Antibody drug conjugates, pharmaceutical compositions and uses thereof

Abstract

The invention discloses an antibody drug conjugate, wherein an anti-CD44v9 antibody or an antigen binding fragment thereof is linked to a belotecan derivative. The conjugates show an excellent anti-tumor effect in various solid tumor models. Also disclosed are pharmaceutical compositions comprising any of these conjugates and methods of using any of these conjugates to treat cancer.

Description

Technical Field

[0001] The present disclosure relates to antibody drug conjugates, and in particular to anti-CD44v9 antibody drug conjugates. The present disclosure also relates to pharmaceutical compositions, preparation methods, uses, and treatment methods of the conjugates. Background Art

[0002] CD44 is a single transmembrane glycoprotein family that participates in homotypic cell-cell, cell-matrix and cell-cytoskeleton interactions. There are several well-known CD44 ligands, including hyaluronic acid (HA), osteopontin (OPN), collagen and matrix metalloproteinases (MMPs). In the human body, CD44 is encoded by 19 exons, of which the first five exons (exons 1-5) and the last five exons (exons 16-20) constantly encode the most common CD44s and the smallest CD44 protein. CD44 variant isoforms (CD44v) are produced by alternative splicing (v2-v10) and have any combination of ten CD44s exons plus the remaining nine variant exons. CD44v isoforms play various roles in cancer and may have an impact on key features of carcinogenesis such as tumor triggering and metastasis. These key features are generally derived from the signal transduction of changes, resistance to oxidative stress, chemical / radioresistance, adhesion, extracellular matrix interactions, tumor matrix formation or the regulation of CSC niche. Furthermore, CD44 variants have been shown to play an important role in Ras activation by forming a co-receptor complex with hepatocyte growth factor (HGF) and its receptor tyrosine kinase Met. Phosphorylation of this co-receptor complex activates Ras signaling. Some isoforms also exhibit anti-apoptotic effects and can block Fas-mediated apoptosis.

[0003] One of the isoforms, CD44v9, a so-called splice variant containing the v9 exon, is overexpressed in a variety of 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, while its expression in normal healthy tissues is very limited. In a meta-analysis, compared with data from CD44v9-negative patients, CD44v9-positive expression was also associated with larger tumor size, deeper tumor invasion, earlier lymph node metastasis, and later clinical stage. Based on these characteristics, CD44v9 is a promising target for the development of potential antibody-derived cancer therapeutics.

[0004] The main categories of CD44 targeted therapies include peptide mimetics, aptamers, pharmacological inhibitors, CD44 decoys, HA oligomers, HyACT (hyaluronic acid chemical transport), neutralizing antibodies and antibody-drug conjugates. Monoclonal CD44-specific antibodies that interfere with the binding of HA to CD44 can inhibit the anchorage-independent growth of mouse breast cancer cells and human colon cancer cells, and may also induce apoptosis. HA oligosaccharides have similar effects and inhibit tumor growth in vivo, which is most likely achieved by interfering with the binding of hHA (high molecular weight HA, about 2000 kDa) to CD44. In addition, CD44 antibodies are used as targeting agents for tumor-specific delivery of radiotherapy or cytotoxic payloads. Several of the above therapeutic compounds have been tested in clinical trials.

[0005] For example, a randomized phase II study of hyaluronic acid-irinotecan (HA-IR) showed improved progression-free survival compared with irinotecan in patients with metastatic colorectal cancer, but unfortunately, these results were not confirmed in a subsequent phase III trial. Another phase IIa trial of HA-irinotecan compared irinotecan and carboplatin in combination with carboplatin in the treatment of extensive-stage small cell lung cancer (SCLC). The study concluded that the therapy was well tolerated, but there was no statistically significant difference in efficacy between the treatment and control groups, although there was a possible trend towards a survival benefit in CD44s-positive tumors. For direct CD44 and specific variant targeting, RG7356, a humanized antibody that binds to the HA-binding proximal domain of all CD44 isoforms, was tested in clinical trials in AML (acute myeloid leukemia) and advanced solid tumors and had limited clinical efficacy but was generally well tolerated. A phase I study of U36, a chimeric monoclonal antibody specific for CD44v6 and labeled with 186Re, was conducted in patients with head and neck squamous cell carcinoma (HNSCC). Studies have shown that the U36 radioconjugate can be safely administered with dose-limiting myelotoxicity and produces a transient therapeutic effect. Although these CD44-targeting drug modalities have yielded only modest clinical efficacy results, they have been generally well tolerated, and some accompanying imaging studies have demonstrated tumor-specific targeting. However, phase I trials of the humanized monoclonal antibody (mAb) bevacizumab (BIWA-4) combined with the potent microtubule inhibitor mertansine to specifically target CD44v6 in patients with HNSCC and metastatic breast cancer have yielded mixed clinical results. Although partial responses were observed in three patients at high dose levels, these clinical trials were interrupted due to significant skin toxicity, with one fatal outcome attributed to the bevacizumab-mertansine conjugate.

[0006] In summary, although a considerable number of CD44-targeting anticancer agents have been investigated in clinical trials, no active compounds with satisfactory therapeutic efficacy and sufficient safety have been successfully developed. Furthermore, none of the compounds investigated in these clinical trials target CD44v9. To date, no antibody-drug conjugates (ADCs) targeting CD44v9 have been approved for clinical trials, nor have any clinical studies been conducted. Unique information regarding the druggability of CD44v9 anticancer agents, particularly ADCs, such as efficacy, safety, pharmacokinetic properties, and therapeutic window, is unknown and requires further investigation. Summary of the Invention

[0007] In one aspect, an antibody drug conjugate having a structure of formula (I) is provided, (TL) γ -mAb(I),

[0008] in:

[0009] T is a cytotoxic drug moiety having the structure of formula (II), wherein R is -C(=O)R x 、-S(=O)R x or -S(=O)2R x , where R x Selected from C 1-6 Alkyl, halogenated C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 3-6 Cycloalkyl, C 3-6 heterocyclic, 6-10 membered aryl and 5-14 membered heteroaryl; preferably, R is -C(=O)R x or -S(=O)2R x , where R x Selected from C 1-6 Alkyl, halogenated C 1-6 Alkyl, C 3-6 Cycloalkyl, C 3-6 heterocyclic, 6-10 membered aryl and 5-14 membered heteroaryl; more preferably, R is -C(=O)R x or -S(=O)2R x , where R x Selected from C 1-6 alkyl, C 3-6 Cycloalkyl and phenyl;

[0010] L is the connector;

[0011] γ is an integer or decimal ranging from 1 to 10, preferably 5 to 8; and

[0012] The mAb is an antibody portion, wherein the antibody is an anti-CD44v9 antibody or an antigen-binding fragment thereof.

[0013] In some embodiments, the linker has the structure of Formula (III), -L1-(L2)m1-(L3)m2-(L4)m3-EG-(III)

[0014] wherein L1 is bonded to T, and G is bonded to mAb, and wherein

[0015] L1 is selected from Lys, Cit, Cit-Val, Val-Ala, Lys-Val, wherein R', R1 and R2 are each independently H (hydrogen), D (deuterium) or C 1-4 alkyl, 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 position 1 of L1; preferably, L1 is selected from Lys, Cit, Cit-Val, Val-Ala, Lys-Val, More preferably, L1 is

[0016] L2 is selected from wherein R3, R4, R5 and R6 are each independently selected from H (hydrogen), D (deuterium) or C 1-4 Alkyl, y1 and y2 are each independently 0, 1, 2, 3, 4, 5, 6, 7 or 8, and L2 is bonded to L1 at position 1 of L2; preferably, L2 is selected from More preferably, L2 is

[0017] L3 is selected from the following groups optionally substituted with one or more R7: amino, N-methylpiperidine, pyrazole or triazole, wherein each R7 is independently selected from H (hydrogen), D (deuterium), halogen, =O, CF3, CN, CH2CN, carboxyl, sulfonic acid group, C 1-4 Alkyl, C 1-4 Alkoxy, C 2-6 Alkenyl or C 2-6 Alkynyl; preferably, L3 is A triazole of the structure of , and bonded to L2 at position 1 of L3;

[0018] L4 is selected from wherein L4 is bonded to E at position 2 of L4; preferably, L4 is selected from More preferably, L4 is

[0019] E is selected from optionally one or more R 12 Substituted pyrimidine, quinoline or pyrrolo[2,3-d]pyrimidine, wherein each R 12 independently selected from H (hydrogen), D (deuterium), halogen, CN, nitro, C 1-2 Alkyl or halogenated C 1-2 Alkyl; preferably, E is optionally replaced by one or more R 12 Substituted pyrimidinyl, wherein each R 12 is independently H (hydrogen) or D (deuterium);

[0020] G is a thioether bond; and

[0021] 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.

[0022] In some embodiments, the cytotoxic drug moiety T is selected from Preferably, the cytotoxic drug moiety is And more preferably, the cytotoxic drug moiety is

[0023] In a preferred embodiment, the antibody drug conjugate has a structure selected from the group consisting of:

[0024] wherein γ is an integer or decimal ranging from 5 to 8, and mAb is an anti-CD44v9 antibody or an antigen-binding fragment thereof.

[0025] In some embodiments, the G of the linker moiety is formed by a thiol group of a Cys residue in the mAb; preferably, the thiol group of the Cys residue in the mAb is a thiol group formed by opening a disulfide bond in the mAb.

[0026] In some embodiments, the mAb binds to a CD44v9 epitope comprising or consisting essentially of SEQ ID NO. 55 or 56.

[0027] 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 one of (a) to (d):

[0028] (a) HCDR1, HCDR2, and HCDR3 of the heavy chain variable region of SEQ ID NO: 47; and LCDR1, LCDR2, and LCDR3 of the light chain variable region of SEQ ID NO: 48;

[0029] (b) 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;

[0030] (c) 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

[0031] (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; and

[0032] Wherein HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are preferably defined according to any CDR definition scheme selected from Chothia, Kabat, IMGT, AbM and Contact.

[0033] In some embodiments, the mAb comprises:

[0034] (a) a heavy chain variable region comprising: a HCDR1 having the amino acid sequence of SEQ ID NO: 1, a HCDR2 having the amino acid sequence of SEQ ID NO: 2, and a HCDR3 having the amino acid sequence of SEQ ID NO: 3, and a light chain variable region comprising: a LCDR1 having the amino acid sequence of SEQ ID NO: 4, a LCDR2 having the amino acid sequence of SEQ ID NO: 5, and a LCDR3 having the amino acid sequence of SEQ ID NO: 6, according to the IMGT definition of CDRs; or

[0035] (b) a heavy chain variable region comprising: a HCDR1 having an amino acid sequence of SEQ ID NO: 31, a HCDR2 having an amino acid sequence of SEQ ID NO: 32, and a HCDR3 having an amino acid sequence of SEQ ID NO: 33, and a light chain variable region comprising: a LCDR1 having an amino acid sequence of SEQ ID NO: 34, a LCDR2 having an amino acid sequence of SEQ ID NO: 35, and a LCDR3 having an amino acid sequence of SEQ ID NO: 36, according to the IMGT definition of CDRs.

[0036] In some embodiments, the mAb comprises:

[0037] (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;

[0038] (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;

[0039] (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

[0040] (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.

[0041] 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.

[0042] In a preferred embodiment, an antibody drug conjugate is provided, which is selected from:

[0043] wherein γ is an integer or decimal ranging 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 and a light chain variable region, the heavy chain variable region comprising HCDR1, HCDR2, and HCDR3, and the 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):

[0044] (a) HCDR1, HCDR2, and HCDR3 of the heavy chain variable region of SEQ ID NO: 47; and LCDR1, LCDR2, and LCDR3 of the light chain variable region of SEQ ID NO: 48;

[0045] (b) 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;

[0046] (c) 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

[0047] (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; and

[0048] Wherein HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are preferably defined according to any CDR definition scheme selected from Chothia, Kabat, IMGT, AbM and Contact.

[0049] In a preferred embodiment, an antibody drug conjugate having the following structure is provided:

[0050] wherein γ is an integer or decimal ranging from 5 to 8, and mAb is an anti-CD44v9 monoclonal antibody or an antigen-binding fragment thereof, wherein the thioether bond attached to the mAb is formed by a thiol group of a Cys residue in the mAb.

[0051] 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 a free Cys residue contained in the mAb.

[0052] In some embodiments, the mAb specifically binds to a CD44v9 epitope comprising or consisting essentially of SEQ ID NO. 55 or 56.

[0053] 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 one of (a) to (d):

[0054] (a) HCDR1, HCDR2, and HCDR3 of the heavy chain variable region of SEQ ID NO: 47; and LCDR1, LCDR2, and LCDR3 of the light chain variable region of SEQ ID NO: 48;

[0055] (b) 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;

[0056] (c) 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

[0057] (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; and

[0058] Wherein HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are preferably defined according to any CDR definition scheme selected from Chothia, Kabat, IMGT, AbM and Contact.

