Antibody-drug conjugates, pharmaceutical compositions and uses thereof

Abstract

The present invention relates to an antibody-drug conjugate, a pharmaceutical composition thereof, and a method for treating cancer using the same. The antibody-drug conjugate is a conjugate of a belotecan derivative and an anti-CD44v9 antibody or an antigen-binding fragment thereof. The antibody-drug conjugate exhibits excellent antitumor activity against various solid tumors.

Description

[Technical Field]

[0001] The present disclosure relates to antibody-drug conjugates, particularly anti-CD44v9 antibody-drug conjugates, and to pharmaceutical compositions, methods of making, uses, and methods of treatment for such conjugates. [Background technology]

[0002] CD44 is a family of single-membrane glycoproteins involved in homotypic cell-cell, cell-matrix, and cell-cytoskeleton interactions. Known CD44 ligands include hyaluronan (HA), osteopontin (OPN), collagen, and matrix metalloproteinases (MMPs). In humans, CD44 is encoded by 19 exons, of which the first five exons (exons 1–5) and the last five exons (exons 16–20) consistently encode the most common and smallest CD44 protein, CD44s. CD44 variant isoforms (CD44v) are generated by alternative splicing (v2–v10) and contain any combination of the 10 CD44s exons and the remaining nine variant exons. The CD44v isoform plays diverse roles in cancer and may affect key aspects of carcinogenesis, such as tumor initiation and metastasis. These important features are usually attributed to altered signal transduction, oxidative stress resistance, chemotherapy / radiation resistance, adhesion, interaction with the extracellular matrix, tumor matrix formation, or regulation of the CSC niche. Furthermore, CD44 variants have been shown to be important for Ras activation through the formation of a coreceptor complex with hepatocyte growth factor (HGF) and its receptor tyrosine kinase Met, and phosphorylation of this coreceptor complex activates Ras signaling. Furthermore, some isoforms exhibit anti-apoptotic effects and can block Fas-mediated apoptosis.

[0003] One of these isoforms, CD44v9, named for the splice variant containing the v9 exon, is overexpressed in various solid tumors, including (but not limited to) head and neck cancer, non-small cell lung cancer, esophageal cancer, pancreatic cancer, colorectal cancer, breast cancer, bladder cancer, and gastric cancer, and is also present in limited normal tissues. In a meta-analysis study, CD44v9 expression was associated with larger tumor size, deeper tumor invasion, earlier lymph node metastasis, and more advanced clinical stage compared with data from CD44v9-negative patients. These properties make CD44v9 a promising target for the development of potential antibody-based cancer therapeutics.

[0004] Major categories of CD44-targeting therapies include peptide mimetics, aptamers, pharmacological inhibitors, CD44 decoys, HA oligomers, HyACT (hyaluronic acid transporter), neutralizing antibodies, and antibody-drug conjugates. Monoclonal CD44-specific antibodies that interfere with HA binding to CD44 can inhibit anchorage-independent growth and induce apoptosis in mouse breast cancer cells and human colon cancer cells. HA oligosaccharides also have a similar effect, inhibiting tumor growth in vivo, presumably by interfering with the binding of hHA (high molecular weight HA, approximately 2000 kDa) to CD44. Furthermore, CD44 antibodies have been used as targeting agents for tumor-specific delivery of radiotherapy or cytotoxic payloads. Several of these therapeutic compounds are currently being tested in clinical trials.

[0005] For example, a randomized phase II study of hyaluronic acid-irinotecan (HA-IR) demonstrated improved progression-free survival compared with irinotecan in patients with metastatic colorectal cancer, but unfortunately, this result was not confirmed in a subsequent phase III trial. Another phase IIa study of the combination of HA-irinotecan and carboplatin compared with irinotecan and carboplatin in extensive-stage small cell lung cancer (SCLC) was conducted. The study concluded that while the treatment was well tolerated, there was a trend toward improved survival in CD44s-positive tumors, but no statistically significant difference was observed between the treatment and control groups. Regarding direct targeting of CD44 and specific variants, the humanized antibody RG7356, which binds to the HA-binding proximal domain of all CD44 isoforms, has been tested in clinical trials for acute myeloid leukemia (AML) and advanced solid tumors. It demonstrated limited clinical efficacy but was generally well tolerated. A phase I trial of the 186Re-labeled chimeric monoclonal antibody U36, specific for CD44v6, was conducted in patients with head and neck squamous cell carcinoma (HNSCC). This study demonstrated that the U36 radioconjugate can be safely administered with dose-limiting bone marrow toxicity and provides a transient therapeutic response. Although the above CD44-targeted drug therapy demonstrated modest clinical efficacy results, it was generally well tolerated, and several accompanying imaging studies suggest that tumor-specific targeting was achieved. However, a phase I trial of the humanized monoclonal antibody (mAb) bivatuzumab (BIWA-4) in combination with the potent microtubule inhibitor mertansine, which specifically targets CD44v6, in patients with HNSCC and metastatic breast cancer, yielded different clinical results. At higher dose levels, partial responses were seen in three patients, but these clinical trials were discontinued due to significant skin toxicity, including one fatal outcome, attributed to the bivatuzumab-mertansine conjugate.

[0006] In summary, although numerous anticancer drugs targeting CD44 have been investigated in clinical trials, no active ingredients with satisfactory therapeutic efficacy and sufficient safety have been successfully developed, and none of the compounds investigated in clinical trials target CD44v9. To date, no antibody-drug conjugates (ADCs) targeting CD44v9 have been approved or investigated in clinical trials. Specific information regarding the druggability of CD44v9 anticancer drugs, especially ADCs, such as their efficacy, safety, pharmacokinetic properties, and therapeutic window, remains unclear and requires further research and establishment. Summary of the Invention

[0007] In one aspect, an antibody-drug conjugate is provided having the structure of formula (I): (TL) γ -mAb (I) where: T is a cytotoxic drug moiety having the structure of formula (II): [ka] where R is -C(=O)R x , -S(=O)R x or -S(=O)2R x where R x is C 1~6 Alkyl groups, halogenated C 1~6 Alkyl group, C 2~6 Alkenyl group, C 2~6 Alkynyl group, C 3~6 Cycloalkyl groups, C 3~6 R is selected from the group consisting of a heterocyclyl group, a 6- to 10-membered aryl group, and a 5- to 14-membered heteroaryl group, and preferably R is —C(═O)R x or -S(=O)2R x where R x is C 1~6 Alkyl groups, halogenated C 1~6 Alkyl group, C 3~6 Cycloalkyl groups, C 3~6R is selected from the group consisting of a heterocyclyl group, a 6- to 10-membered aryl group, and a 5- to 14-membered heteroaryl group, and more preferably R is —C(═O)R x or -S(=O)2R x where R x is C 1~6 Alkyl group, C 3~6 selected from the group consisting of cycloalkyl groups and phenyl groups; L is a linker, γ is an integer or decimal number from 1 to 10, preferably from 5 to 8, and A mAb is an antibody portion, wherein the antibody is an anti-CD44v9 antibody or an antigen-binding fragment thereof.

[0008] In some embodiments, the linker has the structure of formula (III): -L1-(L2)m1-(L3)m2-(L4)m3-EG- (III) wherein L1 is bound to T and G is bound to the mAb, and wherein L1 is Lys, Cit, Cit-Val, Val-Ala, Lys-Val, [ka] wherein R', R1 and R2 are each independently selected from the group consisting of (hydrogen), D (deuterium) or C 1~4 an alkyl group, Z1 is Cit, Lys, Cit-Val, Cit-Ala, Val-Ala, or Lys-Val, x1 and x3 are each independently 0, 1, or 2, and L1 is bonded to T at the 1-position of L1, preferably L1 is Lys, Cit, Cit-Val, Val-Ala, Lys-Val, [ka] and more preferably, L1 is selected from the group consisting of [ka] and L2 is selected from the group consisting of: [ka] wherein R3, R4, R5 and R6 each independently represent H (hydrogen), D (deuterium) or C 1~4 alkyl groups, y1 and y2 are each independently 0, 1, 2, 3, 4, 5, 6, 7, or 8, and L2 is bonded to L1 at the 1-position of L2, preferably L2 is selected from the group consisting of: [ka] More preferably, L2 is: [ka] L3 is selected from the group consisting of an amino group, an N-methylpiperidine group, a pyrazolylene group, or a triazolylene group, optionally substituted by one or more R7, wherein each R7 is H (hydrogen), D (deuterium), a halogen, =O, CF3, CN, CH2CN, a carboxy group, a sulfonic acid group, C 1~4 Alkyl group, C 1~4 Alkoxy group, C 2~6 Alkenyl group or C 2~6 L3 is independently selected from alkynyl groups, preferably a triazolylene group having the structure: [ka] and L3 is bound to L2 at position 1, L4 is selected from the group consisting of: [ka] wherein L4 is attached to E at position 2 of L4, and preferably L4 is selected from the group consisting of: [ka] More preferably, L4 is: [ka] E optionally comprises one or more R 12 wherein each R is selected from the group consisting of a pyrimidylene group, a quinolylene group, or a pyrrolo[2,3-d]pyrimidylene group substituted by 12 is H (hydrogen), D (deuterium), halogen, CN, nitro group, C 1~2 Alkyl or halogenated C 1~2 alkyl groups, and preferably E is optionally one or more R 12 wherein each R 12 are independently H (hydrogen) or D (deuterium), G is a thioether bond, and m1 is 0, 1, 2 or 3, preferably m1 is 1, m2 is 0, 1 or 2, preferably m2 is 0 or 1, and m3 is 0, 1, 2 or 3, preferably m3 is 1.

[0009] In some embodiments, the cytotoxic drug moiety, T, is selected from the group consisting of: [ka] Preferably, the cytotoxic drug moiety is: [ka] And more preferably, the cytotoxic drug moiety is: [ka]

[0010] In a preferred embodiment, the antibody-drug conjugate has a structure selected from the group consisting of: [ka] [ka] [ka] [ka] [ka] [ka] where γ is an integer or decimal number between 5 and 8, and the mAb is an anti-CD44v9 antibody or antigen-binding fragment thereof.

[0011] In some embodiments, G of the linker moiety is formed from a thiol group of a Cys residue in the mAb, and preferably, the thiol group of the Cys residue in the mAb is a thiol group formed by opening a disulfide bond in the mAb.

[0012] In some embodiments, the mAb binds to a CD44v9 epitope comprising or consisting essentially of SEQ ID NO:55 or 56.

[0013] In some embodiments, the mAb comprises a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3, and a light chain variable region comprising LCDR1, LCDR2, and LCDR3, wherein HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are the same as any of (a)-(d); (a) HCDR1, HCDR2, and HCDR3 of a heavy chain variable region of SEQ ID NO: 47 and LCDR1, LCDR2, and LCDR3 of a light chain variable region of SEQ ID NO: 48; (b) HCDR1, HCDR2, and HCDR3 of a heavy chain variable region of SEQ ID NO: 49 and LCDR1, LCDR2, and LCDR3 of a light chain variable region of SEQ ID NO: 50; (c) HCDR1, HCDR2, and HCDR3 of a heavy chain variable region of SEQ ID NO: 51 and LCDR1, LCDR2, and LCDR3 of a light chain variable region of SEQ ID NO: 52; and (d) HCDR1, HCDR2, and HCDR3 of a heavy chain variable region of SEQ ID NO: 53 and LCDR1, LCDR2, and LCDR3 of a light chain variable region of SEQ ID NO: 54, and Here, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are preferably defined according to any of the CDR definition schemes selected from the group consisting of Chothia, Kabat, IMGT, AbM and Contact.

[0014] In some embodiments, the mAb (a) a heavy chain variable region comprising an HCDR1 having the amino acid sequence of SEQ ID NO: 1, an HCDR2 having the amino acid sequence of SEQ ID NO: 2, and an HCDR3 having the amino acid sequence of SEQ ID NO: 3, and a light chain variable region comprising an LCDR1 having the amino acid sequence of SEQ ID NO: 4, an LCDR2 having the amino acid sequence of SEQ ID NO: 5, and an LCDR3 having the amino acid sequence of SEQ ID NO: 6, according to the CDR definition of IMGT; or (b) A heavy chain variable region comprising an HCDR1 having the amino acid sequence of SEQ ID NO: 31, an HCDR2 having the amino acid sequence of SEQ ID NO: 32, and an HCDR3 having the amino acid sequence of SEQ ID NO: 33, according to the CDR definition of IMGT; and a light chain variable region comprising an LCDR1 having the amino acid sequence of SEQ ID NO: 34, an LCDR2 having the amino acid sequence of SEQ ID NO: 35, and an LCDR3 having the amino acid sequence of SEQ ID NO: 36.

[0015] In some embodiments, the mAb (a) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 47 and a light chain variable region having the amino acid sequence of SEQ ID NO: 48; (b) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 49 and a light chain variable region having the amino acid sequence of SEQ ID NO: 50; (c) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 51 and a light chain variable region having the amino acid sequence of SEQ ID NO: 52; or (d) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 53 and a light chain variable region having the amino acid sequence of SEQ ID NO: 54.

[0016] In some embodiments, the mAb comprises a heavy chain constant region having the amino acid sequence of SEQ ID NO:45 and a light chain constant region having the amino acid sequence of SEQ ID NO:46.

[0017] In a preferred embodiment, an antibody drug conjugate is provided that is selected from the group consisting of: [ka] [ka] [ka] [ka] wherein γ is an integer or decimal number from 5 to 8, and the mAb is an anti-CD44v9 monoclonal antibody or an antigen-binding fragment thereof, wherein the mAb is linked via a thiol group of a Cys residue in the mAb, and the mAb comprises a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3, and a light chain variable region comprising LCDR1, LCDR2, and LCDR3, and wherein HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are the same as any of (a) to (d); (a) HCDR1, HCDR2, and HCDR3 of a heavy chain variable region of SEQ ID NO: 47 and LCDR1, LCDR2, and LCDR3 of a light chain variable region of SEQ ID NO: 48; (b) HCDR1, HCDR2, and HCDR3 of a heavy chain variable region of SEQ ID NO: 49 and LCDR1, LCDR2, and LCDR3 of a light chain variable region of SEQ ID NO: 50; (c) HCDR1, HCDR2, and HCDR3 of a heavy chain variable region of SEQ ID NO: 51 and LCDR1, LCDR2, and LCDR3 of a light chain variable region of SEQ ID NO: 52; and (d) HCDR1, HCDR2, and HCDR3 of a heavy chain variable region of SEQ ID NO: 53 and LCDR1, LCDR2, and LCDR3 of a light chain variable region of SEQ ID NO: 54, and Here, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are preferably defined according to any of the CDR definition schemes selected from the group consisting of Chothia, Kabat, IMGT, AbM and Contact.

[0018] In a preferred embodiment, an antibody-drug conjugate is provided having the structure: [ka] wherein γ is an integer or decimal number between 5 and 8, and the mAb is an anti-CD44v9 monoclonal antibody or an antigen-binding fragment thereof, wherein the thioether bond linked to the mAb is formed from the thiol group of a Cys residue in the mAb.

[0019] In some embodiments, the thiol group of a Cys residue in the mAb is a thiol group formed by opening a disulfide bond in the mAb, or is a free Cys residue contained in the mAb.

[0020] In some embodiments, the mAb specifically binds to a CD44v9 epitope comprising or consisting essentially of SEQ ID NO:55 or 56.

[0021] In some embodiments, the mAb comprises a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3, and a light chain variable region comprising LCDR1, LCDR2, and LCDR3, wherein HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are the same as any of (a)-(d); (a) HCDR1, HCDR2, and HCDR3 of a heavy chain variable region of SEQ ID NO: 47 and LCDR1, LCDR2, and LCDR3 of a light chain variable region of SEQ ID NO: 48; (b) HCDR1, HCDR2, and HCDR3 of a heavy chain variable region of SEQ ID NO: 49 and LCDR1, LCDR2, and LCDR3 of a light chain variable region of SEQ ID NO: 50; (c) HCDR1, HCDR2, and HCDR3 of a heavy chain variable region of SEQ ID NO: 51 and LCDR1, LCDR2, and LCDR3 of a light chain variable region of SEQ ID NO: 52; and (d) HCDR1, HCDR2, and HCDR3 of a heavy chain variable region of SEQ ID NO: 53 and LCDR1, LCDR2, and LCDR3 of a light chain variable region of SEQ ID NO: 54, and Here, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are preferably defined according to any of the CDR definition schemes selected from the group consisting of Chothia, Kabat, IMGT, AbM and Contact.

