Anti-her2 antibody-drug conjugates and uses thereof

Abstract

This invention discloses novel anti-HER2 ADCs that combine with camptothecin derivatives for therapeutic use. The antitumor activity of ADCs of formula (PL-A) (such as compounds MB-2a and MB-3a (trastuzumab-medotecan)) makes these compounds suitable for treating proliferative diseases, such as cancer.

Description

[Technical Field]

[0001] Cross-reference to related applications

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 153,530, filed on February 25, 2021, which is incorporated herein by reference in its entirety. [Previous Technology]

[0003] HER2 (human epidermal growth factor receptor 2) has become an important therapeutic target due to its role in the development of cancers such as breast cancer. For example, overexpression of the HER2 gene has been found in up to 25-30% of breast cancer cases and some gastric and esophageal cancer cases. Since HER2 overexpression may be associated with faster growth rates and poorer prognosis, new and effective anti-HER2 therapies are still needed. [Summary of the Invention]

[0004] Antibody-drug conjugates (ADCs) have attracted considerable interest as a novel class of therapeutic agents. For example, ADCs utilize monoclonal antibodies (MAbs) to target and deliver cytotoxic agents to tumor cells, thereby allowing the use of highly cytotoxic drugs that are not accessible via conventional non-targeting modalities. The design of ADCs (typically characterized by the conjugation of a cytotoxic agent to an antibody, usually via a linker) involves consideration of various factors, including the presence of a binding stem on the drug for linking to the linker and the linking technique used to conditionally stabilize the drug to the antibody. Suboptimal design can lead to reduced ADC potency, insufficient immunospecificity of the conjugate, and increased toxicity due to non-specific release of the drug from the conjugate.

[0005] This invention is at least in part based on the antitumor therapeutic effect of an anti-HER2 ADC that is combined with a camptothecin derivative for therapeutic use. The ADC described herein is suitable for treating proliferative diseases, such as cancer.

[0006] In embodiments, the present invention provides compounds having the structure of formula PL-A: (PL-A), or a pharmaceutically acceptable salt thereof, wherein E is a peptide comprising 2 to 8 amino acids; wherein E is substituted with one or more polyols as appropriate; and wherein the N-terminus of the peptide is covalently linked to Z'; Z' is -C(=O)-L1-Y', or; wherein m represents an integer from 1 to 10 and * indicates a site covalently linked to the C; L1 is -(C1-C10 alkyl)-, -CH2CH2(OCH2CH2)nN(R1)C(=O)-L2-* or -CH2(OCH2CH2)nN(R1)C(=O)-L2-*; wherein n represents an integer from 1 to 10; and wherein * indicates a site covalently linked to the Y'; L2 is -(C1-C10 alkyl)-; R1 is -H or -CH3; Y' is a group formed by the reaction of an electrophilic group with a reactive nucleophilic group present on the cell binder C; and p is the drug-to-antibody ratio (DAR), which is an average of about 2-10; and C is a binder targeting Her2, comprising: a heavy chain comprising an amino acid sequence having at least about 80% similarity to SEQ ID NO:1; and a light chain comprising an amino acid sequence having at least about 80% similarity to SEQ ID NO:2.

[0007] In the embodiments, C is a binder comprising a heavy chain comprising an amino acid sequence having at least about 85%, 90%, or 95% consistency with SEQ ID NO:1; and a light chain comprising an amino acid sequence having at least about 85%, 90%, or 95% consistency with SEQ ID NO:2.

[0008] In the embodiments, C is a binder that comprises the heavy chain amino acid sequence of SEQ ID NO:1 and the light chain amino acid sequence of SEQ ID NO:2.

[0009] Exemplary compounds according to formula (PL-A) include MB-2a and MB-3a (trastuzumab meditecan) as described herein.

[0010] In the examples, C is a binding agent, which is an antibody or its antigen-binding fragment.

[0011] In the embodiments, C is a binding agent, which is trastuzumab (Herceptin), trastuzumab-dkst (Ogivri), trastuzumab-pkrb (Herzuma), trastuzumab-dttb (Ontruzant), trastuzumab-qyyp (Trazimera) or trastuzumab-anns (Kanjinti).

[0012] In the embodiments, L1 is -(C1-C10 alkyl)-.

[0013] In the embodiment, L1 is -CH2CH2(OCH2CH2)nN(R1)C(=O)-L2-* or -CH2(OCH2CH2)nN(R1)C(=O)-L2-*, where n represents an integer from 1 to 10; and * indicates a site covalently connected to Y'.

[0014] In the embodiments, L1 is -CH2CH2CH2CH2CH2-, -CH2CH2-, -CH2-, -CH2CH2OCH2CH2OCH2CH2NHC(=O)CH2CH2-* or -CH2OCH2CH2OCH2CH2NHC(=O)CH2CH2-*; wherein * indicates a site covalently linked to Y'.

[0015] In the embodiments, Y' is formed from a Michael acceptor group, succinimide, epoxide or halogen.

[0016] In the embodiments, Y' is formed from the following: R2, R3, R4, R5, or R6; wherein R2 and R3 are each independently -H or C1-C3 alkyl.

[0017] In the embodiments, Y' is , , or ; wherein R2 and R3 are each independently -H or C1-C3 alkyl, and * indicates the site covalently linked to the C.

[0018] In the embodiments, Z' is formed by the following: , , , or.

[0019] In the embodiment, Z' is: , , , , , or; where * indicates a site covalently connected to C.

[0020] In the embodiment, Z' is; where * indicates a covalent connection with C, and n is an integer from 2 to 10.

[0021] In the embodiment, Z' is; where * indicates a covalent connection with C.

[0022] In an embodiment, Z' is, where * indicates a site covalently connected to the C. In an embodiment, m is 1. In an embodiment, m is 2. In an embodiment, m is 3. In an embodiment, m is 4. In an embodiment, m is 5. In an embodiment, m is 6. In an embodiment, m is 7. In an embodiment, m is 8. In an embodiment, m is 9. In an embodiment, m is 10.

[0023] In an embodiment, Z' is, where * indicates a site covalently connected to the C. In an embodiment, m is 1. In an embodiment, m is 2. In an embodiment, m is 3. In an embodiment, m is 4. In an embodiment, m is 5. In an embodiment, m is 6. In an embodiment, m is 7. In an embodiment, m is 8. In an embodiment, m is 9. In an embodiment, m is 10.

[0024] In the embodiments, E is a peptide of 2, 3, 4 or 5 amino acids.

[0025] In the embodiments, E is a peptide of 2, 3 or 4 amino acids.

[0026] In the embodiments, each amino acid in the peptide is an L amino acid, or at least one amino acid in the peptide is a D amino acid.

[0027] In an embodiment, E comprises one or more amino acids selected from glycine, alanine, valine, glutamic acid, glutamic acid, phenylalanine and leucine, wherein the glutamic acid or glutamic acid is substituted with a polyol as appropriate.

[0028] In the embodiments, E comprises an amino acid selected from glycine, alanine, valine, glutamic acid, glutamic acid, phenylalanine and leucine, wherein the glutamic acid or glutamic acid is substituted with a polyol as appropriate.

[0029] In the embodiments, E comprises an amino acid having the following structure: wherein R4 is -H or C1-C6 alkyl.

[0030] In the embodiments, E comprises an amino acid having the following structure:

[0031] In an embodiment, E is selected from the group consisting of: -Ala-Val-*, -Val-Ala-*, -Gly-Gly-*, -Val-Cit-*, -Cit-Val-*, -Leu-Ala-*, -Ala-Leu-*, -Leu-Cit-*, -Cit-Leu-*, -Leu-Ala-*, -Ala-Leu-*, -Lys-Lys-*, -Ala-Lys-*, -Lys-Ala-*, -Val-Lys-*, -Lys-Val-*, -Tyr-Arg-*, -Arg-Tyr-*, -Arg-Arg-*, -Ala-Ala-*, -Phe-Lys-*, -Lys-Phe-*, -Thr-Thr-*, -Thr-Met-*, -Met-Thr-*, -Met-Tyr-*, -Tyr-Met-*, -Phe-Gln-*, -Gln-Phe-*, -Gly-Ser-*, -Leu-Gln-*, -Gln-Leu-*, -Ser-Ala-*, -Ser-Gly-*, -Val-Thr-*, -Thr-Val-*, -Val-Gln-*, -Ser-Val-*, -Val-Ser-*, -Ala-Met-*, -Met-Ala-*, -Val-Arg-*, -Arg-Val-*, -Phe-Ala-*, -Ala-Phe-*, -Cit-Val-*, -Gln-Val-*, -Phe-Arg-*, -Arg-Phe-*, -Ala-Ala-Ala-*, -Gly-Gly-Gly-*, -Ala-Val-Ala-*, -Gly-Val-Gly-*, -Ala-Val-Gly-*, -Gly-Phe-Lys-*, -Lys-Phe-Gly-*, -Leu-Ala-Leu-*, -Val-Ala-Leu-*, -Leu-Ala-Val-*, -Val-Ala-Val-*, -Ala-Val-Ala-Gly-* (SEQ ID NO: 10), -Gly-Phe-Gly-Gly-* (SEQ ID NO: 11), -Gly-Gly-Phe-Gly-* (SEQ ID NO: 12), -Ala-Val-Gly-Gly-* (SEQ ID NO: 13), -Ala-Ala-Ala-Ala-* (SEQ ID NO: 14), -Ala-Val-Ala-Ala-* (SEQ ID NO: 15), -Ala-Leu-Ala-Leu-* (SEQ ID NO: 16), -Leu-Ala-Leu-Ala-* (SEQ IDNO: 17), -Gly-Phe-Leu-Gly-* (SEQ ID NO: 18) and -Gly-Leu-Phe-Gly-* (SEQ ID NO: 19), wherein * indicates covalently linked to the N-terminus of the peptide of Z'.

[0032] In an embodiment, E is selected from the group consisting of: -L-Ala-D-Val-*, -L-Val-D-Ala-*, -L-Val-D-Lys-*, -L-Val-D-Arg-*, -L-Val-D-Cit-*, -L-Val-D-Arg-*, -L-Val-D-Cit-*, -L-Val-D-Lys-*, -L-Val-D-Arg-*, - L-Arg-D-Arg-*, -L-Ala-D-Ala-*, -L-Ala-D-Lys-*, -L-Ala-D-Arg-*, -L-Ala-D-Ala-L-Ala-*, -L-Ala-D-Val-L-Ala-*, -L-Ala-D-Ala-Gly-* and -L-Ala-D-Val-Gly-*, where * indicates covalently linked to the N-terminus of the peptide of Z'.

[0033] In an embodiment, E comprises -(L-Val)-(L-Ala)-. In an embodiment, E further comprises an amino acid having the following structure:

[0034] In the embodiments, one of the following structures is provided, wherein * indicates covalently linked to the N-terminus of the peptide of Z': , , , or.

[0035] In the embodiments, it is formed by one of the following structures: , , , or.

[0036] In the embodiment, it is one of the following structures, where * indicates the connection point with C: , , , or.

[0037] In the embodiments, the compound has one of the following structures, or a pharmaceutically acceptable salt thereof: , , , or , and wherein p is about 2-10, 4-8 or 7-8.

[0038] In the embodiments, the compound has one of the following structures, or a pharmaceutically acceptable salt thereof: or, wherein p is about 4-8, or 7-8.

[0039] In the embodiment, p is about 7-8.

[0040] In the embodiment, p is 8.

[0041] In another embodiment, the present invention provides a pharmaceutical composition comprising any of the compounds described herein or a pharmaceutically acceptable salt thereof.

[0042] In another embodiment, the present invention provides a method for treating cancer, the method comprising administering to an individual in need any of the compounds described herein or a medically acceptable salt thereof.

[0043] In the embodiments, the compound is, or a pharmaceutically acceptable salt thereof,

[0044] In the embodiments, p is about 4-8, or 7-8.

[0045] In the embodiment, p is about 7-8.

[0046] In the embodiment, p is 8.

[0047] In the embodiments, the cancer is lung cancer, urothelial carcinoma, colorectal cancer, prostate cancer, ovarian cancer, pancreatic cancer, breast cancer, bladder cancer, gastric cancer, gastrointestinal stromal tumor, cervical cancer, esophageal cancer, squamous cell carcinoma, peritoneal cancer, liver cancer, hepatocellular carcinoma, colorectal cancer, rectal cancer, endometrial cancer, uterine cancer, salivary gland cancer, kidney cancer, vulvar cancer, thyroid cancer, penile cancer, leukemia, malignant lymphoma, plasmacytoma, myeloma, or sarcoma.

