture was allowed to react for another 30 minutes at 22° C.

[0183] Purification of antibody-drug conjugate: The above reaction solution was purified by the method of operation D in Example 5 to obtain antibody-drug conjugate L1H5-DXd. Characterization of antibody-drug conjugates: The resulting antibody-drug conjugates were analyzed by procedure E(ε) of Example 5. D,280 =5178 and ε D,370 =20217), procedure F, and procedure G were used to characterize the samples.

[0184] The concentration of the antibody-drug conjugate was calculated to be 5.17 mg / mL by procedure E, and the average number of conjugated payloads per antibody was calculated to be 6.54 by procedure E. Figure 25A shows the aggregate detection profile, with an aggregate content of 2.02% for antibody-drug conjugate L1H5-DXd as measured by procedure F. Figure 25B shows the hydrophobic chromatography detection profile of antibody-drug conjugate L1H5-DXd, with a retention time of 7.807 minutes for antibody-drug conjugate L1H5-DXd as measured by procedure G.

[0185] Comparative Example 4: Preparation of antibody-drug conjugate Dxd isotype control ADC Antibody reduction: The antibody was reduced to 1.35 mL mg as per procedure B of Example 5 (the extinction coefficient of the antibody at 280 nm). -1 cm -1The cells were then subjected to a medium exchange using PBS 7.0 / EDTA according to procedure C, with the antibody concentration after the medium exchange being 10 mg / mL. To 2.45 mL of an aqueous solution of isotype control antibody human IgG (purchased from Beijing Solarbio Science & Technology Co., Ltd., product model P001), 326.67 μL of 5 mM TCEP solution (equivalent to 10 times the antibody content) was added, and 700 μL of 50 mM PBS 7.0 and 23.33 μL of deionized water were simultaneously added. The mixture was allowed to react at 37°C for 2 hours.

[0186] Conjugation of antibody to linker-payload: The above mixture was incubated for 10 minutes at 4° C. The linker-payload GGFG-DXd was dissolved in DMA and added to the mixture in a volume of 294 μL (equivalent to 18 times the antibody content), and the mixture was allowed to react for another 30 minutes at 22° C.

[0187] Purification of antibody-drug conjugate: The above reaction solution was purified by the method of procedure D in Example 5 to obtain antibody-drug conjugate Dxd isotype control ADC. Characterization of antibody-drug conjugates: The resulting antibody-drug conjugates were analyzed by procedure E(ε) of Example 5. D,280 =5178 and ε D,370 =20217), procedure F, and procedure G were used to characterize the samples.

[0188] The concentration of the antibody-drug conjugate was calculated to be 8.20 mg / mL as measured by Procedure E, and the average number of conjugated payloads per antibody was calculated to be 7.08 as measured by Procedure E. Figure 26A shows the aggregate detection profile, with the aggregate content being 4.25% for the antibody-drug conjugate Dxd isotype control ADC as measured by Procedure F. Figure 26B shows the hydrophobic chromatography detection profile of the antibody-drug conjugate Dxd isotype control ADC, with the retention time being 8.222 minutes for the antibody-drug conjugate Dxd isotype control ADC as measured by Procedure G.

[0189] Comparative Example 5: Preparation of antibody-drug conjugate LP3 isotype control ADC Antibody reduction: The antibody was reduced to 1.35 mL mg as per procedure B of Example 5 (the extinction coefficient of the antibody at 280 nm). -1 cm -1 The cells were then subjected to a medium exchange using PBS 7.0 / EDTA according to procedure C, with the antibody concentration after the medium exchange being 10 mg / mL. To 4 mL of an aqueous solution of isotype control antibody human IgG, 672 μL of 5 mM TCEP solution (equivalent to 7 times the antibody content) was added, and 1440 μL of 50 mM PBS 7.0 and 1088 μL of deionized water were simultaneously added thereto. The mixture was reacted at 37°C for 2 hours.

[0190] Conjugation of antibody to linker-payload: The above mixture was incubated for 10 minutes at 4° C. Linker-payload LP3 was dissolved in DMA and added to the mixture in a volume of 672 μL (equivalent to 14 times the antibody content), and the mixture was allowed to react for an additional 30 minutes at 22° C.

[0191] Purification of antibody-drug conjugate: The above reaction solution was purified by the method of procedure D in Example 5 to obtain antibody-drug conjugate LP3 isotype control ADC. Characterization of antibody-drug conjugates: The resulting antibody-drug conjugates were analyzed by procedure E(ε) of Example 5. D,280 =5186 and ε D,370 =13688), procedure F, and procedure G.

[0192] The concentration of the antibody-drug conjugate was calculated to be 7.42 mg / mL as measured by Procedure E, and the average number of conjugated payloads per antibody was calculated to be 7.28 as measured by Procedure E. Figure 27A shows the aggregate detection profile, with the aggregate content being 5.37% for the antibody-drug conjugate LP3 isotype control ADC as measured by Procedure F. Figure 27B shows the hydrophobic chromatography detection profile of the antibody-drug conjugate LP3 isotype control ADC, with the retention time being 6.450 minutes for the antibody-drug conjugate LP3 isotype control ADC as measured by Procedure G.

[0193] Assay Example 1: Flow cytometry of cells expressing Mu4O3 antibody and varying amounts of HER3 Four cell lines, MCF-7 (human breast cancer cells, purchased from the National Collection of Authenticated Cell Cultures of the Chinese Academy of Sciences), SW620 (human colon cancer cells, purchased from Zhejiang Meisen Cell Technology Co., Ltd.), SK-BR-3 (human breast cancer cells, purchased from Shanghai Xunqing Biotechnology Co., Ltd.), and MDA-MB-231 (human breast cancer cells, purchased from Saibaikang (Shanghai) Biotechnology Co., Ltd.), were separately digested with trypsin in culture dishes. The digested cells were centrifuged at 1000 rpm for 5 minutes and then blocked with 10% goat serum (purchased from Beijing Solarbio Science & Technology Co., Ltd., catalog number SL038) for 30 minutes. After blocking, the Mu4O3 antibody prepared in Example 4 was added at a final concentration of 20 μg / mL, incubated for 1 hour, and washed twice with phosphate-buffered saline (PBS, pH 7.2-7.4). FITC-labeled goat anti-mouse secondary antibody (purchased from Jackson ImmunoResearch, catalog number 111-545-003) was added at a ratio of 1:500, and the mixture was incubated for 1 hour and washed twice with PBS. The results were analyzed using a flow cytometer.

[0194] The results are shown in Figure 5. The Mu4O3 antibody was positive in all three HER3-positive cell lines (MCF-7, SW620, and SK-BR-3), but the assay result was negative in the HER3-negative cell line (MDA-MB-231), and no fluorescent signal was detected. The results demonstrate the specificity of the Mu4O3 antibody.