[0059] In some embodiments, the mAb comprises:

[0060] (a) a heavy chain variable region comprising: a HCDR1 having the amino acid sequence of SEQ ID NO: 1, a HCDR2 having the amino acid sequence of SEQ ID NO: 2, and a HCDR3 having the amino acid sequence of SEQ ID NO: 3, and a light chain variable region comprising: a LCDR1 having the amino acid sequence of SEQ ID NO: 4, a LCDR2 having the amino acid sequence of SEQ ID NO: 5, and a LCDR3 having the amino acid sequence of SEQ ID NO: 6, according to the IMGT definition of CDRs; or

[0061] (b) a heavy chain variable region comprising: a HCDR1 having an amino acid sequence of SEQ ID NO: 31, a HCDR2 having an amino acid sequence of SEQ ID NO: 32, and a HCDR3 having an amino acid sequence of SEQ ID NO: 33, and a light chain variable region comprising: a LCDR1 having an amino acid sequence of SEQ ID NO: 34, a LCDR2 having an amino acid sequence of SEQ ID NO: 35, and a LCDR3 having an amino acid sequence of SEQ ID NO: 36, according to the IMGT definition of CDRs.

[0062] In some embodiments, the mAb comprises:

[0063] (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;

[0064] (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;

[0065] (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

[0066] (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.

[0067] 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.

[0068] In another aspect, provided is a pharmaceutical composition for treating cancer, comprising the antibody drug conjugate as disclosed herein and a pharmaceutically acceptable carrier.

[0069] In yet another aspect, provided is a method for treating cancer in a subject, the method comprising administering to the subject an effective amount of an antibody drug conjugate as disclosed herein.

[0070] In yet another aspect, an antibody drug conjugate as disclosed herein for use in treating cancer is provided.

[0071] In yet another aspect, there is provided use of an antibody drug conjugate as disclosed herein in the preparation of a medicament for treating cancer.

[0072] Beneficial effects of the present invention

[0073] The antibody drug conjugates of the present disclosure specifically target CD44v9 and induce cytotoxicity in cells ectopically expressing CD44v9, but have no obvious toxic side effects on cells normally expressing CD44v9.

[0074] The antibody drug conjugates of the present disclosure have shown encouraging preclinical in vivo efficacy in various 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.

[0075] The antibody drug conjugate hHTS033-KL disclosed herein was well tolerated in non-human primate toxicology studies (HNSTD = 30 mg / kg), and no severe skin toxicity was observed. hHTS033-KL may have a broad therapeutic window in clinical trials and may benefit cancer patients with high unmet medical needs. BRIEF DESCRIPTION OF THE DRAWINGS

[0076] Figure 1 .RP-HPLC chromatogram of hHTS033-KL.

[0077] Figure 2 SEC chromatogram of hHTS033-KL.

[0078] Figure 3 .Binding affinity of hHTS033-KL on tumor cell lines.

[0079] Figure 4 Internalization of hHTS033-KL on tumor cell lines.

[0080] Figure 5 .Tumor growth curve in the mouse subcutaneous MDA-MB-468 model—ADC drug research.

[0081] Figure 6.The average body weight change curve of tumor-bearing mice in the subcutaneous MDA-MB-468CDX mouse model.

[0082] Figure 7 .Tumor growth curve in mouse subcutaneous 5637 model-ADC drug research.

[0083] Figure 8 .The average body weight change curve of tumor-bearing mice in the subcutaneous 5637CDX mouse model.

[0084] Figure 9 .Tumor growth curve of LD1-0025-361336 PDX model.

[0085] Figure 10 .Relative changes in body weight of LD1-0025-361336 PDX model after treatment.

[0086] Figure 11 Tumor growth curve of the LD1-0017-361443 gastric cancer PDX model.

[0087] Figure 12 .Relative changes in body weight of LD1-0017-361443 gastric cancer PDX model after treatment.

[0088] Figure 13 .Tumor growth curve of liver cancer PDX model LD1-0011-360763.

[0089] Figure 14 Relative changes in body weight of the liver cancer PDX model LD1-0011-360763 after treatment. DETAILED DESCRIPTION

[0090] definition

[0091] It should be noted that the term "a" or "an" entity refers to one or more of that entity; for example, "truncated non-signaling variant" should be understood to represent one or more truncated non-signaling variants. Therefore, the terms "a" (or "an"), "one or more" and "at least one" are used interchangeably herein.

[0092] As used herein, the term "antibody drug conjugate" is a compound in which an antibody or an 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 sometimes referred to as "conjugate" specifically refers to a compound in which an anti-CD44v9 antibody or an antigen-binding fragment thereof is linked to a belotecan derivative via a linker.

[0093] As used herein, the term "linker" refers to a fragment that connects the cytotoxic drug portion and the antibody portion. The linker may be cleavable or non-cleavable. For example, the linker may be cleaved in a specific environment (e.g., a low pH environment within a cell) or under a specific action (e.g., the action of a lysosomal protease), thereby dissociating the cytotoxic drug portion from the antibody portion.

[0094] As used herein, the term "antibody" refers to a fully monoclonal antibody or a multispecific antibody (e.g., a bispecific antibody). Thus, an "anti-CD44v9 antibody" includes fully anti-CD44v9 monoclonal antibodies and fully multispecific antibodies having one specificity for CD44v9 and the remaining specificity for one or more different antigens (e.g., tumor-specific antigens or tumor-associated antigens) or different epitopes of the same antigen.

[0095] As used herein, the term "monoclonal antibody" refers to an antibody from a group of substantially homogeneous antibodies (i.e., the antibodies constituting the group are identical antibodies except for a small amount of possible natural mutations). Monoclonal antibodies are highly specific to a determinant (epitope) of an antigen, while relative polyclonal antibodies contain different antibodies directed against different antigenic determinants (epitopes). In addition to specificity, the monoclonal antibody also has the advantage of not being contaminated by other antibodies during synthesis. In the context of the present disclosure, monoclonal antibodies also particularly include chimeric antibodies, i.e., a portion of a heavy chain and / or light chain is identical or homologous to the type, class or subclass of the antibody, while the remainder is identical or homologous to another type, class or subclass of the antibody, provided that the antibody has the desired biological activity. Chimeric antibodies available in the present disclosure include antibodies containing variable region antigen-binding sequences from rodents (e.g., rats or mice) and human constant region sequences.

[0096] The term "multispecific antibody" includes, for example, bispecific antibodies or trispecific antibodies, which are antibodies with two and three different antigen-binding specificities, respectively. Similarly, a tetraspecific antibody is an antibody 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 plain LC, tandAbs, IgG-scFv, scFv2-Fc, etc.

[0097] The term "bispecific antibody", also known as "bifunctional antibody conjugate", refers to an antibody formed by a first antibody (fragment) and a second antibody (fragment) through a coupling arm. Bispecific antibodies retain the activity of each antibody and therefore have bifunctional and bispecific properties. The dual anti-ADCs currently under investigation can be divided into two main categories: bispecific ADCs targeting dual tumor-associated antigens (TAAs) (e.g., MUC1 / EGFR-ADC), and bispecific ADCs targeting dual epitopes (e.g., HER2 / HER2-ADC).

[0098] As used herein, the term "antigen" refers to a molecule that triggers the production of antibodies by inducing an immune response. Typical antigens are proteins or extracellular domains or portions thereof that are present on the surface 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.

[0099] The term "complete antibody" refers to an antibody that does not undergo cutting and can vary according to the class to which the antibody belongs. Complete antibodies can be divided into different "classes" according to the amino acid sequence of the heavy chain constant region. Five main classes are IgA, IgD, IgE, IgG and IgM, and several of them can also be further divided into different "subclasses" (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2. For example, the complete antibody of IgG type will be an antibody containing 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 region and constant region can be natural (for example, people's natural constant region sequence) or humanized (for example, humanized sequences from mouse sequences via CDR transplantation). However, in the case of heavy chain antibodies, complete antibodies are antibodies that only contain VH, CH2 and CH3.

[0100] As used interchangeably herein, the terms "antibody fragment" or "antigen-binding fragment" of an antibody refer to a fragment of a complete antibody that contains the antigen-binding region of an antibody. Examples of antibody fragments include Fab, Fab', F(ab')2, Fv, scFv, and the like. Regardless of the structure, the antibody fragment binds to the same antigen that is recognized by the complete 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.

[0101] "Specific binding," "specificity," or "specific for..." generally means that an antibody binds to an epitope via its antigen binding domain, and that this binding requires some complementarity between the antigen binding domain and the epitope. According to this definition, an antibody is said to "specifically bind" to an epitope when it binds to the epitope via its antigen binding domain more readily than it binds to a random, unrelated epitope. The term "specificity" is used herein to qualify the relative affinity with which an antibody binds to an epitope. For example, antibody "A" may be said to have a higher specificity for a given epitope than for antibody "B," or antibody "A" may be said to bind to epitope "C" with a higher specificity than to a related epitope "D."

[0102] As used herein, "cancer" or "tumor," as used interchangeably herein, means a group of diseases that can be treated according to the present disclosure and involve abnormal cell growth that has the potential to invade or spread to other parts of the body. Not all tumors are cancerous; benign tumors do not spread to other parts of the body. Possible signs and symptoms include: new lumps, abnormal bleeding, long-term cough, unexplained weight loss, and changes in bowel movements, among others. There are more than 100 different cancers known to affect humans. As used herein, "cancer" includes, but is not limited to, solid cancers (e.g., tumors) and hematological malignancies. "Hematological 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 (such as 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 thrombocythemia, primary myelofibrosis, and chronic myeloid leukemia), lymphomas, multiple myeloma, MGUS and similar diseases, Hodgkin lymphoma, non-Hodgkin 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 cancer” includes, but is not limited to, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing sarcoma, leiomyosarcoma, rhabdomyosarcoma, colon cancer, colorectal cancer, renal cancer, pancreatic cancer, bone cancer, breast cancer, ovarian cancer, prostate cancer, esophageal cancer, gastric cancer, oral cancer, nasal cancer, pharyngeal cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland cancer, sebaceous gland cancer, papillary carcinoma, and ulcerative colitis. Adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, 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, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, skin cancer, melanoma, neuroblastoma, retinoblastoma.

[0103] As used herein, the term "treatment" refers to both therapeutic treatment and preventive or defensive measures, wherein the goal is to prevent or slow down (mitigate) undesirable physiological changes or conditions, such as cancer progression. Beneficial or desired clinical outcomes include, but are not limited to, the alleviation of detectable or undetectable symptoms, the weakening of the degree of disease, the stable (i.e., non-exacerbated) state of the disease, the delay or slowing of disease progression, the improvement or alleviation of the disease state, and (partial or complete) relief. "Treatment" may also mean extending survival compared to the expected survival period without treatment. Those requiring treatment include those already suffering from the patient's condition or condition, and those prone to the condition or condition, or those requiring prevention of the patient's condition or condition.

[0104] As used herein, phrases such as "a patient in need of treatment" or "a subject in need of treatment" include subjects (such as mammalian subjects) who would benefit from administration of a conjugate or composition of the present disclosure, e.g., for detection, diagnostic procedures, and / or therapy.

[0105] 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 result. A therapeutically effective amount may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the therapeutic agent or combination of therapeutic agents to elicit a desired response in the individual. Exemplary indicators of an effective therapeutic agent or combination of therapeutic agents include, for example, an improvement in the patient's health status, a decrease in tumor burden, stagnant or slowed growth of a tumor, and / or the absence of metastasis of cancer cells to other locations in the body.

[0106] So-called "conservative mutations" refer to one or more changes of an amino acid residue to a homologous residue (such as isoleucine to leucine, aspartic acid to glutamic acid, or cysteine ​​to serine) that are not expected to significantly interfere with the protein. In addition, nucleotide or amino acid substitutions, deletions, or insertions may be performed to result in conservative mutations or changes in "non-essential" amino acid regions. For example, a polypeptide or amino acid sequence derived from a specified protein may be identical to the starting sequence except for one or more individual amino acid substitutions, insertions, or deletions, such as one, two, three, four, five, six, seven, eight, nine, ten, fifteen, twenty, or more individual amino acid substitutions, insertions, or deletions. In certain embodiments, a polypeptide or amino acid sequence derived from a specified protein has one to five, one to ten, one to fifteen, or one to twenty individual amino acid substitutions, insertions, or deletions relative to the starting sequence.