[0048] In the embodiment, the cancer is breast cancer.

[0049] In the embodiment, the cancer is gastric cancer.

[0050] In the embodiment, the cancer is lung cancer (e.g., non-small cell lung cancer).

[0051] In this embodiment, the cancer is ovarian cancer.

[0052] In the embodiment, the cancer is metastatic.

[0053] In the embodiments, the cancer is characterized by low Her2 expression.

[0054] In the embodiment, the cancer is characterized by intermediate Her2 expression.

[0055] In the embodiments, cancer is characterized by high Her2 expression.

[0056] In the embodiments, the cancer is trastuzumab resistant.

Implementation Method

[0068] This invention relates to antibody-drug conjugates comprising an antitumor drug that binds to an anti-HER2 antibody via a linker structural portion. Such compounds are particularly suitable for treating cancers (e.g., breast and gastric cancer) characterized by HER2 overexpression and / or metastatic and / or trastuzumab resistance. Definition

[0069] Unless otherwise stated, the following terms and phrases as used herein are intended to have the following meanings. When a trademark is used herein, unless the context otherwise indicates, a trademark includes product formulations of the trademarked product, generic drugs, and one or more active pharmaceutical ingredients.

[0070] As used herein, the term "antibody" refers to an agent that specifically binds to a particular antigen. In some specific examples, the term encompasses any polypeptide or polypeptide complex that includes sufficient to confer specific binding immunoglobulin structural elements. Exemplary antibodies include, but are not limited to, monoclonal or polyclonal antibodies. In some embodiments, an antibody may include one or more constant region sequences characteristic of mouse, rabbit, primate, or human antibodies. In some embodiments, an antibody may include one or more humanized, primate-like, chimeric, or other sequence elements known in the art. In many embodiments, the term "antibody" is used to refer to one or more constructs or forms known or developed in the art for utilizing the structural and functional characteristics of antibodies in alternative presentations. For example, in embodiments, the antibodies used according to the present invention are selected from, but not limited to, the following formats: intact IgA, IgG, IgE, or IgM antibodies; bispecific or multispecific antibodies (e.g., Zybodies®); antibody fragments, such as Fab fragments, Fab' fragments, F(ab')2 fragments, Fd' fragments, Fd fragments, and isolated CDRs or collections thereof; single-chain Fv; peptide-Fc fusions; single-domain antibodies (e.g., shark single-domain antibodies, such as IgNAR or fragments thereof); camel antibodies; masking antibodies (e.g., Probodies®); Small Modular antibodies. ImmunoPharmaceuticals ("SMIPs™"); single-chain or tandem bifunctional antibodies (TandAb®); VHH; Anticalins®; Nanobodies® microantibodies; BiTE®s; ankylosing spicules or DARPINs®; Avimers®; DARTs; TCR-like antibodies; Adnectins®; Affilins®; Trans-bodies®; Affibodies®; TrimerX®; MicroProteins; Fynomers®, Centyrins®; and KALBITOR®s. In some embodiments, the antibody may lack the covalent modifications (e.g., linked glycans) that it may have in natural production. In some embodiments, the antibody may contain covalent modifications (e.g., linked glycans, loadings [e.g., detectable moieties, therapeutic moieties, catalytic moieties, etc.], or other side groups [e.g., polyethylene glycol, etc.]). In many embodiments, the antibody is or comprises a polypeptide whose amino acid sequence includes one or more structural elements recognized by those skilled in the art as complementarity-determining regions (CDRs); in some embodiments, the antibody is or comprises a polypeptide whose amino acid sequence includes at least one CDR (e.g., at least one heavy chain CDR and / or at least one light chain CDR) substantially identical to a CDR found in a reference antibody. In some specific examples, the antibody agent is or comprises a polypeptide whose amino acid sequence includes structural elements recognized by those skilled in the art as immunoglobulin variable domains.In some embodiments, the antibody agent is a polypeptide protein having a binding domain that is homologous or substantially homologous to an immunoglobulin binding domain.

[0071] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous population of antibodies, meaning that the individual antibodies constituting that population are identical except for the possibility of naturally occurring mutations that may be present in small amounts. Monoclonal antibodies are highly specific for a single antigenic site. The modifier "monoclonal" indicates that the antibody is obtained from a substantially homogeneous population of antibodies and should not be construed as requiring any specific method to produce the antibody.

[0072] As used herein, the term "human antibody" is intended to include antibodies having variable and constant regions generated (or assembled) from human immunoglobulin sequences. In some embodiments, an antibody (or antibody component) may be considered "human" even if its amino acid sequence includes, for example, residues or elements in one or more CDRs and especially CDR3 that are not encoded by human germline immunoglobulin sequences (e.g., including sequence variants that may be (initially) induced by random or site-specific mutations in vivo or introduced by somatic mutations in vivo).

[0073] As is known in the art, the term "humanized" is commonly used to refer to an antibody (or antibody component) whose amino acid sequence includes the VH and VL region sequences of a reference antibody produced in a non-human species (e.g., mouse), and also includes modifications in those sequences to make them more "human-like," i.e., more similar to human germline sequences, relative to the reference antibody. In some embodiments, a "humanized" antibody (or antibody component) is an antibody that binds specifically to an associated antigen and has a framework (FR) region having an amino acid sequence substantially like that of a human antibody, and a complementarity-determining region (CDR) having an amino acid sequence substantially like that of a non-human antibody. A humanized antibody comprises at least one and typically substantially all of two variable domains (Fab, Fab', F(ab')2, FabC, Fv), wherein all or substantially all of the CDR regions correspond to the CDR regions of non-human immunoglobulins (i.e., donor immunoglobulins) and all or substantially all of the framework regions are framework regions having a common sequence of human immunoglobulins. In some embodiments, the humanized antibody also includes at least a portion of the immunoglobulin constant region (Fc), typically at least a portion of the human immunoglobulin constant region. In some embodiments, the humanized antibody includes both a light chain and at least a variable domain of the heavy chain. The antibody may also include the CH1, hinge, CH2, CH3, and, where applicable, CH4 regions of the heavy chain constant region. In some embodiments, the humanized antibody contains only the humanized VL region. In some embodiments, the humanized antibody contains only the humanized VH region. In some specific embodiments, the humanized antibody contains both the humanized VH and VL regions.

[0074] An "intact antibody" is an antibody that contains an antigen-binding variable region suitable for its antibody class, as well as a light chain constant domain (CL) and heavy chain constant domains CH1, CH2, CH3, and CH4. The constant domains may be natural sequence constant domains (e.g., human natural sequence constant domains) or variants of their amino acid sequences.

[0075] An "antibody fragment" comprises a portion of a complete antibody, including its antigen-binding region or variable region. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments that bind immunospecifically to a target antigen (e.g., cancer cell antigen, viral antigen, or microbial antigen), bifunctional antibodies, trifunctional antibodies, tetrafunctional antibodies, linear antibodies, single-chain antibody molecules, scFv, scFv-Fc, multispecific antibody fragments formed from antibody fragments, fragments generated from Fab expression libraries, or antigen-determinant binding fragments of any of the above.

[0076] Antigen is an entity that specifically binds to an antibody.

[0077] It should be understood that, as used herein, the term "binding" generally refers to a non-covalent association between or among two or more entities. "Direct" binding involves physical contact between entities or parts; indirect binding involves physical interaction through physical contact with one or more intermediate entities. Binding between two or more entities can generally be evaluated in any of a variety of contexts: including when the interacting entities or parts are studied in isolation or in more complex systems (e.g., when covalently or otherwise bound to a carrier entity and / or in a biological system or cell). In some embodiments, "binding" refers to a type of non-covalent interaction occurring between an immunoglobulin molecule and an antigen to which the immunoglobulin is specific. The strength or affinity of an immunobinding interaction can be expressed as the dissociation constant (Kd) of the interaction, where a smaller Kd indicates a larger affinity. The immunobinding properties of selected peptides can be quantified using methods well known in the art. One such method requires measuring the rates of antigen binding site / antigen complex formation and dissociation, where these rates depend on the concentration of the complex, the affinity of the interaction, and geometric parameters that equally affect the rates in both directions. Therefore, the "association rate constant" (Kon) and the "dissociation rate constant" (Koff) can both be determined by calculating the concentration and the actual rates of association and dissociation (see Nature 361:186-87 (1993)). The ratio of Koff / Kon cancels out all parameters unrelated to affinity and is equal to the dissociation constant Kd (see Davies et al. (1990) Annual Rev Biochem 59:439-473).

[0078] The terms "specific binding" and "specifically binding" mean that an antibody or antibody derivative binds to the corresponding antigenic determinant of a target antigen in a highly selective manner and does not bind to a multitude of other antigens. Typically, the antibody or antibody derivative binds with an affinity of at least about 1 × 10⁻⁷ M, and preferably 10⁻⁸ M to 10⁻⁹ M, 10⁻¹⁰ M, 10⁻¹¹ M or 10⁻¹² M, and binds to the target antigen with an affinity at least twice as high as that bound to nonspecific antigens (e.g., BSA, casein) other than the predetermined antigen or closely related antigens. The term "specific" refers to the ability of a cell binder (e.g., as described herein, such as an antibody or fragment thereof) to specifically bind to a given target antigen, such as a human target antigen (e.g., to which an immune response is elicited).

[0079] Generally speaking, a "protein" is a polypeptide (i.e., a chain of at least two amino acids linked together by peptide bonds). Proteins may include portions other than amino acids (e.g., glycoproteins) and / or may be otherwise processed or modified. Those skilled in the art will understand that a "protein" may be a complete polypeptide chain, such as that produced by cells (with or without signaling sequences), or may be its functional portion. Those skilled in the art will further understand that proteins may sometimes include more than one polypeptide chain, linked together, for example, by one or more disulfide bonds or otherwise.

[0080] The term "inhibit / inhibition of" means to reduce a measurable quantity or to completely prevent it.

[0081] The term “substantial” or “truly” means the majority of a group, mixture or sample, i.e., >50%, preferably more than 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the group.

[0082] The term "cytotoxic activity" refers to the cytotoxic effect of a drug, a camptothecin conjugate, or a camptothecin conjugate's intracellular metabolite. Cytotoxic activity can be expressed as the IC50 value, which is the concentration per unit volume (moles or mass) at which half of the cells survive.

[0083] The term "cell inhibitory activity" refers to the antiproliferative effect of a drug or camptothecin conjugate or its intracellular metabolites.

[0084] As used herein, the term "cytotoxic agent" means a substance that has cytotoxic activity and causes cell destruction. The term is intended to include chemotherapeutic agents and toxins, such as small molecule toxins or enzyme-active toxins derived from bacteria, fungi, plants or animals, including their synthetic analogues and derivatives.

[0085] As used herein, the term "cell inhibitor" refers to a substance that inhibits cellular function, including cell growth or proliferation. Cell inhibitors include inhibitors, such as protein inhibitors, for example, enzyme inhibitors. Cell inhibitors have cell-inhibiting activity.

[0086] The terms “cancer” and “cancerous” refer to or describe a physiological condition or disease in mammals that is typically characterized by uncontrolled cell growth. A “tumor” contains one or more cancer cells.

[0087] As used herein, the terms "patient" or "individual" refer to any organism to which, for example, the provided compound or the compound described herein is administered for experimental, diagnostic, preventative, and / or therapeutic purposes. Typical individuals include animals. The term "animal" refers to any member of the animal kingdom. In some embodiments, "animal" refers to a human being at any stage of development. In some embodiments, "animal" refers to a non-human animal at any stage of development. In some embodiments, the non-human animal is a mammal (e.g., rodent, mouse, rat, rabbit, monkey, dog, cat, sheep, cow, primate, and / or pig). In some embodiments, animals include, but are not limited to, mammals, birds, reptiles, amphibians, fish, insects, and / or worms. In some embodiments, an animal may be a genetically modified animal, a genetically engineered animal, and / or a purebred. In embodiments, an animal is a mammal, such as a mouse, rat, rabbit, non-human primate, and human; an insect; a worm; etc. In embodiments, an individual is a human. In some embodiments, an individual may have and / or be susceptible to diseases, conditions and / or symptoms (such as cancer). As used herein, “patient population” or “individual population” refers to a plurality of patients or individuals.