[0195] Assay Example 2: Immunofluorescence assay of Mu4O3 antibody in HER3-GFP tagged overexpressing 293T cells 293T cells (purchased from the National Collection of Authenticated Cell Cultures of the Chinese Academy of Sciences) were seeded into 24-well cell culture plates to achieve a cell density of 70%. The GFP-tagged human HER3 recombinant plasmid HER3-GFP Tag (purchased from Hunan Keai Medical Devices Co., Ltd., catalog number G109862) was transfected into the cells using the cationic polymer polyethyleneimine (PEI) transfection method. Specifically, 0.5 μg of the plasmid and 1.5 μg of PEI were mixed in 500 μL of Opti-MEM medium (purchased from Thermo Fisher Scientific (China) Co., Ltd., catalog number 31985070). After stabilization at room temperature for 15 minutes, the mixture was slowly added to the cell culture well and returned to a 37°C incubator. After 24 hours, the cells were washed with PBS and fixed with 4% tissue fixative (PFA) for 10 minutes. After washing with PBS, the cells were blocked with 10% goat serum (purchased from Beijing Solarbio Science & Technology Co., Ltd., catalog number SL038) for 30 minutes. After blocking, Mu4O3 antibody was added at a final concentration of 20 μg / mL, incubated for 1 hour, and washed twice with PBS. APC-labeled goat anti-mouse secondary antibody (purchased from Jackson ImmunoResearch, catalog number 111-585-003) was added at a 1:500 ratio, and the mixture was incubated for 1 hour and washed twice with PBS. DAPI (4',6-diamino-2-phenylindole) solution was added, and the mixture was incubated for 5 minutes, washed twice with PBS, and subjected to fluorescence microscopy and image capture. Figure 6 shows immunofluorescence images of Mu4O3 antibody in HER3-GFP Tag-overexpressing 293T cells. The results demonstrate significant colocalization of HER3 protein and Mu4O3 antibody staining.

[0196] In Figure 6, the photograph labeled GFP shows only green fluorescence, indicating successful transfection of HER3 protein into 293T cells with expression in both the cytoplasm and the cell membrane. The photograph labeled Mu4O3 shows only red fluorescence, indicating successful staining of overexpressing cells with the Mu4O3 antibody, with membrane-localized staining. The photograph labeled DAPI shows only blue fluorescence, indicating nuclear staining. The photograph labeled MERGED is a merged image obtained by overlaying the three separate staining images described above, in which a portion of the membrane-localized green light can be completely overlapped with the membrane-localized red light, and the fluorescence intensity remains the same, thus demonstrating that the Mu4O3 antibody specifically stains HER3-overexpressing protein. Furthermore, the red fluorescent staining of Mu4O3 binding is not observed in cells without overexpression (blue staining only in the cell nucleus, no green emission), indicating that the Mu4O3 antibody does not have nonspecific staining properties.

[0197] Assay Example 3: In vitro killing assay of SW620 cells by Mu4O3 antibody SW620 cells (human colon cancer cells, purchased from Zhejiang Meisen Cell Technology Co., Ltd.) were cultured to achieve a cell density of 80%. The cells were harvested and plated in 96-well plates at a cell density of 2–5 × 10. 4The solution was adjusted to 1 / mL. Cells were plated at 100 μL per well. The Mu4O3 antibody or mouse anti-IgG (mouse IgG, purchased from Abmart Shanghai Co., Ltd.) prepared in Example 4 was serially diluted 3-fold from a starting concentration of 300 nM. After dilution was complete, it was added to the cell culture. 2 μg / mL of MMAE-conjugated goat anti-mouse secondary antibody (purchased from Abmart Shanghai Co., Ltd., catalog number B30008) was simultaneously added. The mixture was incubated and left undisturbed for 5 days. During this period, apoptosis was periodically observed. After 5 days, 15 μL of CCK-8 stock solution was added directly to the 96-well plate. The mixture was incubated in an incubator at 37°C for 0.5 to 2 hours, and the absorbance at 450 nm was measured. Cell survival curves were plotted according to the assay results and the antibody dilution gradient.

[0198] The results are shown in Figure 7, which demonstrate that the Mu4O3 antibody has a significant specific cell killing effect. Assay Example 4: Flow affinity assay of Hu4O3 antibody candidate molecules in MDA-MB-453 and SW620 cell lines SW620 (human colon cancer cells) and MDA-MB-453 (human breast cancer cells, purchased from Zhejiang Meisen Cell Technology Co., Ltd.) were separately digested with trypsin in culture dishes. The digested cells were centrifuged at 1000 rpm for 5 minutes and then blocked with 10% goat serum (purchased from Beijing Solarbio Science & Technology Co., Ltd., catalog number SL038) for 30 minutes. Hu4O3 antibody was serially diluted 3-fold from a starting concentration of 500 nM. After completion, the serially diluted Hu4O3 antibody was added to the cells for 1 hour of incubation. After washing the mixture twice with PBS, FITC-labeled goat anti-human secondary antibody (purchased from Jackson ImmunoResearch, catalog number 109-545-008) was added at a ratio of 1:500. The mixture was incubated for 1 hour and washed twice with PBS. Results were analyzed using a flow cytometer.

[0199] Figures 8 and 9 show flow affinity assay data for Hu4O3 antibody candidate molecules L1H4, L1H5, L2H4, L2H5, L4H4, L4H5, and L4H7 in MDA-MB-453 and SW620 cell lines. The results show that all candidate molecules specifically bind to both cell lines with varying binding affinities. L1H4 and L1H5 have EC50 binding affinities of 71.5 nM and 121.7 nM, respectively. 50 L1H4, L1H5, L4H4, and L4H7 showed excellent affinity for MDA-MB-453 cells with EC values ​​of 193.5 nM, 146.7 nM, 90.0 nM, and 21.9 nM, respectively. 50 L2H4 showed excellent affinity for SW620 cells with an EC of 1203 nM. 50 It was.

[0200] Assay Example 5: In vitro cell proliferation inhibition assay of L1H5 antibody and reference antibodies on SK-BR-3 cell line SK-BR-3 (human breast cancer cells) were cultured to achieve a cell density of 80%. The cells were harvested and plated in 96-well plates at a cell density of 2–5 × 10. 4 The antibody was added to the cell culture at a final concentration of 100 μg / mL. Cells were plated at 100 μL per well. The mixture was incubated for 5 days. Apoptosis was periodically observed during this period. After 5 days, 15 μL of CCK-8 stock solution was added to the 96-well plate, and the mixture was incubated in an incubator at 37°C for 0.5 to 2 hours. The absorbance at 450 nm was then measured. Cell viability was measured based on the assay results and a blank control.

[0201] 10 shows the cell proliferation inhibition rates of the L1H5 antibody prepared in Example 4, the reference antibody, and the blank control in the SK-BR-3 cell line. The results show that the in vitro cell proliferation inhibitory effect of L1H5 on SK-BR-3 is better than that of the reference antibody.

[0202] Assay Example 6: In vivo tumor inhibition assay in the mouse breast cancer CDX model Six-week-old Balb / c nude mice were purchased from Jiangsu Gempharmatech Co., Ltd. Five million BT-474 cells (human breast ductal carcinoma cells) were subcutaneously inoculated into each mouse. The average tumor volume was approximately 150 mm. 3 Once the tumor volume reached 1000 mg / kg, L1H5 prepared in Example 4, a reference antibody (positive control), and blank control PBS (negative control) were subcutaneously injected separately at a dose of 20 mg / kg. Treatment was performed once a week for a total of 2 weeks. Tumor volume was measured twice a week after administration using a Vernier caliper and calculated according to the following formula: TV = (length x width). 2 / 2.

[0203] Figure 11 shows the curve of tumor volume change over time in an in vivo BT-474 breast cancer mouse model. The results show that L1H5 can inhibit tumor growth, comparable to a positive reference antibody.