[0107] The abbreviation " CDR " stands for " complementary determining region ", it is a part of the variable chain in the immunoglobulin (antibody) produced by B cells and the T cell receptor produced by T cells, and these molecules are combined with their specific antigen in this region. The heavy chain variable region and the light chain variable region each contain three CDRs, and these CDRs are respectively named as HCDR1, HCDR2 and HCDR3 (for heavy chain) in the present disclosure, and LCDR1, LCDR2 and LCDR3 (for light chain). For antigen recognition and combination, a total of six CDRs from both the light chain variable region and the heavy chain variable region are needed. However, nano antibodies or heavy chain antibodies only need the three CDRs in the heavy chain variable region to recognize and combine the epitope on the specific antigen. Each of the heavy chain variable region and the light chain variable region can be expressed as FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 (N-terminal towards C-terminal), wherein the CDR districts are distributed in four less-variable framework regions (FR), i.e. FR1, FR2, FR3 and FR4.

[0108] There are various methods and tools that can be used for predicting the CDR in antibodies according to various CDR definition schemes (including but not limited to Chothia, Kabat, IMGT, AbM and Contact) in the art. The Kabat definition is based on sequence variability and is the most commonly used. The Chothia definition is based on the position of the structural loop region. The Contact definition is based on the analysis of available complex crystal structures. This definition may be the most useful for people who wish to carry out mutagenesis to change the affinity of the antibody because these residues participate in the interaction with the antigen. Various online tools can be used for automatically annotating the CDR in the antibody submitted to the tool webpage. Then, the definition of CDR in the variable region will produce the definition of FR1, FR2, FR3 and FR4 in the variable region.

[0109] "Subject" or "individual" or "animal" or "patient" or "mammal" means any subject, particularly a mammalian subject, for whom diagnosis, prognosis, or treatment is desired. Mammalian subjects include humans, livestock, farm animals, and zoo, sport, or pet animals, such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, cows, and the like.

[0110] As used herein, the term "C 1-6 "Alkyl" refers to a straight or branched chain alkyl group containing 1 to 6 carbon atoms, including, for example, "C 1-4 Alkyl", "C 1-3 Alkyl" and "C 1-2Specific examples include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, 2-methylbutyl, neopentyl, 1-ethylpropyl, n-hexyl, isohexyl, 3-methylpentyl, 2-methylpentyl, 1-methylpentyl, 3,3-dimethylbutyl, 2,2-dimethylbutyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 1,3-dimethylbutyl, 2,3-dimethylbutyl, 2-ethylbutyl, and 1,2-dimethylpropyl.

[0111] As used herein, the term "C 2-6 "Alkenyl" refers to a straight chain, branched chain or cyclic alkenyl group containing at least one double bond and 2 to 6 carbon atoms, including for example "C 2-4 Examples 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.

[0112] As used herein, the term "C 2-6 "Alkynyl" refers to a straight or branched chain alkynyl group containing at least one triple bond and 2 to 6 carbon atoms, including, for example, "C 2-4 Examples include, but are not limited to, ethynyl, propynyl, 2-butynyl, 2-pentynyl, 3-pentynyl, 4-methyl-2-pentynyl, 2-hexynyl, 3-hexynyl, and 5-methyl-2-hexynyl.

[0113] As used herein, the term "halogen" includes fluorine, chlorine, bromine, and iodine.

[0114] As used herein, the term "C 3-6 "Cycloalkyl" refers to a saturated cyclic alkyl group containing 3 to 6 carbon atoms. Specific examples include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl.

[0115] As used herein, the term "3-6 membered heterocyclyl" refers to a cyclic group containing 3 to 6 ring atoms, wherein at least one ring atom is a heteroatom, such as a nitrogen atom, an oxygen atom, or a sulfur atom. Examples include, but are not limited to, oxiranyl, oxocyclobutyl, pyrrolidinyl, tetrahydrofuranyl, piperidinyl, and piperazinyl.

[0116] As used herein, the term "C 1-6 "Alkoxy" refers to a group having C 1-6 Alkyl-O- structure group, where C 1-6Alkyl is as defined above. Specific examples include, but are not limited to, methoxy, ethoxy, propoxy, isopropoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, tert-butoxy, pentyloxy, and hexyloxy.

[0117] As used herein, the term "6-10 membered aryl group" refers to a monocyclic or polycyclic hydrocarbon group having aromaticity containing 6 to 10 ring atoms, such as phenyl and naphthyl.

[0118] As used herein, the term "5-14 membered heteroaryl" refers to a cyclic group having aromaticity 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, but are not limited to, furyl, thienyl, pyrrolyl, thiazolyl, isothiazolyl, thiadiazolyl, oxazolyl, isoxazolyl, oxadiazolyl, imidazolyl, pyrazolyl, 1,2,3-triazolyl, 1,2,4-triazolyl, 1,2,3-oxadiazolyl, 1,2,4-oxadiazolyl, 1,2,5-oxadiazolyl, 1,3,4-oxadiazolyl, pyridyl, 2-pyridin ... oxazinyl, 4H-1,2-oxazinyl, 6H-1,2-oxazinyl, 4H-1,3-oxazinyl, 6H-1,3-oxazinyl, 4H-1,4-oxazinyl, pyridazinyl, pyrazinyl, 1,2,3-oxazinyl, 1,3,5-triazinyl and 1,2,4,5-tetrazinyl.

[0119] anti-CD44v9 antibody

[0120] An antibody that can be used in the antibody drug conjugates described herein is an anti-CD44v9 antibody or an antigen-binding fragment thereof.

[0121] In some embodiments, the anti-CD44v9 antibody specifically binds to CD44v9 expressed on tumor cells and is internalized by the tumor cells.

[0122] In some embodiments, the anti-CD44v9 antibody binds to an epitope of CD44v9 comprising or consisting essentially of the amino acid sequence of 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 of SEQ ID NO: 55. In some embodiments, the anti-CD44v9 antibody binds to an epitope of CD44v9 consisting of the amino acid sequence of SEQ ID NO: 56.

[0123] In some embodiments, an 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 one of (a) to (d):

[0124] (a) HCDR1, HCDR2, and HCDR3 of the heavy chain variable region of SEQ ID NO: 47; and LCDR1, LCDR2, and LCDR3 of the light chain variable region of SEQ ID NO: 48;

[0125] (b) 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;

[0126] (c) 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

[0127] (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; and

[0128] Wherein HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are preferably defined according to any CDR definition scheme selected from Chothia, Kabat, IMGT, AbM and Contact.

[0129] 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 the HCDR1, HCDR2 and HCDR3 are identical to the HCDR1, HCDR2 and HCDR3 of the heavy chain variable region of SEQ ID NO:49, and wherein the LCDR1, LCDR2 and LCDR3 are identical to the LCDR1, LCDR2 and LCDR3 of the light chain variable region of SEQ ID NO:50; wherein HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are preferably defined according to any CDR definition scheme selected from Chothia, Kabat, IMGT, AbM and Contact.

[0130] 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 the HCDR1, HCDR2 and HCDR3 are identical to the HCDR1, HCDR2 and HCDR3 of the heavy chain variable region of SEQ ID NO:51, and wherein the LCDR1, LCDR2 and LCDR3 are identical to the LCDR1, LCDR2 and LCDR3 of the light chain variable region of SEQ ID NO:52; wherein HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are preferably defined according to any CDR definition scheme selected from Chothia, Kabat, IMGT, AbM and Contact.

[0131] In a preferred embodiment, the anti-CD44v9 antibody comprises: a heavy chain variable region comprising: a HCDR1 having the amino acid sequence of SEQ ID NO: 1, a HCDR2 having the amino acid sequence of SEQ ID NO: 2, and a HCDR3 having the amino acid sequence of SEQ ID NO: 3, and a light chain variable region comprising: a LCDR1 having the amino acid sequence of SEQ ID NO: 4, a LCDR2 having the amino acid sequence of SEQ ID NO: 5, and a LCDR3 having the amino acid sequence of SEQ ID NO: 6, according to the IMGT definition of CDRs.

[0132] In a preferred embodiment, the anti-CD44v9 antibody comprises: a heavy chain variable region comprising: a HCDR1 having an amino acid sequence of SEQ ID NO:31, a HCDR2 having an amino acid sequence of SEQ ID NO:32, and a HCDR3 having an amino acid sequence of SEQ ID NO:33, and a light chain variable region comprising: a LCDR1 having an amino acid sequence of SEQ ID NO:34, a LCDR2 having an amino acid sequence of SEQ ID NO:35, and a LCDR3 having an amino acid sequence of SEQ ID NO:36, according to the IMGT definition of CDRs.

[0133] 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.

[0134] 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.

[0135] 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.

[0136] 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.

[0137] 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.

[0138] 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.

[0139] 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.

[0140] In any of the above embodiments, the anti-CD44v9 antibody is preferably of IgG type, more preferably of IgG1 subtype. Preferably, the anti-CD44v9 antibody is of IgG1 type and comprises: a heavy chain variable region comprising: HCDR1 having the amino acid sequence of SEQ ID NO: 1, a HCDR2 having the amino acid sequence of SEQ ID NO: 2, and a HCDR3 having the amino acid sequence of SEQ ID NO: 3, and a light chain variable region comprising: LCDR1 having the amino acid sequence of SEQ ID NO: 4, a LCDR2 having the amino acid sequence of SEQ ID NO: 5, and a LCDR3 having the amino acid sequence of SEQ ID NO: 6, according to the IMGT definition of CDRs. Preferably, the anti-CD44v9 antibody is of 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 of 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.

[0141] Methods for producing and purifying anti-CD44v9 antibodies are disclosed in the art (see WO 2022 / 041104, WO 2019 / 161528, WO 2015 / 076425, and WO 2011 / 007853, the disclosures of each of which are herein incorporated by reference in their entirety).

[0142] belotecan derivatives

[0143] The cytotoxic drug moiety that can be used in the antibody drug conjugates as described herein is a belotecan derivative having the structure of formula (II),

[0144] wherein R is -C(=O)R x 、-S(=O)R x or -S(=O)2R x , where R x Selected from C 1-6 Alkyl, halogenated C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 3-6 Cycloalkyl, C 3-6 heterocyclic, 6-10 membered aryl and 5-14 membered heteroaryl; preferably, R is -C(=O)R x or -S(=O)2R x , where R x Selected from C1-6 Alkyl, halogenated C 1-6 Alkyl, C 3-6 Cycloalkyl, C 3-6 heterocyclic, 6-10 membered aryl and 5-14 membered heteroaryl; more preferably, R is -C(=O)R x or -S(=O)2R x , where R x Selected from C 1-6 Alkyl, C 3-6 Cycloalkyl and phenyl.

[0145] In some embodiments, the cytotoxic drug moiety useful in the antibody drug conjugates described herein is selected from

[0146] In a preferred embodiment, the cytotoxic drug moiety useful in the antibody drug conjugates described herein is

[0147] In a more preferred embodiment, the cytotoxic drug moiety useful in the antibody drug conjugates described herein is

[0148] Methods for preparing and purifying belotecan derivatives as described herein are available in the art (see, for example, WO 2020 / 156189, the entire disclosure of which is incorporated herein by reference).

[0149] connector

[0150] The anti-CD44v9 antibody is linked to the cytotoxic drug via a suitable linker to form an antibody drug conjugate as described herein. The present disclosure contemplates that any linker conventionally used in the field of antibody drug conjugates can be used with the antibody drug conjugates as described herein.

[0151] In some embodiments, the linker has the structure of Formula (III),

[0152] -L1-(L2)m1-(L3)m2-(L4)m3-EG-(III)

[0153] wherein L1 is bonded to the cytotoxic drug portion, and G is bonded to the anti-CD44v9 antibody portion, and wherein

[0154] L1 is selected from Lys, Cit, Cit-Val, Val-Ala, Lys-Val, wherein R', R1 and R2 are each independently H (hydrogen), D (deuterium) or C 1-4alkyl, 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 the cytotoxic drug moiety at position 1 of L1; preferably, L1 is selected from Lys, Cit, Cit-Val, Val-Ala, Lys-Val, More preferably, L1 is

[0155] L2 is selected from wherein R3, R4, R5 and R6 are each independently selected from H (hydrogen), D (deuterium) or C 1-4 Alkyl, y1 and y2 are each independently 0, 1, 2, 3, 4, 5, 6, 7 or 8, and L2 is bonded to L1 at position 1 of L2; preferably, L2 is selected from More preferably, L2 is

[0156] L3 is selected from the following groups optionally substituted with one or more R7: amino, N-methylpiperidine, pyrazole or triazole, wherein each R7 is independently selected from H (hydrogen), D (deuterium), halogen, =O, CF3, CN, CH2CN, carboxyl, sulfonic acid group, C 1-4 Alkyl, C 1-4 Alkoxy, C 2-6 Alkenyl or C 2-6 Alkynyl; preferably, L3 is A triazole of the structure of , and bonded to L2 at position 1 of L3;

[0157] L4 is selected from wherein L4 is bonded to E at position 2 of L4; preferably, L4 is selected from More preferably, L4 is

[0158] E is selected from optionally one or more R 12 Substituted pyrimidine, quinoline or pyrrolo[2,3-d]pyrimidine, wherein each R 12 independently selected from H (hydrogen), D (deuterium), halogen, CN, nitro, C 1-2 Alkyl or halogenated C 1-2Alkyl; preferably, E is optionally replaced by one or more R 12 Substituted pyrimidinyl, wherein each R 12 is independently H (hydrogen) or D (deuterium);

[0159] G is a thioether bond; and

[0160] 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.