[0022] In some embodiments, the mAb (a) a heavy chain variable region comprising an HCDR1 having the amino acid sequence of SEQ ID NO: 1, an HCDR2 having the amino acid sequence of SEQ ID NO: 2, and an HCDR3 having the amino acid sequence of SEQ ID NO: 3, and a light chain variable region comprising an LCDR1 having the amino acid sequence of SEQ ID NO: 4, an LCDR2 having the amino acid sequence of SEQ ID NO: 5, and an LCDR3 having the amino acid sequence of SEQ ID NO: 6, according to the CDR definition of IMGT; or (b) A heavy chain variable region comprising an HCDR1 having the amino acid sequence of SEQ ID NO: 31, an HCDR2 having the amino acid sequence of SEQ ID NO: 32, and an HCDR3 having the amino acid sequence of SEQ ID NO: 33, according to the CDR definition of IMGT; and a light chain variable region comprising an LCDR1 having the amino acid sequence of SEQ ID NO: 34, an LCDR2 having the amino acid sequence of SEQ ID NO: 35, and an LCDR3 having the amino acid sequence of SEQ ID NO: 36.

[0023] In some embodiments, the mAb (a) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 47 and a light chain variable region having the amino acid sequence of SEQ ID NO: 48; (b) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 49 and a light chain variable region having the amino acid sequence of SEQ ID NO: 50; (c) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 51 and a light chain variable region having the amino acid sequence of SEQ ID NO: 52; or (d) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 53 and a light chain variable region having the amino acid sequence of SEQ ID NO: 54.

[0024] In some embodiments, the mAb comprises a heavy chain constant region having the amino acid sequence of SEQ ID NO:45 and a light chain constant region having the amino acid sequence of SEQ ID NO:46.

[0025] In another aspect, there is provided a pharmaceutical composition for treating cancer comprising an antibody-drug conjugate disclosed herein and a pharmaceutically acceptable carrier.

[0026] In a further aspect, there is provided a method of treating cancer in a subject, comprising administering to the subject an effective amount of an antibody drug conjugate disclosed herein.

[0027] In a further aspect, there is provided an antibody drug conjugate as disclosed herein for use in the treatment of cancer.

[0028] In a further aspect, there is provided the use of an antibody-drug conjugate disclosed herein in the manufacture of a medicament for the treatment of cancer. [Effects of the Invention]

[0029] (Advantages of the present invention) The antibody-drug conjugates of the present disclosure specifically target CD44v9 and induce cytotoxicity in cells that ectopically express CD44v9, while causing no obvious toxicity or side effects in cells that normally express CD44v9.

[0030] The antibody drug conjugates of the present disclosure have shown promising preclinical in vivo efficacy in multiple solid tumor models, including but not limited to NSCLC (non-small cell lung cancer), HNSCC (head and neck squamous cell carcinoma), ESCC (esophageal squamous cell carcinoma), GC (gastric cancer), BC (bladder cancer), TNBC (triple-negative breast cancer), and liver cancer.

[0031] The antibody-drug conjugate hHTS033-KL of the present disclosure was well tolerated in a non-human primate toxicology study (HNSTD = 30 mg / kg) with no significant dermal toxicity observed. hHTS033-KL may have a wide therapeutic window in clinical trials and may benefit cancer patients with high unmet medical needs. [Brief explanation of the drawings]

[0032] [Figure 1] RP-HPLC chromatograph of hHTS033-KL. [Figure 2] SEC chromatograph of hHTS033-KL. [Figure 3] Binding affinity of hHTS033-KL in tumor cell lines. [Figure 4] Internalization of hHTS033-KL in tumor cell lines. [Figure 5] 1 shows tumor growth curves for ADC drug studies using a mouse subcutaneous MDA-MB-468 model. [Figure 6] 1 shows the change curve of the average body weight of tumor-bearing mice in the subcutaneous MDA-MB-468 CDX mouse model. [Figure 7] 1 shows tumor growth curves for ADC drug studies using a mouse subcutaneous 5637 model. [Figure 8] 1 shows the change curve of the average body weight of tumor-bearing mice in the subcutaneous 5637 CDX mouse model. [Figure 9] Tumor growth curve of LD1-0025-361336 PDX model. [Figure 10] Relative change in body weight after treatment in the LD1-0025-361336 PDX model. [Figure 11] Tumor growth curve of the LD1-0017-361443 gastric cancer PDX model. [Figure 12] Relative change in body weight after treatment in the LD1-0017-361443 gastric cancer PDX model. [Figure 13]Tumor growth curve of liver cancer PDX model LD1-0011-360763. [Figure 14] Relative change in body weight after treatment in liver cancer PDX model LD1-0011-360763. DETAILED DESCRIPTION OF THE INVENTION

[0033] (definition) It should be noted that the term "one" or "one kind" of entity refers to one or more of that entity. For example, "truncated non-signaling variant" is understood to refer to one or more truncated non-signaling variants. Thus, the terms "one" (or "one kind"), "one or more," and "at least one" are used interchangeably herein.

[0034] As used herein, the term "antibody-drug conjugate" refers to a compound in which an antibody or antigen-binding fragment thereof is linked to a cytotoxic drug via a linker. In the context of the present disclosure, the term "antibody-drug conjugate," or alternatively "conjugate," specifically refers to a compound in which an anti-CD44v9 antibody or antigen-binding fragment thereof is linked to a belotecan derivative via a linker.

[0035] As used herein, the term "linker" refers to a fragment that connects a cytotoxic drug moiety and an antibody moiety. The linker may be cleavable or non-cleavable. For example, the linker is cleaved under certain circumstances (e.g., the low pH environment within a cell) or under certain actions (e.g., the action of a lysosomal protease), thereby dissociating the cytotoxic drug moiety from the antibody moiety.

[0036] As used herein, the term "antibody" refers to a complete monoclonal antibody or a multispecific antibody (e.g., a bispecific antibody). Thus, "anti-CD44v9 antibody" includes a complete anti-CD44v9 monoclonal antibody and a complete multispecific antibody having one specificity for CD44v9 and a remaining specificity for one or more different antigens (e.g., tumor-specific or tumor-associated antigens) or different epitopes of the same antigen.

[0037] As used herein, the term "monoclonal antibody" refers to an antibody that belongs to a substantially homogeneous antibody population. That is, the antibodies in the population are identical except for a few possible natural variations. Monoclonal antibodies have high specificity for a single determinant (epitope) on an antigen, whereas comparable polyclonal antibodies contain different antibodies for different determinants (epitopes). In addition to specificity, monoclonal antibodies also have the advantage of being free from contamination by other antibodies during synthesis. In the context of this disclosure, monoclonal antibodies specifically include chimeric antibodies. Chimeric antibodies are antibodies in which a portion of the heavy and / or light chain is identical or homologous to an antibody of one type, class, or subclass, and the remaining portion is identical or homologous to an antibody of another type, class, or subclass, provided that the required biological activity is maintained. Chimeric antibodies that can be used in this disclosure include antibodies containing variable region antigen-binding sequences derived from a rodent (e.g., rat or mouse) and human constant region sequences.

[0038] The term "multispecific antibody" includes, for example, bispecific antibodies and trispecific antibodies, which are antibodies with two or three different antigen-binding specificities, respectively. Similarly, tetraspecific antibodies are antibodies with four different antigen-binding specificities. Various forms of multispecific antibodies are available in the art, including, but not limited to, BiTE, DART, Trimab, CrossMab, knob-in-hole IgG common LC, tandAbs, IgG-scFv, scFv2-Fc, etc.

[0039] The term "bispecific antibody," also known as a "bifunctional antibody conjugate," refers to an antibody formed by combining a first antibody (fragment) and a second antibody (fragment) via their binding arms. Bispecific antibodies retain the activity of each antibody, thus possessing dual functionality and specificity. Currently under investigation, dual anti-ADCs can be divided into two main categories: bispecific ADCs that target dual tumor-associated antigens (TAAs) (e.g., MUC1 / EGFR-ADCs) and bispecific ADCs that target dual epitopes (e.g., HER2 / HER2-ADCs).

[0040] As used herein, the term "antigen" refers to a molecule that induces an immune response and thereby stimulates the production of antibodies. Typical antigens are proteins present on the surface or extracellular domains or portions thereof of pathogens such as bacteria, fungi, viruses, and other foreign particles. When these harmful substances enter the body, they induce an immune response in the body to produce antibodies.

[0041] The term "intact antibody" refers to an antibody that has not undergone cleavage and may vary depending on the class to which the antibody belongs. Intact antibodies can be classified into different "classes" based on the amino acid sequence of their heavy chain constant region. The five major classes are IgA, IgD, IgE, IgG, and IgM, with some of these further divided into different "subclasses" (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. For example, an IgG-type intact antibody is an antibody that contains a heavy chain variable region (VH), a light chain variable region (VL), a light chain constant region (CL), and a heavy chain constant region (CH, including CH1, CH2, and CH3). The variable and constant regions are either native (e.g., human native constant region sequences) or humanized (e.g., humanized sequences from mouse sequences by CDR grafting). However, in the case of heavy chain antibodies, an intact antibody is an antibody that contains only VH, CH2, and CH3.

[0042] The terms "antibody fragment" or "antigen-binding fragment" of an antibody, used interchangeably herein, refer to a fragment of an intact antibody that contains the antigen-binding region of the antibody. Examples of antibody fragments include Fab, Fab', F(ab')2, Fv, scFv, etc. Regardless of structure, an antibody fragment binds to the same antigen that is recognized by the intact antibody. The term "antibody fragment" also includes aptamers, spiegelmers, and diabodies. The term "antibody fragment" also includes any synthetic or genetically engineered protein that acts like an antibody by binding to a specific antigen to form a complex.

[0043] "Specifically binds," "specific for," or "having specificity for" generally mean that an antibody binds to an epitope via its antigen-binding domain, and that the binding involves some complementarity between the antigen-binding domain and the epitope. According to this definition, an antibody is said to "specifically bind" to an epitope if it binds to that epitope via its antigen-binding domain more readily than it would bind to a random, unrelated epitope. As used herein, the term "specificity" is used to refer to the relative affinity of a particular antibody for binding to a particular epitope. For example, antibody "A" may be considered to have higher specificity for a given epitope than antibody "B," and antibody "A" may be said to bind epitope "C" with greater specificity than to the related epitope "D."

[0044] "Cancer" or "tumor," as used interchangeably herein, refer to a group of diseases involving abnormal cell growth that can invade or spread to other parts of the body and that can be treated according to the present disclosure. Not all tumors are cancerous; benign tumors do not spread to other parts of the body. Possible signs and symptoms include a new lump, abnormal bleeding, persistent cough, unexplained weight loss, and changes in bowel movements. There are over 100 known types of cancer that affect humans. As used herein, "cancer" includes, but is not limited to, solid cancers (e.g., tumors) and hematologic malignancies. "Hematologic malignancies," also known as blood cancers, are cancers that originate in blood-forming tissues, such as the bone marrow, or other cells of the immune system. Hematological malignancies include, but are not limited to, leukemias (acute myeloid leukemia (ANIL), acute promyelocytic leukemia, acute lymphoblastic leukemia (ALL), acute mixed lineage leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia (CLL), hairy cell leukemia and large granular lymphocytic leukemia, myelodysplastic syndromes (MDS), myeloproliferative disorders (polycythemia vera, essential thrombocytosis, primary myelofibrosis and chronic myelogenous leukemia), lymphomas, multiple myeloma, MGUS and similar disorders, Hodgkin's lymphoma, non-Hodgkin's lymphoma (NHL), primary mediastinal large B-cell lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, transformed follicular lymphoma, splenic marginal zone lymphoma, lymphocytic lymphoma, T-cell lymphoma, and other B-cell malignancies."Solid cancers" include fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, chordoma, angiosarcoma, endothelial tumor, lymphangiosarcoma, lymphangioendothelial tumor, synovium, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, colorectal cancer, kidney cancer, pancreatic cancer, bone cancer, breast cancer, ovarian cancer, prostate cancer, esophageal cancer, stomach cancer, oral cancer, nasal cancer, pharyngeal cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland cancer, sebaceous gland cancer, papillary carcinoma, and papillary adenocarcinoma. These include, but are not limited to, cystadenocarcinoma, medullary carcinoma, bronchial carcinoma, renal cell carcinoma, hepatocellular carcinoma, cholangiocarcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, uterine cancer, testicular cancer, small cell lung cancer, bladder cancer, lung cancer, epithelial carcinoma, glioma, glioblastoma multiforme, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, skin cancer, melanoma, neuroblastoma, and retinoblastoma.

[0045] As used herein, the term "treatment" refers to both therapeutic treatment and prophylactic or preventative measures, the purpose of which is to prevent or slow (alleviate) an undesirable physiological change or disorder, such as the progression of cancer. Beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, reduction in the extent of disease, a state of stabilized disease (i.e., not worsening), delay or slowing of disease progression, improvement or palliation of the disease state, and remission (partial or complete), whether detectable or undetectable. "Treatment" can also mean prolonging survival as compared to expected survival in the absence of treatment. Subjects in need of treatment include those already suffering from a condition or disorder, as well as those susceptible to a condition or disorder, or those in whom a condition or disorder is to be prevented.

[0046] As used herein, phrases such as "patient in need of treatment" or "subject in need of treatment" include subjects, such as mammalian subjects, who would benefit from the administration of a conjugate or composition of the present disclosure for use in detection, diagnostic procedures, and / or treatment.

[0047] A "therapeutically effective amount" or "effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic effect. A therapeutically effective amount may vary depending on factors such as the individual's medical condition, age, sex, and weight, and the ability of the therapeutic agent or combination of therapeutic agents to elicit a desired response in the individual. Indicators of an effective therapeutic agent or combination of therapeutic agents include, for example, an improvement in the patient's health, a decrease in tumor burden, a halt or slowing of tumor growth, and / or a lack of metastasis of cancer cells to other sites in the body.

[0048] A "conservative mutation" refers to changing one or more amino acid residues to homologous residues (e.g., isoleucine to leucine, aspartic acid to glutamic acid, or cysteine ​​to serine), and is not expected to result in significant changes to the protein. Additionally, nucleotide or amino acid substitutions, deletions, or insertions leading to conservative mutations or changes in "non-essential" amino acid regions may be made. For example, a polypeptide or amino acid sequence derived from a designated protein may be identical to the starting sequence except for one or more individual amino acid substitutions, insertions, or deletions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or more individual amino acid substitutions, insertions, or deletions). In certain embodiments, a polypeptide or amino acid sequence derived from a designated protein has 1 to 5, 1 to 10, 1 to 15, or 1 to 20 individual amino acid substitutions, insertions, or deletions relative to the starting sequence.

[0049] The abbreviation "CDR" stands for "complementarity-determining region," and is the portion of the variable chain of immunoglobulins (antibodies) produced by B cells and T cell receptors produced by T cells where these molecules bind to specific antigens. Each heavy and light chain variable region contains three CDRs, designated in the present disclosure as HCDR1, HCDR2, and HCDR3 (for heavy chains) and LCDR1, LCDR2, and LCDR3 (for light chains). A total of six CDRs from both the light and heavy chain variable regions are required to recognize and bind to an antigen. However, nanobodies or heavy chain antibodies require only three CDRs in the heavy chain variable region to recognize and bind to an epitope on a specific antigen. Each of the heavy and light chain variable regions can be represented as FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 (N-terminus to C-terminus), where the CDR regions are distributed among four less variable framework regions (FR), i.e., FR1, FR2, FR3 and FR4.