[0204] Assay Example 7: In vitro killing assay of SW620 cells by Hu4O3 antibody candidate molecule-drug conjugates SW620 (human colon cancer cells) were cultured to achieve a cell density of 80%. The cells were harvested and plated in 96-well plates at a cell density of 2–5 × 10. 4 The concentration was adjusted to 1 / mL. Cells were plated at 100 μL per well. The ADC molecule was serially diluted 3-fold from a starting concentration of 300 nM. After dilution was complete, it was added to the cell cultures. The mixture was incubated and left undisturbed for 5 days. During this period, apoptosis was periodically observed. After 5 days, 15 μL of CCK-8 stock solution was added to the 96-well plate, and the mixture was incubated in an incubator at 37°C for 0.5 to 2 hours. The absorbance at 450 nm was then measured. Cell survival curves were plotted according to the assay results and the ADC dilution gradient.

[0205] 12 shows cell viability curves for the antibody-drug conjugates L1H4-LP1, L1H5-LP1, and L4H4-LP1 prepared in Example 7 and the antibody-drug conjugate positive control reference ADC prepared in Comparative Example 1 in the SW620 cell line. The results show that the in vitro cell-killing effects of L1H4-LP1, L1H5-LP1, and L4H4-LP1 on the SW620 cell line are better than those of the reference ADC, and the cell growth inhibitory effect of L1H5-LP1 on the SW620 cell line is optimal.

[0206] Assay Example 8: In vitro cell killing assay of L1H5 antibody, L1H5-LP3, reference antibody, and reference ADC against SK-BR-3 and BXPC-3 cell lines SK-BR-3 (human breast cancer cells) and BXPC-3 (human pancreatic cancer cells, purchased from the National Collection of Authenticated Cell Cultures of the Chinese Academy of Sciences) were cultured to achieve a cell density of 80%. The cells were harvested and plated in 96-well plates at a cell density of 2–5 × 10. 4 The antibody and ADC molecules were serially diluted 3-fold from a starting concentration of 300 nM. Cells were plated at 100 μL per well. After dilution was complete, they were added to the cell cultures. The mixture was incubated and left undisturbed for 5 days. During this period, apoptosis was periodically observed. After 5 days, 15 μL of CCK-8 stock solution was added to the 96-well plate. The mixture was incubated in an incubator at 37°C for 0.5 to 2 hours, and the absorbance at 450 nm was measured. Cell survival curves were plotted according to the assay results and the ADC and antibody dilution gradients.

[0207] 13 and 14 show cell viability curves for the L1H5 antibody prepared in Example 4, the antibody-drug conjugate L1H5-LP3 prepared in Example 6, a reference antibody (positive control), and the antibody-drug conjugate reference ADC (positive control) prepared in Comparative Example 1 in the SK-BR-3 and BXPC-3 cell lines, respectively. The results show that the in vitro cell-killing effect of L1H5-LP3 on the two cell lines was significantly better than that of the reference ADC, the cell growth inhibitory effect of the L1H5 antibody on the SK-BR-3 cell line was better than that of the reference antibody, and that the L1H5 antibody and the reference antibody did not exhibit in vitro cell-killing effect on the BXPC-3 cell line.

[0208] Assay Example 9: In vitro killing assay of HCT-15 cells by L1H5-LP1 and L1H5-LP2 antibodies HCT-15 (human colon cancer cells) were cultured to achieve a cell density of 80%. The cells were harvested and plated in 96-well plates at a cell density of 2–5 × 10. 4 The concentration was adjusted to 1 / mL. Cells were plated at 100 μL per well. The ADC molecule was serially diluted 3-fold from a starting concentration of 300 nM. After dilution was complete, it was added to the cell cultures. The mixture was incubated and left undisturbed for 5 days. During this period, apoptosis was periodically observed. After 5 days, 15 μL of CCK-8 stock solution was added to the 96-well plate, and the mixture was incubated in an incubator at 37°C for 0.5 to 2 hours. The absorbance at 450 nm was then measured. Cell survival curves were plotted according to the assay results and the ADC dilution gradient.

[0209] 15 shows cell viability curves for the antibody-drug conjugate L1H5-LP1 prepared in Example 7, L1H5-LP2 prepared in Example 8, and the antibody-drug conjugate positive control reference ADC prepared in Comparative Example 1 in the HCT-15 cell line. The results show that the in vitro cell killing effects of L1H5-LP1 and L1H5-LP2 on the HCT-15 cell line are significantly better than those of the reference ADC.

[0210] Assay Example 10: In vivo tumor inhibition assay in a mouse colon cancer CDX model Six-week-old Balb / c nude mice were purchased from Jiangsu Gempharmatech Co., Ltd. Five million HCT-15 cells were subcutaneously inoculated into each mouse. The average tumor volume was approximately 150 mm. 3 Once the tumor volume reached 1000 mg / kg, the reference ADC prepared in Comparative Example 1, L1H5-LP2 prepared in Example 8, and L1H5-LP1 prepared in Example 7 were each intravenously injected at a dose of 10 mg / kg. A blank control (negative control) group was also administered an equal dose of PBS intravenously. Administration was performed once a week for a total of three times. Tumor volume was measured twice a week using a Vernier caliper and calculated according to the following formula: TV = (length x width). 2 16 shows the curves of tumor volume change over time in an in vivo HCT-15 colon cancer mouse model. The results show that both L1H5-LP1 and L1H5-LP2 can inhibit tumor growth, and do so slightly better than the positive reference ADC.

[0211] Assay Example 11: In vivo tumor inhibition assay in a mouse colon PDX model NU / NU mice, female, 6 weeks old, weighing 18-21 g, were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. Mice were subcutaneously inoculated with colon cancer tumor masses (colon cancer tissue specimen number 361795, Xi'an Lide Biotechnology Co., Ltd.). The average tumor volume was 150 mm 3When tumor volume reached 100 mg / kg, the reference ADC prepared in Comparative Example 1, L1H5-LP3 prepared in Example 6, L1H5-LP1 prepared in Example 7, Dxd isotype control ADC prepared in Comparative Example 4, and LP3 isotype control ADC prepared in Comparative Example 5 were each intravenously injected at a dose of 10 mg / kg. Additionally, a blank control (negative control) group was intravenously injected with an equal volume of PBS. Administration was performed once a week for a total of three times. Tumor volume was measured twice a week after administration using Vernier calipers and calculated according to the following formula: TV = (length x width). 2 / 2.

[0212] Figure 17 shows the curves of tumor volume change over time in an in vivo colon cancer model mouse. The results show that L1H5-LP3 and L1H5-LP1 significantly inhibit tumor growth compared to the positive control reference ADC.

[0213] Assay Example 12: In vivo tumor inhibition assay in a murine EGFR-TKI-resistant lung adenocarcinoma PDX model NU / NU mice, female, 6 weeks old, weighing 18-21 g, were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. Each mouse was subcutaneously inoculated with an EGFR-TKI-resistant lung adenocarcinoma tumor mass (EGFR-TKI-resistant lung adenocarcinoma tissue specimen number 0025-200717, Xi'an Lide Biotechnology Co., Ltd.). The average tumor volume was 150 mm. 3 When tumor volume reached 100 mg / kg, the reference ADC prepared in Comparative Example 1, L1H5-LP3 prepared in Example 6, L1H5-LP1 prepared in Example 7, Dxd isotype control ADC prepared in Comparative Example 4, and LP3 isotype control ADC prepared in Comparative Example 5 were each intravenously injected at a dose of 10 mg / kg. In addition, a vehicle control group was set up to receive an equal volume of intravenous PBS. Administration was performed once a week for a total of two times. Tumor volume was measured twice a week after administration using Vernier calipers and calculated according to the following formula: TV = (length x width).2 / 2.