[0161] In some embodiments, the linker has the structure of Formula (III),

[0162] -L1-(L2)m1-(L3)m2-(L4)m3-EG-(III)

[0163] wherein L1 is bonded to the cytotoxic drug portion, and G is bonded to the anti-CD44v9 antibody portion, and wherein

[0164] L1 is selected from Lys, Cit, Cit-Val, Val-Ala, Lys-Val, and L1 is bonded to the cytotoxic drug moiety at position 1 of L1;

[0165] L2 is selected from L2 is bonded to L1 at position 1 of L2;

[0166] L3 has A triazole of the structure of , and bonded to L2 at position 1 of L3;

[0167] L4 is selected from and L4 is bonded to E at position 2 of L4;

[0168] E is optionally replaced by one or more R 12 Substituted pyrimidinyl, wherein each R 12 are independently H (hydrogen) or D (deuterium);

[0169] G is a thioether bond; and

[0170] 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.

[0171] In some embodiments, the linker has the structure of Formula (III),

[0172] -L1-(L2)m1-(L3)m2-(L4)m3-EG-(III)

[0173] wherein L1 is bonded to the cytotoxic drug portion, and G is bonded to the anti-CD44v9 antibody portion, and wherein

[0174] L1 is and is bonded to the cytotoxic drug moiety at position 1 of L1;

[0175] L2 is and is bonded to L1 at position 1 of L2;

[0176] L3 has A triazole of the structure of , and bonded to L2 at position 1 of L3;

[0177] L4 is and is bonded to E at position 2 of L4;

[0178] E is a pyrimidinyl group;

[0179] G is a thioether bond; and

[0180] m1 is 1; m2 is 0 or 1; and m3 is 1.

[0181] It should be noted that the thioether bond (G) in the present disclosure is formed by the thiol group of the Cys residue in the anti-CD44v9 antibody, which Cys residue can be formed by opening the disulfide bond in the antibody, or is a free Cys residue contained in the antibody. As is well known in the art, according to different classification schemes, the thioether bond (G) can also be considered not to be part of the linker, but rather to be part of the anti-CD44v9 antibody, as seen from the final conjugate synthesized. For example, in some embodiments, the linker portion does not contain a thioether bond and the sulfur atom forms part of the Cys residue of the anti-CD44v9 antibody.

[0182] Antibody Drug Conjugates

[0183] In one aspect, the present disclosure provides an antibody drug conjugate having a structure of formula (I), (TL) γ -mAb(I),

[0184] in:

[0185] T is a cytotoxic drug moiety having the structure of formula (II), wherein R is -C(=O)R x、-S(=O)R x or -S(=O)2R x , where R x Selected from C 1-6 Alkyl, halogenated C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 3-6 Cycloalkyl, C 3-6 heterocyclic, 6-10 membered aryl and 5-14 membered heteroaryl; preferably, R is -C(=O)R x or -S(=O)2R x , where R x Selected from C 1-6 Alkyl, halogenated C 1-6 Alkyl, C 3-6 Cycloalkyl, C 3-6 heterocyclic, 6-10 membered aryl and 5-14 membered heteroaryl; more preferably, R is -C(=O)R x or -S(=O)2R x , where R x Selected from C 1-6 alkyl, C 3-6 Cycloalkyl and phenyl;

[0186] L is the joint part;

[0187] γ is an integer or decimal ranging from 1 to 10; and

[0188] The mAb is an antibody portion, wherein the antibody is an anti-CD44v9 antibody or an antigen-binding fragment thereof.

[0189] In some embodiments, an antibody drug conjugate having the structure of Formula (I) is provided, (TL) γ -mAb(I),

[0190] in:

[0191] T is a cytotoxic drug moiety as defined above for the belotecan derivative moiety;

[0192] L is a linker moiety as defined above for a linker moiety;

[0193] γ is an integer or decimal ranging from 1 to 10; and

[0194] The mAb is an antibody portion, wherein the antibody is an anti-CD44v9 antibody or an antigen-binding fragment thereof.

[0195] In some embodiments, an antibody drug conjugate having the structure of Formula (I) is provided, (TL)γ -mAb(I),

[0196] in:

[0197] T is a cytotoxic drug moiety as defined above for the belotecan derivative moiety;

[0198] L is the joint part;

[0199] γ is an integer or decimal ranging from 1 to 10; and

[0200] The mAb is an anti-CD44v9 antibody or antigen-binding fragment thereof as defined above in the anti-CD44v9 antibody section.

[0201] In some embodiments, an antibody drug conjugate having the structure of Formula (I) is provided, (TL) γ -mAb(I),

[0202] in:

[0203] T is a cytotoxic drug moiety as defined above for the belotecan derivative moiety;

[0204] L is a linker moiety as defined above for a linker moiety;

[0205] γ is an integer or decimal ranging from 1 to 10; and

[0206] The mAb is an anti-CD44v9 antibody or antigen-binding fragment thereof as defined above in the anti-CD44v9 antibody section.

[0207] In any of the above embodiments, γ is preferably an integer or decimal range from 5 to 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 called DAR (Drug to Antibody Ratio), which is one of the most important quality parameters of antibody drug conjugates and is expressed as the average number of drug molecules conjugated to the antibody.

[0208] A variety of analytical methods 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 coupled with electrospray ionization mass spectrometry (LC-ESI-MS). In some embodiments, the value of the parameter γ of the antibody drug conjugate disclosed herein is determined by RP-HPLC.

[0209] In a preferred embodiment, the antibody drug conjugate disclosed herein is selected from:

[0210] wherein γ is an integer or decimal ranging from 5 to 8, and mAb is an antibody portion, wherein the antibody is an anti-CD44v9 antibody or an antigen-binding fragment thereof.

[0211] In a preferred embodiment, the anti-CD44v9 antibody is as defined above in the anti-CD44v9 antibody section.

[0212] As described above, in the antibody drug conjugates disclosed herein, the thioether bond (G) is formed by the thiol group of a Cys residue in the anti-CD44v9 antibody, which can be formed by opening the disulfide bonds in the antibody, or is a free Cys residue contained in the antibody.

[0213] In a further preferred embodiment, the antibody drug conjugate disclosed herein is selected from:

[0214] wherein γ is an integer or decimal ranging 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 and a light chain variable region, the heavy chain variable region comprising HCDR1, HCDR2, and HCDR3, and the 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):

[0215] (a) HCDR1, HCDR2, and HCDR3 of the heavy chain variable region of SEQ ID NO: 47; and LCDR1, LCDR2, and LCDR3 of the light chain variable region of SEQ ID NO: 48;

[0216] (b) 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;

[0217] (c) 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

[0218] (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; and

[0219] Wherein HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are preferably defined according to any CDR definition scheme selected from Chothia, Kabat, IMGT, AbM and Contact.

[0220] 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: a HCDR1 having an amino acid sequence of SEQ ID NO: 1, a HCDR2 having an amino acid sequence of SEQ ID NO: 2, and a HCDR3 having an amino acid sequence of SEQ ID NO: 3, and a light chain variable region comprising: a LCDR1 having an amino acid sequence of SEQ ID NO: 4, a LCDR2 having an amino acid sequence of SEQ ID NO: 5, and a LCDR3 having an amino acid sequence of SEQ ID NO: 6, according to the IMGT definition of CDRs.

[0221] 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: a HCDR1 having an amino acid sequence of SEQ ID NO:31, a HCDR2 having an amino acid sequence of SEQ ID NO:32, and a HCDR3 having an amino acid sequence of SEQ ID NO:33, and a light chain variable region comprising: a LCDR1 having an amino acid sequence of SEQ ID NO:34, a LCDR2 having an amino acid sequence of SEQ ID NO:35, and a LCDR3 having an amino acid sequence of SEQ ID NO:36, according to the IMGT definition of CDRs.

[0222] In a preferred embodiment, the mAb is an anti-CD44v9 monoclonal antibody or an 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.

[0223] In a preferred embodiment, the mAb is an anti-CD44v9 monoclonal antibody or an 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.

[0224] In a preferred embodiment, the mAb is an anti-CD44v9 monoclonal antibody or an 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.

[0225] In a preferred embodiment, the mAb is an anti-CD44v9 monoclonal antibody or an 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.

[0226] In a preferred embodiment, the mAb is an anti-CD44v9 monoclonal antibody or an 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.

[0227] In a preferred embodiment, the mAb is an anti-CD44v9 monoclonal antibody or an 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.

[0228] In a more preferred embodiment, the antibody drug conjugate disclosed herein has the structure

[0229] wherein γ is an integer or decimal ranging from 5 to 8, and mAb is an anti-CD44v9 monoclonal antibody or an antigen-binding fragment thereof, wherein the thioether bond attached to the mAb is formed by a 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 section.

[0230] In some embodiments, the mAb specifically binds to a CD44v9 epitope comprising or consisting essentially of SEQ ID NO. 55 or 56.

[0231] In a more preferred embodiment, the mAb comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3,

[0232] wherein HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are the same as any one of (a) to (d):

[0233] (a) HCDR1, HCDR2, and HCDR3 of the heavy chain variable region of SEQ ID NO: 47; and LCDR1, LCDR2, and LCDR3 of the light chain variable region of SEQ ID NO: 48;

[0234] (b) 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;

[0235] (c) 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

[0236] (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;

[0237] Wherein HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are preferably defined according to any CDR definition scheme selected from Chothia, Kabat, IMGT, AbM and Contact.

[0238] In a more preferred embodiment, the mAb comprises: a heavy chain variable region comprising: a HCDR1 having the amino acid sequence of SEQ ID NO: 1, a HCDR2 having the amino acid sequence of SEQ ID NO: 2, and a HCDR3 having the amino acid sequence of SEQ ID NO: 3, and a light chain variable region comprising: a LCDR1 having the amino acid sequence of SEQ ID NO: 4, a LCDR2 having the amino acid sequence of SEQ ID NO: 5, and a LCDR3 having the amino acid sequence of SEQ ID NO: 6, according to the IMGT definition of CDRs.

[0239] In a preferred embodiment, the mAb comprises: a heavy chain variable region comprising: a HCDR1 having an amino acid sequence of SEQ ID NO:31, a HCDR2 having an amino acid sequence of SEQ ID NO:32, and a HCDR3 having an amino acid sequence of SEQ ID NO:33, and a light chain variable region comprising: a LCDR1 having an amino acid sequence of SEQ ID NO:34, a LCDR2 having an amino acid sequence of SEQ ID NO:35, and a LCDR3 having an amino acid sequence of SEQ ID NO:36, according to the IMGT definition of CDRs.

[0240] 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.

[0241] 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.

[0242] 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.

[0243] In a more preferred embodiment, the mAb is an anti-CD44v9 monoclonal antibody or an 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.

[0244] In a more preferred embodiment, the mAb is an anti-CD44v9 monoclonal antibody or an 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.

[0245] In any of the above embodiments, γ is preferably an integer or decimal range from 5 to 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 called DAR (Drug to Antibody Ratio), which is one of the most important quality parameters of antibody drug conjugates and is expressed as the average number of drug molecules conjugated to the antibody.

[0246] A variety of analytical methods 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 coupled with electrospray ionization mass spectrometry (LC-ESI-MS). In some embodiments, the value of the parameter γ of the antibody drug conjugate disclosed herein is determined by RP-HPLC.

[0247] In any of the more preferred embodiments, gamma is preferably 7.8 as determined by RP-HPLC.

[0248] In a preferred embodiment, the antibody drug conjugate disclosed herein has the structure

[0249] 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 to the mAb is formed by a thiol group of a Cys residue in the mAb, and the mAb comprises: a heavy chain variable region comprising a HCDR1 having an amino acid sequence of SEQ ID NO: 1, a HCDR2 having an amino acid sequence of SEQ ID NO: 2, and a HCDR3 having an amino acid sequence of SEQ ID NO: 3, and a light chain variable region comprising a LCDR1 having an amino acid sequence of SEQ ID NO: 4, a LCDR2 having an amino acid sequence of SEQ ID NO: 5, and a LCDR3 having an amino acid sequence of SEQ ID NO: 6, according to the IMGT definition of CDRs.

[0250] In a preferred embodiment, the antibody drug conjugate disclosed herein has the structure

[0251] wherein gamma 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 to the mAb is formed by a 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.

[0252] In a preferred embodiment, the antibody drug conjugate disclosed herein has the structure

[0253] wherein gamma 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 to the mAb is formed by a 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.

[0254] Methods for preparing linker moieties are known in the art and are also exemplified in the examples disclosed herein. Detailed descriptions of the synthesis of linkers and intermediates used therein are discussed in, for example, EP 3725798, the entire disclosure of which is incorporated herein by reference.