[0050] Various methods and tools are available in the art for predicting antibody CDRs according to various CDR definition schemes, including, but not limited to, Chothia, Kabat, IMGT, AbM, and Contact. The Kabat definition is based on sequence variability and is the most commonly used. The Chothia definition is based on the location of structural loop regions. The Contact definition is based on analysis of available complex crystal structures. This definition is considered most useful for practitioners who wish to perform mutagenesis to alter the affinity of their antibodies, as these are the residues involved in interactions with the antigen. Various online tools are available that automatically annotate the CDRs of antibodies submitted to the tool's webpage. Defining the CDRs within a variable region defines FR1, FR2, FR3, and FR4 within that variable region.

[0051] "Subject" or "individual" or "animal" or "patient" or "mammal" refers to any subject for whom diagnosis, prognosis, or treatment is desired, particularly a mammalian subject. Mammalian subjects include humans, livestock, farm animals, and zoo, sport, or pet dogs, cats, guinea pigs, rabbits, rats, mice, horses, cows, and the like.

[0052] As used herein, "C 1~6 The term "alkyl group" refers to a straight or branched chain alkyl group containing 1 to 6 carbon atoms, e.g., "C 1~4 alkyl group," "C 1~3 Alkyl group" and "C 1~2 Specific examples include, but are not limited to, a methyl group, an ethyl group, an n-propyl group, an isopropyl group, an n-butyl group, an isobutyl group, a sec-butyl group, a tert-butyl group, an n-pentyl group, an isopentyl group, a 2-methylbutyl group, a neopentyl group, a 1-ethylpropyl group, an n-hexyl group, an isohexyl group, a 3-methylpentyl group, a 2-methylpentyl group, a 1-methylpentyl group, a 3,3-dimethylbutyl group, a 2,2-dimethylbutyl group, a 1,1-dimethylbutyl group, a 1,2-dimethylbutyl group, a 1,3-dimethylbutyl group, a 2,3-dimethylbutyl group, a 2-ethylbutyl group, and a 1,2-dimethylpropyl group.

[0053] As used herein, "C 2~6 The term "alkenyl group" refers to a straight, branched or cyclic alkenyl containing at least one double bond and 2 to 6 carbon atoms, e.g., "C 2~4Examples of alkenyl groups include, but are not limited to, ethenyl, 1-propenyl, 2-propenyl, 1-butenyl, 2-butenyl, 1,3-butadienyl, 1-pentenyl, 2-pentenyl, 3-pentenyl, 1,3-pentadienyl, 1,4-pentadienyl, 1-hexenyl, 2-hexenyl, 3-hexenyl, 1,4-hexadienyl, cyclopentenyl, 1,3-cyclopentadienyl, cyclohexenyl, and 1,4-cyclohexadienyl.

[0054] As used herein, "C 2~6 The term "alkynyl group" refers to a straight or branched chain alkynyl group containing at least one triple bond and 2 to 6 carbon atoms, e.g., "C 2~4 Examples of alkynyl groups include, but are not limited to, ethynyl, propynyl, 2-butynyl, 2-pentynyl, 3-pentynyl, 4-methyl-2-pentynyl, 2-hexynyl, 3-hexynyl, and 5-methyl-2-hexynyl groups.

[0055] As used herein, the term "halogen" includes fluorine, chlorine, bromine and iodine.

[0056] As used herein, "C 3~6 The term "cycloalkyl group" refers to a saturated cyclic alkyl group containing 3 to 6 carbon atoms. Specific examples include, but are not limited to, a cyclopropyl group, a cyclobutyl group, a cyclopentyl group, and a cyclohexyl group.

[0057] The term "3- to 6-membered heterocyclyl group" as used herein refers to a cyclic group containing 3 to 6 ring-forming atoms, at least one of which is a heteroatom such as a nitrogen atom, oxygen atom, or sulfur atom. Examples include, but are not limited to, oxiranyl, oxocyclobutyl, pyrrolidinyl, tetrahydrofuranyl, piperidinyl, and piperazinyl groups.

[0058] As used herein, "C 1~6 The term "alkoxy group" refers to 1~6 It refers to a group having the structure alkyl-O-, where C 1~6 The alkyl group is as defined above. Specific examples include, but are not limited to, a methoxy group, an ethoxy group, a propoxy group, an isopropoxy group, an n-propoxy group, an isopropoxy group, an n-butoxy group, an isobutoxy group, a tert-butoxy group, a pentoxy group, and a hexyloxy group.

[0059] As used herein, the term "6- to 10-membered aryl group" refers to a monocyclic or polycyclic hydrocarbon containing 6 to 10 ring atoms and having aromatic character, such as a phenyl group and a naphthyl group.

[0060] The term "5- to 14-membered heteroaryl group" as used herein refers to a cyclic group having aromaticity and containing 5 to 14 ring atoms, wherein at least one ring atom is a heteroatom such as a nitrogen atom, an oxygen atom, or a sulfur atom. Specific examples include a furyl group, a thienyl group, a pyrrolyl group, a thiazolyl group, an isothiazolyl group, a thiadiazolyl group, an oxazolyl group, an isoxazolyl group, an oxadiazolyl group, an imidazolyl group, a pyrazolyl group, a 1,2,3-triazolyl group, a 1,2,4-triazolyl group, a 1,2,3-oxadiazolyl group, a 1,2,4-oxadiazolyl group, a 1,2,5-oxadiazolyl group, a 1,3,4-oxadiazolyl group, a pyridyl group, a 2-pyridone group, a 4-pyridone ... Examples of the aryl group include, but are not limited to, cyclohexyl, cyclohexyl, cyclopentyl, cyclopentyl, cyclohexyl, cyclohexyl groups ...

[0061] (Anti-CD44v9 antibody) The antibody useful in the antibody drug conjugates described herein is an anti-CD44v9 antibody or an antigen-binding fragment thereof.

[0062] In some embodiments, the anti-CD44v9 antibody specifically binds to CD44v9 expressed on tumor cells and is internalized by tumor cells.

[0063] In some embodiments, the anti-CD44v9 antibody binds to an epitope of CD44v9 comprising or consisting essentially of the amino acid sequence set forth in SEQ ID NO: 55 or 56. In some embodiments, the anti-CD44v9 antibody binds to an epitope of CD44v9 consisting of the amino acid sequence set forth in SEQ ID NO: 55. In some embodiments, the anti-CD44v9 antibody binds to an epitope of CD44v9 consisting of the amino acid sequence set forth in SEQ ID NO: 56.

[0064] In some embodiments, the anti-CD44v9 antibody comprises a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3, and a light chain variable region comprising LCDR1, LCDR2, and LCDR3, wherein HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are the same as any of (a)-(d); (a) HCDR1, HCDR2, and HCDR3 of a heavy chain variable region of SEQ ID NO: 47 and LCDR1, LCDR2, and LCDR3 of a light chain variable region of SEQ ID NO: 48; (b) HCDR1, HCDR2, and HCDR3 of a heavy chain variable region of SEQ ID NO: 49 and LCDR1, LCDR2, and LCDR3 of a light chain variable region of SEQ ID NO: 50; (c) HCDR1, HCDR2, and HCDR3 of a heavy chain variable region of SEQ ID NO: 51 and LCDR1, LCDR2, and LCDR3 of a light chain variable region of SEQ ID NO: 52; and (d) HCDR1, HCDR2, and HCDR3 of a heavy chain variable region of SEQ ID NO: 53 and LCDR1, LCDR2, and LCDR3 of a light chain variable region of SEQ ID NO: 54, and Here, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are preferably defined according to any of the CDR definition schemes selected from the group consisting of Chothia, Kabat, IMGT, AbM and Contact.

[0065] In some embodiments, the anti-CD44v9 antibody comprises a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3, and a light chain variable region comprising LCDR1, LCDR2, and LCDR3, wherein HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are the same as HCDR1, HCDR2, and HCDR3 of the heavy chain variable region of SEQ ID NO: 49, and LCDR1, LCDR2, and LCDR3 of the light chain variable region of SEQ ID NO: 50, and wherein HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are preferably defined according to any of the CDR definition schemes selected from the group consisting of Chothia, Kabat, IMGT, AbM, and Contact.

[0066] In some embodiments, the anti-CD44v9 antibody comprises a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3, and a light chain variable region comprising LCDR1, LCDR2, and LCDR3, wherein HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are the same as HCDR1, HCDR2, and HCDR3 of the heavy chain variable region of SEQ ID NO: 51, and LCDR1, LCDR2, and LCDR3 of the light chain variable region of SEQ ID NO: 52, and wherein HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are preferably defined according to any of the CDR definition schemes selected from the group consisting of Chothia, Kabat, IMGT, AbM, and Contact.

[0067] In a preferred embodiment, the anti-CD44v9 antibody comprises a heavy chain variable region comprising an HCDR1 having the amino acid sequence of SEQ ID NO: 1, an HCDR2 having the amino acid sequence of SEQ ID NO: 2, and an HCDR3 having the amino acid sequence of SEQ ID NO: 3, according to the CDR definitions of IMGT; and a light chain variable region comprising an LCDR1 having the amino acid sequence of SEQ ID NO: 4, an LCDR2 having the amino acid sequence of SEQ ID NO: 5, and an LCDR3 having the amino acid sequence of SEQ ID NO: 6.

[0068] In a preferred embodiment, the anti-CD44v9 antibody comprises a heavy chain variable region comprising an HCDR1 having the amino acid sequence of SEQ ID NO: 31, an HCDR2 having the amino acid sequence of SEQ ID NO: 32, and an HCDR3 having the amino acid sequence of SEQ ID NO: 33, according to the CDR definitions of IMGT; and a light chain variable region comprising an LCDR1 having the amino acid sequence of SEQ ID NO: 34, an LCDR2 having the amino acid sequence of SEQ ID NO: 35, and an LCDR3 having the amino acid sequence of SEQ ID NO: 36.

[0069] In a preferred embodiment, the anti-CD44v9 antibody comprises a heavy chain variable region having the amino acid sequence of SEQ ID NO:47 and a light chain variable region having the amino acid sequence of SEQ ID NO:48.

[0070] In a preferred embodiment, the anti-CD44v9 antibody comprises a heavy chain variable region having the amino acid sequence of SEQ ID NO:49 and a light chain variable region having the amino acid sequence of SEQ ID NO:50.

[0071] In a preferred embodiment, the anti-CD44v9 antibody comprises a heavy chain variable region having the amino acid sequence of SEQ ID NO:51 and a light chain variable region having the amino acid sequence of SEQ ID NO:52.

[0072] In a preferred embodiment, the anti-CD44v9 antibody comprises a heavy chain variable region having the amino acid sequence of SEQ ID NO:53 and a light chain variable region having the amino acid sequence of SEQ ID NO:54.

[0073] In a preferred embodiment, the anti-CD44v9 antibody comprises a heavy chain constant region having the amino acid sequence of SEQ ID NO:45 and a light chain constant region having the amino acid sequence of SEQ ID NO:46.

[0074] In a preferred embodiment, the anti-CD44v9 antibody comprises a heavy chain variable region having the amino acid sequence of SEQ ID NO:49, a light chain variable region having the amino acid sequence of SEQ ID NO:50, a heavy chain constant region having the amino acid sequence of SEQ ID NO:45, and a light chain constant region having the amino acid sequence of SEQ ID NO:46.

[0075] In a preferred embodiment, the anti-CD44v9 antibody comprises a heavy chain variable region having the amino acid sequence of SEQ ID NO:51, a light chain variable region having the amino acid sequence of SEQ ID NO:52, a heavy chain constant region having the amino acid sequence of SEQ ID NO:45, and a light chain constant region having the amino acid sequence of SEQ ID NO:46.

[0076] In any of the above embodiments, the anti-CD44v9 antibody is preferably an IgG type, more preferably an IgG1 subtype. Preferably, the anti-CD44v9 antibody is an IgG1 type and comprises a heavy chain variable region comprising, according to the IMGT CDR definition, an HCDR1 having the amino acid sequence of SEQ ID NO: 1, an HCDR2 having the amino acid sequence of SEQ ID NO: 2, and an HCDR3 having the amino acid sequence of SEQ ID NO: 3, and a light chain variable region comprising an LCDR1 having the amino acid sequence of SEQ ID NO: 4, an LCDR2 having the amino acid sequence of SEQ ID NO: 5, and an LCDR3 having the amino acid sequence of SEQ ID NO: 6. Preferably, the anti-CD44v9 antibody is an IgG1 type and comprises a heavy chain variable region having the amino acid sequence of SEQ ID NO: 49 and a light chain variable region having the amino acid sequence of SEQ ID NO: 50. Preferably, the anti-CD44v9 antibody is an IgG1 type and comprises a heavy chain variable region having the amino acid sequence of SEQ ID NO: 51 and a light chain variable region having the amino acid sequence of SEQ ID NO: 52.

[0077] Methods for producing and purifying anti-CD44v9 antibodies have been disclosed in the art (see WO2022 / 041104, WO2019 / 161528, WO2015 / 076425, and WO2011 / 007853, the disclosures of each of which are incorporated herein by reference in their entireties).

[0078] (Belotecan derivative) Cytotoxic drug moieties useful in the antibody-drug conjugates described herein are belotecan derivatives having the structure of formula (II): [ka] (II) where R is -C(=O)R x , -S(=O)R x or -S(=O)2R x where R x is C 1~6 Alkyl groups, halogenated C 1~6 Alkyl group, C 2~6 Alkenyl group, C 2~6 Alkynyl group, C 3~6 Cycloalkyl groups, C 3~6 R is selected from the group consisting of a heterocyclyl group, a 6- to 10-membered aryl group, and a 5- to 14-membered heteroaryl group, and preferably R is —C(═O)R x or -S(=O)2R x where R x is C 1~6 Alkyl groups, halogenated C 1~6 Alkyl group, C 3~6 Cycloalkyl groups, C 3~6 R is selected from the group consisting of a heterocyclyl group, a 6- to 10-membered aryl group, and a 5- to 14-membered heteroaryl group, and more preferably R is —C(═O)R x or -S(=O)2R x where R x is C 1~6 Alkyl group, C 3~6 It is selected from the group consisting of a cycloalkyl group and a phenyl group.

[0079] In some embodiments, the cytotoxic drug moiety useful in the antibody drug conjugates described herein is selected from the group consisting of: [ka]

[0080] In preferred embodiments, the cytotoxic drug moieties useful in the antibody drug conjugates described herein are: [ka]

[0081] In more preferred embodiments, the cytotoxic drug moieties useful in the antibody drug conjugates described herein are: [ka]

[0082] Methods for producing and purifying the belotecan derivatives described herein are available in the art (see, for example, WO2020 / 156189, the entire disclosure of which is incorporated herein by reference).

[0083] (Linker) The anti-CD44v9 antibody is linked to a cytotoxic drug via a suitable linker to form the antibody-drug conjugate described herein. The present disclosure contemplates that any linker conventionally used in the field of antibody-drug conjugates can be used in the antibody-drug conjugates described herein.

[0084] In some embodiments, the linker has the structure of formula (III): -L1-(L2)m1-(L3)m2-(L4)m3-EG- (III) wherein L1 is conjugated to a cytotoxic drug moiety, and G is conjugated to an anti-CD44v9 antibody moiety, and wherein: L1 is Lys, Cit, Cit-Val, Val-Ala, Lys-Val, [ka] is selected from the group consisting of where R', R1 and R2 are each independently H (hydrogen), D (deuterium) or C 1~4an alkyl group, Z1 is Cit, Lys, Cit-Val, Cit-Ala, Val-Ala, or Lys-Val, x1 and x3 are each independently 0, 1, or 2, and L1 is attached to a cytotoxic drug moiety at the 1-position of L1, preferably L1 is Lys, Cit, Cit-Val, Val-Ala, Lys-Val, [ka] and more preferably, L1 is: [ka] L2 is selected from the group consisting of: [ka] wherein R3, R4, R5, and R6 are each independently H (hydrogen), D (deuterium), or C 1~4 alkyl groups, y1 and y2 are each independently 0, 1, 2, 3, 4, 5, 6, 7, or 8, and L2 is bonded to L1 at the 1-position of L2, preferably L2 is selected from the group consisting of: [ka] More preferably, L2 is: [ka] L3 is selected from the group consisting of an amino group, an N-methylpiperidine group, a pyrazolylene group, or a triazolylene group, optionally substituted by one or more R7, wherein each R7 is H (hydrogen), D (deuterium), a halogen, =O, CF3, CN, CH2CN, a carboxy group, a sulfonic acid group, C 1~4 Alkyl group, C 1~4 Alkoxy group, C 2~6 Alkenyl group or C 2~6 L3 is independently selected from alkynyl groups, preferably a triazolylene group having the structure: [ka] and L3 is bound to L2 at position 1, L4 is selected from the group consisting of: [ka] wherein L4 is attached to E at position 2 of L4, and preferably L4 is selected from the group consisting of: [ka] More preferably, L4 is: [ka] E optionally comprises one or more R 12 wherein each R is selected from the group consisting of a pyrimidylene group, a quinolylene group, or a pyrrolo[2,3-d]pyrimidylene group substituted by 12 is H (hydrogen), D (deuterium), halogen, CN, nitro group, C 1~2 Alkyl or halogenated C 1~2 alkyl groups, and preferably E is optionally one or more R 12 wherein each R 12 are independently H (hydrogen) or D (deuterium), G is a thioether bond, and m1 is 0, 1, 2 or 3, preferably m1 is 1, m2 is 0, 1 or 2, preferably m2 is 0 or 1, and m3 is 0, 1, 2 or 3, preferably m3 is 1.