[0214] Figure 18 shows the time course of tumor volume changes in an in vivo EGFR-TKI-resistant lung adenocarcinoma model mouse. The results show that L1H5-LP3 and L1H5-LP1 significantly inhibit tumor growth compared to the positive control reference ADC.

[0215] Assay Example 13: In vivo tumor inhibition assay in a mouse colon cancer CDX model Six-week-old Balb / c nude mice were purchased from Jiangsu Gempharmatech Co., Ltd. Five million SW620 cells were subcutaneously inoculated into each mouse. The average tumor volume was approximately 150 mm. 3 Once the tumor volume reached 100 mg / kg, L1H5-DXd prepared in Comparative Example 3, the reference antibody-LP3 prepared in Comparative Example 2, L1H5-LP3 prepared in Example 6, the reference ADC prepared in Comparative Example 1, and the negative control PBS were each intravenously injected at a dose of 10 mg / kg. Treatment was performed once a week for a total of 3 weeks. Tumor volume was measured twice a week using Vernier calipers after administration and calculated according to the following formula: TV = (length x width). 2 / 2.

[0216] Figure 19 shows the curves of tumor volume change over time in an in vivo mouse colon cancer CDX model, showing that L1H5-DXd, L1H5-LP3, and the reference antibody-LP3 all outperform the positive control reference ADC.

[0217] Figure 20 is a partial enlargement of Figure 19, with the negative control PBS group removed. As can be seen from the figure, the four active molecules are ranked from highest to lowest according to their overall tumor-inhibitory effect in vivo: L1H5-LP3 > L1H5-DXd > Reference antibody-LP3 > Reference ADC.

[0218] Assay Example 14: In vivo tumor inhibition assay in a mouse pancreatic cancer CDX model NU / NU mice, female, 6 weeks old, weighing 18-21 g, were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. Each mouse was subcutaneously inoculated with a pancreatic cancer tumor mass (sample number 0033-361319, Xi'an Lide Biotechnology Co., Ltd.). The average tumor volume was 150 mm. 3 When tumor volume reached 1000 mg / kg, the reference ADC prepared in Comparative Example 1, L1H5-LP3 prepared in Example 6, L1H5-LP1 prepared in Example 7, Dxd isotype control ADC prepared in Comparative Example 4, and LP3 isotype control ADC prepared in Comparative Example 5 were intravenously injected at a dose of 10 mg / kg. In addition, a blank control group was intravenously injected with an equal dose of PBS. Administration was performed once a week for a total of three times. Tumor volume was measured twice a week using Vernier calipers and calculated according to the following formula: TV = (length x width). 2 / 2.

[0219] Figure 21 shows the in vivo tumor inhibition curves in a pancreatic cancer mouse model. The results show that the positive control reference ADC, L1H5-LP3, and L1H5-LP1 were all able to significantly inhibit tumor growth in the early stages of administration, and the tumor inhibition effects of L1H5-LP3 and L1H5-LP1 were significantly better than those of the positive control reference ADC during the administration period, and furthermore, the duration of their tumor inhibition was significantly longer than that of the reference ADC.

[0220] The antibody-drug conjugates prepared in accordance with the present disclosure have superior in vivo antitumor efficacy to ADCs conjugated with GGFG-DXd, and L1H5-LP3 exhibits better in vivo efficacy than reference ADCs in multiple tumor models, and L1H5-LP3 exhibits more durable tumor-inhibiting effects in individual models. Furthermore, L1H5-LP3 has a maximum tolerated dose (MTD) comparable to that of GGFG-DXd ADC, and is predicted to have a wider therapeutic window than ADCs conjugated with GGFG-DXd. The antibody-drug conjugates provided by the present disclosure have excellent safety profiles.

Claims

1. An anti-HER3 antibody or antigen-binding fragment thereof comprising at least one light chain variable region (VL) and a heavy chain variable region (VH), CDR1 of the VL comprises the amino acid sequence set forth in SEQ ID NO: 1, CDR2 of the VL comprises the amino acid sequence set forth in SEQ ID NO: 2, and CDR3 of the VL comprises the amino acid sequence set forth in SEQ ID NO: 3; An anti-HER3 antibody or antigen-binding fragment thereof, wherein CDR1 of the VH comprises the amino acid sequence set forth in SEQ ID NO:4, CDR2 of the VH comprises an amino acid sequence having 5, 4, 3, 2, 1 or less, or 0 mutations compared to the amino acid sequence set forth in SEQ ID NO:5, and CDR3 of the VH comprises the amino acid sequence set forth in SEQ ID NO:

6.

2. CDR2 of the VH is (a) and (b): (a) C52N, T62N, T66K, and G67S; (b) C52Y, T62N, T66K, and G67S comprising one or more mutations selected from The anti-HER3 antibody or antigen-binding fragment thereof according to claim 1.

3. CDR2 of the VH is (a) or (b): (a) C52N, T62N, T66K, and G67S; (b) C52Y, T62N, T66K, and G67S including mutations of The anti-HER3 antibody or antigen-binding fragment thereof according to claim 1 or 2.

4. The combination of VH and VL contained in the anti-HER3 antibody or antigen-binding fragment thereof is selected from one of groups (c) to (j). (c) the VL and VH comprise amino acid sequences having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequences set forth in SEQ ID NO:9 and SEQ ID NO:10, respectively; (d) the VL and VH comprise amino acid sequences having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequences set forth in SEQ ID NO:11 and SEQ ID NO:14, respectively; (e) the VL and VH comprise amino acid sequences having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequences set forth in SEQ ID NO: 11 and SEQ ID NO: 15, respectively; (f) the VL and VH comprise amino acid sequences having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequences set forth in SEQ ID NO: 12 and SEQ ID NO: 14, respectively; (g) the VL and VH comprise amino acid sequences having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequences set forth in SEQ ID NO: 12 and SEQ ID NO: 15, respectively; (h) the VL and VH comprise amino acid sequences having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequences set forth in SEQ ID NO: 13 and SEQ ID NO: 14, respectively; (i) the VL and VH comprise amino acid sequences having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequences set forth in SEQ ID NO: 13 and SEQ ID NO: 15, respectively; (j) the VL and VH comprise amino acid sequences having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequences set forth in SEQ ID NO: 13 and SEQ ID NO: 16, respectively; The anti-HER3 antibody or antigen-binding fragment thereof according to any one of claims 1 to 3.