[0088] As used herein, the term "normal" when used to modify the terms "individual" or "subject" refers to an individual or group of individuals who does not have a specific disease or symptom and is not a carrier of the disease or symptom. The term "normal" is also used herein to define a biological sample or specimen isolated from a normal or wild-type individual or individual, such as a "normal biological sample".

[0089] An individual who has “suffered” a disease, condition and / or symptom (such as any cancer described herein) has been diagnosed with or is showing one or more symptoms of a disease, condition and / or symptom.

[0090] An individual who is “susceptible” to a disease, condition, and / or symptom has not been diagnosed with and / or may not exhibit symptoms of the disease, condition, and / or symptom. In some embodiments, an individual who is susceptible to a disease, condition, and / or symptom (e.g., cancer) may be characterized by one or more of the following: (1) a gene mutation associated with the disease, condition, and / or symptom; (2) a gene polymorphism associated with the disease, condition, and / or symptom; (3) an increased and / or decreased expression and / or activity of a protein associated with the disease, condition, and / or symptom; (4) habits and / or lifestyles associated with the disease, condition, and / or symptom; (5) a family history of the disease, condition, and / or symptom; (6) a response to certain bacteria or viruses; (7) exposure to certain chemicals. In some embodiments, an individual who is susceptible to a disease, condition, and / or symptom will develop the disease, condition, and / or symptom. In some embodiments, an individual susceptible to a disease, condition, and / or symptom will not develop that disease, condition, and / or symptom.

[0091] Unless the context otherwise requires, the terms "treat" or "treatment" refer to any administration of a therapeutic molecule (such as any compound described herein) that partially or completely relieves, improves, alleviates, inhibits one or more symptoms or features of a particular disease, condition, and / or symptom (e.g., cancer), delays its onset, delays its progression, reduces its severity, and / or reduces its incidence. Such treatment may be administered to subjects who do not exhibit signs of the relevant disease, condition, and / or symptom and / or subjects who only exhibit early signs of the disease, condition, and / or symptom. Alternatively or additionally, such treatment may be administered to subjects exhibiting one or more established signs of the relevant disease, condition, and / or symptom. Alternatively, the pharmacological and / or physiological effects may be prophylactic, i.e., the complete or partial prevention of the disease or its symptoms (e.g., delaying the onset or slowing the progression of the disease or its symptoms). In this regard, the method of the present invention includes administration of a "preventively effective amount" of binder. "Preventively effective amount" refers to an amount that effectively achieves the desired preventive outcome at the necessary dose and time. Therefore, the purpose of treatment (including preventative treatment) is to suppress or slow down (alleviate) undesirable physiological changes or conditions, such as the development or spread of cancer. For the purposes of this invention, beneficial or desired clinical outcomes include, but are not limited to, symptom relief, reduction of disease severity, stabilization of disease symptoms (i.e., no worsening), slowing or decelerating disease progression, improvement or mitigation and remission of disease symptoms (partial or complete), whether detectable or undetectable. Treatment may also include extending survival compared to expected survival without treatment. Patients requiring treatment include those already suffering from symptoms or conditions and those susceptible to developing symptoms or conditions.

[0092] In the case of cancer, the term "treatment" includes any or all of the following: killing tumor cells; inhibiting the growth of tumor cells, cancer cells, or tumors; inhibiting the replication of tumor cells or cancer cells; reducing the total tumor burden or the number of cancer cells; and improving one or more symptoms associated with the disease.

[0093] In the case of autoimmune diseases, the term "treatment" includes any or all of the following: inhibiting the replication of cells (including but not limited to cells that produce autoimmune antibodies) associated with the symptoms of autoimmune diseases, reducing the autoimmune antibody load, and improving one or more symptoms of autoimmune diseases.

[0094] The terms "therapeutic effective amount" or "effective amount" refer to the amount of a combination that is effective in treating or preventing a disease or symptom in mammals (such as those described herein). In the case of cancer, a therapeutically effective amount of the combination may reduce the number of cancer cells; reduce tumor size; inhibit (i.e., to some extent slow down and better stop) the infiltration of cancer cells into surrounding organs; inhibit (i.e., to some extent slow down and better stop) tumor metastasis; inhibit tumor growth to some extent; and / or alleviate one or more symptoms associated with cancer to some extent. To achieve the extent that the drug can inhibit the growth of existing cancer cells and / or kill existing cancer cells, the drug may have cell growth inhibitory properties and / or cytotoxicity. Regarding cancer therapy, efficacy may be measured, for example, by assessing disease progression (TTP) and / or measuring response rate (RR).

[0095] As used herein, the term "pharmaceutically acceptable form" refers to the form of the disclosed compound, including but not limited to its pharmaceutically acceptable salts, esters, hydrates, solvates, polymorphs, isomers, prodrugs, and isotopically labeled derivatives. In one embodiment, "pharmaceutically acceptable form" includes, but is not limited to, its pharmaceutically acceptable salts, esters, prodrugs, and isotopically labeled derivatives. In another embodiment, "pharmaceutically acceptable form" includes, but is not limited to, its pharmaceutically acceptable isomers and stereoisomers, prodrugs, and isotopically labeled derivatives.

[0096] In the embodiments, the pharmaceutically acceptable form is a pharmaceutically acceptable salt. As used herein, the term "pharmaceutically acceptable salt" refers to a pharmaceutically acceptable organic or inorganic salt of a compound (e.g., camptothecin, camptothecin loading, or camptothecin conjugate). In some forms, the compound may contain at least one amino group, and thus an acid addition salt may be formed from that amino group. Exemplary salts include, but are not limited to, sulfates, trifluoroacetates, citrates, acetates, oxalates, chlorides, bromides, iodides, nitrates, hydrogen sulfates, phosphates, acid phosphates, isonicotinates, lactates, salicylates, acid citrates, tartrates, oleates, tannates, pantothenates, hydrogen tartrates, ascorbic acid salts, succinates, maleate, fumarates, gluconates, glucurons, glucuronoses, formates, benzoates, glutamates, methanesulfonates, ethanesulfonates, benzenesulfonates, p-toluenesulfonates, and bis(hydroxynaphthyl)ates (i.e., 1,1'-methylene-bis(2-hydroxy-3-naphthylcarbamate)). Pharmaceutically acceptable salts may involve another molecule, such as an acetate ion, a succinate ion, or other relative ions. The relative ion may be any organic or inorganic moiety that stabilizes the charge on the parent compound. Furthermore, pharmaceutically acceptable salts may have more than one charged atom in their structure. In cases where multiple charged atoms constitute a portion of a pharmaceutically acceptable salt, multiple relative ions may be present. Therefore, pharmaceutically acceptable salts may have one or more charged atoms and / or one or more relative ions.

[0097] As used herein, the term "pharmaceutical composition" means a composition in which an active agent (e.g., a compound according to any one of formulas (I)-(III) as described herein) is formulated together with one or more pharmaceutically acceptable carriers. In some embodiments, the active agent is present in a treatment regimen at a dose suitable for administration to a given dose, which, when administered to a relevant population, shows a statistically significant probability of achieving a predetermined therapeutic effect. In some embodiments, the pharmaceutical composition may be specifically formulated for administration in solid or liquid form, including pharmaceutical compositions suitable for: oral administration, such as a single-dose medication (aqueous or non-aqueous solution or suspension), tablets (e.g., tablets targeted for buccal, sublingual, and systemic absorption), pills, powders, granules, pastes for tongue application; non-enteral administration, such as administration by subcutaneous, intramuscular, intravenous, or epidural injection in the form of, for example, a sterile solution or suspension or a sustained-release formulation; topical application, such as in the form of creams, ointments, controlled-release patches, or sprays applied to the skin, lungs, or mouth; intravaginal or rectal administration, such as in the form of a pessary, cream, or foam; sublingual; ocular; transdermal; or via the nose, lungs, and other mucosal surfaces.

[0098] As used herein, "carrier" or "pharmaceutically acceptable carrier" means a diluent, adjuvant, excipient, or mediator administered together with the composition. In some exemplary embodiments, the carrier may include sterile liquids, such as (e.g.) water and oils, including petroleum, animal, plant, or synthetic oils, such as (e.g.) peanut oil, soybean oil, mineral oil, sesame oil, and the like. In some embodiments, the carrier is or comprises one or more solid components. In some specific examples, the carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol and the like), and suitable mixtures thereof. Appropriate flowability may be maintained, for example, by using a coating such as lecithin, by maintaining the desired particle size in the case of a dispersion, and by using surfactants. Prevention of microbial action may be achieved by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenols, ascorbic acid, thimerosal, and the like. In some cases, it may be desirable to include isotonic agents in the composition, such as sugars, polyols (e.g., mannitol, sorbitol), and sodium chloride. Extended absorption of injectable compositions can be achieved by including agents that delay absorption (e.g., aluminum monostearate and gelatin) in the composition.

[0099] As used herein, the term "kit" means any delivery system for delivering substances. Such delivery systems may include systems that allow multiple diagnostic or therapeutic agents (e.g., oligonucleotides, enzymes, etc. in appropriate containers) and / or supporting substances (e.g., buffers, written instructions for performing the analysis, etc.) to be stored, transported, or delivered from one location to another. For example, a kit includes one or more shells (e.g., boxes, tubes, bottles, ampoules, etc.) containing the relevant reaction reagents and / or supporting substances. As used herein, the term "segmented kit" means a delivery system comprising two or more separate containers, each containing a sub-part of the total kit components. The containers may be delivered together or separately to the intended recipient. For example, the first container may contain an enzyme for analysis, while the second container contains an oligonucleotide. The term "segmented kit" is intended to cover, but is not limited to, kits containing analyte-specific reagents (ASRs) regulated under Part 520(e) of the Federal Food, Drug, and Cosmetic Act. In practice, any delivery system comprising two or more separate containers, each containing a sub-part of the overall kit components, is included in the term "segmented kit." In contrast, "combined kit" refers to a delivery system containing all components in a single container (e.g., in a single box holding all the required components). The term "kit" includes both segmented and combined kits.

[0100] As used herein, the term "administration" generally refers to the administration of a composition to an individual or system to achieve delivery of the composition or a pharmaceutical agent included in the composition. Those skilled in the art will recognize that a variety of routes of administration can be used to administer the composition to an individual (e.g., a human) in suitable settings. Examples of administration routes include non-enteric, such as intravenous, intradermal, subcutaneous, oral (e.g., inhalation), percutaneous (i.e., local), mucosal, and rectal administration. For example, in some embodiments, administration may be ocular, oral, non-enteric, local, etc. In an embodiment, administration is non-enteric (e.g., intravenous administration). In an embodiment, intravenous administration is intravenous infusion. In some specific embodiments, administration may be via the bronchus (e.g., via bronchial instillation), cheek, skin (which may be one or more of, such as local dermis, intradermis, interdermis, percutaneous, etc.), intestine, artery, intradermis, stomach, intramedullary, muscle, nose, peritoneum, intrasheath, vein, ventricle, specific organ (e.g., liver), mucosa, nose, oral, rectum, subcutaneous, sublingual, local, trachea (e.g., via tracheal instillation), vagina, vitreous body, etc.

[0101] As used herein, the term "nucleophile" refers to an electron-rich, non-shared electron pair that acts as a reactive site and reacts with positively charged or electron-deficient sites. Examples of nucleophiles suitable for use in this invention include, but are not limited to, amines (e.g., primary amines, secondary amines, hydroxylamines and / or hydrazines), thiols, phenols, and alcohols. In embodiments, the nucleophilic functional group comprises: an amino group, a hydrazine group, a hydroxylamine group, a hydroxyl group, or a thiol group. In embodiments, the nucleophilic functional group is methylamine, N-hydroxymethylamine, carboxymethylhydrazine, or a guanidine group. In embodiments, the nucleophilic group is a thiol group or contains a thiol group. Some nucleophilic groups must be activated with a base in order to react with electrophilic groups. For example, when a nucleophilic thiol group and a hydroxyl group are present in a polyfunctional compound, the compound must be mixed with an aqueous base to remove a proton and provide a thiolate or hydroxylated anion to enable reaction with the electrophilic group. Unless the base is required to participate in the reaction, a non-nucleophilic base is preferred. In some embodiments, the base may be present as a component of the buffer solution.