[0085] In some embodiments, the linker has the structure of formula (III): -L1-(L2)m1-(L3)m2-(L4)m3-EG- (III) wherein L1 is conjugated to a cytotoxic drug moiety, and G is conjugated to an anti-CD44v9 antibody moiety, and wherein: L1 is Lys, Cit, Cit-Val, Val-Ala, Lys-Val, [ka] and L1 is attached to the cytotoxic drug moiety at position 1 of L1; L2 is selected from the group consisting of: [ka] L2 is bound to L1 at position 1 of L2, L3 is a triazolylene group having the structure: [ka] and L3 is bound to L2 at position 1, L4 is selected from the group consisting of: [ka] and L4 is bonded to E at position 2 of L4; E optionally comprises one or more R 12 wherein each R 12 are independently H (hydrogen) or D (deuterium), G is a thioether bond, and m1 is 0, 1, 2 or 3, preferably m1 is 1, m2 is 0, 1 or 2, preferably m2 is 0 or 1, and m3 is 0, 1, 2 or 3, preferably m3 is 1.

[0086] In some embodiments, the linker has the structure of formula (III): -L1-(L2)m1-(L3)m2-(L4)m3-EG- (III) wherein L1 is conjugated to a cytotoxic drug moiety, and G is conjugated to an anti-CD44v9 antibody moiety, and wherein: L1 is: [ka] and linked to a cytotoxic drug moiety at position 1 of L1; L2 is: [ka] and L2 is bound to L1 at position 1, L3 is a triazolylene group having the structure: [ka] and L3 is bound to L2 at position 1, L4 is: [ka] and L4 is bound to E at position 2, E is a pyrimidinyl group; G is a thioether bond, and m1 is 1, m2 is 0 or 1, and m3 is 1.

[0087] It should be noted that the thioether bond (G) in the present disclosure is formed from the thiol group of a Cys residue in the anti-CD44v9 antibody, which may be formed by opening a disulfide bond in the antibody or may be a free Cys residue contained in the antibody. As is readily known in the art, depending on various partitioning methods, the thioether bond (G) may be considered to belong to the anti-CD44v9 antibody, rather than to the linker, as found in the final synthesized conjugate. For example, in some embodiments, the linker portion does not contain a thioether bond, and the sulfur atom forms part of the Cys residue in the anti-CD44v9 antibody.

[0088] (antibody drug conjugates) The present disclosure provides, in one aspect, an antibody-drug conjugate having the structure of formula (I): (TL) γ -mAb (I) where: T is a cytotoxic drug moiety having the structure of formula (II): [ka] where R is -C(=O)R x , -S(=O)R x or -S(=O)2R x where R x is C 1~6 Alkyl groups, halogenated C 1~6 Alkyl group, C 2~6 Alkenyl group, C 2~6 Alkynyl group, C 3~6 Cycloalkyl groups, C 3~6 R is selected from the group consisting of a heterocyclyl group, a 6- to 10-membered aryl group, and a 5- to 14-membered heteroaryl group, and preferably R is —C(═O)R x or -S(=O)2R x where R x is C 1~6 Alkyl groups, halogenated C 1~6 Alkyl group, C 3~6 Cycloalkyl groups, C 3~6 R is selected from the group consisting of a heterocyclyl group, a 6- to 10-membered aryl group, and a 5- to 14-membered heteroaryl group, and more preferably R is —C(═O)R x or -S(=O)2R x where R x is C 1~6 Alkyl group, C 3~6 selected from the group consisting of cycloalkyl groups and phenyl groups; L is a linker moiety, γ is an integer or decimal number between 1 and 10, and A mAb is an antibody portion, wherein the antibody is an anti-CD44v9 antibody or an antigen-binding fragment thereof.

[0089] In some embodiments, an antibody-drug conjugate is provided having the structure of Formula (I): (TL) γ -mAb (I) where: T is a cytotoxic drug moiety as defined herein above in the belotecan derivatives section; L is a linker moiety as defined above in the Linker section of this specification; γ is an integer or decimal number between 1 and 10, and A mAb is an antibody portion, wherein the antibody is an anti-CD44v9 antibody or an antigen-binding fragment thereof.

[0090] In some embodiments, an antibody-drug conjugate is provided having the structure of Formula (I): (TL) γ -mAb (I) where: T is a cytotoxic drug moiety as defined herein above in the belotecan derivatives section; L is a linker moiety, γ is an integer or decimal number between 1 and 10, and The mAb is an anti-CD44v9 antibody or antigen-binding fragment thereof as defined above in the anti-CD44v9 antibody portion section of this specification.

[0091] In some embodiments, an antibody-drug conjugate is provided having the structure of Formula (I): (TL) γ -mAb (I) where: T is a cytotoxic drug moiety as defined herein above in the belotecan derivatives section; L is a linker moiety as defined above in the Linker section of this specification; γ is an integer or decimal number between 1 and 10, and The mAb is an anti-CD44v9 antibody or antigen-binding fragment thereof as defined above in the anti-CD44v9 antibody portion section of this specification.

[0092] In any of the above embodiments, γ is preferably an integer or decimal number between 5 and 8, for example, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, or 8. The parameter γ is also known as the DAR (drug-antibody ratio), and is one of the most important quality parameters of an antibody-drug conjugate, and is expressed as the average number of drug molecules conjugated to an antibody.

[0093] A variety of analytical techniques for DAR assessment are available in the art, including, but not limited to, ultraviolet-visible (UV / Vis) spectroscopy, hydrophobic interaction chromatography (HIC), reversed-phase high-performance liquid chromatography (RP-HPLC), and liquid chromatography with electrospray ionization mass spectrometry (LC-ESI-MS). In some embodiments, for the antibody-drug conjugates disclosed herein, the value of parameter γ is determined by RP-HPLC.

[0094] In preferred embodiments, the antibody drug conjugate disclosed herein is selected from the group consisting of: [ka] [ka] [ka] [ka] [ka] [ka] where γ is an integer or decimal number between 5 and 8, and mAb is an antibody moiety, where the antibody is an anti-CD44v9 antibody or an antigen-binding fragment thereof.

[0095] In a preferred embodiment, the anti-CD44v9 antibody is as defined above in the anti-CD44v9 antibody portion section herein.

[0096] As described above, in the antibody-drug conjugates disclosed herein, the thioether bond (G) is formed from the thiol group of a Cys residue in the anti-CD44v9 antibody, which may be formed by opening a disulfide bond in the antibody or may be a free Cys residue contained in the antibody.

[0097] In a further preferred embodiment, the antibody drug conjugate disclosed herein is selected from the group consisting of: [ka] [ka] [ka] [ka] wherein γ is an integer or decimal number from 5 to 8, and the mAb is an anti-CD44v9 monoclonal antibody or an antigen-binding fragment thereof, wherein the mAb is linked via a thiol group of a Cys residue in the mAb, and the mAb comprises a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3, and a light chain variable region comprising LCDR1, LCDR2, and LCDR3, and wherein HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are the same as any of (a) to (d); (a) HCDR1, HCDR2, and HCDR3 of a heavy chain variable region of SEQ ID NO: 47 and LCDR1, LCDR2, and LCDR3 of a light chain variable region of SEQ ID NO: 48; (b) HCDR1, HCDR2, and HCDR3 of a heavy chain variable region of SEQ ID NO: 49 and LCDR1, LCDR2, and LCDR3 of a light chain variable region of SEQ ID NO: 50; (c) HCDR1, HCDR2, and HCDR3 of a heavy chain variable region of SEQ ID NO: 51 and LCDR1, LCDR2, and LCDR3 of a light chain variable region of SEQ ID NO: 52; and (d) HCDR1, HCDR2, and HCDR3 of a heavy chain variable region of SEQ ID NO: 53 and LCDR1, LCDR2, and LCDR3 of a light chain variable region of SEQ ID NO: 54, and Here, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are preferably defined according to any of the CDR definition schemes selected from the group consisting of Chothia, Kabat, IMGT, AbM and Contact.

[0098] In a preferred embodiment, the mAb is an anti-CD44v9 monoclonal antibody or an antigen-binding fragment thereof, comprising a heavy chain variable region comprising an HCDR1 having the amino acid sequence of SEQ ID NO: 1, an HCDR2 having the amino acid sequence of SEQ ID NO: 2, and an HCDR3 having the amino acid sequence of SEQ ID NO: 3, according to the CDR definition of IMGT, and a light chain variable region comprising an LCDR1 having the amino acid sequence of SEQ ID NO: 4, an LCDR2 having the amino acid sequence of SEQ ID NO: 5, and an LCDR3 having the amino acid sequence of SEQ ID NO: 6.

[0099] In a preferred embodiment, the mAb is an anti-CD44v9 monoclonal antibody or an antigen-binding fragment thereof, comprising a heavy chain variable region comprising an HCDR1 having the amino acid sequence of SEQ ID NO: 31, an HCDR2 having the amino acid sequence of SEQ ID NO: 32, and an HCDR3 having the amino acid sequence of SEQ ID NO: 33, according to the CDR definition of IMGT, and a light chain variable region comprising an LCDR1 having the amino acid sequence of SEQ ID NO: 34, an LCDR2 having the amino acid sequence of SEQ ID NO: 35, and an LCDR3 having the amino acid sequence of SEQ ID NO: 36.

[0100] In a preferred embodiment, the mAb is an anti-CD44v9 monoclonal antibody or antigen-binding fragment thereof comprising a heavy chain variable region having the amino acid sequence of SEQ ID NO:47 and a light chain variable region having the amino acid sequence of SEQ ID NO:48.

[0101] In a preferred embodiment, the mAb is an anti-CD44v9 monoclonal antibody or antigen-binding fragment thereof comprising a heavy chain variable region having the amino acid sequence of SEQ ID NO:49 and a light chain variable region having the amino acid sequence of SEQ ID NO:50.

[0102] In a preferred embodiment, the mAb is an anti-CD44v9 monoclonal antibody or antigen-binding fragment thereof comprising a heavy chain variable region having the amino acid sequence of SEQ ID NO:51 and a light chain variable region having the amino acid sequence of SEQ ID NO:52.

[0103] In a preferred embodiment, the mAb is an anti-CD44v9 monoclonal antibody or antigen-binding fragment thereof comprising a heavy chain variable region having the amino acid sequence of SEQ ID NO:53 and a light chain variable region having the amino acid sequence of SEQ ID NO:54.

[0104] In a preferred embodiment, the mAb is an anti-CD44v9 monoclonal antibody or antigen-binding fragment thereof comprising a heavy chain variable region having the amino acid sequence of SEQ ID NO:49, a light chain variable region having the amino acid sequence of SEQ ID NO:50, a heavy chain constant region having the amino acid sequence of SEQ ID NO:45, and a light chain constant region having the amino acid sequence of SEQ ID NO:46.

[0105] In a preferred embodiment, the mAb is an anti-CD44v9 monoclonal antibody or antigen-binding fragment thereof comprising a heavy chain variable region having the amino acid sequence of SEQ ID NO:51, a light chain variable region having the amino acid sequence of SEQ ID NO:52, a heavy chain constant region having the amino acid sequence of SEQ ID NO:45, and a light chain constant region having the amino acid sequence of SEQ ID NO:46.

[0106] In a more preferred embodiment, the antibody drug conjugate disclosed herein has the structure: [ka] wherein γ is an integer or decimal number from 5 to 8, and the mAb is an anti-CD44v9 monoclonal antibody or an antigen-binding fragment thereof, wherein the thioether bond linked to the mAb is formed from the thiol group of a Cys residue in the mAb, and the mAb is an anti-CD44v9 antibody or an antigen-binding fragment thereof as defined above in the anti-CD44v9 antibody moiety section of this specification.

[0107] In some embodiments, the mAb specifically binds to a CD44v9 epitope comprising or consisting essentially of SEQ ID NO:55 or 56.

[0108] In a more preferred embodiment, the mAb comprises a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3, and a light chain variable region comprising LCDR1, LCDR2, and LCDR3, wherein HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are the same as any one of (a) to (d), (a) HCDR1, HCDR2, and HCDR3 of a heavy chain variable region of SEQ ID NO: 47 and LCDR1, LCDR2, and LCDR3 of a light chain variable region of SEQ ID NO: 48; (b) HCDR1, HCDR2, and HCDR3 of a heavy chain variable region of SEQ ID NO: 49 and LCDR1, LCDR2, and LCDR3 of a light chain variable region of SEQ ID NO: 50; (c) HCDR1, HCDR2, and HCDR3 of a heavy chain variable region of SEQ ID NO: 51 and LCDR1, LCDR2, and LCDR3 of a light chain variable region of SEQ ID NO: 52; and (d) HCDR1, HCDR2, and HCDR3 of the heavy chain variable region of SEQ ID NO: 53 and LCDR1, LCDR2, and LCDR3 of the light chain variable region of SEQ ID NO: 54 Here, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are preferably defined according to any of the CDR definition schemes selected from the group consisting of Chothia, Kabat, IMGT, AbM and Contact.

[0109] In a more preferred embodiment, the mAb comprises a heavy chain variable region comprising an HCDR1 having the amino acid sequence of SEQ ID NO: 1, an HCDR2 having the amino acid sequence of SEQ ID NO: 2, and an HCDR3 having the amino acid sequence of SEQ ID NO: 3, according to the IMGT CDR definition, and a light chain variable region comprising an LCDR1 having the amino acid sequence of SEQ ID NO: 4, an LCDR2 having the amino acid sequence of SEQ ID NO: 5, and an LCDR3 having the amino acid sequence of SEQ ID NO: 6.

[0110] In a preferred embodiment, the mAb comprises a heavy chain variable region comprising an HCDR1 having the amino acid sequence of SEQ ID NO: 31, an HCDR2 having the amino acid sequence of SEQ ID NO: 32, and an HCDR3 having the amino acid sequence of SEQ ID NO: 33, according to the IMGT CDR definition, and a light chain variable region comprising an LCDR1 having the amino acid sequence of SEQ ID NO: 34, an LCDR2 having the amino acid sequence of SEQ ID NO: 35, and an LCDR3 having the amino acid sequence of SEQ ID NO: 36.

[0111] In a more preferred embodiment, the mAb comprises (a) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 47 and a light chain variable region having the amino acid sequence of SEQ ID NO: 48; (b) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 49 and a light chain variable region having the amino acid sequence of SEQ ID NO: 50; or (c) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 51 and a light chain variable region having the amino acid sequence of SEQ ID NO: 52; or (d) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 53 and a light chain variable region having the amino acid sequence of SEQ ID NO: 54.

[0112] In a preferred embodiment, the mAb comprises (a) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 49 and a light chain variable region having the amino acid sequence of SEQ ID NO: 50, or (b) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 51 and a light chain variable region having the amino acid sequence of SEQ ID NO: 52.