5. The combination of VH and VL contained in the anti-HER3 antibody or antigen-binding fragment thereof is selected from one of groups (c) to (j). (c) the VL comprises the amino acid sequence set forth in SEQ ID NO: 9 and conservatively substituted variants thereof, and the VH comprises the amino acid sequence set forth in SEQ ID NO: 10 and conservatively substituted variants thereof; (d) the VL comprises the amino acid sequence set forth in SEQ ID NO: 11 and conservatively substituted variants thereof, and the VH comprises the amino acid sequence set forth in SEQ ID NO: 14 and conservatively substituted variants thereof; (e) the VL comprises the amino acid sequence set forth in SEQ ID NO: 11 and conservatively substituted variants thereof, and the VH comprises the amino acid sequence set forth in SEQ ID NO: 15 and conservatively substituted variants thereof; (f) the VL comprises the amino acid sequence set forth in SEQ ID NO: 12 and conservatively substituted variants thereof, and the VH comprises the amino acid sequence set forth in SEQ ID NO: 14 and conservatively substituted variants thereof; (g) the VL comprises the amino acid sequence set forth in SEQ ID NO: 12 and conservatively substituted variants thereof, and the VH comprises the amino acid sequence set forth in SEQ ID NO: 15 and conservatively substituted variants thereof; (h) the VL comprises the amino acid sequence set forth in SEQ ID NO: 13 and conservatively substituted variants thereof, and the VH comprises the amino acid sequence set forth in SEQ ID NO: 14 and conservatively substituted variants thereof; (i) the VL comprises the amino acid sequence set forth in SEQ ID NO: 13 and conservatively substituted variants thereof, and the VH comprises the amino acid sequence set forth in SEQ ID NO: 15 and conservatively substituted variants thereof; (j) the VL comprises the amino acid sequence set forth in SEQ ID NO: 13 and conservatively substituted variants thereof, and the VH comprises the amino acid sequence set forth in SEQ ID NO: 16 and conservatively substituted variants thereof; An anti-HER3 antibody or antigen-binding fragment thereof according to any one of claims 1 to 4.

6. The anti-HER3 antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, which specifically binds to human HER3.

7. A nucleic acid encoding the anti-HER3 antibody or antigen-binding fragment thereof of any one of claims 1 to 6.

8. A vector comprising the nucleic acid of claim 7.

9. A host cell comprising a nucleic acid according to claim 7 and / or a vector according to claim 8.

10. Formula I Ab-(LD)n (I) or an isomer, isotopic variant, pharmaceutically acceptable salt, prodrug, or solvate thereof, or a combination thereof. wherein Ab is an anti-HER3 antibody or antigen-binding fragment thereof according to any one of claims 1 to 6; L is a linker covalently connecting Ab and D; D is the payload, where n is a number from 1 to 10).

11. The antibody-drug conjugate of claim 10, wherein the linker is a cleavable linker and a non-cleavable linker.

12. The antibody-drug conjugate of claim 10 or 11, wherein the linker comprises a cleavable peptide.

13. The antibody-drug conjugate of claim 12, wherein the cleavable peptide is enzymatically cleavable.

14. The antibody-drug conjugate of claim 13 , wherein the enzyme comprises cathepsin B.

15. The antibody-drug conjugate of any one of claims 10 to 14, wherein the cleavable peptide or L comprises an amino acid unit.

16. The antibody-drug conjugate of claim 15, wherein the amino acid unit comprises a dipeptide, tripeptide, tetrapeptide, or pentapeptide.

17. the amino acid unit is selected from any one or combination of Val-Cit, Val-Ala, Glu-Val-Cit, Ala-Ala-Asn, Gly-Val-Cit, Gly-Gly-Gly, and Gly-Gly-Phe-Gly; The antibody-drug conjugate of claim 16.

18. The antibody-drug conjugate of any one of claims 10 to 17, wherein the linker comprises a self-immolative spacer.

19. 19. The antibody-drug conjugate of claim 18, wherein the self-immolative spacer comprises p-aminobenzoxycarbonyl (PABC) or p-aminobenzyl (PAB).

20. 20. The antibody-drug conjugate of any one of claims 12 to 19, wherein the cleavable peptide is spliced ​​directly onto the self-immolative spacer.

21. The linker is -L １ -L ２ -L ３ - (In the formula, L １ is -(succinimide-3-yl-N)-(CH ２ ) m １ -C(=O)-, -CH ２ -C(=O)-NH-(CH ２ ) m ２ -C(=O)-, or -C(=O)-(CH ２ ) m ３ -C(=O)-(in the formula, m １ represents an integer of 2 to 8, m ２ represents an integer of 1 to 8, m ３ represents an integer of 1 to 8), L ２ represents an amino acid unit, L ３ represents a self-immolative spacer) Including the structure of The antibody-drug conjugate of any one of claims 10 to 20.