[0255] The methods for preparing the antibody drug conjugates disclosed herein can be readily performed according to conventional practice in the art and are also exemplified in the Examples herein. Detailed descriptions of the synthesis of antibody drug conjugates having the same cytotoxic drug moiety and linker moiety but with different antibodies are discussed, for example, in EP 3725798, supra.

[0256] The present disclosure has demonstrated that an antibody drug conjugate (hHTS033-KL) can be internalized upon binding to CD44v9 on the surface of tumor cells, with subsequent payload release, interference with topoisomerase I function, and ultimately leading to apoptotic cell death.

[0257] Pharmaceutical composition

[0258] In another aspect, the present disclosure provides pharmaceutical compositions comprising an antibody drug conjugate as disclosed herein and a pharmaceutically acceptable carrier. These compositions are suitable for veterinary or human administration.

[0259] The compositions of the present invention can be any form that allows the composition to be applied to the patient. For example, the composition can be in solid, liquid or gas (aerosol) form. Typical routes of administration include, but are not limited to, oral, topical, parenteral, sublingual, rectal, vaginal, ocular, intratumoral and intranasal. Parenteral administration includes subcutaneous injection, intravenous injection, intramuscular injection, intrasternal injection or infusion technology. In one aspect, these compositions are parenteral. In yet another aspect, these compositions are intravenously administered.

[0260] The pharmaceutical composition can be formulated so that the conjugate becomes bioavailable after administration of the composition to a patient. The composition can take the form of one or more dosage units, where, for example, a tablet can be a single dosage unit, and a container of the conjugate in aerosol form can hold multiple dosage units.

[0261] The materials used in preparing the pharmaceutical composition can be non-toxic in the amounts used. It is apparent to those of ordinary skill in the art that the optimal dosage of the active ingredient in the pharmaceutical composition depends on a variety of factors. Relevant factors include, but are not limited to, the type of animal (e.g., human), the specific form of the conjugate, the mode of administration, and the composition employed.

[0262] The pharmaceutically acceptable carrier or vehicle can be in a granular form, such that the composition is in the form of, for example, a tablet or powder. The carrier can be a liquid, such that the composition is, for example, an oral syrup or an injectable liquid. In addition, the carrier can be gaseous or granular, so as to provide an aerosol composition that can be used, for example, for inhalation administration.

[0263] The composition can be in liquid form, such as an elixir, syrup, solution, emulsion or suspension. The liquid can be used for oral administration or delivered by injection. When intended for oral administration, the composition can include one or more of a sweetener, a preservative, a dye / colorant and a flavor enhancer. In compositions for administration by injection, one or more of a surfactant, a preservative, a wetting agent, a dispersant, a suspending agent, a buffer, a stabilizer and an isotonic agent can also be included.

[0264] Liquid compositions, whether they are solutions, suspensions or other similar forms, may include one or more of the following: a sterile diluent, such as water for injection, saline solution, preferably physiological saline, Ringer's solution, isotonic sodium chloride; a fixed oil, such as synthetic monoglycerides or diglycerides, polyethylene glycol, glycerol, cyclodextrin, propylene glycol or other solvents that can be used as a solvent or suspending medium; an antibacterial agent, such as benzyl alcohol or methyl paraben; an antioxidant, such as ascorbic acid or sodium bisulfite; a chelating agent, such as ethylenediaminetetraacetic acid; a buffer, such as acetate, citrate or phosphate; and an agent for adjusting tonicity, such as sodium chloride or glucose. Parenteral compositions may be packaged 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.

[0265] The amount of the conjugate effective in treating a particular disorder or condition will depend on the nature of the disorder or condition and can be determined by standard clinical techniques. In addition, in vitro or in vivo assays may optionally be employed to help identify the optimal dosage range. The precise dose employed in the composition also depends on the route of administration and the severity of the disease or condition and should be determined according to the physician's judgment and each patient's circumstances.

[0266] The composition comprises an effective amount of the conjugate so as to obtain a suitable dosage. Typically, this amount is at least about 0.01% of the conjugate by weight of the composition. When intended for oral administration, this amount may vary in the range of about 0.1% to about 80% by weight of the composition. In one aspect, the oral composition may comprise about 4% to about 50% of the conjugate by weight of the composition. In yet another aspect, the composition of the present invention is prepared so that a parenteral dosage unit contains about 0.01% to about 2% by weight of the conjugate.

[0267] For intravenous administration, the composition can contain from about 0.01 mg conjugate / kg body weight to about 100 mg conjugate / kg body weight of the animal. In one aspect, the composition can contain from about 1 mg conjugate / kg body weight to about 100 mg conjugate / kg body weight of the animal. In another aspect, the amount administered will be in the range of from about 0.1 mg conjugate / kg body weight to about 25 mg conjugate / kg body weight.

[0268] Typically, the dosage of the conjugate administered to a patient is typically from about 0.01 mg / kg animal body weight to about 2000 mg / kg animal body weight. In one aspect, the dosage administered to a patient is from about 0.01 mg / kg animal body weight to about 10 mg / kg animal body weight, in another aspect, the dosage administered to a patient is from about 0.1 mg / kg animal body weight to about 250 mg / kg animal body weight, in yet another aspect, the dosage administered to a patient is from about 0.1 mg / kg animal body weight to about 20 mg / kg animal body weight, in yet another aspect, the dosage administered is from about 0.1 mg / kg animal body weight to about 10 mg / kg animal body weight, and in yet another aspect, the dosage administered is from about 1 mg / kg animal body weight to about 10 mg / kg animal body weight.

[0269] The conjugate or composition can be administered by any convenient route, such as by infusion or bolus injection, absorbed through the epithelium or mucocutaneous lining (e.g., oral mucosa, rectal and intestinal mucosa, etc.). Administration can be systemic or local. Various delivery systems are known, such as encapsulated in liposomes, microparticles, microcapsules, capsules, etc., and can be used to administer the conjugate or composition. In certain embodiments, more than one conjugate or composition is administered to the patient.

[0270] In a specific embodiment, it may be necessary to apply one or more conjugates or compositions topically to the area in need of treatment. This can be achieved by, for example, but not limited to, local infusion during surgery, topical application (e.g., in combination with a postoperative wound dressing), by injection, by a catheter, by a suppository, or by an implant, which is a porous, non-porous, or gel-like material, including a membrane such as a sialic acid membrane or fiber. In one embodiment, it can be applied by direct injection at the site (or previous site) of a cancer, tumor, or neoplastic tissue or neoplastic anterior tissue. In another embodiment, it can be applied by direct injection at the site (or previous site) where an autoimmune disease is present.

[0271] In certain embodiments, it may be desirable to introduce one or more conjugates or compositions into the central nervous system by any suitable route, including intraventricular injection and intrathecal injection. Intraventricular injection can be facilitated, for example, by a ventricular catheter connected to a reservoir (such as an Ommaya reservoir).

[0272] Pulmonary administration can also be employed, for example, by use of an inhaler or nebulizer and formulation with an aerosolizing agent, or by instillation of a fluorocarbon or synthetic lung surfactant.

[0273] In yet another embodiment, the conjugate or composition can be delivered in a controlled release system, such as, but not limited to, a pump or various polymeric materials. In yet another embodiment, the controlled release system can be placed near the target (e.g., brain) of the conjugate or composition, thus requiring only a fraction of the systemic dose.

[0274] The term "carrier" refers to a diluent, adjuvant, or vehicle used in conjunction with the conjugate. Such pharmaceutical carriers can be liquids, such as water and oils, including those of petroleum, animal, plant, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. These carriers can be saline, gum arabic, gelatin, starch paste, talc, keratin, colloidal silicon dioxide, urea, etc. In addition, adjuvants, stabilizers, thickeners, lubricants, and colorants can be used. In one embodiment, when administered to a patient, the conjugate or composition and the pharmaceutically acceptable carrier are sterile. When the conjugate is administered intravenously, water is an exemplary carrier. Saline solutions, as well as aqueous glucose solutions and glycerol solutions can also be used as liquid carriers, particularly for injectable solutions. Suitable carriers also include carriers such as starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol, etc. If desired, the present compositions may also contain minor amounts of wetting or emulsifying agents, or pH buffering agents.

[0275] In one embodiment, the conjugate is formulated into a pharmaceutical composition suitable for intravenous administration to animals (particularly humans) according to conventional methods. Typically, the carrier or solvent for intravenous administration is a sterile isotonic aqueous buffer solution. If necessary, the composition may also include a solubilizing agent. The composition for intravenous administration may optionally include a local anesthetic (such as lidocaine) to relieve pain at the injection site. Typically, these ingredients are provided alone or mixed together in unit dosage form, for example, as a lyophilized powder or anhydrous concentrate in a sealed container (such as an ampoule or a pouch) to indicate the amount of the active agent. When the conjugate is administered by infusion, it can be distributed, for example, with an infusion bottle containing sterile pharmaceutical grade water or saline. When the conjugate is administered by injection, an ampoule of sterile water for injection or saline can be provided to mix the ingredients before use.

[0276] The composition may comprise various materials that alter the physical form of a solid or liquid dosage unit. For example, the composition may comprise a material that forms a coating shell around the active ingredient. The material forming the coating shell is generally inert and may be selected from, for example, sugar, shellac, and other enteric coating agents. Alternatively, the active ingredient may be encapsulated in a gelatin capsule.

[0277] The composition may be composed of gaseous dosage units, for example, which may be in the form of an aerosol. The term aerosol is used to denote a variety of systems ranging from those with colloidal properties to systems consisting of pressurized packages. Delivery may be by liquefied or compressed gas or by a suitable pump system for dispensing the active ingredient.

[0278] The compositions of the present invention may comprise an agent useful in treating cancer, whether the agent is in solid, liquid or gaseous form.

[0279] Uses and therapies

[0280] The conjugates can be used to inhibit the proliferation of tumor cells or cancer cells, induce apoptosis of tumor cells or cancer cells, or be used to treat cancer in patients. Therefore, the conjugates can be used in various settings for treating animal cancers. The conjugates can be used to deliver drugs or drug units to tumor cells or cancer cells. Without being bound by theory, in one embodiment, the antibody portion of the conjugates disclosed herein binds to or associates with a CD44v9 antigen that is expressed on or associated with the cell surface of a cancer cell or tumor cell, and the conjugate can be taken up into the tumor cell or cancer cell via receptor-mediated endocytosis. The CD44v9 antigen can be attached to a tumor cell or cancer cell, or can be an extracellular matrix protein associated with a tumor cell or cancer cell. Once inside the cell, one or more specific peptide sequences within the linker portion are hydrolytically cleaved, resulting in the release of the cytotoxic drug or drug-linker compound. The released cytotoxic drug or drug-linker compound then migrates freely within the cell and induces cytotoxic or cytostatic activity. In an alternative embodiment, the cytotoxic drug or drug-linker compound is cleaved from the conjugate outside the tumor cell or cancer cell, and the cytotoxic drug or drug-linker compound then penetrates the cell.

[0281] In yet another aspect, the present disclosure provides a method for treating or preventing cancer in a subject, comprising administering to the subject an effective amount of the antibody drug conjugate disclosed herein.

[0282] The cancer can be a solid cancer or a hematological malignancy that is positive for the CD44v9 antigen."Hematological 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 (such as 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 thrombocythemia, primary myelofibrosis, and chronic myeloid leukemia), lymphomas, multiple myeloma, MGUS and similar diseases, Hodgkin lymphoma, non-Hodgkin 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 cancer” includes, but is not limited to, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing sarcoma, leiomyosarcoma, rhabdomyosarcoma, colon cancer, colorectal cancer, renal cancer, pancreatic cancer, bone cancer, breast cancer, ovarian cancer, prostate cancer, esophageal cancer, gastric cancer, oral cancer, nasal cancer, pharyngeal cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland cancer, sebaceous gland cancer, papillary carcinoma, and ulcerative colitis. Adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, 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, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, skin cancer, melanoma, neuroblastoma, retinoblastoma. Preferably, the cancers treatable by the present disclosure include 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), colorectal cancer and liver cancer.

[0283] 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 has not been found to be refractory to the treatment of cancer. In another embodiment, the chemotherapeutic agent is one that has been found to be refractory to the treatment of cancer. The conjugate can be administered to patients who have also undergone surgery to treat the cancer.

[0284] In a specific embodiment, the conjugate is administered concurrently with a chemotherapeutic agent or with radiotherapy. In another specific embodiment, the chemotherapeutic agent or radiotherapy is administered before or after the conjugate is administered, in one aspect, the chemotherapeutic agent or radiotherapy is administered at least one hour, five hours, 12 hours, one day, one week, one month before or after the conjugate is administered, in another aspect, the chemotherapeutic agent or radiotherapy is administered several months (e.g., at most three months) before or after the conjugate is administered.