[0113] In a preferred embodiment, the mAb comprises a heavy chain constant region having the amino acid sequence of SEQ ID NO:45 and a light chain constant region having the amino acid sequence of SEQ ID NO:46.

[0114] In a more preferred embodiment, the mAb is an anti-CD44v9 monoclonal antibody or antigen-binding fragment thereof comprising a heavy chain variable region having the amino acid sequence of SEQ ID NO:49, a light chain variable region having the amino acid sequence of SEQ ID NO:50, a heavy chain constant region having the amino acid sequence of SEQ ID NO:45, and a light chain constant region having the amino acid sequence of SEQ ID NO:46.

[0115] In a more preferred embodiment, the mAb is an anti-CD44v9 monoclonal antibody or antigen-binding fragment thereof comprising a heavy chain variable region having the amino acid sequence of SEQ ID NO:51, a light chain variable region having the amino acid sequence of SEQ ID NO:52, a heavy chain constant region having the amino acid sequence of SEQ ID NO:45, and a light chain constant region having the amino acid sequence of SEQ ID NO:46.

[0116] In any of the above embodiments, γ is preferably an integer or decimal number between 5 and 8, for example, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, or 8. The parameter γ is also known as the DAR (drug-antibody ratio), and is one of the most important quality parameters of an antibody-drug conjugate, and is expressed as the average number of drug molecules conjugated to an antibody.

[0117] A variety of analytical techniques for DAR assessment are available in the art, including, but not limited to, ultraviolet-visible (UV / Vis) spectroscopy, hydrophobic interaction chromatography (HIC), reversed-phase high-performance liquid chromatography (RP-HPLC), and liquid chromatography with electrospray ionization mass spectrometry (LC-ESI-MS). In some embodiments, for the antibody-drug conjugates disclosed herein, the value of parameter γ is determined by RP-HPLC.

[0118] In any of the more preferred embodiments, γ is preferably 7.8 as determined by RP-HPLC.

[0119] In a preferred embodiment, the antibody drug conjugate disclosed herein has the structure: [ka] wherein γ is 7.8 as determined by RP-HPLC, and the mAb is an anti-CD44v9 antibody or an antigen-binding fragment thereof, wherein the thioether bond linked to the mAb is formed from the thiol group of a Cys residue in the mAb, and the mAb comprises a heavy chain variable region comprising an HCDR1 having the amino acid sequence of SEQ ID NO: 1, an HCDR2 having the amino acid sequence of SEQ ID NO: 2, and an HCDR3 having the amino acid sequence of SEQ ID NO: 3, according to the CDR definition of IMGT, and a light chain variable region comprising an LCDR1 having the amino acid sequence of SEQ ID NO: 4, an LCDR2 having the amino acid sequence of SEQ ID NO: 5, and an LCDR3 having the amino acid sequence of SEQ ID NO: 6.

[0120] In a preferred embodiment, the antibody drug conjugate disclosed herein has the structure: [ka] wherein γ is 7.8 as determined by RP-HPLC, and the mAb is an anti-CD44v9 antibody or an antigen-binding fragment thereof, wherein the thioether bond linked to the mAb is formed from the thiol group of a Cys residue in the mAb, and the mAb comprises a heavy chain variable region having the amino acid sequence of SEQ ID NO:49 and a light chain variable region having the amino acid sequence of SEQ ID NO:50.

[0121] In a preferred embodiment, the antibody drug conjugate disclosed herein has the structure: [ka] wherein γ is 7.8 as determined by RP-HPLC, and the mAb is an anti-CD44v9 antibody or antigen-binding fragment thereof, wherein the thioether bond linked to the mAb is formed from the thiol group of a Cys residue in the mAb, and the mAb comprises a heavy chain variable region having the amino acid sequence of SEQ ID NO: 49, a light chain variable region having the amino acid sequence of SEQ ID NO: 50, a heavy chain constant region having the amino acid sequence of SEQ ID NO: 45, and a light chain constant region having the amino acid sequence of SEQ ID NO: 46.

[0122] Methods for preparing linker moieties are well known in the art and are exemplified in the Examples disclosed herein. Detailed descriptions of the synthesis of linkers and intermediates used therein are described, for example, in EP 3725798, the disclosure of which is incorporated herein by reference in its entirety.

[0123] The method for producing the antibody-drug conjugate disclosed herein can be easily carried out according to conventional methods in the art, and is also exemplified in the Examples of the present specification. Detailed descriptions of the synthesis of antibody-drug conjugates using different antibodies but having the same cytotoxic drug moiety and linker moiety are described, for example, in the aforementioned EP3725798.

[0124] This disclosure demonstrates that an antibody-drug conjugate (hHTS033-KL) can bind to CD44v9 on the surface of tumor cells and then be internalized, after which the payload is released and is believed to interfere with the function of the topoisomerase I enzyme, ultimately inducing cell death by apoptosis.

[0125] (Pharmaceutical composition) In another aspect, the present disclosure provides pharmaceutical compositions comprising an antibody-drug conjugate as disclosed herein and a pharmaceutically acceptable carrier, these compositions being suitable for veterinary or human administration.

[0126] The composition can be in any form that allows for administration to a patient. For example, it can be in the form of a solid, liquid, or gas (aerosol). Typical routes of administration include, but are not limited to, oral, topical, parenteral, sublingual, rectal, vaginal, intraocular, intratumoral, and intranasal. Parenteral administration includes subcutaneous injection, intravenous injection, intramuscular injection, intrasternal injection, or infusion techniques. In one embodiment, the composition is administered parenterally. In yet another embodiment, the composition is administered intravenously.

[0127] The pharmaceutical composition can be formulated so that the conjugate is bioavailable after administration to a patient. The composition can be in the form of one or more dosage units, for example, a tablet can be a single dosage unit, and a container of the conjugate in aerosol form can hold multiple dosage units.

[0128] The materials used in preparing the pharmaceutical composition may be non-toxic in the amounts used. It will be apparent to those skilled in the art that the optimal dosage of the active ingredient in the pharmaceutical composition will depend on a variety of factors. Relevant factors include, but are not limited to, the type of animal (e.g., human), the particular form of the conjugate, the method of administration, and the composition used.

[0129] The pharmaceutically acceptable carrier or vehicle may be particulate, so that the composition is, for example, in tablet or powder form. The carrier may be liquid, so that the composition is, for example, in oral syrup or injectable liquid. Furthermore, the carrier may be gaseous or particulate, so that it provides an aerosol composition useful, for example, for inhalation administration.

[0130] The composition can be in the form of a liquid, such as an elixir, syrup, solution, emulsion, or suspension. The liquid is useful for oral administration or delivery by injection. For oral administration, the composition can contain one or more of a sweetener, a preservative, a dye / colorant, and a flavor enhancer. For injection, the composition can contain one or more of a surfactant, a preservative, a wetting agent, a dispersing agent, a suspending agent, a buffer, a stabilizer, and an isotonic agent.

[0131] Liquid compositions, whether in solution, suspension, or other similar form, may contain one or more of the following: a sterile diluent, such as water for injection or saline, preferably saline, Ringer's solution, or isotonic sodium chloride; fixed oils such as synthetic monoglycerides or diglycerides, polyethylene glycol, glycerin, cyclodextrin, propylene glycol, or other solvents that can serve as solvents or suspending media; antibacterial agents such as benzyl alcohol or methylparabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates, or phosphates; and tonicity adjusters such as sodium chloride or glucose. Parenteral compositions can be enclosed in ampoules, disposable syringes, or multiple-dose vials made of glass, plastic, or other materials. Physiological saline is an exemplary adjuvant. Injectable compositions are preferably sterile.

[0132] The amount of the conjugate that is effective in the treatment of a particular disorder or condition will depend on the nature of the disorder or condition, and can be determined by standard clinical techniques. Additionally, in vitro or in vivo assays can optionally be performed to identify optimal dosage ranges. The precise dose to be employed in the compositions will also depend on the route of administration and the seriousness of the disease or disorder, and should be decided according to the judgment of the practitioner and each patient's circumstances.

[0133] The composition contains an effective amount of the complex so that a suitable dosage is obtained. Typically, this amount is at least about 0.01% of the complex by weight of the composition. For oral administration, this amount can vary from about 0.1% to about 80% by weight of the composition. In one embodiment, the oral composition can contain from about 4% to about 50% of the complex by weight of the composition. In yet another embodiment, the composition is prepared so that a parenteral dosage unit contains from about 0.01% to about 2% of the complex.

[0134] For intravenous administration, the composition can contain about 0.01 mg to about 100 mg of the complex per kg of animal body weight. In one embodiment, the composition can contain about 1 mg to about 100 mg of the complex per kg of animal body weight. In another embodiment, the dosage ranges from about 0.1 to about 25 mg of the complex per kg of body weight.

[0135] In general, the dosage of the conjugate administered to a patient is typically about 0.01 mg to about 2000 mg per kg of animal body weight. In one embodiment, the dosage administered to a patient is about 0.01 mg to about 10 mg per kg of animal body weight, in another embodiment, the dosage administered to a patient is about 0.1 mg to about 250 mg per kg of animal body weight, in yet another embodiment, the dosage administered to a patient is about 0.1 mg to about 20 mg per kg of animal body weight, in yet another embodiment, the dosage administered to a patient is about 0.1 mg to about 10 mg per kg of animal body weight, and in yet another embodiment, the dosage administered to a patient is about 1 mg to about 10 mg per kg of animal body weight.

[0136] The conjugate or composition can be administered by any convenient route, for example, by infusion or bolus injection, or by absorption through epithelial or mucocutaneous layers (e.g., oral, rectal, and intestinal mucosa, etc.). Administration can be systemic or local. Various delivery systems are well known and can be used to administer the conjugate or composition, such as encapsulation in liposomes, microparticles, microcapsules, capsules, etc. In certain embodiments, multiple conjugates or compositions are administered to the patient.

[0137] In specific embodiments, it may be desirable to administer one or more complexes or compositions locally to the site in need of treatment. This may be achieved, for example, but not limited to, by local infusion during surgery, topical application such as in conjunction with a wound dressing after surgery, injection, catheter, suppository, or implant composed of porous, non-porous, or gelatinous materials, including membranes such as sialastic membranes or fibers. In one embodiment, administration may be by direct injection at the site (or former site) of a cancer, tumor, or neoplastic or pre-neoplastic tissue. In another embodiment, administration may be by direct injection at the site (or former site) of an autoimmune disease manifestation.

[0138] In certain embodiments, it may be desirable to introduce one or more complexes or compositions into the central nervous system by any suitable route, including intraventricular and intraspinal injection, which may be facilitated, for example, by an intraventricular catheter attached to a reservoir, such as an Ommaya reservoir.

[0139] Pulmonary administration can also be by use of an inhaler or nebulizer, by formulation with an aerosolizing agent, or by perfusion in a fluorocarbon or synthetic pulmonary surfactant.

[0140] In yet another embodiment, the complex or composition can be delivered using a controlled release system, such as, but not limited to, a pump or various polymeric materials, hi yet another embodiment, the controlled release system can be placed near the target of the complex or composition, e.g., the brain, thereby requiring only a fraction of the systemic dose.

[0141] The term "carrier" refers to a diluent, adjuvant, or carrier used in administering the conjugate. Such pharmaceutical carriers can be liquids, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, for example, peanut oil, soybean oil, mineral oil, sesame oil, and the like. Carriers include saline, gum acacia, gelatin, starch paste, talc, keratin, colloidal silica, urea, and the like. Additionally, adjuvants, stabilizers, thickeners, lubricants, and coloring agents can be used. In one embodiment, the conjugate or composition and pharmaceutically acceptable carrier are sterile when administered to a patient. Water is an exemplary carrier when the conjugate is administered intravenously. Saline solutions, aqueous dextrose solutions, and aqueous glycerol solutions can also be used as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical carriers also include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, nonfat dry milk, glycerol, propylene glycol, water, ethanol, etc. The present compositions, if desired, can also contain minor amounts of wetting agents, emulsifying agents, or pH buffering agents.

[0142] In one embodiment, the conjugate is formulated according to conventional procedures as a pharmaceutical composition suitable for intravenous administration to animals, particularly humans. Typically, the carrier or vehicle for intravenous administration is a sterile isotonic aqueous buffer solution. Where necessary, the composition can also contain a solubilizing agent. Compositions for intravenous administration can optionally contain a local anesthetic, such as lignocaine, to reduce pain at the injection site. Generally, the ingredients are supplied separately or mixed together in unit dosage form, for example, as a lyophilized powder or water-free concentrate in a hermetically sealed container, such as an ampoule or sachet indicating the quantity of active ingredient. For infusion administration, the conjugate can be dispensed, for example, using an infusion bottle containing sterile pharmaceutical-grade water or saline. For injection administration, an ampoule of sterile water for injection or saline can be provided so that the ingredients can be mixed prior to administration.

[0143] The composition can contain various materials that modify the physical form of the solid or liquid dosage unit. For example, the composition can contain materials that form a coating shell around the active ingredient. The coating shell material is typically inert and can be selected from, for example, sugar, shellac, and other enteric coating agents. Alternatively, the active ingredient can be encapsulated in a gelatin capsule.

[0144] The composition may be in the form of a gaseous dosage unit, such as, for example, aerosol form.The term aerosol is used to refer to various systems, from those with colloidal properties to those consisting of pressurized packaging.Delivery can be by liquefied gas or compressed gas, or by a suitable pump system that dispenses the active ingredient.

[0145] Whether in solid, liquid, or gaseous form, the composition can include a pharmacological agent used in the treatment of cancer.

[0146] (Use and Treatment) These conjugates are useful for inhibiting tumor or cancer cell proliferation, inducing apoptosis in tumor or cancer cells, or treating cancer in patients. Therefore, these conjugates can be used in a variety of settings for treating cancer in animals. These conjugates can be used to deliver drugs or drug units to tumor or cancer cells. Without being bound by theory, in one embodiment, the antibody portion of the conjugates disclosed herein binds to or associates with the CD44v9 antigen expressed on or associated with the cell surface of cancer or tumor cells, and the conjugates can be internalized into tumor or cancer cells by receptor-mediated endocytosis. The CD44v9 antigen can be attached to or an extracellular matrix protein associated with tumor or cancer cells. Once inside the cell, one or more specific peptide sequences within the linker moiety are hydrolytically cleaved, releasing the cytotoxic drug or drug-linker compound. The released cytotoxic drug or drug-linker compound is then free to move within the cell and induce cytotoxic or cytostatic activity. In an alternative embodiment, the cytotoxic drug or drug-linker compound is cleaved from the conjugate outside the tumor or cancer cell, after which the cytotoxic drug or drug-linker compound penetrates into the cell.

[0147] In a further aspect, the present disclosure provides a method of treating or preventing cancer in a subject, comprising administering to the subject an effective amount of an antibody-drug conjugate disclosed herein.

[0148] The cancer may be a CD44v9 antigen-positive solid cancer or a hematological malignancy. A "hematological malignancy," also known as a blood cancer, is a cancer originating in blood-forming tissues such as bone marrow or other cells of the immune system. Hematological malignancies include leukemias (acute myeloid leukemia (ANIL), acute promyelocytic leukemia, acute lymphoblastic leukemia (ALL), acute mixed lineage leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia (CLL), hairy cell leukemia and large granular lymphocytic leukemia, myelodysplastic syndromes (MDS), myeloproliferative disorders (polycythemia vera, essential thrombocytosis, primary myelofibrosis and chronic myelogenous leukemia), lymphomas, multiple myeloma, MGUS and similar disorders, Hodgkin's lymphoma, non-Hodgkin's lymphoma (NHL), and others. L), primary mediastinal large B-cell lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, transformed follicular lymphoma, splenic marginal zone lymphoma, lymphocytic lymphoma, T-cell lymphoma, and other B-cell malignancies. "Solid cancer" includes, but is not limited to, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, chordoma, angiosarcoma, endothelial sarcoma, lymphangiosarcoma, lymphangioendothelial sarcoma, synovium, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, Colorectal cancer, kidney cancer, pancreatic cancer, bone cancer, breast cancer, ovarian cancer, prostate cancer, esophageal cancer, stomach cancer, oral cancer, nasal cancer, pharyngeal cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchial cancer, renal cell carcinoma, hepatocellular carcinoma, bile duct cancer, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, uterine cancer, testicular cancer, small cell lung cancer, bladder cancer, lung cancer, epithelial cancer, glioma, glioblastoma multiforme, astrocytoma, medulloblastoma Cancers treatable by the present disclosure include, but are not limited to, NSCLC (non-small cell lung cancer), HNSCC (head and neck squamous cell carcinoma), esophageal cancer such as ESCC (esophageal squamous cell carcinoma), GC (gastric cancer), BC (bladder cancer), breast cancer such as TNBC (triple-negative breast cancer), colon cancer, and liver cancer.