[0102] As used herein, the term "electrophile" refers to a reactive group that is susceptible to nucleophilic attack; that is, it readily reacts with the nucleophilic group that enters. The electrophilic group can be selected to make it possible to react with the nucleophilic group of the paired reactant. For example, when the nucleophilic reactive group is an amine, the electrophilic group can be selected to react with the amine. Similarly, when the nucleophilic reactive group is a thiol moiety, the corresponding electrophilic group can be a thiol reactive group or a similar group. Examples of electrophilic groups suitable for use in this invention include, but are not limited to, carboxylic acid esters, acetal groups, acid anhydrides, isocyanates, thioisocyanates, epoxides, activated hydroxyl groups, succinimidyl esters, sulfosuccinimidyl esters, maleic anhydrides, and ethylene sulfonyl groups. In the examples, the electrophilic group is an aldehyde, α-haloketone, maleic anhydride, succinic anhydride, hydroxysuccinic anhydride, isothiocyanate, isocyanate, azid, sulfonyl chloride, toluenesulfonate, glyoxal, epoxide, ethylene oxide, carbonate, imine ester, acid anhydride, fluorophenyl ester, hydroxymethylphosphine derivative, carbonate, haloacetyl, trichloroacetyl, haloacetyl, alkyl halide, aziridine, acrylonitrile derivative, ketone, carboxylic acid, ester, acetyl chloride, or acetic anhydride. In the examples, the electrophilic group is or contains a maleic anhydride or succinic anhydride group. The carboxylic acid group can be activated to react with a nucleophile, including reacting with a suitable hydroxyl-containing compound in the presence of a dehydrating agent such as dicyclohexylcarbodiimide (DCC) or dicyclohexylurea (DHU). For example, carboxylic acids can react with alkoxy-substituted N-hydroxybutyridine or N-hydroxysulfonbutylbutyridine in the presence of DCC to form reactive electrophilic N-hydroxybutyridine esters and N-hydroxysulfonbutylbutyridine esters, respectively. Carboxylic acids can also be activated by reacting with acetohalides, such as acetochloro (e.g., acetochloro), to provide reactive anhydride groups. In another example, carboxylic acids can be converted to acetochloro groups using, for example, thionyl chloride or acetochloro that is capable of undergoing an exchange reaction.

[0103] Unless otherwise specified, the term "alkyl" itself, or as part of another term, refers to a substituted or unsubstituted straight-chain or branched saturated or unsaturated hydrocarbon having the indicated number of carbon atoms (e.g., "-C1-C8 alkyl" or "-C1-C10" alkyl refers to an alkyl having 1 to 8 or 1 to 10 carbon atoms, respectively). When the number of carbon atoms is not specified, an alkyl has 1 to 8 carbon atoms. Representative straight-chain "-C1-C8 alkyl" groups include, but are not limited to, -methyl, -ethyl, -n-propyl, -n-butyl, -n-pentyl, -n-hexyl, -n-heptyl, and -n-octyl; while branched-chain "-C3-C8 alkyl" groups include, but are not limited to, -isopropyl, -dibutyl, -isobutyl, -tert-butyl, -isopentyl, and -2-methylbutyl; unsaturated "-C2-C8 alkyl" groups include, but are not limited to, -vinyl, -allyl, -1-butenyl, -2-butenyl, -isobutenyl, -1-pentenyl, -2-pentenyl, -3-methyl-1-butenyl, -2-methyl-2-butenyl, -2,3-dimethyl-2-butenyl, -1-hexyl, 2-hexyl, -3-hexyl, -ethynyl, -propynyl, -1-butynyl, -2-butynyl, -1-pentynyl, -2-pentynyl, and -3-methyl-1-butynyl. Sometimes the alkyl group is unsubstituted. The alkyl group may be substituted with one or more groups. In other states, the alkyl group will be saturated.

[0104] Unless otherwise indicated, "alkylene" itself, or as part of another term, refers to a substituted or unsubstituted saturated, branched, straight, or cyclic hydrocarbon group having the stated number of carbon atoms (typically 1-10 carbon atoms) and having two monovalent group centers obtained by removing two hydrogen atoms from the same or two different carbon atoms of the parent alkane. Typical alkylene groups include, but are not limited to: methylene (—CH2—), 1,2-alkyleneethyl (—CH2CH2—), 1,3-alkylenepropyl (—CH2CH2CH2—), 1,4-alkylenebutyl (—CH2CH2CH2CH2—), and similar groups. In a preferred embodiment, the alkylene group is a branched or straight-chain hydrocarbon (i.e., it is not a cyclic hydrocarbon).

[0105] Unless otherwise specified, "aryl" itself, or as part of another term, means a monovalent carbocyclic aromatic group of the specified number of carbon atoms (usually 6-20 carbon atoms), substituted or unsubstituted, which is derived by removing a hydrogen atom from a single carbon atom of the parent aromatic ring system. Some aryl groups are represented by exemplary structures such as "Ar". Typical aryl groups include, but are not limited to, groups derived from benzene, substituted benzene, naphthalene, anthracene, biphenyl, and the like. An exemplary aryl group is phenyl.

[0106] Unless otherwise specified, "extrinyl" itself or as part of another term is an aryl group as defined above, having two covalent bonds (i.e., it is divalent) and being ortho, meta or para oriented.

[0107] Unless otherwise specified, "C3-C8 heterocycle" itself, or as part of another term, refers to a monovalent substituted or unsubstituted aromatic or non-aromatic monocyclic or bicyclic system having 3 to 8 carbon atoms (also called ring members) and one to four independent heteroatom ring members selected from N, O, P, or S, and is derived by removing a hydrogen atom from a ring atom of the parent ring system. One or more N, C, or S atoms in the heterocycle may be oxidized. The ring including the heteroatoms may be aromatic or non-aromatic. A heterocycle in which all ring atoms are associated with aromaticity is called a heteroaryl and is also called a heterocarbon ring. Unless otherwise indicated, the heterocycle is attached to its side groups at any heteroatom or carbon atom that produces a stable structure. Thus, a heteroaryl may be bonded via the aromatic carbons of its aromatic ring system, called a C-linked heteroaryl, or via non-double-bonded N atoms (i.e., non-=N—) of its aromatic ring system, called an N-linked heteroaryl. Therefore, the nitrogen-containing heterocycle can be C-linked or N-linked and includes pyrrole moieties such as pyrrole-1-yl (N-linked) and pyrrole-3-yl (C-linked); and imidazole moieties such as imidazole-1-yl and imidazole-3-yl (both N-linked), and imidazole-2-yl, imidazole-4-yl and imidazole-5-yl moieties (all C-linked).

[0108] Unless otherwise specified, “C3-C8 heteroaryl” refers to an aromatic C3-C8 heterocycle, wherein the subscript indicates the total number of carbons in the cyclic system of the heterocycle or the total number of aromatic carbons in the aromatic ring system of the heteroaryl and does not imply the size of the ring system or the presence or absence of ring fusion. Representative examples of C3-C8 heterocycles include, but are not limited to, pyrrolidyl, azacyclic butyl, piperidinyl, morpholinyl, tetrahydrofuranyl, tetrahydropiperanyl, benzofuranyl, benzothiophene, indolyl, benzopyrazolyl, pyrrolyl, thiophene (thiophene), furanyl, thiazolyl, imidazolyl, pyrazolyl, pyrimidinyl, pyridinyl, pyryl, dayl, isothiazolyl, and isozolyl. When explicitly given, the size of the ring system of the heterocycle or heteroaryl is indicated by the total number of atoms in the ring. For example, specifying a 5- or 6-membered heteroaryl indicates the total number of aromatic atoms (i.e., 5 or 6) in the heteroaryl ring system, but does not imply the number of aromatic heteroatoms or aromatic carbons in that ring system. Fused heteroaryls are explicitly defined or implied by the context and are generally indicated by the number of aromatic atoms in the aromatic rings of the fused heteroaryl ring system formed by their fusion. For example, a 5,6-membered heteroaryl is an aromatic 5-membered ring fused to an aromatic 6-membered ring, wherein one or both of these rings have one or more aromatic heteroatoms or wherein the two rings share a heteroatom.

[0109] A heterocycle fused to an aryl or heteroaryl group such that the heterocycle remains non-aromatic and forms part of the larger structure via attachment to the non-aromatic portion of the fused ring system is an example of a heterocycle that is substituted, wherein the heterocycle is substituted by ring fusion with the aryl or heteroaryl group. Similarly, an aryl or heteroaryl group fused to a heterocycle or carbocyclic group that forms part of the larger structure via attachment to the aromatic portion of the fused ring system is an example of an aryl or heterocycle that is substituted, wherein the aryl or heterocycle is substituted by ring fusion with the heterocycle or carbocyclic group.

[0110] Unless otherwise specified, "C3-C8 heterocycle" itself or as part of another term means a C3-C8 heterocycle as defined above, wherein one of the hydrogen atoms of the heterocycle is substituted by a single bond (i.e., it is divalent). Unless otherwise specified, "C3-C8 heteroaryl" itself or as part of another term means a C3-C8 heteroaryl as defined above, wherein one of the hydrogen atoms of the heteroaryl group is substituted by a single bond (i.e., it is divalent).

[0111] Unless otherwise specified, “C3-C8 carbon ring” alone or as part of another term refers to a 3-, 4-, 5-, 6-, 7-, or 8-membered monovalent, substituted, or unsubstituted saturated or unsaturated non-aromatic monocyclic or bicyclic carbon ring derived by removing a hydrogen atom from a ring atom in the parent ring system. Representative C3-C8 carbon rings include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentadienyl, cyclohexyl, cyclohexenyl, 1,3-cyclohexadienyl, 1,4-cyclohexadienyl, cycloheptyl, 1,3-cycloheptadienyl, 1,3,5-cycloheptanetrienyl, cyclooctyl, and cyclooctadienyl.

[0112] Unless otherwise specified, “C3-C8 carbon ring” itself or as part of another term refers to a C3-C8 carbon ring group as defined above, wherein the hydrogen atom of the other carbon ring group is replaced by a single bond (i.e., it is divalent).

[0113] Unless otherwise specified, the term "heteroalkyl" on its own or in combination with another term, unless otherwise specified, means a stable straight-chain or branched hydrocarbon or combination thereof that is fully saturated or contains 1 to 3 degrees of unsaturation and consists of a specified number of carbon atoms and one to ten, preferably one to three, heteroatoms selected from the group consisting of O, N, Si, and S, wherein the nitrogen and sulfur atoms may be oxidized and the nitrogen heteroatoms may be quaternized. Heteroatoms O, N, and S may be located at any internal position of the heteroalkyl group or at the position where the alkyl group is attached to the rest of the molecule. Heteroatoms Si may be located at any position of the heteroalkyl group, including the position where the alkyl group is attached to the rest of the molecule. Examples include —CH2—CH2—O—CH3, —CH2—CH2—NH—CH3, —CH2—CH2—N(CH3)—CH3, —CH2—S—CH2—CH3, —CH2—CH2—S(O)—CH3, —NH—CH2—CH2—NH—C(O)—CH2—CH3, —CH2—CH2—S(O)2—CH3, —CH=CH—O—CH3, —Si(CH3)3, —CH2—CH=N—O—CH3, and —CH=CH—N(CH3)—CH3. At most two heteroatoms can be consecutive, such as —CH2—NH—OCH3 and —CH2—O—Si(CH3)3. Typically, C1 to C4 heteroalkyl or heteroalkyl groups have 1 to 4 carbon atoms and 1 or 2 heteroatoms, and C1 to C3 heteroalkyl or heteroalkyl groups have 1 to 3 carbon atoms and 1 or 2 heteroatoms. In some samples, the heteroalkyl groups are saturated.

[0114] Unless otherwise specified, the term "hexaalkyl" on its own or in combination with another term means a divalent group derived from a heteroalkyl group (as discussed above), as exemplified by —CH2—CH2—S—CH2—CH2— and —CH2—S—CH2—CH2—NH—CH2—. For heteroalkyl groups, the heteroatom may also occupy one or both chain ends. Furthermore, the orientation of the linking group is not implied for heteroalkyl groups and heteroalkyl linking groups.