[0285] Chemotherapeutic agents can be administered over a range of time periods. Suitable chemotherapeutic agents include, but are not limited to, methotrexate, paclitaxel, L-asparaginase, mercaptopurine, thioguanine, hydroxyurea, cytarabine, cyclophosphamide, ifosfamide, nitrosoureas, cisplatin, carboplatin, mitomycin, dacarbazine, procarbazine, topotecan, nitrogen mustard, cyclophosphamide, etoposide, 5-fluorouracil, BCNU, irinotecan, camptothecin, bleomycin, doxorubicin, idarubicin, daunorubicin, actinomycin, plicamycin, mitoxantrone, asparaginase, vinblastine, vincristine, vinorelbine, paclitaxel, and docetaxel. With respect to radiation, any radiotherapy regimen can be used depending on the type of cancer to be treated. For example, but not limited to, X-ray radiation can be administered; in particular, high-energy megavolt (radiation with energy greater than 1 MeV) can be used for deep tumors, and electron beam and positive voltage X-ray radiation can be used for skin cancer. Gamma-emitting radioisotopes, such as those of radium, cobalt, and other elements, may also be administered.

[0286] In addition, methods of treating cancer with the conjugates are provided as an alternative to chemotherapy or radiation therapy, where the chemotherapy or radiation therapy has proven or may prove to be too toxic, e.g., resulting in unacceptable or intolerable side effects for the treated subject. Depending on which treatment is found to be acceptable or tolerable, the treated animal may optionally be treated with another cancer treatment such as surgery, radiation therapy, or chemotherapy.

[0287] The conjugates can also be used in vitro or ex vivo, such as for the treatment of certain cancers, including but not limited to leukemias and lymphomas, where such treatments involve autologous stem cell transplantation. This may involve a multi-step process in which the animal's autologous hematopoietic stem cells are harvested and cleared of all cancer cells. The animal's remaining bone marrow cell population is then eradicated by administering high doses of the conjugate with or without high doses of radiation therapy, and the stem cell transplant is infused back into the animal. Supportive care is then provided while bone marrow function is restored and the animal recovers.

[0288] Equivalently, the present disclosure also provides an antibody drug conjugate as disclosed herein for treating a subject's cancer. Equivalently, the present disclosure also provides the use of an antibody drug conjugate as disclosed herein in the preparation of a medicament for treating a subject's cancer. In two equivalent aspects, the cancer is preferably a solid cancer as defined above, and 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), breast cancer such as TNBC (triple negative breast cancer), colorectal cancer and liver cancer.

[0289] sequence

[0290] Table 1. Sequences of the variable regions of anti-CD44v9 antibody HTS033 (mouse antibody)

[0291] Table 2. Sequences of the variable regions of the anti-CD44v9 antibody hHTS033 25-1 (humanized antibody)

[0292] Table 3. Sequences of the variable regions of the anti-CD44v9 antibody hHTS033 25-3 (humanized antibody)

[0293] Table 4. Sequences of the variable regions of anti-CD44v9 antibody mAb116 (mouse antibody)

[0294] Table 5. Light / heavy chain variable region sequences of HTS033, hHTS033 25-1, hHTS033 25-3, and mAb116

[0295] Table 6: Sequences of the heavy and light chain constant regions of hHTS033 25-1 and hHTS033 25-3

[0296] CD44v9 epitopes bound by HTS033, hHTS033 25-1, hHTS033 25-3, and mAb116:

[0297] CD44v9 epitope: SHEGLEEDKD (SEQ ID NO: 55)

[0298] CD44v9 epitope variant: SHEGLEEDKDH (SEQ ID NO: 56) Example

[0299] Example 1: Synthesis of (S)-N-(2-(4-ethyl-4-hydroxy-3,14-diketo-3,4,12,14-tetrahydro-1H-pyrano[3',4',6,7]indolizino[1,2-b]quinolin-11-yl)ethyl)-N-isopropylmethanesulfonamide (Compound T030)

[0300] Methanesulfonyl 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 and the filter cake was washed three times with dichloromethane (3 mL) to obtain the title compound (2.2 g).

[0301] The structural characterization data are as follows:

[0302] 1 H NMR(400MHz,DMSO-d6)δ8.32(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).

[0303] ESI-MS (m / z): 512.2 [M+H] + .

[0304] [α] D 20 It is +28.19° (c=0.101 g / 100 mL, CH3CN).

[0305] 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-nonoxa-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")

[0306] Step 1: Synthesis of methyl 6-(2-(methylthio)pyrimidin-5-yl)-5-hexynoate (Compound 3-2)

[0307] At room temperature, 5-hexynoic acid methyl ester (500 mg, 3.97 mmol) and 5-bromo-2-methylthiopyrimidine were dissolved in N,N-dimethylformamide (3 mL), and triethylamine (3 mL), cuprous iodide (75 mg, 0.4 mmol) and bis(triphenylphosphine) palladium (II) dichloride (279 mg, 0.4 mmol) were added in sequence. The resulting mixture was heated to 95 ° C. under nitrogen protection and stirred for 6 hours, quenched with water, and extracted with ethyl acetate (20 mL × 3). The organic phases were combined, washed with saturated brine (20 mL × 2), and dried over anhydrous sodium sulfate. The desiccant was removed by filtration, and the solvent was evaporated under reduced pressure. Purification was carried out by preparative liquid chromatography to give the title compound (300 mg). ESI-MS (m / z): 251.3 [M + H] + .

[0308] Preparative liquid chromatography:

[0309] Chromatographic column: Waters SunFire C18 5μm 19×250mm

[0310] Mobile phase A: acetonitrile; mobile phase B: water containing 0.05% formic acid

[0311] Time: 0 min-16 min; Mobile phase A: 10%-90%; Flow rate: 28 mL / min

[0312] Step 2: Synthesis of 6-(2-(methylthio)pyrimidin-5-yl)-5-hexynoic acid (Compound 3-3)

[0313] At room temperature, compound 3-2 (200 mg, 0.8 mmol) was dissolved in a mixed solution of tetrahydrofuran and water (4 mL / 4 mL), lithium hydroxide monohydrate (235 mg, 5.6 mmol) was added, and the reaction was stirred at room temperature for 4 hours, then diluted with water and extracted with ethyl acetate (20 mL × 2). The aqueous phase was adjusted to pH = 3 with 1N hydrochloric acid and extracted with ethyl acetate (20 mL × 3), and the organic phases were combined, washed with saturated brine (20 mL × 2), and dried over anhydrous sodium sulfate. The desiccant was removed by filtration, and the solvent was evaporated under reduced pressure to obtain the title compound (120 mg).

[0314] Step 3: Synthesis of 6-(2-(methylsulfonyl)pyrimidin-5-yl)-5-hexynoic acid (Compound 3-4)

[0315] At room temperature, compound 3-3 (20 mg, 0.085 mmol) was dissolved in dichloromethane (4 mL), and m-chloroperbenzoic acid (22 mg, 0.127 mmol) was added, and the reaction was stirred at room temperature overnight. Purification by preparative liquid chromatography gave the title compound (20 mg).

[0316] ESI-MS (m / z): 269.1 [M+H] + .

[0317] Preparative liquid chromatography:

[0318] Chromatographic column: Waters SunFire C18 5μm 19×250mm

[0319] Mobile phase A: acetonitrile; mobile phase B: water containing 0.05% formic acid

[0320] Time: 0 min-16 min; Mobile phase A: 10%-90%; Flow rate: 28 mL / min

[0321] Step 4: Synthesis of 6-(2-(methylsulfonyl)pyrimidin-5-yl)-N-(prop-2-yn-1-yl)hex-5-ynamide

[0322] At 25 ° C, 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), and then N, N-diisopropylethylamine (738 mg, 5.67 mmol) and O-(7-azabenzotriazole-1-yl)-N, N, N', N'-tetramethyluronium hexafluorophosphate (1.63 g, 4.25 mmol) were added in sequence and stirred for 2 hours. The reaction solution was concentrated under reduced pressure, and the residue was purified by silica gel flash column chromatography (ethyl acetate / petroleum ether = 3 / 1) to give the title compound (700 mg).

[0323] ESI-MS (m / z): 306.1 [M+H] + .

[0324] 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

[0325] At 25 ° C and under nitrogen protection, T-030 (250 mg, 0.49 mmol) was dissolved in dichloromethane (10 mL) and cooled to 0 ° C, and then a solution of 4-dimethylaminopyridine (478 mg, 3.91 mmol) in dichloromethane (3 mL) was added, followed by slow dropwise addition of a solution of triphosgene (72 mg, 0.24 mmol) in dichloromethane (10 mL) and stirring at 0 ° C for 20 minutes. The reaction solution was bubbled with nitrogen for 20 minutes, then a solution of (S)-2-(3,2-azido-5-oxo-3,9,12,15,18,21,24,27,30-nonoxa-6-azatriacetamido)-N-(4-(hydroxymethyl)phenyl)-6(((4-methoxyphenyl)diphenylmethyl)amino)acetamide (518 mg, 0.49 mmol) in dichloromethane (7 mL) was added, and the reaction 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 give the title compound (500 mg).

[0326] ESI-MS (m / z): 1597.5 [M+H] + .

[0327] Preparative liquid chromatography:

[0328] Chromatographic column: Daisogel C18 10μm 100×250mm

[0329] Mobile phase A: water; mobile phase B: acetonitrile

[0330] Step 6: Synthesis of (S)-4-ethyl-11-(2-(N-isopropylmethylsulfonyl)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-nonyloxy-3,9-diazapentatriacontamido)benzyl)carbonate

[0331] 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 stirring for 1 hour. Purification by preparative high performance liquid chromatography afforded the title compound (30 mg).

[0332] ESI-MS(m / z):815.9[(M-273) / 2+H] + .

[0333] Preparative liquid chromatography:

[0334] Chromatographic column: Daisogel C18 10μm 50×250mm

[0335] Mobile phase A: water; mobile phase B: acetonitrile

[0336] 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-nonoxa-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)

[0337] Compound 33-2 (30 mg, 0.02 mmol) was dissolved in dichloromethane (1.0 mL), trifluoroacetic acid (0.2 mL) was added to the reaction solution, and the mixture was reacted at room temperature for 30 minutes. Purification by preparative high performance liquid chromatography gave the title compound as a trifluoroacetate salt (20.0 mg).

[0338] Preparative liquid chromatography:

[0339] Chromatographic column: Daisogel C18 10μm 50×250mm

[0340] Mobile phase A: water containing 0.1% trifluoroacetic acid; Mobile phase B: acetonitrile

[0341] The title compound was identified as follows:

[0342] 1H NMR (400MHz, DMSO-d6) δ10.18(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=8.5 6Hz,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,32H),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 =7.08Hz,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).

[0343] ESI-MS (m / z): 816.0 [M / 2+H] + .

[0344] [α] D 20 It is -19.55° (c=1.000 g / 100 mL, CH3CN).

[0345] Example 3. Preparation of anti-CD44v9 antibody drug conjugate hHTS033-KL

[0346] 0.3 mL of the antibody hHTS033 25-1 (33.5 mg / mL) was diluted with 0.25 mL of a solution containing 20 mM phosphate buffered saline (PB), 150 mM NaCl, and 20 mM sodium ethylenediaminetetraacetic acid (pH 7.6). 0.45 mL of a solution containing 20 mM PB and 150 mM NaCl (pH 7.6) was then added and mixed thoroughly. The mixture was adjusted to pH 7.5 with 1 M Na2HPO4 solution, followed by the addition of 6.0 equivalents of 10 mM tris(2-carboxyethyl)phosphine (TCEP) solution and mixing. The mixture was allowed to stand at room temperature for 120 minutes. 10 equivalents of TL033 trifluoroacetate dissolved in dimethyl sulfoxide were added to the solution and mixed thoroughly. The resulting mixture was allowed to stand at room temperature for 90 minutes. The reaction was then terminated by the addition of 6.1 mL of 100 mM cysteine. Finally, the buffer was replaced with a histidine buffer solution at pH 6 and passed through a G-25 gel column to obtain a coupling product of TL033 and antibody hHTS033 25-1, named hHTS033-KL.

[0347] Example 4. Average number of drug molecules linked to each antibody (DAR value)

[0348] The average number of drug molecules conjugated to each antibody molecule in an antibody drug conjugate can be determined using the reverse phase HPLC (RP-HPLC) analytical method described below.

[0349] Reversed-phase chromatography separates components based on differences in hydrophobicity. After reduction to break any interchain disulfide bonds not reduced during ADC preparation, heavy or light chains conjugated to different numbers of drug molecules exhibit varying hydrophobicity. Species with a higher number of conjugated drugs exhibit higher hydrophobicity, resulting in better retention and longer retention times on reversed-phase columns. The sample is retained on the column under low organic phase conditions; the components are then eluted using an organic phase gradient that increases in hydrophobicity from low to high, and detected using a UV detector. The relative abundance of heavy or light chains conjugated to different numbers of drugs is used to calculate the DAR.