[0149] In some embodiments, methods for treating or preventing cancer are provided, comprising administering to a patient in need thereof an effective amount of a conjugate disclosed herein and a chemotherapeutic agent. In one embodiment, the chemotherapeutic agent is one that is not recognized as refractory in the treatment of cancer. In another embodiment, the chemotherapeutic agent is one that is recognized as refractory in the treatment of cancer. These conjugates can be administered to patients who have also undergone surgery as a treatment for cancer.

[0150] In a specific embodiment, the conjugate is administered simultaneously with a chemotherapeutic agent or with radiation therapy. In another specific embodiment, the chemotherapeutic agent or radiation therapy is administered before or after administration of the conjugate, in one aspect at least 1 hour, 5 hours, 12 hours, 1 day, 1 week, 1 month, and in a further aspect, several months (e.g., up to 3 months) before or after administration.

[0151] Chemotherapeutic agents can be administered over multiple sessions. Suitable chemotherapeutic agents include, but are not limited to, methotrexate, taxol, L-asparaginase, mercaptopurine, thioguanine, hydroxyurea, cytarabine, cyclophosphamide, ifosfamide, nitrosourea, cisplatin, carboplatin, mitomycin, dacarbazine, procarbidine, topotecan, nitrogen mustard, cytoxan, etoposide, 5-fluorouracil, BCNU, irinotecan, camptothecin, bleomycin, doxorubicin, idarubicin, daunorubicin, dactinomycin, plicamycin, mitoxantrone, asparaginase, vinblastine, vincristine, vinorelbine, paclitaxel, and docetaxel. Regarding radiation therapy, any radiation therapy protocol can be used depending on the type of cancer being treated. Examples include, but are not limited to, X-ray irradiation. In particular, high-energy megavoltage (radiation with an energy greater than 1 MeV) can be used for deep tumors, and electron beam and orthovoltage X-ray irradiation can be used for skin cancer. Gamma-ray-emitting radioisotopes, such as radioisotopes of radium, cobalt, and other elements, can also be administered.

[0152] Additionally, the method of treating cancer using the conjugates provides an alternative to chemotherapy or radiation therapy when chemotherapy or radiation therapy has proven or may prove to be excessively toxic to the treated subject, e.g., when it causes unacceptable or intolerable side effects. The treated animal can optionally undergo other cancer treatments, such as surgery, radiation therapy, or chemotherapy, depending on the treatment determined to be acceptable or tolerable.

[0153] These conjugates can also be used in vitro or ex vivo, for example, to treat certain cancers, including, but not limited to, leukemia and lymphoma. Such treatments include autologous stem cell transplantation, a multi-step process that involves harvesting an animal's autologous hematopoietic stem cells, removing all cancer cells, administering high doses of the conjugates with or without high-dose radiation therapy to eradicate any remaining bone marrow cell populations, and reinfusing the stem cell graft into the animal. Supportive care is then administered until bone marrow function is restored and the animal recovers.

[0154] Similarly, the present disclosure also provides an antibody-drug conjugate disclosed herein for use in treating cancer in a subject. Similarly, the present disclosure also provides use of an antibody-drug conjugate disclosed herein in the manufacture of a medicament for treating cancer in a subject. In both equivalent aspects, the cancer is preferably a solid cancer as defined above, more preferably NSCLC (non-small cell lung cancer), HNSCC (head and neck squamous cell carcinoma), esophageal cancer such as ESCC (esophageal squamous cell carcinoma), GC (gastric cancer), BC (bladder cancer), breast cancer such as TNBC (triple-negative breast cancer), colon cancer, and liver cancer. (array) [Table 1] [Table 2] [Table 3] [Table 4] [Table 5-1] [Table 5-2] [Table 6] CD44v9 epitopes bound by HTS033, hHTS033 25-1, hHTS033 25-3, and mAb116: CD44v9 epitope: SHEGLEEDKD (SEQ ID NO: 55) CD44v9 epitope variant: SHEGLEEDKDH (SEQ ID NO: 56)

[0155] (Example) Example 1: Synthesis of (S)—N-(2-(4-ethyl-4-hydroxy-3,14-dione-3,4,12,14-tetrahydro-1H-pyrano[3′,4′,6,7]indolizino[1,2-b]quinolin-11-yl)ethyl)-N-isopropylmethanesulfonamide (Compound T030) [ka] Methylsulfonyl chloride (462 mg, 12.77 mmol, purity: approximately 70%) was added dropwise to a solution of belotecan hydrochloride (3 g, 6.38 mmol) and triethylamine (2.58 g, 25.54 mmol) in dichloromethane (40 mL). The resulting mixture was reacted at room temperature for 2 hours. The mixture was filtered under suction, and the filter cake was washed three times with dichloromethane (3 mL) to obtain the title compound (2.2 g).

[0156] The structural property data are as follows: 1 H NMR(400MHz,DMSO-d6)δ 8.32(d,J=8.4Hz,1H),8.20(dd,J=8.4,1.2Hz,1H),7.93-7.84(m,1H),7.79(t,J=7.6Hz,1H),7.35(s,1H),6.56(s,1H),5.44(d,J=9.2Hz,4H), 3.98(p,J=6.7Hz,1H),3.50(t,J=8.0Hz,2H),3.42-3.35(m,2H),3.00(s,3H),1.93-1.82(m,2H),1.15(d,J=6.7Hz,6H),0.88(t,J=7.3Hz,3H). ESI-MS(m / z):512.2[M+H] + . [α] D20 =+28.19°(c=0.101g / 100mL,CH3CN).

[0157] Example 2 Preparation of 4-((S)-2-(4-aminobutyl)-35-(4-((6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynamido)methyl)-1H-1,2,3-triazol-1-yl)-4,8-dioxo-6,12,15,18,21,24,27,30,33-nonaoxa-3,9-diazapentatriacontamido)benzyl ((S)-4-ethyl-11-(2-(N-isopropylmethylsulfonamido)ethyl)-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl)carbonate (Compound “TL033”) [ka] Step 1: Synthesis of methyl 6-(2-(methylthio)pyrimidin-5-yl)-5-hexynoate (compound 3-2) Methyl 5-hexynoate (500 mg, 3.97 mmol) and 5-bromo-2-methylthiopyrimidine were dissolved in N,N-dimethylformamide (3 mL) at room temperature, and triethylamine (3 mL), cuprous iodide (75 mg, 0.4 mmol), and bis(triphenylphosphine)palladium(II) dichloride (279 mg, 0.4 mmol) were added sequentially. The resulting mixture was heated to 95 °C under nitrogen protection and stirred for 6 hours. The reaction was quenched with water and extracted with ethyl acetate (20 mL × 3). The combined organic phase was washed with saturated brine (20 mL × 2) and dried over anhydrous sodium sulfate. The drying agent was removed by filtration, and the solvent was evaporated under reduced pressure. The product was purified by preparative liquid chromatography to give the title compound (300 mg). ESI-MS (m / z): 251.3 [M+H] + .

[0158] Preparative Liquid Chromatography: Chromatography column: Waters SunFire C18 5μm 19×250mm Mobile phase A: acetonitrile, Mobile phase B: water containing 0.05% formic acid Time: 0 min to 16 min, Mobile phase A: 10% to 90%, Flow rate: 28 mL / min

[0159] Step 2: Synthesis of 6-(2-(methylthio)pyrimidin-5-yl)-5-hexynoic acid (compound 3-3) Compound 3-2 (200 mg, 0.8 mmol) was dissolved in a mixture of tetrahydrofuran and water (4 mL:4 mL) at room temperature, and lithium hydroxide monohydrate (235 mg, 5.6 mmol) was added. The mixture was stirred at room temperature for 4 hours, then diluted with water and extracted with ethyl acetate (20 mL x 2). The aqueous phase was adjusted to pH 3 with 1N hydrochloric acid and extracted with ethyl acetate (20 mL x 3). The combined organic phase was washed with saturated brine (20 mL x 2) and dried over anhydrous sodium sulfate. The desiccant was removed by filtration, and the solvent was evaporated under reduced pressure to give the title compound (120 mg).

[0160] Step 3: Synthesis of 6-(2-(methylsulfonyl)pyrimidin-5-yl)-5-hexynoic acid (compound 3-4) Compound 3-3 (20 mg, 0.085 mmol) was dissolved in dichloromethane (4 mL) at room temperature, m-chloroperoxybenzoic acid (22 mg, 0.127 mmol) was added, and the mixture was stirred overnight at room temperature. The mixture was purified by preparative liquid chromatography to obtain the title compound (20 mg). ESI-MS(m / z): 269.1 [M + H] + .

[0161] Preparative Liquid Chromatography: Chromatography column: Waters SunFire C18 5μm 19×250mm Mobile phase A: acetonitrile, Mobile phase B: water containing 0.05% formic acid Time: 0 min to 16 min, Mobile phase A: 10% to 90%, Flow rate: 28 mL / min

[0162] Step 4: Synthesis of 6-(2-(methylsulfonyl)pyrimidin-5-yl)-N-(prop-2-yn-1-yl)hex-5-ynamide Prop-2-ynyl-1-amine (189 mg, 3.4 mmol) and compound 3-4 (800 mg, 2.83 mmol) were dissolved in dichloromethane (10 mL) at 25 °C, and N,N-diisopropylethylamine (738 mg, 5.67 mmol) and O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (1.63 g, 4.25 mmol) were added sequentially. The mixture was stirred for 2 hours. The reaction solution was concentrated under reduced pressure, and the residue was purified by flash silica gel column chromatography (ethyl acetate:petroleum ether = 3:1) to give the title compound (700 mg). ESI-MS(m / z):306.1[M+H] + .

[0163] Step 5: Synthesis of 4-((S)-35-azido-2-(4-(((4-methoxyphenyl)benzhydryl)amino)butyl)-4,8-dioxo-6,12,15,18,21,24,27,30,33-nonazo-3,9-diazapentatriacontamido)benzyl ((S)-4-ethyl-11-(2-(N-isopropylmethanesulfonamido)ethyl)-3,14-dioxo-3,4,12,14-tetrahydro-2H-pyrano[2,3-b]-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-4-yl)carbonate At 25°C under nitrogen protection, T-030 (250 mg, 0.49 mmol) was dissolved in dichloromethane (10 mL) and cooled to 0°C. After that, a solution of 4-dimethylaminopyridine (478 mg, 3.91 mmol) in dichloromethane (3 mL) was added, followed by the slow dropwise addition of a solution of triphosgene (72 mg, 0.24 mmol) in dichloromethane (10 mL). The mixture was allowed to react for 20 minutes at 0°C with stirring. After bubbling nitrogen through the reaction solution for 20 minutes, a solution of (S)-2-(32-azido-5-oxo-3,9,12,15,18,21,24,27,30-nonaoxa-6-azatriacetamido)-N-(4-(hydroxymethyl)phenyl)-6(((4-methoxyphenyl)benzhydryl)amino)acetamide (518 mg, 0.49 mmol) in dichloromethane (7 mL) was added and the mixture was stirred at 0°C for 1 hour. The reaction solution was concentrated under reduced pressure, and the residue was purified by preparative high-performance liquid chromatography to obtain the title compound (500 mg). ESI-MS(m / z):1597.5[M+H] + .

[0164] Preparative Liquid Chromatography: Chromatography column: Daisogel C18 10 μm 100 × 250 mm Mobile phase A: water, Mobile phase B: acetonitrile [Table 7]

[0165] Step 6: Synthesis of (S)-4-ethyl-11-(2-(N-isopropylmethanesulfonamido)ethyl)-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-4-yl (4-((S)-2-(4-(((4-methoxyphenyl)diphenylmethyl)amino)butyl)-35-(4-((6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynamido)methyl)-1H-1,2,3-triazol-1-yl)-4,8-dioxo-6,12,15,18,21,24,27,30,33-nonoxy-3,9-diazapentatriacontamido)benzyl) carbonate Compound 33-1 (14 mg, 0.05 mmol) was dissolved in dimethyl sulfoxide and water (2.0 mL: 0.5 mL) at room temperature, followed by the addition of cuprous bromide (11 mg, 0.08 mmol) and the mixture was stirred for 1 hour. The mixture was purified by preparative high-performance liquid chromatography to give the title compound (30 mg). ESI-MS(m / z):815.9[(M-273) / 2+H] + .

[0166] Preparative Liquid Chromatography: Chromatography column: Daisogel C18 10 μm 50 × 250 mm Mobile phase A: water, Mobile phase B: acetonitrile [Table 8]

[0167] Step 7: Synthesis of 4-((S)-2-(4-aminobutyl)-35-(4-((6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynamido)methyl)-1H-1,2,3-triazol-1-yl)-4,8-dioxo-6,12,15,18,21,24,27,30,33-nonaoxa-3,9-diazapentatriacontamido)benzyl ((S)-4-ethyl-11-(2-(N-isopropylmethylsulfonamido)ethyl)-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-4-yl)carbonate (compound TL033) Compound 33-2 (30 mg, 0.02 mmol) was dissolved in dichloromethane (1.0 mL), and trifluoroacetic acid (0.2 mL) was added to the reaction solution, followed by reaction at room temperature for 30 minutes. Purification by preparative high-performance liquid chromatography afforded the trifluoroacetate salt of the title compound (20.0 mg).

[0168] Preparative Liquid Chromatography: Chromatography column: Daisogel C18 10 μm 50 × 250 mm Mobile phase A: water containing 0.1% trifluoroacetic acid, Mobile phase B: acetonitrile [Table 9]

[0169] The title compound is identified as follows: 1H NMR(400MHz,DMSO-d6)δ 10.18(s,1H),9.10(s,2H),8.38(t,J=5.56Hz,1H),8.32(d,J=8.40Hz,1H),8.22-8.20(m,2H),8.09(t ,J=5.68Hz,1H),7.91-7.87(m,2H),7.82-7.78(m,1H),7.69(brs,3H),7.61(d,J=8.56Hz,2H),7.32(d, J=8.56Hz,2H),7.06(s,1H),5.56(d,J=16.96Hz,1H),5.51(d,J=16.96Hz,1H),5.47(d,J=19.28Hz,1H ),5.42(d,J=19.28Hz,1H),5.14(d,J=12.20Hz,1H),5.07(d,J=12.16Hz,1H),4.48(t,J=5.24Hz,2H),4 .46-4.43(m,1H),4.29(d,J=5.60Hz,2H),4.08-3.95(m,5H),3.79(t,J=5.28Hz,2H),3.51-3.43(m,32 H),3.40(s,3H),3.39-3.35(m,2H),3.30-3.26(m,2H),3.00(s,3H),2.82-2.74(m,2H),2.56(t,J=7.08 Hz,2H),2.29(t,J=7.36Hz,2H),2.23-2.13(m,2H),1.82(p,J=7.24Hz,2H),1.78-1.63(m,2H),1.61-1 .49(m,2H),1.42-1.27(m,2H),1.15(d,J=6.80Hz,3H),1.13(d,J=6.76Hz,3H),0.90(t,J=7.32Hz,3H). ESI-MS (m / z): 816.0 [M / 2+H] + . [α] D 20 =-19.55°(c=1.000g / 100mL,CH3CN).