[0115] Unless otherwise specified, "aminoalkyl" on its own or in combination with another term means heteroalkyl, wherein the alkyl moiety as defined herein is substituted with an amino, alkylamino, dialkylamino, or cycloalkylamino group. Exemplary, non-limiting aminoalkyl groups are —CH2NH2, —CH2CH2NH2, —CH2CH2NHCH3, and —CH2CH2N(CH3)2, and further include branched species such as —CH(CH3)NH2 and —C(CH3)CH2NH2 in (R)- or (S)- configurations. Alternatively, an aminoalkyl group is an alkyl moiety, group, or substituent as defined herein, wherein the sp3 carbons other than the group carbons are replaced by an amino or alkylamino moiety, wherein the sp3 nitrogen replaces the sp3 carbon of the alkyl group, subject to the condition that at least one sp3 carbon is retained. When the aminoalkyl moiety refers to a substituent of a larger structure or another part, the aminoalkyl group is covalently linked to that structure or part via a carbon group of the alkyl moiety of the aminoalkyl group.

[0116] Unless otherwise specified, "alkylamino" and "cycloalkylamino" on their own or in combination with other terms mean alkyl or cycloalkyl as described herein, wherein the group carbon of the alkyl or cycloalkyl group has been replaced by a nitrogen group, provided that at least one sp3 carbon is retained. In cases where the alkylamino group is substituted at its nitrogen by another alkyl moiety, the resulting substituted group is sometimes referred to as a dialkylamino moiety, group, or substituent, wherein the alkyl moiety replacing the nitrogen is independently selected. Illustrative and non-limiting amino, alkylamino, and dialkylamino substituents include substituents having a -N(R')2 structure, wherein in such examples R' is independently selected from hydrogen or C1-6 alkyl, typically hydrogen or methyl, and in cycloalkylamines included in heterocyclic alkyl groups, the two R's together with the nitrogen to which they are attached define the heterocycle. When both R's are hydrogen or alkyl, the moiety is sometimes described as a primary amino group and a tertiary amino group, respectively. When one R' is hydrogen and the other is alkyl, the moiety is sometimes described as a secondary amino group. Primary and secondary alkylamine moieties are more reactive as nucleophiles targeting electron-philic carbonyl centers, while tertiary amines are more basic.

[0117] "Substituted alkyl" and "substituted aryl" refer to alkyl and aryl groups, respectively, in which one or more hydrogen atoms (usually one) are independently substituted. Typical substituents include, but are not limited to, —X, —R', —OH, —OR', —SR', —N(R')2, —N(R')3, =NR', —CX3, —CN, —NO2, —NR'C(=O)R', —C(=O)R', —C(=O)N(R')2, —S(=O)2R', —S(=O)2NR, —S(=O)R', —OP(=O)(OR')2, —P(=O)(OR')2, —PO3=, PO3H2, —C(=O)R', —C( =S)R', —CO2R', —CO2-, —C(=S)OR', —C(=O)SR', —C(=S)SR', —C(=O)N(R')2, —C(=S)N(R)2 and —C(=NR)N(R')2, wherein each X is independently selected from the group consisting of —F, —CI, —Br and —I; and each R' is independently selected from the group consisting of —H, —C1-C20 alkyl, —C6-C20 aryl, —C3-C14 heterocycle, protecting group and prodrug moiety.

[0118] More generally, the substituents are chosen from the group consisting of: —X, —R', —OH, —OR', —SR', —N(R')2, —N(R')3, =NR', —NR'C(=O)R, —C(=O)R', —C(=O)N(R')2, —S(=O)2R', —S(=O)2NR', —S(=O)R', —C(=O)R', —C(=S)R, —C(=O)N(R')2, —C(=S)N(R')2 and —C(=NR)N(R')2, wherein each X is independently chosen from the group consisting of —F and —CI, or from the group consisting of: —X, —R, —OH, —OR', —N(R')2, —N(R')3, —NR' C(=O)R', —C(=O)N(R')2, —S(=O)2R', —S(=O)2NR', —S(=O)R', —C(=O)R', —C(=O)N(R')2, —C(=NR)N(R')2, protecting group, and prodrug moiety, wherein each X is —F; and each R' is independently selected from the group consisting of: hydrogen, —C1-C20 alkyl, —C6-C20 aryl, —C3-C14 heterocycle, protecting group, and prodrug moiety. In some states, the alkyl substituents are selected from the group consisting of: —N(R')2, —N(R')3, and —C(=NR)N(R')2, wherein R is selected from the group consisting of hydrogen and —C1-C20 alkyl. In other states, the alkyl group is partially substituted with a series of ethylened ethyloxy groups to define the PEG unit. As mentioned above, alkylene, carbocyclic, carbocyclic, aryl, heteroalkyl, heteroalkylene, heterocyclic, heterocyclic, heteroaryl and heteroaryl can also be similarly substituted.

[0119] As used herein, a "protecting group" means a portion that prevents or reduces the ability of the atom or functional group to which it is attached to to participate in undesirable reactions. Typical protecting groups for atoms or functional groups are given in Greene (1999), "Protective Groups in Organic Synthesis, 3rd Edition", Wiley Interscience. Protecting groups for heteroatoms such as oxygen, sulfur, and nitrogen are used in some cases to minimize or avoid undesirable reactions with electrophilic compounds. In other cases, protecting groups are used to reduce or eliminate the nucleophilicity and / or basicity of unprotected heteroatoms. Non-limiting examples of protected oxygen are given by —ORPR, where RPR is a protecting group for a hydroxyl group, which is typically protected as an ester (e.g., acetate, propionate, or benzoate). Other protecting groups for the hydroxyl group prevent interference with the nucleophilicity of organometallic reagents or other highly basic reagents. The hydroxyl group is typically protected in the form of an ether, including alkyl or heterocyclic alkyl ethers (e.g., methyl or tetrahydropiperanyl ethers), alkoxymethyl ethers (e.g., methoxymethyl or ethoxymethyl ethers), substituted aryl ethers as appropriate, and silyl ethers (e.g., trimethylsilyl (TMS), triethylsilyl (TES), tributyldiphenylsilyl (TBDPS), tributyldimethylsilyl (TBS / TBDMS), triisopropylsilyl (TIPS), and [2-(trimethylsilyl)ethoxy]-methylsilyl (SEM)). Nitrogen protecting groups include protecting groups of primary or secondary amines, such as in —NHRPR or —N(RPR)2—, where at least one of the RPRs is a nitrogen-atom protecting group, or both RPRs together contain a protecting group.

[0120] A suitable protecting group is one that prevents or avoids unwanted side reactions or premature loss of the protecting group under the reaction conditions required to achieve the desired chemical transformation elsewhere in the molecule and during the purification of the newly formed molecule as desired, and that can be removed without adversely affecting the structural or stereochemical integrity of the newly formed molecule. By way of example and not limitation, suitable protecting groups may include those previously described for protecting functional groups. Suitable protecting groups are sometimes those used in peptide coupling reactions.

[0121] "Aromatic alcohol" refers, either directly or as part of a larger structure, to an aromatic ring system substituted with a hydroxyl functional group (-OH). Therefore, an aromatic alcohol refers to any aryl, heteroaryl, extended aryl, and extended heteroaryl moiety as described herein, having a hydroxyl functional group bonded to the aromatic carbon of its aromatic ring system. An aromatic alcohol may be part of a larger portion, where its aromatic ring system is a substituent of this portion, or may be inserted into that larger portion by ring fusion, and may be partially substituted as described herein, including one or more other hydroxyl substituents. Phenolic alcohols are aromatic alcohols having a phenolic group as an aromatic ring.

[0122] “Aliphatic alcohol” refers, either on its own or as part of a larger structure, to a portion of a non-aromatic carbon bonded to a hydroxyl functional group –OH. The carbon carrying the hydroxyl group may be unsubstituted (i.e., methanol) or may have one, two, or three, as appropriate, substituted branched or unbranched alkyl substituents to define a primary or secondary or tertiary aliphatic alcohol within a straight-chain or cyclic structure. When part of a larger structure, the alcohol may be substituented by means of a hydroxyl-containing carbon, a carbon bonded to the hydroxyl-containing carbon via an alkyl or other portion as described herein, or a substituent of that alkyl or other portion. Aliphatic alcohols encompass non-aromatic cyclic structures (i.e., carbocyclic and heterocyclic rings, as appropriate, substituted), in which a hydroxyl functional group is bonded to a non-aromatic carbon of its cyclic ring system.

[0123] As used herein, “arylalkyl” or “heteroarylalkyl” means a substituent, part or group, wherein the aryl moiety is bonded to the alkyl moiety, i.e., aryl-alkyl-, wherein the alkyl and aryl groups are as described above, for example C6H5—CH2— or C6H5—CH(CH3)CH2—. The arylalkyl or heteroarylalkyl is associated with a larger structure or part via the sp3 carbon of its alkyl moiety.

[0124] As used herein, "succinyldiimidium moiety" refers to the organic moiety consisting of a succinyldiimidium ring system, present in a type Y' of a compound of formula (PL-A), which is typically further composed of an alkyl-containing portion of the succinyldiimidium nitrogen bound to the ring system. The succinyldiimidium moiety is typically generated by Michael addition of a thiol group of a cell binder to the cis-butenyldiimidium ring system of a camptothecin-supported compound (Formula II). Thus, the succinyldiimidium moiety consists of a thiolated succinyldiimidium ring system and, when present in a camptothecin conjugate, its succinyldiimidium nitrogen is substituted by the remaining portion of the cell binder of the camptothecin conjugate and, where applicable, by a substituent present on the cis-butenyldiimidium ring system of the compound of formula II.

[0125] As used herein, "acid-acetylamine moiety" refers to succinic acid having an acetylamine substituent arising from a thiolated succinimine ring system of the succinimine moiety, which has undergone hydrolysis involving the breaking of one of its carbonyl-nitrogen bonds. The hydrolysis resulting in the succinic acid-acetylamine moiety provides a linker that is less likely to suffer premature loss of the linker it is bound to by eliminating the thiolated substituent. It is anticipated that the hydrolysis of the succinimine ring system of the thiolated succinimine moiety will provide regiochemical isomers of the acid-acetylamine moiety, which are due to the difference in reactivity of the two carbonyl carbons of the succinimine ring system, which can be attributed at least in part to any substituent present in the cis-butenimine ring system of the compound of formula II and to the thiolated substituent introduced by the targeted ligand.

[0126] As used herein, the term "prodrug" refers to a less biologically active or inactive compound that is converted in vivo into a more biologically active compound through a chemical or biological process (i.e., a chemical reaction or enzymatic biotransformation). Typically, a biologically active compound is partially chemically modified by a prodrug to exhibit less biological activity (i.e., converted into a prodrug). In some cases, the prodrug is a type II prodrug, which is bioactivated extracellularly (e.g., in digestive fluids) or in the body's circulatory system (e.g., in the blood). Exemplary prodrugs are esters and (β-D-glucopyranoside).

[0127] In many cases, the components of conjugates, linkers, and components described herein refer to reactive groups. A "reactive group" or RG is a group containing a reactive site (RS) capable of forming a bond with a linker component of a camptothecin-loaded or camptothecin-conjugated compound, or with camptothecin itself. RS is a reactive site within a reactive group (RG). Reactive groups include thiols for forming disulfide or thioether bonds; aldehydes, ketones, or hydrazines for forming hydrazone bonds; carboxyl or amino groups for forming peptide bonds; carboxyl or hydroxyl groups for forming ester bonds; sulfonic acids for forming sulfonylurea bonds; alcohols for forming carbamate bonds; and amines for forming sulfonylurea or carbamate bonds. The following table illustrates reactive groups, reactive sites, and exemplary functional groups that can be formed after a reaction at a reactive site. This table is not restrictive. Those skilled in the art will understand that the R' and R" portions mentioned in the table are effectively any organic portion (e.g., alkyl, aryl, heteroaryl; or substituted alkyl, aryl, or heteroaryl) compatible with the bond formation provided in converting RG into one of the exemplary functional groups. It should also be understood that, when applied to embodiments of the invention, R' may, depending on the specific circumstances, represent one or more components of a self-stabilizing linker or, as appropriate, a secondary linker, and R" may, depending on the specific circumstances, represent one or more components of, as appropriate, a secondary linker, camptothecin, a stabilizing unit, or a detection unit.

[0128] The combinations of substituents and variables contemplated by the present invention are only combinations that result in the formation of stable compounds. As used herein, the term "stable" means a compound having sufficient stability to allow for manufacture and maintaining its integrity for a sufficient period of time for the purposes detailed herein (e.g., administration to an individual for treatment or prophylaxis).