[0350] The peak sequence of each component is LC0 (light chain not coupled to any linker-payload), LC1 (light chain coupled to 1 linker-payload), HC0 (heavy chain not coupled to any linker-payload), HC1 (heavy chain coupled to 1 linker-payload), HC2 (heavy chain coupled to 2 linkers-payload), HC3 (heavy chain coupled to 3 linkers-payload). The content percentage 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:

[0351] 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

[0352] The RP-HPLC chromatogram of hHTS033-KL is shown in Figure 1 middle. Figure 1 It was shown that the average number of payloads coupled to each antibody was 7.8 as measured and calculated by RP-HPLC.

[0353] Example 5. Measurement of Aggregates in Antibody Drug Conjugates

[0354] Aggregates in antibody-drug conjugates were detected using size exclusion chromatography followed by HPLC. The method is as follows.

[0355] High-performance liquid chromatography system: Agilent 1260 Infinity II HPLC system

[0356] Detector: UV absorption spectrometer (detection wavelength: 280nm)

[0357] Column type: TOSOH TSKgel G3000SWXL (7.8×300mm, 5μm)

[0358] Mobile phase: 200 mmol / L KHPO4, 150 mmol / L NaCl, 15% (v / v) isopropanol, pH 7.0

[0359] Flow rate: 0.75 mL / min

[0360] Analysis time: 18 minutes

[0361] Column temperature: room temperature

[0362] Sample volume: 50 μg

[0363] Data Analysis: Matter

[0364] The size exclusion chromatogram of hHTS033-KL is shown in Figure 2 middle. Figure 2 A detection curve graph of aggregates is shown. The aggregate content in the antibody drug conjugate hHTS033-KL measured by this method is 0.7%.

[0365] Example 6. Binding affinity evaluation of hHTS033-KL on tumor cell lines

[0366] To evaluate the binding affinity of the ADC, tumor cells from NCI-H292 (non-small cell lung cancer, BMCR, Cat#SCSP-582), A253 (submandibular salivary gland carcinoma, Meisen CTCC, Cat#CTCC-007-0437), A549 (non-small cell lung cancer, iCell, Cat#iCell-h011), PC-9 (non-small cell lung cancer, CELLCOOK, Cat#CC0204), and Detroit 562 (pharyngeal carcinoma, BMCR, Cat#TCHu231) were detached with 1 mM trypsin-EDTA and centrifuged at 1200 rpm for 5 minutes. The cell pellet was resuspended in PBS containing 5% BSA. The cells were plated at 5×10 5 50 μL / well was added to a 96-well assay plate and incubated at 2°C to 8°C for 30 minutes. ADC was diluted to the indicated final concentration and incubated with cells at 4°C for 1 hour, then the cells were washed and incubated with anti-human IgG (Fc specific)-FITC (fluorescein isothiocyanate) antibody (Sigma, Cat#F9512-2mL) as a secondary antibody for 45 minutes in a 4°C incubator. TM Data were analyzed using a flow cytometer (MACSQuant X) and Graphpad Prism 8 software.

[0367] like Figure 3 As shown, hHTS033-KL specifically binds to NCI-H292, PC-9, A253, and Detroit 562 cell lines, among which EC 50 The values ​​were 0.6734 nM, 2.613 nM, 2.179 nM, and 3.197 nM, respectively (Table 7). However, it did not bind to the A549 cell line, indicating that A549 does not express CD44v9. The isotype control ADC (human IgG-KL-DAR8, Sichuan Kelun Botai Biopharmaceutical Co., Ltd.) did not bind to any of the above cell lines.

[0368] Table 7. EC values ​​for hHTS033-KL binding to NCI-H292, A253, A549, PC-9, and Detroit 562 50 (nM) NCI-H292 A253 A549 PC-9 Detroit 562 EC50 (nM) 0.6734 2.179 n / d 2.613 3.197

[0369] Example 7. Internalization of hHTS033-KL in tumor cell lines

[0370] PC-9, A253, NCI-H292, and Detroit 562 cell cultures were maintained in vitro as independent monolayer cultures at 37°C in a 5% CO atmosphere. The cells were detached with PBS containing 1 mM EDTA, 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. The cells were then fixed with 4% PFA (paraformaldehyde) for 15 minutes at room temperature. The cells were washed and resuspended in PBS and incubated at 4°C overnight. The cells were then stained with a FITC-conjugated anti-human antibody for 45 minutes and analyzed using FACS by calculating the surface MFI (mean fluorescence intensity).

[0371] like Figure 4 As shown, hHTS033-KL can be internalized by PC-9, A253, NCI-H292 and Detroit 562 cell lines. Table 8 shows the internalization of hHTS033-KL on each cell line. 1 / 2 (Hour).

[0372] Table 8. T cells internalized by hHTS033-KL in PC-9, A235, NCI-H292, and Detroit 562 tumor cell lines. 1 / 2 (Hour) cell PC-9 A235 NCI-H292 Detroit 562 <![CDATA[T 1 / 2 (hours)]]> 1.284 1.857 1.497 0.6895

[0373] Example 8. Study on the anti-tumor activity of hHTS033-KL in mice bearing subcutaneous xenografts of human triple-negative breast cancer cells MDA-MB-468

[0374] Human triple-negative breast cancer cells MDA-MB-468 (Cell Bank of the Chinese Academy of Sciences, Cat#TCHu136) were cultured in RPMI-1640 medium containing 10% fetal bovine serum in a 5% CO2 incubator at 37°C. The cells were separated using 0.25% trypsin and 0.02% EDTA solution, collected, and plated at 5×10 6 Cells / animal were implanted subcutaneously into the right armpit of female NCG mice (6 to 8 weeks old, purchased from Jiangsu Jicui Pharmaceutical Biotechnology Co., Ltd.). When the average tumor volume reached approximately 200 mm 3Mice were randomly divided into groups at 32 days after tumor inoculation. On day 32 after tumor inoculation, mice were injected intravenously with 10 mg / kg of hHTS033-KL, hHTS033 25-1, and isotype control ADC (human IgG-KL-DAR8). Body weight and tumor volume were measured twice a week throughout the study. Tumor volume was calculated as follows: V (mm 3 )=0.5×length(mm)×width 2 (mm). Differences between and within groups were analyzed using unpaired two-tailed t-tests in GraphPad Prism version 6.0.

[0375] The curves and statistical analysis of tumor volumes in mice injected with a single intravenous dose of hHTS033-KL, hHTS033 25-1, and isotype control ADC are shown in Figure 5 and Table 9. 10 mg / kg of hHTS033-KL induced tumor regression (p < 0.0001), and all 5 mice achieved tumor elimination at D50, and a sustained anti-tumor effect was observed until the end of the study. Animal body weight ( Figure 6 ) tended to increase, no mortality occurred, and the drug was well tolerated during the administration period.

[0376] Table 9. Tumor volume in CDX (MDA-MB-468) (mean ± SEM) Note: **** indicates p < 0.0001; *** indicates p < 0.001; ** indicates p < 0.01; p < 0.05 indicates a significant difference; ns indicates p > 0.05, no significant difference.

[0377] Example 9. Study on the anti-tumor activity of hHTS033-KL in a subcutaneous xenograft mouse model of human bladder cancer cell 5637

[0378] Human bladder cancer cells 5637 (Shanghai Xunqing Biotechnology Co., Ltd.) were cultured in RPMI-1640 medium containing 10% fetal bovine serum in a 5% CO2 incubator at 37°C. The cells were collected and plated at 10 7 Cells / animal were implanted subcutaneously into the right axilla of female Balb / c nude mice (6 to 8 weeks old, purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd.). When the average tumor volume reached approximately 200 mm 3The animals were randomly divided into six groups, with 5 mice in each group. On day 4 after tumor inoculation, 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 Botai Biopharmaceutical Co., Ltd.) (5 mg / kg or 2.5 mg / kg), or vehicle. Tumor volume was calculated as follows: V (mm 3 )=0.5×length(mm)×width 2 Body weight and tumor volume were measured 2 to 3 times per week throughout the study. Inter-group and intra-group differences were analyzed using unpaired two-tailed t-tests in GraphPad Prism version 6.0.

[0379] The curves and statistical analysis of tumor volume are shown in Figure 7 and Table 10. Treatment with 2.5 mg / kg and 5 mg / kg of hHTS033-KL resulted in 173.4% and 192.3% TGI, respectively, while treatment with 5 mg / kg of hHTS033 25-1 had no antitumor activity. Both 2.5 mg / kg and 5 mg / kg of hHTS033-KL treatment achieved sustained tumor regression. During the administration period, the body weight of the animals ( Figure 8 ) tended to increase, and no mortality occurred, indicating that hHTS033-KL at both 2.5 mg / kg and 5 mg / kg dose levels was well tolerated in the 5637 xenograft model.

[0380] Table 10. Tumor volume in CDX(5637) (mean ± SEM) Note: **** indicates p < 0.0001; *** indicates p < 0.001; ** indicates p < 0.01; p < 0.05 indicates a significant difference; ns indicates p > 0.05, no significant difference.

[0381] Example 10. Study on the anti-tumor activity of hHTS033-KL in NSCLC PDX mouse model

[0382] The tumors (shown in Table 11) were cut into 3 mm × 3 mm × 3 mm (approximately 45 mg to 60 mg) fragments and these fragments were subcutaneously implanted into the right flank of mice (female NCG mice, 35 to 42 days old, purchased from Jicui Yaokang Biotechnology Co., Ltd.). When the average tumor size reached 161.42 mm 3At 4 hr, mice were randomly divided into two groups based on tumor volume. 10 mg / kg of hHTS033-KL and isotype control ADC (human IgG-KL-DAR8, Sichuan Kelun Botai Biopharmaceutical Co., Ltd.) were intravenously administered to mice on day 0 (the first day of administration), day 7, and day 14, respectively. Tumor size was measured twice a week in two dimensions using a caliper, and tumor volume (in mm) was expressed using the following formula: 3 ):V=0.5a×b 2 , where a and b are the long and short diameters of the tumor, respectively. Animal body weight was recorded twice weekly, and t-tests were performed to compare differences between the two groups. All data were analyzed using GraphPad Prism 8, and p < 0.05 indicated statistical significance between the groups.

[0383] Table 11. LD1-0025-361336 lung cancer PDX model information Model ID Cancer type Patient gender Patient age describe Model generation LD1-0025-361336 lung cancer M 59 Moderately differentiated to well-differentiated squamous cell carcinoma FP4+3 Note: FP4+3 (frozen passage 4+3) indicates that the tumor was at passage 4 when frozen and was passaged 3 after recovery.

[0384] The results are shown in Figure 9 and Figure 10 As shown in Table 12, hHTS033-KL (10 mg / kg, QW*3) treatment demonstrated statistically significant antitumor effects on the lung cancer PDX model LD1-0025-361336 compared to the isotype control ADC (10 mg / kg, QW*3) group. All mice tolerated the treatment in this study well, as assessed by body weight changes.

[0385] Table 12. Summary of efficacy endpoints for the test article in the LD1-0025-361336 lung cancer PDX model

[0386] Example 11. Study on the efficacy of hHTS033-KL in the gastric cancer PDX model LD1-0017-361443

[0387] The tumors (shown in Table 13) were cut into 3 mm × 3 mm × 3 mm (approximately 45 mg to 60 mg) fragments and implanted subcutaneously into the right flank of mice (female Nu / Nu mice, 35 to 42 days old, purchased from Beijing Weitonglihua Experimental Animal Technology Co., Ltd.). The average tumor size reached 163.38 mm on day 15 after inoculation. 3At 4 hr, mice were randomly divided into 3 groups based on tumor volume. On day 0 (the first administration day) and day 7, vehicle control and 10 mg / kg of hHTS033-KL and isotype control ADC (human IgG-KL-DAR8, Sichuan Kelun Botai Biopharmaceutical Co., Ltd.) were intravenously administered to mice. Tumor size was measured twice a week and the following formula was used to express tumor volume (unit: mm) 3 ):V=0.5a×b 2 , where a and b are the long and short diameters of the tumor, respectively. Animal body weight was recorded twice weekly. Tumor volume in each group was compared with that in the vehicle group using one-way analysis of variance. Differences between the two groups were compared using the t-test. All data were analyzed using GraphPad Prism 8, and p < 0.05 indicated statistical significance between groups.

[0388] Table 13. LD1-0017-361443 Gastric Cancer PDX Model Information Model ID Cancer type Patient gender Patient age describe Model generation LD1-0017-361443 Gastric cancer F -- Poorly to moderately differentiated adenocarcinoma FP2+3 Note: FP2+3 (frozen passage 2+3) indicates that the tumor was at passage 2 when frozen and was passaged 3 after recovery.