[0170] Example 3: Production of anti-CD44v9 antibody complex hHTS033-KL 0.3 mL of antibody hHTS033 25-1 (33.5 mg / mL) was diluted with 0.25 mL of a solution containing 20 mM phosphate buffer (PB), 150 mM NaCl, and 20 mM sodium edetate (pH 7.6), followed by 0.45 mL of a solution containing 20 mM PB and 150 mM NaCl (pH 7.6) and mixing to homogeneity. The mixture was adjusted to pH 7.5 with 1 M NaHPO, followed by addition of 6.0 equivalents of 10 mM tris(2-carboxyethyl)phosphine (TCEP) solution and mixing to homogeneity. The mixture was then allowed to stand at room temperature for 120 minutes. 10 equivalents of TL033 trifluoroacetate dissolved in dimethyl sulfoxide were added to this solution and mixed to homogeneity. The resulting mixture was allowed to stand at room temperature for 90 minutes. The reaction was then stopped by adding 6.1 mL of 100 mM cysteine. Finally, the buffer was replaced with a histidine buffer at pH 6 using a G-25 gel column to obtain a coupling product of TL033 and antibody hHTS033 25-1, which was designated hHTS033-KL.

[0171] Example 4: Average number of drug molecules linked to each antibody (DAR value) The average number of drug molecules bound to each antibody molecule in an antibody-drug conjugate can be determined using the reverse-phase HPLC (RP-HPLC) analytical method described below.

[0172] Reverse-phase chromatography separates components according to their hydrophobicity. After unreduced ADCs are reduced to cleave the interchain disulfide bonds during manufacture, heavy or light chains carrying different numbers of drug molecules differ in hydrophobicity. Higher drug-loaded species exhibit higher hydrophobicity, resulting in better retention and longer retention times on the reverse-phase column. Under low organic phase conditions, the sample is retained on the column, and then components are eluted with an increasing organic phase gradient, from less hydrophobic to more hydrophobic, and detected with a UV detector. The relative abundance of heavy or light chains carrying different numbers of drugs is used to calculate the DAR.

[0173] The peak sequences of each component were: LC0 (light chain not bound to linker-payload), LC1 (light chain bound to one linker-payload), HC0 (heavy chain not bound to linker-payload), HC1 (heavy chain bound to one linker-payload), HC2 (heavy chain bound to two linker-payloads), and HC3 (heavy chain bound to three linker-payloads). The content of each component can be obtained by measuring the peak area ratio of each peak. The DAR of the corresponding sample is then calculated as follows: DAR=(LC0[Area]×0+LC1[Area]×1) / (LC0[Area]+LC1[Area])×2+(HC0[Area]×0+HC1[Area]×1+HC2[Area]×2+HC3[Area]×3) / (HC0[Area]×0+HC1[Area]+HC2[Area]+HC3[Area])×2

[0174] The RP-HPLC chromatogram of hHTS033-KL is shown in Figure 1. Figure 1 showed that the average number of conjugated payloads per antibody measured and calculated by RP-HPLC was 7.8.

[0175] Example 5: Measurement of aggregates in antibody drug conjugates Aggregates in the antibody-drug conjugate were detected using size exclusion chromatography in high-performance liquid chromatography. The method was as follows.

[0176] High-performance liquid chromatography system: Agilent 1260 Infinity II HPLC system Detector: UV absorption spectrometer (detection wavelength: 280 nm) Column type: TOSOH TSKgel G3000SWXL (7.8 x 300 mm, 5 μm) Mobile phase: 200 mmol / L KHPO4, 150 mmol / L NaCl, 15% (v / v) isopropyl alcohol, pH 7.0 Flow rate: 0.75mL / min Analysis time: 18 minutes Column temperature: room temperature Injection amount: 50μg Data Analysis: Substances

[0177] The size exclusion chromatogram of hHTS033-KL is shown in Figure 2. Figure 2 shows the aggregate detection graph, and the aggregate content in the antibody-drug conjugate hHTS033-KL measured by this method was 0.7%.

[0178] Example 6: Binding affinity evaluation of hHTS033-KL in tumor cell lines To evaluate the binding affinity of the ADCs, NCI-H292 (non-small cell lung cancer, BMCR, Catalog No. SCSP-582), A253 (submandibular salivary gland cancer, Meisen CTCC, Catalog No. CTCC-007-0437), A549 (non-small cell lung cancer, iCell, Catalog No. iCell-h011), PC-9 (non-small cell lung cancer, CELLCOOK, Catalog No. CC0204), and Detroit 562 (pharyngeal cancer, BMCR, Catalog No. TCHu231) tumor cells were detached with 1 mM trypsin-EDTA and centrifuged at 1200 rpm for 5 minutes. The cell pellet was resuspended in 5% BSA in PBS. 5 × 10 cells were plated in a 96-well assay plate. 5 50 μL / well of ADC was added and incubated at 2°C to 8°C for 30 minutes. The ADC was diluted to the final concentration indicated and incubated with the cells at 4°C for 1 hour. The cells were washed and incubated with anti-human IgG (Fc-specific)-FITC (fluorescein isothiocyanate) antibody (Sigma, catalog number F9512, 2 mL) as the secondary antibody for 45 minutes in a 4°C incubator. Data were analyzed using a FACSAria™ flow cytometer (MACSQuant X) and Graphpad Prism 8 software.

[0179] As shown in Figure 3, hHTS033-KL specifically bound to NCI-H292, PC-9, A253, and Detroit 562 cell lines, and inhibited EC 50The values ​​were 0.6734 nM, 2.613 nM, 2.179 nM, and 3.197 nM, respectively (Table 7), but there was no binding to the A549 cell line, indicating that A549 does not express CD44v9. An isotype control ADC (human IgG-KL-DAR8, Sichuan Kelun-Biotech Biopharmacuetical Co., Ltd.) did not bind to any of the above cell lines. [Table 10]

[0180] Example 7: Internalization of hHTS033-KL in tumor cell lines PC-9, A253, NCI-H292, and Detroit 562 cell cultures were maintained in vitro as independent monolayer cultures at 37°C under a 5% CO2 atmosphere. The cells were detached with 1 mM EDTA in PBS, washed twice with PBS, and then collected by centrifugation at 1000 rpm for 5 minutes. The cell pellet was resuspended and incubated with 50 μg / mL hHTS033-KL at 4°C for 1 hour. After washing, the cells were incubated in PBS containing 2% BSA at 37°C for 0, 0.5, 1, 2, 4, and 8 hours, respectively. The cells were then fixed in 4% PFA (paraformaldehyde) for 15 minutes at room temperature. The cells were washed, resuspended in PBS, and incubated overnight at 4°C. The cells were then stained with an anti-human antibody conjugated to FITC for 45 minutes and analyzed by calculating the surface MFI (mean fluorescence intensity) using FAC.

[0181] As shown in Figure 4, hHTS033-KL is internalized by PC-9, A253, NCI-H292, and Detroit 562 cell lines. Table 8 shows the T of hHTS033-KL internalization in each tumor cell line. 1 / 2 (h) is shown. [Table 11]

[0182] Example 8: Antitumor activity study of hHTS033-KL in human triple-negative breast cancer cell MDA-MB-468 subcutaneous xenograft mice Human triple-negative breast cancer cells MDA-MB-468 (Cell Bank of the Chinese Academy of Sciences, Catalog No. TCHu136) were cultured in RPMI-1640 medium containing 10% fetal bovine serum in a 5% CO2 incubator at 37°C. Cells were detached with 0.25% trypsin and 0.02% EDTA solution and harvested. 5 × 10 cells were then implanted into the right axilla of female NCG mice (6–8 weeks old) purchased from Jiangsu GemPharmatech Co., Ltd. 6 The average tumor volume was approximately 200 mm. 3 At the time when tumor volume reached 1000 mg / kg, mice were randomly assigned to groups. On day 32 after tumor inoculation, mice received a single intravenous injection of 10 mg / kg of hHTS033-KL, hHTS033 25-1, or isotype control ADC (human IgG-KL-DAR8), respectively. Body weight and tumor volume were measured twice weekly throughout the study period. V (mm 3 ) = 0.5 x length (mm) x width 2 Tumor volume was calculated in mm. Differences between and within groups were analyzed using unpaired two-tailed t-tests with GraphPad Prism version 6.0.

[0183] Tumor volume curves and statistical analysis for mice receiving a single intravenous injection of hHTS033-KL, hHTS033 25-1, and isotype control ADC are shown in Figure 5 and Table 9. Tumor regression was observed with administration of 10 mg / kg of hHTS033-KL (p<0.0001), and all five mice showed tumor disappearance by day 50, demonstrating a sustained antitumor effect until the end of the study. During the administration period, the animals tended to gain weight (Figure 6), and no deaths occurred, indicating that the treatment was well tolerated. [Table 12]

[0184] Example 9: Antitumor activity study of hHTS033-KL in a mouse model of human bladder cancer cell 5637 subcutaneous xenografts Human bladder cancer cell line 5637 (Shanghai Xunqing Biotechnology Co., Ltd.) was cultured in RPMI-1640 medium containing 10% fetal bovine serum in a 5% CO2 incubator at 37°C. Cells were harvested and inoculated into the right axilla of female Balb / c nude mice (6-8 weeks old) purchased from Jiangsu GemPharmatech Co., Ltd. at 10 ng / ml. 7 Cells were implanted subcutaneously at 100 cells / animal, with an average tumor volume of approximately 200 mm. 3 At the time of tumor inoculation, the animals were randomly divided into six groups of five. Four days after tumor inoculation, the mice received a single intravenous injection of hHTS033-KL (5 mg / kg or 2.5 mg / kg), hHTS033 25-1 (5 mg / kg), isotype control ADC (human IgG-KL-DAR8, Sichuan Kelun-Biotech Biopharmacuetical Co., Ltd.) (5 mg / kg or 2.5 mg / kg), or vehicle. V (mm 3 ) = 0.5 x length (mm) x width 2 Tumor volume was calculated in mm. Body weight and tumor volume were measured two to three times weekly throughout the study period. Differences between and within groups were analyzed using unpaired two-tailed t-tests in GraphPad Prism version 6.0.

[0185] Tumor volume curves and statistical analysis are shown in Figure 7 and Table 10. Treatment with 2.5 mg / kg and 5 mg / kg hHTS033-KL resulted in TGIs of 173.4% and 192.3%, respectively, whereas treatment with 5 mg / kg hHTS033 25-1 showed no antitumor activity. Treatment with both 2.5 mg / kg and 5 mg / kg hHTS033-KL resulted in sustained tumor regression. Animal weights tended to increase during the treatment period (Figure 8), and no deaths occurred, indicating that the 2.5 mg / kg and 5 mg / kg dose levels of hHTS033-KL were well tolerated in the 5637 xenograft model. [Table 13]

[0186] Example 10: Antitumor activity study of hHTS033-KL in NSCLC PDX mouse model The tumors (see Table 11) were cut into 3 mm x 3 mm x 3 mm (approximately 45-60 mg) fragments and implanted subcutaneously into the right flank of mice (female NCG mice, 35-42 days old) purchased from GemPharmatech Co., Ltd. The average tumor size was 161.42 mm. 3 At the time of tumor volume reaching 1000 mg / kg, mice were randomly divided into two groups based on tumor volume. Mice were intravenously administered hHTS033-KL and isotype control ADC (human IgG-KL-DAR8, Sichuan Kelun-Biotech Biopharmacuetical Co., Ltd.) at 10 mg / kg on days 0 (the day of first drug administration), 7, and 14. Tumor size was measured bidimensionally twice weekly using calipers and calculated using the formula: V = 0.5a × b 2 Tumor volume was calculated using 3 The mean values ​​are expressed in units, where a and b are the long and short diameters of the tumor, respectively. The body weight of each animal was recorded twice weekly, and a t-test was performed to compare the differences between the two groups. All data were analyzed using GraphPad Prism 8, and p<0.05 indicated that the differences between the groups were statistically significant. [Table 14]

[0187] The results are shown in Figures 9, 10, and Table 12. Treatment with hHTS033-KL (10 mg / kg, QW x 3) demonstrated statistically significant antitumor effects in the lung cancer PDX model LD1-0025-361336 compared to the isotype control ADC (10 mg / kg, QW x 3) group. In this study, treatment was well tolerated in all mice, as assessed by body weight change. [Table 15]

[0188] Example 11: Efficacy study of hHTS033-KL in gastric cancer PDX model LD1-0017-361443 The tumors (see Table 13) were cut into 3 mm x 3 mm x 3 mm (approximately 45-60 mg) fragments and subcutaneously implanted into the right flank of mice (female Nu / Nu mice, 35-42 days old) purchased from Beijing Vital River Laboratory Animal Technology Co. Ltd. At 15 days post-inoculation, the average tumor size was 163.38 mm. 3 At the time of tumor volume reaching 100 mg / kg, mice were randomly divided into three groups based on tumor volume. Mice were intravenously administered hHTS033-KL, isotype control ADC (human IgG-KL-DAR8, Sichuan Kelun-Biotech Biopharmacuetical Co., Ltd.) at 10 mg / kg, or vehicle control on days 0 (the first drug administration day) and 7, respectively. Tumor size was measured twice weekly and calculated using the formula: V = 0.5a × b 2 Tumor volume was calculated using 3 The data are expressed in units, where a and b are the long and short diameters of the tumor, respectively. The body weight of each animal was recorded twice weekly. One-way analysis of variance was performed to compare the tumor volume of each group with that of the vehicle group. A t-test was performed to compare the differences between two groups. All data were analyzed using GraphPad Prism 8, and p<0.05 indicated that the difference between groups was statistically significant. [Table 16]

[0189] The results are shown in Figure 11, Figure 12, and Table 14. In summary, treatment with hHTS033-KL 10 mg / kg QW x 2 demonstrated statistically significant antitumor effects in the gastric cancer PDX model LD1-0017-361443 compared to the vehicle control group, and treatment with hHTS033-KL 10 mg / kg QW x 2 demonstrated statistically significant antitumor effects compared to the isotype control ADC 10 mg / kg QW x 2 group. In this study, treatment was well tolerated in all mice as assessed by body weight change. [Table 17]

[0190] Example 12: Efficacy study of hHTS033-KL in liver cancer PDX model LD1-0011-360763 The tumors (see Table 15) were cut into 3 mm x 3 mm x 3 mm (approximately 45-60 mg) fragments and subcutaneously implanted into the right flank of mice (female Nu / Nu mice, 35-42 days old) purchased from Beijing Vital River Laboratory Animal Technology Co. Ltd. On day 34 after inoculation, the average tumor size was approximately 150 mm. 3 At the time of tumor volume reaching 1000 mg / kg, mice were randomly divided into three groups based on tumor volume. Mice were intravenously administered hHTS033-KL, isotype control ADC (Sichuan Kelun-Biotech Biopharmacuetical Co., Ltd.) 10 mg / kg, and vehicle control on days 0 (first drug administration), 7, and 14, respectively. Tumor size and body weight were measured twice weekly. Formula: V = 0.5a × b 2 Tumor volume was calculated using 3 The values ​​are expressed in units, where a and b are the long and short diameters of the tumor, respectively. A one-way analysis of variance was performed to compare the tumor volume of each group with that of the vehicle group. A t-test was performed to compare the differences between the two groups. All data were analyzed using GraphPad Prism 8, and p<0.05 indicated that the difference between the groups was statistically significant. [Table 18]

[0191] The curves and statistical analysis are shown in Figure 13, Figure 14, and Table 16. In summary, treatment with hHTS033-KL at 10 mg / kg QW x 3 demonstrated statistically significant antitumor effects compared to the vehicle control group in the liver cancer PDX model LD1-0011-360763. Treatment with hHTS033-KL at 10 mg / kg QW x 3 also demonstrated statistically significant antitumor effects compared to treatment with an isotype control ADC (human IgG-KL-DAR8, Sichuan Kelun-Biotech Biopharmacuetical Co., Ltd.) at 10 mg / kg QW x 3. In this study, treatment was well tolerated in all mice, as assessed by body weight change. [Table 19]

[0192] Example 13: A 4-week GLP repeat-dose toxicity study of hHTS033-KL in cynomolgus monkeys followed by a 6-week recovery period Repeated administration of hHTS033-KL to cynomolgus monkeys: Forty cynomolgus monkeys (Zhanjiang Prima Biotech Inc.) were randomly divided into four groups, each containing five males and five females. Vehicle, 10 mg / kg hHTS033-KL, 20 mg / kg hHTS033-KL, and 30 mg / kg hHTS033-KL were intravenously administered on days 1, 15, and 29, respectively. During the experiment, animals were observed for abnormalities, and blood samples were collected for hematological examination and analysis of blood biochemistry indicators. Animals for the main necropsy (day 36) and recovery necropsy (day 71) were euthanized, and samples were collected for pathological analysis.