[0129] The compounds of the present invention are preferably separated and purified after their preparation to obtain a composition containing an amount equal to or greater than 95% (“substantially pure”) by weight, which is then used or formulated as described herein.

[0130] As used herein, the term "conjugate" refers to the compound or derivative thereof described herein that is linked to a cell binder.

[0131] As used herein, the term "linkable to cell binders" refers to the compounds or derivatives thereof described herein, which contain at least one linking group or precursor thereof suitable for linking such compounds or derivatives thereof to cell binders.

[0132] The term "precursor" for a given group means any group that can be generated by any deprotection, chemical modification or coupling reaction.

[0133] The term "linked to a cell binder" means a molecule of a combination comprising at least one of the compounds or derivatives described herein, the molecule of which is bonded to a cell binder via a suitable linker group or a precursor thereof.

[0134] The terms “abnormal cell growth” and “proliferative disorder” are used interchangeably in this application. Unless otherwise specified, as used herein, “abnormal cell growth” means cell growth independent of normal regulatory mechanisms (e.g., loss of contact inhibition). This includes, for example, the following abnormal growths: (1) tumor cells (tumors) that proliferate by expressing mutant tyrosine kinase or overexpressing receptor tyrosine kinase; (2) benign and malignant cells of other proliferative disorders that have abnormal tyrosine kinase activation; (3) any tumor that proliferates by receptor tyrosine kinase; (4) any tumor that proliferates by abnormal serine / threonine kinase activation; and (5) benign and malignant cells of other proliferative disorders that have abnormal serine / threonine kinase activation.

[0135] The terms “cancer” and “cancerous” refer to or describe a physiological condition in mammals that is typically characterized by uncontrolled cell growth. A “tumor” includes one or more cancer cells and / or benign or precancerous cells.

[0136] "Therapeutic agents" include biological agents, such as antibodies, peptides, proteins, enzymes or chemotherapeutic agents.

[0137] "Chemotherapy agents" are compounds that are used to treat cancer.

[0138] A "metabolite" is a product produced in vivo by the metabolism of a specified compound, its derivatives, its conjugates, or its salts. Metabolites of compounds, their derivatives, or their conjugates can be identified using conventional techniques known in this art, and their activity can be determined using tests such as those described herein. Such products can be produced, for example, by oxidation, hydroxylation, reduction, hydrolysis, acetylation, deacetylation, esterification, deesterification, enzymatic cleavage, and the like of the compound being applied. Therefore, this invention includes metabolites of the compounds, their derivatives, or their conjugates of the present invention, including compounds, their derivatives, or their conjugates produced by a method comprising contacting the compounds, their derivatives, or their conjugates of the present invention with a mammal for a period sufficient to produce their metabolites.

[0139] As defined herein, a "linker," "linking portion," or "linking group" refers to a portion that links two groups, such as a cell binder and a cytotoxic compound, together. Typically, a linker is substantially inert under the condition that the two groups it links are connected. A bifunctional crosslinker may contain two reactive groups, one at each end of the linking portion, such that one reactive group can first react with the cytotoxic compound to provide a compound carrying the linking portion and a second reactive group, which can then react with the cell binder. Alternatively, one end of the bifunctional crosslinker may first react with the cell binder to provide a cell binder carrying the linking portion and a second reactive group, which can then react with the cytotoxic compound. The linking portion may contain chemical bonds that allow the cytotoxic portion to be released at a specific site. Suitable chemical bonds are those well known in the art and include disulfide bonds, thioether bonds, acid-labile bonds, light-labile bonds, peptidase-labile bonds, and esterase-labile bonds (see, for example, U.S. Patents 5,208,020; 5,475,092; 6,441,163; 6,716,821; 6,913,748; 7,276,497; 7,276,499; 7,368,565; 7,388,026, and 7,414,073). Disulfide bonds, thioether bonds, and peptidase-labile bonds are preferred. Other connectors that may be used in this invention include non-disintegrable connectors, such as those described in U.S. Publication No. 20050169933, or charged or hydrophilic connectors, and described in US 2009 / 0274713, US 2010 / 01293140 and WO 2009 / 134976, each of which is expressly incorporated herein by reference.

[0140] The term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid simulants that function in a manner similar to naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code and those subsequently modified, such as hydroxyproline, γ-carboxyglutamic acid, selenocysteine, and O-phosphoserine. Amino acid analogs refer to compounds having the same basic chemical structure as naturally occurring amino acids (i.e., carbon atoms bonded to hydrogen, carboxyl, amino, and R groups), such as homoserine, oroleucine, methyl methionine, and methyl strontium methionine. These analogs have modified R groups (e.g., oroleucine) or modified peptide backbones, but retain the same basic chemical structure as naturally occurring amino acids. One particularly usable amino acid is citrulline, a derivative of arginine, which participates in the formation of urea in the liver. Amino acid mimics are compounds that have a structure different from the general chemical structure of amino acids, but function in a manner similar to that of naturally occurring amino acids. The term "non-natural amino acid" is intended to refer to the "D" stereochemical form of the twenty naturally occurring amino acids described above. It should be further understood that the term "non-natural amino acid" includes homologs of natural amino acids or their D isomers, and synthetically modified forms of natural amino acids. Synthetically modified forms include, but are not limited to, amino acids having side chains shortened or lengthened by up to two carbon atoms, amino acids containing, where appropriate, substituted aryl groups, and amino acids containing halogenated groups, preferably halogenated alkyl groups and aryl groups, as well as N-substituted amino acids, such as N-methyl-alanine. Amino acids or peptides may be linked to linkers / spacers or cell binders via a terminal amine or terminal carboxylic acid. Amino acids can also be linked to linkers / spacers or cell binders via reactive side-chain groups, such as, but not limited to, the thiol group of cysteine, the ε-amine of lysine, or the hydroxyl group of serine or threonine.

[0141] In the embodiments, the amino acid is represented by NH2-C(Raa'Raaa)-C(=O)OH, wherein Raa and Raa' are each independently H, and are optionally substituted linear, branched, or cyclic alkyl, alkenyl, alkynyl, aryl, heteroaryl, or heterocyclic groups having 1 to 10 carbon atoms, or the R and N terminal nitrogen atoms may together form a heterocycle (e.g., as in proline). The term "amino acid residue" refers to the corresponding residue when a hydrogen atom is removed from an amine and / or a hydroxyl group is removed from the carboxyl terminus of an amino acid, such as -NH-C(Raa'Raa)-C(=O)O-.

[0142] As used herein, amino acids may be L- or D-isomers. Unless otherwise specified, when an amino acid is referred to, it may be an L- or D-isomer or a mixture thereof. In embodiments, unless otherwise specified, when a peptide is referred to by its amino acid sequence, each of the amino acids may be an L- or D-isomer. Unless otherwise specified, if one of the amino acids in a peptide is designated as a D-isomer, the other amino acids are L-isomers. For example, the peptide D-Ala-Ala means D-Ala-L-Ala.

[0143] Amino acids and peptides can be protected by blocking groups. Blocking groups are atoms or chemical parts that protect the N-terminus of an amino acid or peptide from unwanted reactions and can be used during the synthesis of drug-ligand conjugates. They should remain attached to the N-terminus throughout the synthesis and can be removed selectively by chemical or other conditions after the completion of the drug conjugate synthesis. Blocking groups suitable for N-terminal protection are well known in peptide chemistry. Exemplary blocking groups include, but are not limited to, methyl esters, tributyl esters, 9-enylomethylcarbamate (Fmoc), and benzyloxycarbonyl (Cbz).

[0144] The term "peptide that can be cleaved by a protease" refers to a peptide containing a cleavage recognition sequence of a protease. As used herein, a protease is an enzyme that can cleave peptide bonds. A cleavage recognition sequence of a protease is a specific amino acid sequence recognized by the protease during proteolytic cleavage. Many protease cleavage sites are known in this art, and these and other cleavage sites may be included in the linker portion. See, for example, Matayoshi et al., Science 247: 954 (1990); Dunn et al., Meth. Enzymol. 241: 254 (1994); Seidah et al., Meth. Enzymol. 244: 175 (1994); Thornberry, Meth. Enzymol. 244: 615 (1994); Weber et al., Meth. Enzymol. 244: 595 (1994); Smith et al., Meth. Enzymol. 244: 412 (1994); Bouvier et al., Meth. Enzymol. 248: 614 (1995); Hardy et al., AMYLOID PROTEIN PRECURSOR IN DEVELOPMENT, AGING, AND ALZHEIMER'S DISEASE, Masters et al., eds., pp. 190-198 (1994).

[0145] The peptide sequence is selected based on its ability to be cleaved by a protease, non-limiting examples of which include cathepsins B, C, D, H, L, and S, and furin. Preferably, the peptide sequence is capable of being cleaved in vitro by a suitable isolated protease, which can be tested using in vitro protease cleavage assays known in this art.

[0146] In another embodiment, the peptide sequence is selected based on its ability to be cleaved by lysosomal proteases. Lysosomal proteases are proteases primarily located in lysosomes, but may also be located in endosomes. Examples of lysosomal proteases include, but are not limited to, cathepsins B, C, D, H, L, and S, and furin protease.

[0147] In another embodiment, the peptide sequence is selected based on its ability to be cleaved by tumor-associated proteases, such as proteases found on the surface of cancer cells or extracellularly near tumor cells. Non-limiting examples of such proteases include thimet oligopeptidase (TOP), CD10 (enkephalin), matrix metalloproteinases (such as MMP2 or MMP9), type II transmembrane serine proteases (such as hepsin, testisin, TMPRSS4 or interstitial protease / MT-SP1), podin, and enzymes described in the following reference (Current Topics in Developmental Biology: Cell Surface Proteases, Vol. 54, Zucker S. 2003, Boston, MA). The ability of a peptide to be cleaved by tumor-associated proteases can be tested using in vitro protease cleavage assays known in this art.

[0148] The term "cation" refers to a positively charged ion. A cation can be monovalent (e.g., Na+, K+), divalent (e.g., Ca2+, Mg2+, etc.), or polyvalent (e.g., Al3+, etc.). In the examples, the cation is monovalent. HER2

[0149] HER2 is one of the oncogene products of a typical growth factor receptor oncogene identified as a human epidermal growth factor receptor 2-related oncogene, and is a transmembrane receptor protein with a molecular weight of 185 kDa and a tyrosine kinase domain. HER2 is a member of the EGFR family, consisting of HER1 (EGFR, ErbB-1), HER2 (neu, ErbB-2), HER3 (ErbB-3), and HER4 (ErbB-4). It is known to autophosphorylate tyrosine residues in cells through homodimerization or heterodimerization with another EGFR receptor, HER1, HER3, or HER4, and is activated in this manner, thereby playing an important role in the growth, differentiation, and survival of normal and tumor cells.

[0150] For the HER2 protein to be used in this invention, the HER2 protein can be purified and used directly from human or non-human mammalian (such as rat or mouse) cells expressing HER2, or the cell membrane portion of the aforementioned cells can be prepared and used. Furthermore, HER2 can be obtained by its in vitro synthesis or by its production in host cells through genetic engineering. In genetic engineering, specifically, after HER2 cDNA is integrated into a vector capable of expressing HER2 cDNA, the HER2 protein can be obtained by synthesis in a solution containing the enzymes, acceptors, and energy substances required for transcription and translation, or by expressing HER2 in another prokaryotic or eukaryotic transformed host cell. Alternatively, the aforementioned genetically engineered HER2-expressing cells or HER2-expressing cell lines can be used as the HER2 protein.

[0151] The DNA and amino acid sequences of HER2 are disclosed in public databases and may be referred to, for example, by accession number M11730 (GenBank), NP_004439.2 (NCBI) or similar numbers.

[0152] In addition, proteins composed of amino acid sequences are also included in HER2, wherein one or more amino acids are substituted, deleted and / or added in any of the above-mentioned amino acid sequences of HER2 and also have biological activities equivalent to those of the protein.

[0153] The human HER2 protein is composed of the following: a signal sequence consisting of 22 amino acid residues at the N-terminus, an extracellular domain consisting of 630 amino acid residues, a transmembrane domain consisting of 23 amino acid residues, and an intracellular domain consisting of 580 amino acid residues.