22. L, -(succinimide-3-yl-N)-CH ２ CH ２ -C(=O)-GGFG-PABC-; -(succinimide-3-yl-N)-CH ２ CH ２ CH ２ CH ２ CH ２ -C(=O)-GGFG-PABC-; -(succinimide-3-yl-N)-CH ２ CH ２ CH ２ CH ２ CH ２ -C(=O)-GGFG-NH-PABC-; -(succinimide-3-yl-N)-CH ２ CH ２ -C(=O)-NH-CH ２ CH ２ O-CH ２ CH ２ O-CH ２ CH ２ -C(=O)-GGFG-PABC-; -(succinimide-3-yl-N)-CH ２ CH ２ -C(=O)-NH-CH ２ CH ２ O-CH ２ CH ２ O-CH ２ CH ２ O-CH ２ CH ２ O-CH ２ CH ２ -C(=O)-GGFG-PABC-; -CH ２ -C(=O)-NH-CH ２ CH ２ -C(=O)-GGFG-PABC-; -C(=O)-CH ２ CH ２ CH ２ CH ２ CH ２ CH ２ -C(=O)-GGFG-PABC-; -(succinimide-3-yl-N)-CH ２ CH ２ -C(=O)-GGFG-NH-CH ２ CH ２ -C(=O)-; -(succinimide-3-yl-N)-CH ２ CH ２ -C(=O)-GGFG-NH-CH ２ CH ２ CH ２ -C(=O)-; -(succinimide-3-yl-N)-CH ２ CH ２ CH ２ CH ２ CH ２ -C(=O)-GGFG-NH-CH ２ CH ２ -C(=O)-; -(succinimide-3-yl-N)-CH ２ CH ２ CH ２ CH ２ CH ２ -C(=O)-GGFG-NH-CH ２ CH ２ CH ２ -C(=O)-; -(succinimide-3-yl-N)-CH ２ CH ２ CH ２ CH ２ CH ２ -C(=O)-GGFG-NH-CH ２ CH ２ CH ２ CH ２ CH ２ -C(=O)-; -(succinimide-3-yl-N)-CH ２ CH ２ CH ２ CH ２ CH ２ -C(=O)-GGFG-NH-CH ２ -O-CH ２ -C(=O)-; -(succinimide-3-yl-N)-CH ２ CH ２ CH ２ CH ２ CH ２ -C(=O)-GGFG-NH-CH ２ CH ２ -O-CH ２ -C(=O)-; -(succinimide-3-yl-N)-CH ２ CH ２ -C(=O)-NH-CH ２ CH ２ O-CH ２ CH ２ O-CH ２ CH ２ -C(=O)-GGFG-NH-CH ２ CH ２ CH ２ -C(=O)-; -(succinimide-3-yl-N)-CH ２ CH ２ -C(=O)-NH-CH ２ CH ２ O-CH ２ CH ２ O-CH ２ CH ２ -C(=O)-GGFG-NH-CH ２ CH ２ -C(=O)-; -(succinimide-3-yl-N)-CH ２ CH ２ -C(=O)-NH-CH ２ CH ２ O-CH ２ CH ２ O-CH ２ CH ２ O-CH ２ CH ２ O-CH ２ CH ２ -C(=O)-GGFG-NH-CH ２ CH ２ CH ２ -C(=O)-; -(succinimide-3-yl-N)-CH ２ CH ２ -C(=O)-NH-CH ２ CH ２ O-CH ２ CH ２ O-CH ２ CH ２ O-CH ２ CH ２ O-CH ２ CH ２ -C(=O)-GGFG-NH-CH ２ CH ２ -C(=O)-; -CH ２ -C(=O)-NH-CH ２ CH ２ -C(=O)-GGFG-NH-CH ２ CH ２ CH ２ -C(=O)-; -C(=O)-CH ２ CH ２ CH ２ CH ２ CH ２ CH ２ -C(=O)-GGFG-NH-CH ２ CH ２ CH ２ -C(=O)-; -(succinimide-3-yl-N)-CH ２ CH ２ -C(=O)-VA-PABC-; -(succinimide-3-yl-N)-CH2CH2CH2CH2CH2-C(═O)-VA-PABC-; -(succinimide-3-yl-N)-CH ２ CH ２ CH ２ CH ２ CH ２ -C(=O)-VA-NH-PABC-; -(succinimide-3-yl-N)-CH ２ CH ２ -C(=O)-NH-CH ２ CH ２ O-CH ２ CH ２ O-CH ２ CH ２ -C(=O)-VA-PABC-; -(succinimide-3-yl-N)-CHCH-C(=O)-NH-CH ２ CH ２ O-CH ２ CH ２ O-CH ２ CH ２ O-CH ２ CH ２ O-CH ２ CH ２ -C(=O)-VA-PABC-; -CH ２ -C(=O)-NH-CH ２ CH ２ -C(=O)-VA-PABC-; -C(=O)-CH ２ CH ２ CH ２ CH ２ CH ２ CH ２ -C(=O)-VA-PABC-; -(succinimide-3-yl-N)-CH ２ CH ２ -C(=O)-VA-NH-CH ２ CH ２ -C(=O)-; -(succinimide-3-yl-N)-CH ２ CH ２ -C(=O)-VA-NH-CH ２ CH ２ CH ２ -C(=O)-; -(succinimide-3-yl-N)-CH ２ CH ２ CH ２ CH ２ CH ２ -C(=O)-VA-NH-CH ２ CH ２ -C(=O)-; -(succinimide-3-yl-N)-CH ２ CH ２ CH ２ CH ２ CH ２ -C(=O)-VA-NH-CH ２ CH ２ CH ２ -C(=O)-; -(succinimide-3-yl-N)-CH ２ CH ２ CH ２ CH ２ CH ２ -C(=O)-VA-NH-CH ２ CH ２ CH ２ CH ２ CH ２ -C(=O)-; -(succinimide-3-yl-N)-CH ２ CH ２ CH ２ CH ２ CH ２ -C(=O)-VA-NH-CH ２ -O-CH ２ -C(=O)-; -(succinimide-3-yl-N)-CH ２ CH ２ CH ２ CH ２ CH ２ -C(=O)-VA-NH-CH ２ CH ２ -O-CH ２ -C(=O)-; -(succinimide-3-yl-N)-CH ２ CH ２ -C(=O)-NH-CH ２ CH ２ O-CH ２ CH ２ O-CH ２ CH ２ -C(=O)-VA-NH-CH ２ CH ２ CH ２ -C(=O)-; -(succinimide-3-yl-N)-CH ２ CH ２ -C(=O)-NH-CH ２ CH ２ O-CH ２ CH ２ O-CH ２ CH ２ -C(=O)-VA-NH-CH ２ CH ２ -C(=O)-; -(succinimide-3-yl-N)-CH ２ CH ２ -C(=O)-NH-CH ２ CH ２ O-CH ２ CH ２ O-CH ２ CH ２ O-CH ２ CH ２ O-CH ２ CH ２ -C(=O)-VA-NH-CH ２ CH ２ CH ２ -C(=O)-; -(succinimide-3-yl-N)-CH ２ CH ２ -C(=O)-NH-CH ２ CH ２ O-CH ２ CH ２ O-CH ２ CH ２ O-CH ２ CH ２ O-CH ２ CH ２ -C(=O)-VA-NH-CH ２ CH ２ -C(=O)-; -CH ２ -C(=O)-NH-CH ２ CH ２ -C(=O)-VA-NH-CH ２ CH ２ CH ２ -C(=O)-;および -C(=O)-CH ２ CH ２ CH ２ CH ２ CH ２ CH ２ -C(=O)-VA-NH-CH ２ CH ２ CH ２ -C(=O)- The antibody-drug conjugate of any one of claims 10 to 21, selected from:

23. 23. The antibody-drug conjugate of any one of claims 19 to 22, wherein the p-aminobenzyloxycarbonyl (PABC) or p-aminobenzyl (PAB) is linked to a polysarcosine (poly-N-methylglycine) residue or a methylamino.

24. The linker has formula II: 【Chemical 1】 wherein the linker of formula II is bonded to a sulfhydryl group reduced from an interchain disulfide chain of the antibody by a thioether bond via a succinimidyl group; the carbonyl in the ester group of the linker of Formula II is linked to an amino group in the payload; R １ and R ２ are independently selected from hydrogen, methyl, and isopropyl groups; R ３ But-(CR ５ HCONH)n １ - (CH ２ CONH)n ２ represents - or a single bond, R ５ is selected from hydrogen and benzyl, and n １ represents an integer of 0 to 2, and n ２ represents an integer from 0 to 2, R ４ is a methylamino group or -(NCH ３ COCH ２ ) n ３ -NCH ３ COCH ３ represents n ３ represents an integer from 1 to 20) Denoted by The antibody-drug conjugate of any one of claims 10 to 23.

25. In the linker of formula II, R ４ - (NCH ３ COCH ２ ) n ３ -NCH ３ COCH ３ represents n ３ The antibody-drug conjugate of claim 24, wherein represents an integer of 8 to 15.

26. In the linker of formula II, R ４ - (NCH ３ COCH ２ ) n ３ -NCH ３ COCH ３ represents n ３ The antibody-drug conjugate of claim 24 or 25, wherein represents an integer of 10 to 12.

27. In the linker of formula II, R ３ But-(CR ５ HCONH)n １ - (CH ２ CONH)n ２ represents - or a single bond, R ５ is selected from benzyl, and n １ represents 1 or 2, and n ２ represents 1 or 2, R ４ - (NCH ３ COCH ２ ) n ３ -NCH ３ COCH ３ represents n ３ The antibody-drug conjugate of any one of claims 24 to 26, wherein represents an integer of 8 to 15.

28. In the linker of formula II, R ３ represents a single bond, R ４ - (NCH ３ COCH ２ ) n ３ -NCH ３ COCH ３ represents n ３ The antibody-drug conjugate of any one of claims 24 to 27, wherein represents an integer of 8 to 15.

29. In the linker of formula II, R ３ represents a single bond, R ４ The antibody-drug conjugate of any one of claims 24 to 28, wherein represents methylamino.

30. The linker is a group 【Chemistry 2】 30. The antibody-drug conjugate of any one of claims 10 to 29, selected from one or more of:

31. 31. The antibody-drug conjugate of any one of claims 10 to 30, wherein the payload is selected from at least one of the group comprising a cytotoxic agent, a marker, a nucleic acid, a radionuclide, a hormone, an immunomodulatory agent, a prodrug-converting enzyme, a ribonuclease, an agonist antibody, an antagonist antibody, and fragments, fusion proteins, or derivatives thereof.

32. 32. The antibody-drug conjugate of claim 31 , wherein the cytotoxic agent comprises a tubulin inhibitor and / or a topoisomerase inhibitor, wherein the tubulin inhibitor comprises an auristatin or a derivative thereof, or a maytansine or a derivative thereof, and wherein the topoisomerase inhibitor comprises camptothecin or a derivative thereof.