[0193] As shown in Table 17, under the conditions of this study, hHTS033-KL was administered intravenously to cynomolgus monkeys at doses of 10 mg / kg, 20 mg / kg, and 30 mg / kg for 4 weeks (once every 2 weeks, for a total of 3 doses) and was clinically well tolerated. Transient mild irritation and histopathological changes unrelated to the test product were observed at the injection site in all dose groups, but no significant abnormalities were observed in the nervous, cardiovascular, or respiratory systems. The maximum non-serious toxicity dose (HNSTD) of hHTS033-KL was 30 mg / kg, and the main toxic findings were a decrease in reticulocytes and pigmentation of the skin and / or oral mucosa. After a 6-week recovery period, these changes resolved without significant delayed toxicity. [Table 20]

Claims

1. An antibody-drug conjugate having the structure of formula (I): (T-L) γ -mAb (I) where: (i) T is a cytotoxic drug moiety having the structure of formula (II): 【Chemistry 1】 Here, R is -C(=O)R ｘ , -S(=O)R ｘ Or -S(=O) ２ R ｘ where R ｘ is C １～６ Alkyl groups, halogenated C １～６ Alkyl group, C ２～６ Alkenyl group, C ２～６ Alkynyl group, C ３～６ Cycloalkyl group, C ３～６ R is selected from the group consisting of a heterocyclyl group, a 6- to 10-membered aryl group, and a 5- to 14-membered heteroaryl group, and preferably R is —C(═O)R ｘ Or -S(=O) ２ R ｘ where R ｘ is C １～６ Alkyl groups, halogenated C １～６ Alkyl group, C ３～６ Cycloalkyl group, C ３～６ R is selected from the group consisting of a heterocyclyl group, a 6- to 10-membered aryl group, and a 5- to 14-membered heteroaryl group, more preferably R is —C(═O)R ｘ Or -S(=O) ２ R ｘ where R ｘ is C １～６ Alkyl group, C ３～６ selected from the group consisting of cycloalkyl groups and phenyl groups; (ii) L is a linker; (iii) γ is an integer or decimal number from 1 to 10, preferably from 5 to 8, and (iv) mAb is an antibody moiety, wherein the antibody is an anti-CD44v9 antibody or an antigen-binding fragment thereof; Antibody-drug conjugates.

2. The linker has the structure of formula (III): -L １ -(L ２ )m １ -(L ３ )m ２ -(L ４ )m ３ -E-G- (III) Here, L １ is bound to T and G is bound to the mAb, and (i)L １ Lys, Cit, Cit-Val, Val-Al, Lys-Val, 【Chemistry 2】 wherein R′, R １ and R ２ are each independently H (hydrogen), D (deuterium), or C １～４ is an alkyl group, and Z １ is Cit, Lys, Cit-Val, Cit-Ala, Val-Ala, or Lys-Val, x1 and x3 are each independently 0, 1, or 2, and L １ Is L １ is attached to T at position 1 of １ are Lys, Cit, Cit-Val, Val-Ala, Lys-Val, 【Transformation 3】 and more preferably, L １ is the following: 【Chemistry 4】 (ii) L ２ is selected from the group consisting of: 【Transformation 5】 Here, R ３ , R ４ , R ５ and R ６ are each independently H (hydrogen), D (deuterium), or C １～４ alkyl groups, y1 and y2 are each independently 0, 1, 2, 3, 4, 5, 6, 7, or 8, and L ２ Is L ２ In first place is L １ and preferably L ２ is selected from the group consisting of: 【Transformation 6】 More preferably, L ２ is the following: 【Transformation 7】 (iii) L ３ optionally one or more R ７ wherein each R is selected from the group consisting of an amino group, an N-methylpiperidine group, a pyrazolylene group, or a triazolylene group, substituted by ７ are H (hydrogen), D (deuterium), halogen, ═O, CF ３ , C.N., C.H. ２ CN, carboxy group, sulfonic acid group, C １～４ Alkyl group, C １～４ Alkoxy group, C ２～６ Alkenyl group or C ２～６ alkynyl groups, preferably L ３ is a triazolylene group having the structure 【Transformation 8】 And L ３ In first place is L ２ is combined with (iv) L ４ is selected from the group consisting of: 【Chemistry 9】 Here, L ４ Is L ４ is bonded to E at the 2-position of ４ is selected from the group consisting of: 【Chemistry 10】 More preferably, L ４ is the following: 【Chemistry 11】 (v) E optionally comprises one or more R １２ wherein each R is selected from the group consisting of a pyrimidylene group, a quinolylene group, or a pyrrolo[2,3-d]pyrimidylene group substituted by １２ are H (hydrogen), D (deuterium), halogen, CN, nitro group, C １～２ Alkyl group or halogenated C １～２ alkyl groups, and preferably E is optionally one or more R １２ and a pyrimidinyl group substituted by １２ are independently H (hydrogen) or D (deuterium), (vi) G is a thioether bond, and (vii) m １ is 0, 1, 2 or 3, and preferably, m １ is 1, and m ２ is 0, 1 or 2, and preferably, m ２ is 0 or 1, and m ３ is 0, 1, 2 or 3, and preferably, m ３ The antibody-drug conjugate of claim 1 , wherein

3. said cytotoxic drug moiety T is selected from the group consisting of: 【Chemistry 12】 Preferably, the cytotoxic drug moiety is: 【Chemistry 13】 And more preferably, the cytotoxic drug moiety is: 【Chemistry 14】 The antibody-drug conjugate according to claim 1 or 2.

4. The antibody-drug conjugate has a structure selected from the group consisting of: 【Chemistry 15】 【Chemistry 16】 【Chemistry 17】 [Chemistry 18] 【Chemistry 19】 【Chemistry 20】 The antibody-drug conjugate of any one of claims 1 to 3, wherein γ is an integer or decimal number from 5 to 8, and the mAb is an anti-CD44v9 antibody or an antigen-binding fragment thereof.

5. The antibody-drug conjugate according to any one of claims 2 to 4, wherein G of the linker moiety is formed from a thiol group of a Cys residue in the mAb, and preferably the thiol group of the Cys residue in the mAb is a thiol group formed by opening a disulfide bond in the mAb.

6. The antibody-drug conjugate of any one of claims 1 to 5, wherein the mAb binds to a CD44v9 epitope comprising or consisting essentially of SEQ ID NO:55 or 56.

7. the mAb comprises a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3, and a light chain variable region comprising LCDR1, LCDR2, and LCDR3, wherein the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are the same as any one of (a) to (d); (a) HCDR1, HCDR2, and HCDR3 of a heavy chain variable region of SEQ ID NO: 47 and LCDR1, LCDR2, and LCDR3 of a light chain variable region of SEQ ID NO: 48; (b) HCDR1, HCDR2, and HCDR3 of a heavy chain variable region of SEQ ID NO: 49 and LCDR1, LCDR2, and LCDR3 of a light chain variable region of SEQ ID NO: 50; (c) HCDR1, HCDR2, and HCDR3 of a heavy chain variable region of SEQ ID NO: 51 and LCDR1, LCDR2, and LCDR3 of a light chain variable region of SEQ ID NO: 52; and (d) HCDR1, HCDR2, and HCDR3 of a heavy chain variable region of SEQ ID NO: 53 and LCDR1, LCDR2, and LCDR3 of a light chain variable region of SEQ ID NO: 54; and The antibody-drug conjugate of any one of claims 1 to 6, wherein the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are preferably defined according to any one of the CDR definition schemes selected from the group consisting of Chothia, Kabat, IMGT, AbM, and Contact.

8. The mAb (a) a heavy chain variable region comprising an HCDR1 having the amino acid sequence of SEQ ID NO: 1, an HCDR2 having the amino acid sequence of SEQ ID NO: 2, and an HCDR3 having the amino acid sequence of SEQ ID NO: 3, and a light chain variable region comprising an LCDR1 having the amino acid sequence of SEQ ID NO: 4, an LCDR2 having the amino acid sequence of SEQ ID NO: 5, and an LCDR3 having the amino acid sequence of SEQ ID NO: 6, according to the IMGT CDR definitions; or (b) The antibody-drug conjugate of any one of claims 1 to 7, comprising a heavy chain variable region comprising HCDR1 having the amino acid sequence of SEQ ID NO: 31, HCDR2 having the amino acid sequence of SEQ ID NO: 32, and HCDR3 having the amino acid sequence of SEQ ID NO: 33, according to the IMGT CDR definitions; and a light chain variable region comprising LCDR1 having the amino acid sequence of SEQ ID NO: 34, LCDR2 having the amino acid sequence of SEQ ID NO: 35, and LCDR3 having the amino acid sequence of SEQ ID NO:

36.

9. The mAb (a) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 47 and a light chain variable region having the amino acid sequence of SEQ ID NO: 48; (b) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 49 and a light chain variable region having the amino acid sequence of SEQ ID NO: 50; (c) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 51 and a light chain variable region having the amino acid sequence of SEQ ID NO: 52; or (d) the antibody-drug conjugate according to any one of claims 1 to 8, comprising a heavy chain variable region having the amino acid sequence of SEQ ID NO: 53 and a light chain variable region having the amino acid sequence of SEQ ID NO:

54.

10. 10. The antibody-drug conjugate of claim 9, wherein the mAb comprises a heavy chain constant region having the amino acid sequence of SEQ ID NO: 45 and a light chain constant region having the amino acid sequence of SEQ ID NO:

46.

11. An antibody-drug conjugate selected from the group consisting of: 【Chemistry 21】 【Chemistry 22】 【Chemistry 23】 【Chemistry 24】 wherein γ is an integer or decimal number from 5 to 8, and the mAb is an anti-CD44v9 monoclonal antibody or an antigen-binding fragment thereof, wherein the mAb is linked via a thiol group of a Cys residue in the mAb, and the mAb comprises a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3, and a light chain variable region comprising LCDR1, LCDR2, and LCDR3, and wherein the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are the same as any of (a) to (d); (a) HCDR1, HCDR2, and HCDR3 of a heavy chain variable region of SEQ ID NO: 47 and LCDR1, LCDR2, and LCDR3 of a light chain variable region of SEQ ID NO: 48; (b) HCDR1, HCDR2, and HCDR3 of a heavy chain variable region of SEQ ID NO: 49 and LCDR1, LCDR2, and LCDR3 of a light chain variable region of SEQ ID NO: 50; (c) HCDR1, HCDR2, and HCDR3 of a heavy chain variable region of SEQ ID NO: 51 and LCDR1, LCDR2, and LCDR3 of a light chain variable region of SEQ ID NO: 52; and (d) HCDR1, HCDR2, and HCDR3 of a heavy chain variable region of SEQ ID NO: 53 and LCDR1, LCDR2, and LCDR3 of a light chain variable region of SEQ ID NO: 54; and wherein the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are preferably defined according to any of the CDR definition schemes selected from the group consisting of Chothia, Kabat, IMGT, AbM, and Contact.

12. 1. An antibody-drug conjugate having the structure: 【Chemistry 25】 wherein γ is an integer or decimal number from 5 to 8, and the mAb is an anti-CD44v9 monoclonal antibody or an antigen-binding fragment thereof, and wherein the thioether bond linked to the mAb is formed from the thiol group of a Cys residue in the mAb.

13. The antibody-drug conjugate of claim 12, wherein the thiol group of the Cys residue in the mAb is a thiol group formed by opening a disulfide bond in the mAb or a free Cys residue contained in the mAb.

14. The antibody-drug conjugate of claim 12 or 13, wherein the mAb specifically binds to a CD44v9 epitope comprising or consisting essentially of SEQ ID NO: 55 or 56.

15. the mAb comprises a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3, and a light chain variable region comprising LCDR1, LCDR2, and LCDR3, wherein the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are the same as any one of (a) to (d); (a) HCDR1, HCDR2, and HCDR3 of a heavy chain variable region of SEQ ID NO: 47 and LCDR1, LCDR2, and LCDR3 of a light chain variable region of SEQ ID NO: 48; (b) HCDR1, HCDR2, and HCDR3 of a heavy chain variable region of SEQ ID NO: 49 and LCDR1, LCDR2, and LCDR3 of a light chain variable region of SEQ ID NO: 50; (c) HCDR1, HCDR2, and HCDR3 of a heavy chain variable region of SEQ ID NO: 51 and LCDR1, LCDR2, and LCDR3 of a light chain variable region of SEQ ID NO: 52; and (d) HCDR1, HCDR2, and HCDR3 of a heavy chain variable region of SEQ ID NO: 53 and LCDR1, LCDR2, and LCDR3 of a light chain variable region of SEQ ID NO: 54; and The antibody-drug conjugate of any one of claims 12 to 14, wherein the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are preferably defined according to any one of the CDR definition schemes selected from the group consisting of Chothia, Kabat, IMGT, AbM, and Contact.

16. The mAb (a) a heavy chain variable region comprising an HCDR1 having the amino acid sequence of SEQ ID NO: 1, an HCDR2 having the amino acid sequence of SEQ ID NO: 2, and an HCDR3 having the amino acid sequence of SEQ ID NO: 3, and a light chain variable region comprising an LCDR1 having the amino acid sequence of SEQ ID NO: 4, an LCDR2 having the amino acid sequence of SEQ ID NO: 5, and an LCDR3 having the amino acid sequence of SEQ ID NO: 6, according to the IMGT CDR definitions; or (b) The antibody-drug conjugate of claim 15, comprising a heavy chain variable region comprising an HCDR1 having the amino acid sequence of SEQ ID NO: 31, an HCDR2 having the amino acid sequence of SEQ ID NO: 32, and an HCDR3 having the amino acid sequence of SEQ ID NO: 33, according to the CDR definitions of IMGT, and a light chain variable region comprising an LCDR1 having the amino acid sequence of SEQ ID NO: 34, an LCDR2 having the amino acid sequence of SEQ ID NO: 35, and an LCDR3 having the amino acid sequence of SEQ ID NO:

36.

17. The mAb (a) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 47 and a light chain variable region having the amino acid sequence of SEQ ID NO: 48; (b) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 49 and a light chain variable region having the amino acid sequence of SEQ ID NO: 50; (c) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 51 and a light chain variable region having the amino acid sequence of SEQ ID NO: 52; or (d) the antibody-drug conjugate of claim 16, comprising a heavy chain variable region having the amino acid sequence of SEQ ID NO: 53 and a light chain variable region having the amino acid sequence of SEQ ID NO:

54.

18. The antibody-drug conjugate of any one of claims 12 to 17, wherein the mAb comprises a heavy chain constant region having the amino acid sequence of SEQ ID NO: 45 and a light chain constant region having the amino acid sequence of SEQ ID NO:

46.

19. A pharmaceutical composition for cancer treatment, comprising the antibody-drug conjugate according to any one of claims 1 to 18 and a pharmaceutically acceptable carrier.

20. 20. Use of the antibody-drug conjugate of any one of claims 1 to 18 in the manufacture of a medicament for treating cancer, optionally wherein the cancer is a solid tumor, preferably esophageal cancer such as NSCLC (non-small cell lung cancer), HNSCC (head and neck squamous cell carcinoma), ESCC (esophageal squamous cell carcinoma), GC (gastric cancer), BC (bladder cancer), breast cancer such as TNBC (triple-negative breast cancer), colon cancer, and liver cancer.

21. 20. An antibody-drug conjugate according to any one of claims 1 to 18 for use in the treatment of cancer, optionally wherein the cancer is a solid tumor, preferably esophageal cancer such as NSCLC (non-small cell lung cancer), HNSCC (head and neck squamous cell carcinoma), ESCC (esophageal squamous cell carcinoma), breast cancer such as GC (gastric cancer), BC (bladder cancer), TNBC (triple-negative breast cancer), colon cancer and liver cancer.

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