[0154] HER2 is overexpressed in various cancer types, such as breast cancer, gastric cancer, lung cancer (e.g., non-small cell lung cancer), and ovarian cancer, and has been reported as a negative prognostic factor for breast cancer. Anti-HER2 antibody.

[0155] Anti-HER2 antibodies are antibodies capable of targeting tumor cells, that is, possessing the characteristics of recognizing tumor cells, binding to tumor cells, internalizing in tumor cells, cytotoxic activity against tumor cells, or similar characteristics, and can be bound to a drug with antitumor activity via a linker to form an antibody-drug conjugate. Non-limiting anti-HER2 antibodies applicable to the compounds, compositions, and methods described herein include antibodies having any one or any combination of the following exemplary characteristics: (1) An anti-HER2 antibody having the following characteristics: (a) specifically binding to HER2, and (b) having the activity of internalization in cells expressing HER2 by binding to HER2. (2) The antibody according to (1) above, wherein the antibody binds to the extracellular domain of HER2. (3) The antibody according to (1) or (2) above, wherein the antibody is a monoclonal antibody. (4) An antibody according to any one of (1) to (3) above, wherein the antibody has antibody-dependent cytotoxic (ADCC) activity and / or complement-dependent cytotoxic (CDC) activity. (5) An antibody according to any one of (1) to (4) above, wherein the antibody is a mouse monoclonal antibody, a chimeric monoclonal antibody, or a humanized monoclonal antibody. (6) An antibody according to any one of (1) to (5) above, wherein the antibody is a humanized monoclonal antibody.

[0156] Trastuzumab (Herceptin®) is a humanized monoclonal antibody, also known as recombinant humanized anti-HER2 monoclonal antibody (huMAb4D5-8, rhuMAb HER2), which is a mouse anti-HER2 antibody 4D5. Trastuzumab specifically binds to the extracellular domain IV of HER2 and induces antibody-dependent cytotoxicity (ADCC) or exerts its anticancer effect by inhibiting HER2 signal transduction. Trastuzumab is highly effective against tumors that overexpress HER2, and therefore, it was launched in the United States in 1999 and in Japan in 2001 as a treatment for patients with metastatic breast cancer that overexpresses HER2.

[0157] Although trastuzumab is effective in treating breast cancer, some patients with HER2-overexpressing breast cancer do not respond to trastuzumab treatment or respond only weakly. The anti-HER2 antibody used in the anti-HER2 antibody-drug conjugate of the present invention can be derived from any species, with preferred examples including humans, rats, mice, and rabbits. In cases where the antibody is derived from a species other than humans, it is preferably chimeric or humanized using well-known techniques. The antibody of the present invention can be a polyclonal antibody or a monoclonal antibody, and is preferably a monoclonal antibody.

[0158] Trastuzumab comprises a heavy chain consisting of an amino acid sequence represented by SEQ ID NO: 1 and a light chain consisting of an amino acid sequence represented by SEQ ID NO: 2.

[0159] Heavy chain amino acid sequence of trastuzumab: EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDK THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:1)

[0160] Trastuzumab light chain amino acid sequence: DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO:2)

[0161] The CDR sequence of Kabat's trastuzumab is as follows: HCDR1: GFNIKDTYIH (SEQ ID NO:3) HCDR2: RIYPTNGYTRYADSVKG (SEQ ID NO:4) HCDR3: WGGDGFYAMDY (SEQ ID NO:5) LCDR1: RASQDVNTAVA (SEQ ID NO:6) LCDR2: SASFLYS (SEQ ID NO:7) LCDR3: QQHYTTPPT (SEQ ID NO:8)

[0162] In some embodiments, the anti-HER2 antibody comprises one, two, or three sequences selected from: (a) the amino acid sequence of SEQ ID NO: 3, (b) the amino acid sequence of SEQ ID NO: 4, and (c) the amino acid sequence of SEQ ID NO: 5. In the embodiments, the

[0163] In some embodiments, the anti-HER2 antibody comprises a heavy chain containing an amino acid sequence having at least 80% identity with SEQ ID NO: 1. In some embodiments, the anti-HER2 antibody comprises a light chain containing an amino acid sequence having at least 80% identity with SEQ ID NO: 2.

[0164] In some embodiments, the anti-HER2 antibody comprises a heavy chain having an amino acid sequence having at least 80%, 85%, 90%, 95% or 100% similarity to SEQ ID NO: 1, and a light chain having an amino acid sequence having at least 80%, 85%, 90%, 95% or 100% similarity to SEQ ID NO: 2.

[0165] In the embodiments, the antibody does not contain any lysine residues defined by the sequence SEQ ID NO: 1 at the carboxyl terminus of the heavy chain. In the embodiments, the antibody comprises a heavy chain consisting of amino acid sequences composed of amino acid residues 1 to 449 of SEQ ID NO: 1, and a light chain consisting of amino acid sequences composed of amino acid residues 1 to 214 of SEQ ID NO: 2.

[0166] In some embodiments, the anti-HER2 antibody is trastuzumab.

[0167] In some embodiments, the anti-HER2 antibody is a biosimilar of trastuzumab. The ADC of the present invention is also effective against antibody constructs that are highly similar or biosimilar to commercially available or "innovative" antibody constructs. The performance of the biosimilar ADC is expected to be similar to that of the immune conjugate of the novel product (e.g., trastuzumab). As used herein, the term "biosimilar" with respect to a biological product means that the biological product is highly similar to a reference product, but with minor differences in clinically inactive components, and that there are no clinically significant differences between the biological product and the reference product in terms of safety, purity, and potency.

[0168] In some embodiments, the anti-HER2 antibody is a trastuzumab biosimilar. In some embodiments, the anti-HER2 trastuzumab biosimilar is selected from the group consisting of: trastuzumab-dkst (Ogivri), trastuzumab-pkrb (Herzuma), trastuzumab-dttb (Ontruzant), trastuzumab-qyyp (Trazimera), and trastuzumab-anns (Kanjinti).

[0169] In other embodiments, the anti-HER2 antibody may be interchangeable with trastuzumab. In embodiments, the interchangeable anti-HER2 antibody is biosimilar to trastuzumab and is expected to produce the same clinical outcomes as trastuzumab in any given patient.

[0170] In other embodiments, the anti-HER2 antibody is a biomodified version of trastuzumab. In these embodiments, the "biomodified" biologic product belongs to the same class as FDA-approved biologic products, targets the same target protein as the reference biologic, but is not the same, and is improved compared to the reference product in terms of safety, efficacy, stability, and route of administration. Antibody binding and antitumor activity.

[0171] The binding activity of antibodies against tumor cells can be confirmed using flow cytometry. Antibody internalization into tumor cells can be confirmed using the following methods: (1) visualization of the incorporated antibody under a fluorescence microscope using a fluorescently labeled secondary antibody bound to the therapeutic antibody (Cell Death and Differentiation (2008) 15, 751-761), (2) measurement of the fluorescence intensity of the incorporated antibody using a fluorescently labeled secondary antibody bound to the therapeutic antibody (Molecular Biology of the Cell, Vol. 15, 5268-5282, December 2004), or (3) Mab-ZAP analysis using an immunotoxin bound to the therapeutic antibody, where the toxin is released after incorporation to inhibit cell growth (Bio Techniques 28: 162-165, January 2000). As an immunotoxin, a recombinant complex of diphtheria toxin catalytic domain and protein G can be used.

[0172] The antitumor activity of antibodies can be confirmed in vitro by determining their inhibitory activity against cell growth. For example, cancer cell lines that overexpress the target protein of the antibody are cultured, and the antibody is added to the culture system at different concentrations to determine its inhibitory activity against lesion formation, colony formation, and globular growth. Antitumor activity can be confirmed in vivo, for example by administering the antibody to nude mice with transplanted tumor cell lines that highly express the target protein and determining changes in cancer cells.

[0173] Since the compounds bound in antibody-drug conjugates exert antitumor effects, it is preferable, but not essential, that the antibody itself should possess antitumor activity. In order to specifically and selectively exert the cytotoxic activity of the antitumor compounds against tumor cells, it is important and preferably that the antibody should have the property of internalizing and migrating into tumor cells. Production of anti-HER2 antibodies

[0174] The antibody against HER2 of the present invention can be obtained according to methods commonly performed in this art, such as immunization of an animal with HER2 or any polypeptide selected from the amino acid sequence of HER2, and collection and purification of the in vivo produced antibody. The HER2 biospecies used as the antigen is not limited to humans, and the animal can be derived from animals other than humans, such as mice or rats, or immunized with rat p185neu. In this case, by examining the cross-reactivity between the obtained heterologous HER2 and the antibody binding to human HER2, antibodies suitable for human diseases can be selected.

[0175] In addition, monoclonal antibodies can be obtained from fusion tumors, which are established by fusing antibody-producing cells that produce antibodies against HER2 with myeloma cells according to known methods (e.g., Kohler and Milstein, Nature, (1975) 256, pp. 495-497; Kennet, R. ed., Monoclonal Antibodies, pp. 365-367, Plenum Press, NY (1980)).

[0176] HER2, used as an antigen, can be obtained by genetically engineering the expression of the HER2 gene in host cells. Specifically, a vector capable of expressing the HER2 gene is produced, and the resulting vector is transfected into host cells to express the gene, and subsequently, the expressed HER2 is purified.

[0177] Alternatively, the genetically engineered HER2-expressing cells or HER2-expressing cell lines described above can be used as the HER2 protein. Anti-HER2 antibodies can be obtained by procedures known in this art. Methods for obtaining antibodies against HER2 are specifically described below. Preparation of Antigen

[0178] Examples of antigens to be used to generate anti-HER2 antibodies include HER2, or a polypeptide consisting of a partial amino acid sequence comprising at least six consecutive amino acids of HER2, or a derivative obtained by adding a given amino acid sequence or a carrier thereto.

[0179] HER2 can be purified and used directly from human tumor tissues or tumor cells. In addition, HER2 can be obtained by in vitro synthesis or by genetically engineering HER2 in host cells.

[0180] In terms of genetic engineering, specifically, after HER2 cDNA is integrated into a vector capable of expressing HER2 cDNA, HER2 can be obtained by synthesis in a solution containing the enzymes, acceptors and energy substances required for transcription and translation, or by expressing HER2 in a host cell transformed from another prokaryotic or eukaryotic cell.

[0181] Furthermore, antigens can also be obtained in the form of secretory proteins by expressing the fusion protein obtained by linking the extracellular domain of HER2, a membrane protein, with the constant region of the antibody in a suitable host-vector system.

[0182] HER2 cDNA can be obtained by, for example, the so-called PCR method, in which a cDNA library expressing HER2 cDNA is used as a template and a primer specifically amplifying HER2 cDNA is used to perform a polymerase chain reaction (PCR; Saiki, RK et al., Science, (1988) 239, pp. 487-489).

[0183] As an example of in vitro synthesis of polypeptides, the Rapid Translation System (RTS) manufactured by Roche Diagnostics, Inc. can be cited, but is not limited thereto.

[0184] Examples of prokaryotic host cells include *Escherichia coli* and *Bacillus subtilis*. To transform host cells with a target gene, the host cells are transformed using a plastid vector containing a replicon, that is, an origin of replication derived from a host-compatible species, and regulatory sequences. Furthermore, the vector preferably has sequences capable of imposing phenotypic selectivity on the transformed cells.

[0185] Examples of eukaryotic host cells include vertebrate cells, insect cells, and yeast cells. As vertebrate cells, examples commonly used include monkey COS cells (Gluzman, Y., Cell, (1981) 23, pp. 175-182, ATCC CRL-1650; ATCC: American Culture Collection), mouse fibroblast NIH3T3 (ATCC number CRL-1658), and dihydrofolate reductase-deficient strains of Chinese hamster ovary cells (CHO cells; ATCC: CCL-61) (Urlaub, G. and Chasin, LA, Proc. Natl. Acad. Sci. USA (1980) 77, pp. 4126-4220); and analogues thereof, however, the cells are not limited to these.

[0186] The resulting transformant can be cultured according to methods typically performed in this art, and by culturing the transformant, the target polypeptide is produced intracellularly or extracellularly.

[0187] The suitable culture medium to be used for culturing can be selected from various commonly used culture media depending on the host cells used. For example, if Escherichia coli is used, LB medium supplemented with antibiotics such as ampicillin or IPMG as needed can be used.

[0188] Recombinant proteins produced by such culture in or outside the cells via the transformant can be separated and purified by any of the known isolation methods using the physical or chemical properties of the protein.