[0389] The results are shown in Figure 11 、 Figure 12 and Table 14. In summary, hHTS033-KL 10 mg / kg QW*2 treatment produced a statistically significant anti-tumor effect in the gastric cancer PDX model LD1-0017-361443 compared to the vehicle control group; hHTS033-KL 10 mg / kg QW*2 treatment also exhibited a statistically significant anti-tumor effect compared to the isotype control ADC 10 mg / kg QW*2 group. All mice tolerated the treatments in this study well, as assessed by body weight changes.

[0390] Table 14. Summary of efficacy endpoints of the test article in the LD1-0017-361443 gastric cancer PDX model Note: Mice in the vehicle group were sacrificed on day 11, and data analysis was based on tumor size on day 11.

[0391] Example 12. Study on the efficacy of hHTS033-KL in the liver cancer PDX model LD1-0011-360763

[0392] The tumors (shown in Table 15) were cut into 3 mm × 3 mm × 3 mm (approximately 45 mg to 60 mg) fragments and these fragments were implanted subcutaneously into the right flank of mice (female Nu / Nu mice, 35 to 42 days old, purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd.). When the average tumor size reached approximately 150 mm on day 34 after inoculation, the tumor size was significantly increased. 3 At 4 hr, mice were randomly divided into 3 groups based on tumor volume. Vehicle control, 10 mg / kg hHTS033-KL, and isotype control ADC (Sichuan Kelun Botai Biopharmaceutical Co., Ltd.) were intravenously administered to mice on day 0 (the first day of administration), day 7, and day 14, respectively. Tumor size and body weight were measured twice a week. Tumor volume (unit: mm) was expressed using the following formula: 3 ):V=0.5a×b 2 , where a and b represent the long and short diameters of the tumor, respectively. Tumor volumes of each group were compared with those of the vehicle group using one-way analysis of variance. Differences between the two groups were compared using the t-test. All data were analyzed using GraphPad Prism 8. A p < 0.05 value indicated statistical significance between groups.

[0393] Table 15. LD1-0011-360763 liver cancer PDX model information Note: FP2+2 (frozen passage 2+2) indicates that the tumor was at passage 2 when frozen and was passaged 2 after recovery.

[0394] The curves and statistical analysis are shown in Figure 13 、 Figure 14 and Table 16. In summary, hHTS033-KL 10 mg / kg QW*3 treatment produced a statistically significant antitumor effect in the liver cancer PDX model LD1-0011-360763 compared to the vehicle control group; in addition, hHTS033-KL 10 mg / kg QW*3 treatment also showed a statistically significant antitumor effect compared to the isotype control ADC (human IgG-KL-DAR8, Sichuan Kelun Botai Biopharmaceutical Co., Ltd.) 10 mg / kg QW*3 treatment group. All mice tolerated the treatment in this study well, as assessed by body weight changes.

[0395] Table 16. Summary of efficacy endpoints of the test article in the LD1-0011-360763 liver cancer PDX model

[0396] Example 13. GLP Repeated Dosage Toxicity Study of hHTS033-KL in Cynomolgus Monkeys (4 Weeks of Dosage, 6 Weeks of Recovery After Discontinuation)

[0397] Repeated hHTS033-KL administration experiment in cynomolgus monkeys: 40 cynomolgus monkeys (Zhanjiang Prima Biotech Inc.) were randomly divided into 4 groups, each with 5 males and 5 females. Vehicle, 10 mg / kg hHTS033-KL, 20 mg / kg hHTS033-KL, and 30 mg / kg hHTS033-KL were intravenously infused on days 1, 15, and 29, respectively. During the experiment, the animals were observed for any abnormalities, and blood samples were collected for routine blood tests and blood biochemical analysis. Animals for the main phase necropsy (day 36) and the recovery phase necropsy (day 71) were euthanized, and samples were taken for pathological analysis.

[0398] As shown in Table 17, under the conditions of this study, hHTS033-KL, which is clinically well tolerated, was repeatedly administered to cynomolgus monkeys by intravenous infusion 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). Transient mild irritation and non-test article-related histopathological changes occurred at the application site in all dose groups, and no significant abnormalities were observed in the nervous, cardiovascular, and respiratory systems. The highest non-serious toxic dose (HNSTD) of hHTS033-KL was 30 mg / kg, and the main toxicities were reticulocytopenia and pigmentation in the skin and / or oral mucosa. These changes disappeared after a 6-week recovery period, with no significant delayed toxicity.

[0399] Table 17. Summary of toxicology results from repeated administration of hHTS033-KL to cynomolgus monkeys HNSTD*: Maximum dose without severe toxicity.

Claims

1. An antibody drug conjugate having a structure of formula (I), (T-L) γ -mAb(I), in: (i) T is a cytotoxic drug moiety having the structure of formula (II), wherein R is -C(=O)R x 、-S(=O)R x or -S(=O)2R x , where R x Selected from C 1-6 Alkyl, halogenated C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 3-6 Cycloalkyl, C 3-6 heterocyclic, 6-10 membered aryl and 5-14 membered heteroaryl; preferably, R is -C(=O)R x or -S(=O)2R x , where R x Selected from C 1-6 Alkyl, halogenated C 1-6 Alkyl, C 3-6 Cycloalkyl, C 3-6 heterocyclic, 6-10 membered aryl and 5-14 membered heteroaryl; more preferably, R is -C(=O)R x or -S(=O)2R x , where R x Selected from C 1-6 Alkyl, C 3-6 Cycloalkyl and phenyl; (ii) L is a linker; (iii) γ is an integer or decimal ranging from 1 to 10, preferably 5 to 8; and (iv) The mAb is an antibody portion, wherein the antibody is an anti-CD44v9 antibody or an antigen-binding fragment thereof.

2. The antibody drug conjugate according to claim 1, wherein the linker has a structure of formula (III), -L1-(L2)m1-(L3)m2-(L4)m3-EG-(III) wherein L1 is bonded to T, and G is bonded to mAb, and wherein (i) L1 is selected from Lys, Cit, Cit-Val, Val-Ala, Lys-Val, wherein R', R1 and R2 are each independently H (hydrogen), D (deuterium) or C 1-4 alkyl, 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 position 1 of L1; preferably, L1 is selected from Lys, Cit, Cit-Val, Val-Ala, Lys-Val, More preferably, L1 is (ii) L2 is selected from wherein R3, R4, R5 and R6 are each independently selected from H (hydrogen), D (deuterium) or C 1-4 Alkyl, y1 and y2 are each independently 0, 1, 2, 3, 4, 5, 6, 7 or 8, and L2 is bonded to L1 at position 1 of L2; preferably, L2 is selected from More preferably, L2 is (iii) L3 is selected from the following groups optionally substituted with one or more R7: amino, N-methylpiperidine, pyrazole or triazole, wherein each R7 is independently selected from H (hydrogen), D (deuterium), halogen, =O, CF3, CN, CH2CN, carboxyl, sulfonic acid group, C 1-4 Alkyl, C 1-4 Alkoxy, C 2-6 Alkenyl or C 2-6 Alkynyl; preferably, L3 is A triazole of the structure of , and bonded to L2 at position 1 of L3; (iv) L4 is selected from wherein L4 is bonded to E at position 2 of L4; preferably, L4 is selected from More preferably, L4 is (v) E is selected from optionally one or more R 12 Substituted pyrimidine, quinoline or pyrrolo[2,3-d]pyrimidine, wherein each R 12 independently selected from H (hydrogen), D (deuterium), halogen, CN, nitro, C 1-2 Alkyl or halogenated C 1-2 Alkyl; preferably, E is optionally replaced by one or more R 12 Substituted pyrimidinyl, wherein each R 12 is independently H (hydrogen) or D (deuterium); (vi) G is a thioether bond; and (vii) 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.

3. The antibody drug conjugate according to claim 1 or 2, wherein the cytotoxic drug portion T is selected from Preferably, the cytotoxic drug moiety is and More preferably, the cytotoxic drug moiety is 4. The antibody drug conjugate according to any one of claims 1 to 3, wherein the antibody drug conjugate has a structure selected from the group consisting of: wherein γ is an integer or decimal ranging from 5 to 8, and 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 the G of the linker moiety is formed by a thiol group of a Cys residue in the mAb; 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 antibody drug conjugate according to any one of claims 1 to 6, wherein the mAb comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising HCDR1, HCDR2 and HCDR3, the 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 the heavy chain variable region of SEQ ID NO: 47; and LCDR1, LCDR2, and LCDR3 of the light chain variable region of SEQ ID NO: 48; (b) 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; (c) 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 (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; and Wherein said HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are preferably defined according to any CDR definition scheme selected from Chothia, Kabat, IMGT, AbM and Contact.

8. The antibody drug conjugate according to any one of claims 1 to 7, wherein the mAb comprises: (a) a heavy chain variable region comprising: a HCDR1 having the amino acid sequence of SEQ ID NO: 1, a HCDR2 having the amino acid sequence of SEQ ID NO: 2, and a HCDR3 having the amino acid sequence of SEQ ID NO: 3, and a light chain variable region comprising: a LCDR1 having the amino acid sequence of SEQ ID NO: 4, a LCDR2 having the amino acid sequence of SEQ ID NO: 5, and a LCDR3 having the amino acid sequence of SEQ ID NO: 6, according to the IMGT definition of CDRs; or (b) a heavy chain variable region comprising: a HCDR1 having an amino acid sequence of SEQ ID NO: 31, a HCDR2 having an amino acid sequence of SEQ ID NO: 32, and a HCDR3 having an amino acid sequence of SEQ ID NO: 33, and a light chain variable region comprising: a LCDR1 having an amino acid sequence of SEQ ID NO: 34, a LCDR2 having an amino acid sequence of SEQ ID NO: 35, and a LCDR3 having an amino acid sequence of SEQ ID NO: 36, according to the IMGT definition of CDRs.

9. The antibody drug conjugate according to any one of claims 1 to 8, wherein 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; (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.

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, wherein the antibody-drug conjugate is selected from: wherein γ is an integer or decimal ranging 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 and a light chain variable region, the heavy chain variable region comprising HCDR1, HCDR2, and HCDR3, and the 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 the heavy chain variable region of SEQ ID NO: 47; and LCDR1, LCDR2, and LCDR3 of the light chain variable region of SEQ ID NO: 48; (b) 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; (c) 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 (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; and wherein said HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are preferably defined according to any CDR definition scheme selected from Chothia, Kabat, IMGT, AbM and Contact.

12. An antibody drug conjugate having the following structure wherein γ is an integer or decimal ranging from 5 to 8, and the mAb is an anti-CD44v9 monoclonal antibody or an antigen-binding fragment thereof, wherein the thioether bond attached to the mAb is formed by a thiol group of a Cys residue in the mAb. 13 . The antibody drug conjugate according to 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 antibody drug conjugate according to any one of claims 12 to 14, wherein the mAb comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising HCDR1, HCDR2 and HCDR3, the 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 the heavy chain variable region of SEQ ID NO: 47; and LCDR1, LCDR2, and LCDR3 of the light chain variable region of SEQ ID NO: 48; (b) 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; (c) 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 (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; and wherein said HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are preferably defined according to any CDR definition scheme selected from Chothia, Kabat, IMGT, AbM and Contact.

16. The antibody drug conjugate of claim 15, wherein the mAb comprises: (a) a heavy chain variable region comprising: a HCDR1 having the amino acid sequence of SEQ ID NO: 1, a HCDR2 having the amino acid sequence of SEQ ID NO: 2, and a HCDR3 having the amino acid sequence of SEQ ID NO: 3, and a light chain variable region comprising: a LCDR1 having the amino acid sequence of SEQ ID NO: 4, a LCDR2 having the amino acid sequence of SEQ ID NO: 5, and a LCDR3 having the amino acid sequence of SEQ ID NO: 6, according to the IMGT definition of CDRs; or (b) a heavy chain variable region comprising: a HCDR1 having an amino acid sequence of SEQ ID NO: 31, a HCDR2 having an amino acid sequence of SEQ ID NO: 32, and a HCDR3 having an amino acid sequence of SEQ ID NO: 33, and a light chain variable region comprising: a LCDR1 having an amino acid sequence of SEQ ID NO: 34, a LCDR2 having an amino acid sequence of SEQ ID NO: 35, and a LCDR3 having an amino acid sequence of SEQ ID NO: 36, according to the IMGT definition of CDRs.

17. The antibody drug conjugate of claim 16, wherein 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; (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.

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 treating cancer, comprising the antibody-drug conjugate according to any one of claims 1 to 18 and a pharmaceutically acceptable carrier.

20. Use of the antibody drug conjugate according to any one of claims 1 to 18 in the preparation of a medicament for treating cancer; optionally, the cancer is a solid tumor, 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), colorectal cancer and liver cancer.

21. The antibody drug conjugate according to any one of claims 1 to 18, for use in treating cancer; optionally, the cancer is a solid tumor, 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), colorectal cancer and liver cancer.

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