33. The payload is represented by Formula III 【Chemistry 3】 is an exatecan of formula III linked to the linker by the nitrogen atom of the amino group on the cyclohexane ring of the exatecan of formula III; The antibody-drug conjugate of any one of claims 10 to 32.

34. The following groups 【Chemistry 4-1】 【Chemistry 4-2】 (n is 1 to 10 or 4 to 10) The antibody-drug conjugate of any one of claims 10 to 33, wherein the antibody-drug conjugate is selected from one of the following:

35. A method for preparing the antibody-drug conjugate of any one of claims 10 to 34, comprising the steps of: reducing at least a portion of the interchain disulfide bonds of the antibody or antigen-binding fragment thereof by a reduction treatment; and reacting the reactive groups of the linker in the linker-payload to form a compound of formula I Ab-(LD)n (I) or an isomer, isotopic variant, pharmaceutically acceptable salt, prodrug, or solvate thereof, or a combination thereof. wherein Ab is an anti-HER3 antibody or antigen-binding fragment thereof according to any one of claims 1 to 6; L is a linker covalently connecting Ab and D; D is the payload, n is a number from 1 to 10) and obtaining the compound.

36. 36. The method of claim 35, wherein the linker is a cleavable linker and a non-cleavable linker.

37. 37. The method of claim 35 or 36, wherein the linker comprises a cleavable peptide.

38. 38. The method of claim 37, wherein the cleavable peptide is enzymatically cleavable.

39. 39. The method of claim 38, wherein the enzyme comprises cathepsin B.

40. 40. The method of any one of claims 35 to 39, wherein the cleavable peptide or L comprises an amino acid unit.

41. 41. The method of claim 40, wherein the amino acid unit comprises a dipeptide, tripeptide, tetrapeptide, or pentapeptide.

42. the amino acid unit is selected from any one or combination of Val-Cit, Val-Ala, Glu-Val-Cit, Ala-Ala-Asn, Gly-Val-Cit, Gly-Gly-Gly, and Gly-Gly-Phe-Gly; 42. The method of claim 41.

43. 43. The method of any one of claims 35 to 42, wherein the linker comprises a self-immolative spacer.

44. 44. The method of claim 43, wherein the self-immolative spacer comprises p-aminobenzyloxycarbonyl (PABC) or p-aminobenzyl (PAB).

45. 45. The method of any one of claims 37 to 44, wherein the cleavable peptide is spliced ​​directly onto the self-immolative spacer.

46. The linker is -L １ -L ２ -L ３ - (In the formula, L １ is -(succinimide-3-yl-N)-(CH ２ ) m １ -C(=O)-, -CH ２ -C(=O)-NH-(CH ２ ) m ２ -C(=O)-, or -C(=O)-(CH ２ ) m ３ -C(=O)-(in the formula, m １ represents an integer of 2 to 8, m ２ represents an integer of 1 to 8, m ３ represents an integer of 1 to 8), L ２ represents an amino acid unit, L ３ represents a self-immolative spacer) Including the structure of 46. ​​A method of preparation according to any one of claims 35 to 45.

47. L, -(succinimide-3-yl-N)-CH ２ CH ２ -C(=O)-GGFG-PABC-; -(succinimide-3-yl-N)-CH ２ CH ２ CH ２ CH ２ CH ２ -C(=O)-GGFG-PABC-; -(succinimide-3-yl-N)-CH ２ CH ２ CH ２ CH ２ CH ２ -C(=O)-GGFG-NH-PABC-; -(succinimide-3-yl-N)-CH ２ CH ２ -C(=O)-NH-CH ２ CH ２ O-CH ２ CH ２ O-CH ２ CH ２ -C(=O)-GGFG-PABC-; -(succinimide-3-yl-N)-CH ２ CH ２ -C(=O)-NH-CH ２ CH ２ O-CH ２ CH ２ O-CH ２ CH ２ O-CH ２ CH ２ O-CH ２ CH ２ -C(=O)-GGFG-PABC-; -CH ２ -C(=O)-NH-CH ２ CH ２ -C(=O)-GGFG-PABC-; -C(=O)-CH ２ CH ２ CH ２ CH ２ CH ２ CH ２ -C(=O)-GGFG-PABC-; -(succinimide-3-yl-N)-CH ２ CH ２ -C(=O)-GGFG-NH-CH ２ CH ２ -C(=O)-; -(succinimide-3-yl-N)-CH ２ CH ２ -C(=O)-GGFG-NH-CH ２ CH ２ CH ２ -C(=O)-; -(succinimide-3-yl-N)-CH ２ CH ２ CH ２ CH ２ CH ２ -C(=O)-GGFG-NH-CH ２ CH ２ -C(=O)-; -(succinimide-3-yl-N)-CH ２ CH ２ CH ２ CH ２ CH ２ -C(=O)-GGFG-NH-CH ２ CH ２ CH ２ -C(=O)-; -(succinimide-3-yl-N)-CH ２ CH ２ CH ２ CH ２ CH ２ -C(=O)-GGFG-NH-CH ２ CH ２ CH ２ CH ２ CH ２ -C(=O)-; -(succinimide-3-yl-N)-CH ２ CH ２ CH ２ CH ２ CH ２ -C(=O)-GGFG-NH-CH ２ -O-CH ２ -C(=O)-; -(succinimide-3-yl-N)-CH ２ CH ２ CH ２ CH ２ CH ２ -C(=O)-GGFG-NH-CH ２ CH ２ -O-CH ２ -C(=O)-; -(succinimide-3-yl-N)-CH ２ CH ２ -C(=O)-NH-CH ２ CH ２ O-CH ２ CH ２ O-CH ２ CH ２ -C(=O)-GGFG-NH-CH ２ CH ２ CH ２ -C(=O)-; -(succinimide-3-yl-N)-CH ２ CH ２ -C(=O)-NH-CH ２ CH ２ O-CH ２ CH ２ O-CH ２ CH ２ -C(=O)-GGFG-NH-CH ２ CH ２ -C(=O)-; -(succinimide-3-yl-N)-CH ２ CH ２ -C(=O)-NH-CH ２ CH ２ O-CH ２ CH ２ O-CH ２ CH ２ O-CH ２ CH ２ O-CH ２ CH ２ -C(=O)-GGFG-NH-CH ２ CH ２ CH ２ -C(=O)-; -(succinimide-3-yl-N)-CH ２ CH ２ -C(=O)-NH-CH ２ CH ２ O-CH ２ CH ２ O-CH ２ CH ２ O-CH ２ CH ２ O-CH ２ CH ２ -C(=O)-GGFG-NH-CH ２ CH ２ -C(=O)-; -CH ２ -C(=O)-NH-CH ２ CH ２ -C(=O)-GGFG-NH-CH ２ CH ２ CH ２ -C(=O)-; -C(=O)-CH ２ CH ２ CH ２ CH ２ CH ２ CH ２ -C(=O)-GGFG-NH-CH ２ CH ２ CH ２ -C(=O)-; -(succinimide-3-yl-N)-CH ２ CH ２ -C(=O)-VA-PABC-; -(succinimide-3-yl-N)-CH2CH2CH2CH2CH2-C(═O)-VA-PABC-; -(succinimide-3-yl-N)-CH ２ CH ２ CH ２ CH ２ CH ２ -C(=O)-VA-NH-PABC-; -(succinimide-3-yl-N)-CH ２ CH ２ -C(=O)-NH-CH ２ CH ２ O-CH ２ CH ２ O-CH ２ CH ２ -C(=O)-VA-PABC-; -(succinimide-3-yl-N)-CHCH-C(=O)-NH-CH ２ CH ２ O-CH ２ CH ２ O-CH ２ CH ２ O-CH ２ CH ２ O-CH ２ CH ２ -C(=O)-VA-PABC-; -CH ２ -C(=O)-NH-CH ２ CH ２ -C(=O)-VA-PABC-; -C(=O)-CH ２ CH ２ CH ２ CH ２ CH ２ CH ２ -C(=O)-VA-PABC-; -(succinimide-3-yl-N)-CH ２ CH ２ -C(=O)-VA-NH-CH ２ CH ２ -C(=O)-; -(succinimide-3-yl-N)-CH ２ CH ２ -C(=O)-VA-NH-CH ２ CH ２ CH ２ -C(=O)-; -(succinimide-3-yl-N)-CH ２ CH ２ CH ２ CH ２ CH ２ -C(=O)-VA-NH-CH ２ CH ２ -C(=O)-; -(succinimide-3-yl-N)-CH ２ CH ２ CH ２ CH ２ CH ２ -C(=O)-VA-NH-CH ２ CH ２ CH ２ -C(=O)-; -(succinimide-3-yl-N)-CH ２ CH ２ CH ２ CH ２ CH ２ -C(=O)-VA-NH-CH ２ CH ２ CH ２ CH ２ CH ２ -C(=O)-; -(succinimide-3-yl-N)-CH ２ CH ２ CH ２ CH ２ CH ２ -C(=O)-VA-NH-CH ２ -O-CH ２ -C(=O)-; -(succinimide-3-yl-N)-CH ２ CH ２ CH ２ CH ２ CH ２ -C(=O)-VA-NH-CH ２ CH ２ -O-CH ２ -C(=O)-; -(succinimide-3-yl-N)-CH ２ CH ２ -C(=O)-NH-CH ２ CH ２ O-CH ２ CH ２ O-CH ２ CH ２ -C(=O)-VA-NH-CH ２ CH ２ CH ２ -C(=O)-; -(succinimide-3-yl-N)-CH ２ CH ２ -C(=O)-NH-CH ２ CH ２ O-CH ２ CH ２ O-CH ２ CH ２ -C(=O)-VA-NH-CH ２ CH ２ -C(=O)-; -(succinimide-3-yl-N)-CH ２ CH ２ -C(=O)-NH-CH ２ CH ２ O-CH ２ CH ２ O-CH ２ CH ２ O-CH ２ CH ２ O-CH ２ CH ２ -C(=O)-VA-NH-CH ２ CH ２ CH ２ -C(=O)-; -(succinimide-3-yl-N)-CH ２ CH ２ -C(=O)-NH-CH ２ CH ２ O-CH ２ CH ２ O-CH ２ CH ２ O-CH ２ CH ２ O-CH ２ CH ２ -C(=O)-VA-NH-CH ２ CH ２ -C(=O)-; -CH ２ -C(=O)-NH-CH ２ CH ２ -C(=O)-VA-NH-CH ２ CH ２ CH ２ -C(=O)-;および -C(=O)-CH ２ CH ２ CH ２ CH ２ CH ２ CH ２ -C(=O)-VA-NH-CH ２ CH ２ CH ２ -C(=O)- Selected from:

50. The method of any one of claims 35 to 49.

48. 48. The method of any one of claims 44 to 47, wherein the p-aminobenzyloxycarbonyl (PABC) or p-aminobenzyl (PAB) is linked to a polysarcosine (poly-N-methylglycine) residue or methylamino.

49. reducing at least a portion of the interchain disulfide bonds of the anti-HER3 antibody or antigen-binding fragment thereof by a reduction treatment; and 【Chemistry 5】 The linker part of wherein the carbonyl in the ester group of the linker of formula IV is linked to an amino group in the payload; In formula IV, R １ and R ２ are independently selected from hydrogen, methyl, and isopropyl groups; R ３ But-(CR ５ HCONH)n １ - (CH ２ CONH)n ２ represents - or a single bond, R ５ is selected from hydrogen and benzyl, and n １ represents an integer of 0 to 2, and n ２ represents an integer from 0 to 2, R ４ is a methylamino group or -(NCH ３ COCH ２ ) n ３ -NCH ３ COCH ３ represents n ３ represents an integer from 1 to 20) with the carbon atom at the 3-position of the maleimido-N-yl 49. A method of preparation according to any one of claims 35 to 48.

50. 50. The method of claim 49, further comprising reacting the anti-HER3 antibody or antigen-binding fragment thereof with a reducing agent in a buffer solution containing a chelating agent, adding a linker-payload solution, and adjusting the pH.

51. Formula III 【Chemistry 6】 51. The method of claim 49 or 50, further comprising the step of linking the nitrogen atom of the amino group on the cyclohexane ring of the payload, which is exatecan of formula IV, to the carbonyl of the ester of formula IV.

52. A pharmaceutical composition comprising the anti-HER3 antibody or antigen-binding fragment thereof of any one of claims 1 to 6, or the antibody-drug conjugate of any one of claims 10 to 34, or an isomer, isotopic variant, pharmaceutically acceptable salt, prodrug, or solvate thereof, or a combination thereof, and a pharmaceutically acceptable excipient.

53. A kit comprising the anti-HER3 antibody or antigen-binding fragment thereof of any one of claims 1 to 6, or the antibody-drug conjugate of any one of claims 10 to 34, or an isomer, isotopic variant, pharmaceutically acceptable salt, prodrug, or solvate thereof, or a combination thereof.

54. Use of the anti-HER3 antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, the antibody-drug conjugate according to any one of claims 10 to 34, the antibody-drug conjugate prepared by the method according to any one of claims 35 to 51, or the pharmaceutical composition according to claim 52, or the kit according to claim 53, in the preparation of a therapeutic agent for diagnosing, preventing and treating tumor diseases.

55. 55. The use of claim 54, including use in the preparation of a medicament that targets HER3.

56. 56. The use of claim 54 or 55, wherein the tumor comprises a HER3-expressing solid tumor.

57. 57. The use of any one of claims 54 to 56, wherein the tumor disease comprises lung cancer, kidney cancer, urothelial cancer, colorectal cancer, prostate cancer, glioblastoma multiforme, ovarian cancer, pancreatic cancer, breast cancer, metastatic breast cancer, luminal breast cancer, melanoma, liver cancer, bladder cancer, gastric cancer (cancer of the stomach), gastrointestinal stromal tumor, cervical cancer, head and neck cancer, esophageal cancer, epidermoid carcinoma, peritoneal cancer, adult glioblastoma multiforme, liver cancer, hepatocellular carcinoma, colon cancer, rectal cancer, colorectal cancer, endometrial cancer, uterine cancer, salivary gland cancer, renal cancer, vulvar cancer, thyroid cancer, hepatocarcinoma, anal cancer, penile cancer, and lung adenocarcinoma.

58. A method for diagnosing, preventing, and treating a tumor disease, comprising the step of administering to a subject a therapeutic dose of a therapeutic agent comprising the anti-HER3 antibody or antigen-binding fragment thereof of any one of claims 1 to 6, the antibody-drug conjugate of any one of claims 10 to 34, the antibody-drug conjugate prepared by the method of any one of claims 35 to 51, the pharmaceutical composition of claim 52, or the kit of claim 53.

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