[0189] Specific examples of methods include treatment with common protein precipitants, ultrafiltration, various types of liquid chromatography (such as molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography and affinity chromatography), dialysis and combinations thereof.

[0190] Furthermore, by tagging the recombinant protein to be expressed with a six-histidine residue (SEQ ID NO:9), the protein can be efficiently purified using a nickel affinity column. Alternatively, by tagging the IgG Fc region to the recombinant protein to be expressed, the protein can be efficiently purified using a protein A column.

[0191] By combining the above methods, large quantities of target peptides can be easily produced with high yield and high purity.

[0192] The above-mentioned transformants can be used as antigens themselves. Cell lines expressing HER2 can also be used as antigens. Examples of such cell lines include human breast cancer lines SK-BR-3, BT-474, KPL-4, and JIMT-1, human gastric cancer line NCI-N87, and human ovarian cancer line SK-OV-3. The cell lines of the present invention are not limited to these cell lines, as long as they express HER2. Production of anti-HER2 monoclonal antibodies

[0193] Examples of antibodies that specifically bind to HER2 include monoclonal antibodies that specifically bind to HER2, and methods for obtaining such antibodies are described below.

[0194] The production of monoclonal antibodies typically requires the following steps: (a) purifying the biopolymer to be used as an antigen or preparing cells to express the antigen; (b) preparing antibody-producing cells by immunizing animals with the antigen, collecting blood, and analyzing the antibody titer to determine when to remove the spleen; (c) preparing myeloma cells (hereinafter referred to as "myeloma"); (d) fusing the antibody-producing cells with myeloma; (e) screening a group of fusion tumors that produce the desired antibodies; (f) dividing the fusion tumors into single-cell pure lines (selection); (g) culturing the fusion tumor or feeding animals implanted with the fusion tumor, as appropriate, to produce large quantities of monoclonal antibodies; (h) examining the bioactivity and binding specificity of the monoclonal antibodies produced, or analyzing their properties as labeled reagents; and similar.

[0195] In the following description, the method for generating monoclonal antibodies will follow the steps outlined above. However, the method is not limited to this, and antibody-producing cells other than spleen cells and myeloma cells may be used, for example. Antigen purification

[0196] As an antigen, HER2 or a portion thereof prepared by the method described above may be used.

[0197] In addition, membrane portions prepared from recombinant cells expressing HER2 or the recombinant cells themselves expressing HER2, as well as partial peptides of the proteins of the present invention chemically synthesized by methods known to those skilled in the art, can also be used as antigens.

[0198] In addition, cell lines expressing HER2 can also be used as antigens. Preparation of antibody-producing cells.

[0199] The antigen obtained in step (a) is mixed with an adjuvant, such as Freund's complete or incomplete adjuvant or auxiliary agent, such as potassium aluminum sulfate, and the resulting mixture is used as an immunogen for immunizing experimental animals. Another method involves immunizing experimental animals with cells expressing the antigen as an immunogen. As experimental animals, any animal known to be used in fusion tumor production methods can be used without hindrance. Specifically, for example, mice, rats, goats, sheep, cattle, horses, or similar animals can be used. However, from the viewpoint of easy access to myeloma cells fused with extracted antibody-producing cells, mice or rats are preferred as animals to be immunized.

[0200] Furthermore, the mouse or rat strain to be used is not specifically limited, and in the case of mice, various strains such as A, AKR, BALB / c, BDP, BA, CE, C3H, 57BL, C57BL, C57L, DBA, FL, HTH, HT1, LP, NZB, NZW, RF, R III, SJL, SWR, WB and 129 and similar strains may be used, and in the case of rats, strains such as Wistar, Low, Lewis, Sprague, Dawley, ACI, BN, Fischer and similar strains may be used.

[0201] These mice and rats may be commercially available from breeders / distributors of laboratory animals, such as CLEA Japan, Inc. and Charles River Laboratories Japan, Inc.

[0202] As animals to be immunized, in the case of mice, the BALB / c strain is particularly preferred in terms of compatibility with the myeloma cells described below, and in the case of rats, the Wistar and Low strains are particularly preferred.

[0203] In addition, considering the antigenic homology between humans and mice, it is also preferable to use mice with reduced biological function due to the removal of autoantibodies, that is, mice with autoimmune diseases.

[0204] The age of these mice or rats at the time of immunization is preferably 5 to 12 weeks, more preferably 6 to 8 weeks.

[0205] For the purpose of immunizing animals with HER2 or its recombinants, for example, known methods described in detail in, for example, the following: Weir, DM, Handbook of Experimental Immunology, Volumes I, II, III, Blackwell Scientific Publications, Oxford (1987); Kabat, EA and Mayer, MM, Experimental Immunochemistry, Charles C Thomas Publisher Springfield, Ill. (1964) or similar.

[0206] In these immunization methods, the preferred particular method of the present invention is as follows, for example.

[0207] That is, firstly, the membrane protein fraction that serves as the antigen or the cell that expresses the antigen is administered intradermally or intraperitoneally to the animal. However, a combination of the two administration routes is better for increasing immunization efficiency, and immunization efficiency can be particularly increased when intradermal administration is performed in the first half and intraperitoneal administration is performed in the second half or only at the last administration.

[0208] The timing of antigen administration varies depending on the type of animal to be immunized, individual differences, or similar factors. However, in general, an administration frequency of 3 to 6 times with an interval of 2 to 6 weeks is preferred, and an administration frequency of 3 to 4 times with an interval of 2 to 4 weeks is even better.

[0209] In addition, the dosage of the antigen varies depending on the animal type, individual differences or similar factors; however, the dosage is generally set at 0.05 to 5 mg, preferably about 0.1 to 0.5 mg.

[0210] A booster immunization should be administered 1 to 6 weeks, preferably 1 to 4 weeks, and even more preferably 1 to 3 weeks after administration of the antigen as described above. When the immunogen is cells, use 1 × 10⁶ to 1 × 10⁷ cells.

[0211] The dosage of antigen used for booster immunization varies depending on the type, size, or similar factors of the animal. However, in the case of mice, for example, the dosage is generally set at 0.05 to 5 mg, preferably 0.1 to 0.5 mg, and more preferably about 0.1 to 0.2 mg. When the immunogen is cells, 1 × 10⁶ to 1 × 10⁷ cells are used.

[0212] One to ten days, preferably two to five days, or even more preferably two to three days after booster immunization, spleen cells or lymphocytes, including antibody-producing cells, are aseptically removed from the immunized animal. At this time, antibody titers are measured, and subsequent procedures can be performed more efficiently if animals with sufficiently increased antibody titers are used as a source of antibody-producing cells.

[0213] Examples of methods for measuring the antibody titer to be used herein include RIA and ELISA methods, but are not limited to these. For example, if the ELISA method is used, the antibody titer in this invention can be measured according to the procedure described below.

[0214] First, purified or partially purified antigen is adsorbed onto a solid phase, such as the surface of a 96-well disc used for ELISA, and the surface of the solid phase without adsorbed antigen is covered with a protein unrelated to the antigen, such as bovine serum albumin (BSA). After washing the surface, the surface is brought into contact with a serially diluted sample (e.g., mouse serum) as a primary antibody to allow the antibody in the sample to bind to the antigen.

[0215] Furthermore, as a secondary antibody, an enzyme-labeled antibody against a mouse antibody is added and bound to the mouse antibody. After washing, an enzyme acceptor is added, and the absorbance change induced by acceptor degradation or its analogues is measured, and the antibody titer is calculated based on the measured value.

[0216] Antibody-producing cells can be isolated from spleen cells or lymphocytes of autoimmunized animals using known methods (e.g., Kohler et al., Nature (1975), 256, p. 495; Kohler et al., Eur. J. Immunol. (1977), 6, p. 511; Milstein et al., Nature (1977), 266, p. 550; Walsh, Nature (1977), 266, p. 495). For example, in the case of spleen cells, a common method can be used, in which antibody-producing cells are isolated by homogenizing the spleen, obtaining cells by filtration through a stainless steel sieve, and suspending the cells in Eagle's minimum essential medium (MEM). Preparation of myeloma cells (hereinafter referred to as "myeloma")

[0217] The myeloma cells to be used for cell fusion are not particularly restricted and suitable cells may be selected from known cell lines. However, considering the convenience of selecting hybridomas from fusion cells, it is preferable to use HGPRT (hypoxanthine-guanine phosphoribosyltransferase) deficient lines with established selection procedures.

[0218] More specifically, examples of HGPRT-deficient strains include mouse-derived X63-Ag8 (X63), NS1-ANS / 1 (NS1), P3X63-Ag8.U1 (P3U1), X63-Ag8.653 (X63.653), SP2 / 0-Agl4 (SP2 / 0), MPC11-45.6TG1.7 (45.6TG), FO, S149 / 5XXO and BU.1; rat-derived 210.RSY3.Ag.1.2.3 (Y3); and human-derived U266AR (SKO-007), GM1500.GTG-A12 (GM1500), UC729-6, LICR-LOW-HMy2 (HMy2) and 8226AR / NIP4-1 (NP41). These HGPRT defective strains can be purchased from, for example, ATCC or similar sources.

[0219] These cell lines were subcultured in appropriate media such as 8-azaguanine medium [obtained by adding 8-azaguanine to RPMI 1640 medium supplemented with glutamic acid, 2-mercaptoethanol, gentamicin and fetal bovine serum (hereinafter referred to as "FCS")], Isco-modified Durbeco medium (hereinafter referred to as "IMDM"), or Dalberg-modified Eagle medium (hereinafter referred to as "DMEM"). In this case, the cells were subcultured in normal medium (e.g., ASF104 medium containing 10% FCS (manufactured by Ajinomoto Co., Ltd.)) 3 to 4 days prior to cell fusion to ensure a cell count of at least 2 × 10⁷ cells on the day of fusion. Cell fusion

[0220] The fusion between antibody-producing cells and myeloma cells can be carried out appropriately according to known methods (Weir, DM Handbook of Experimental Immunology, Volumes I, II, III, Blackwell Scientific Publications, Oxford (1987); Kabat, EA and Mayer, MM, Experimental Immunochemistry, Charles C Thomas Publisher, Springfield, Ill. (1964), etc.) without excessively reducing cell viability.

[0221] As such methods, for example, chemical methods can be used, in which antibody-producing cells and myeloma cells are mixed in high concentrations in a solution containing a polymer such as polyethylene glycol; physical methods can be used, such as electrical stimulation; or similar methods can be employed. Specific examples of chemical methods are described below.

[0222] That is, when polyethylene glycol is used in a solution containing a high concentration of polymer, antibody-producing cells and myeloma cells are mixed in a polyethylene glycol solution with a molecular weight of 1500 to 6000, more preferably 2000 to 4000, at a temperature of 30 to 40°C, preferably 35 to 38°C, for 1 to 10 minutes, preferably 5 to 8 minutes. Selection of fusion tumor groups.

[0223] There are no specific limitations on the method for selecting fusion tumors obtained by the above-mentioned cell fusion. Usually, the HAT (hypoxanthine, aminopterin, thymidine) selection method is used (Kohler et al., Nature (1975), 256, p. 495; Milstein et al., Nature (1977), 266, p. 550).

[0224] This method is effective when using HGPRT-deficient myeloma cells that cannot survive in the presence of aminopterin to obtain fusion tumors. That is, by culturing unfused cells and fusion tumors in HAT medium, only fusion tumors resistant to aminopterin can selectively survive and proliferate. They then divide into single-cell pure lines (selective colonization).

[0225] Known methods such as the methylcellulose method, soft agarose method, or limiting dilution method can be used as selection methods for fusion tumors (see, for example, Barbara, BM, and Stanley, MS: Selected Methods in Cellular Immunology, WH Freeman and Company, San Francisco (1980)). Among these methods, specifically, three-dimensional culture methods such as the methylcellulose method are preferred. For example, fusion tumors generated by cell fusion are suspended in methylcellulose medium, such as ClonaCell-HY Selective Medium D (manufactured by StemCell Technologies, Inc., #03804) and cultured. Subsequently, the formed fusion tumor colonies are collected to obtain single fusion tumors. The collected individual fusion tumor colonies are cultured, and fusion tumors that have been confirmed to have stable antibody titers in the obtained fusion tumor culture supernatant are selected as fusion tumor strains for producing HER2 monoclonal antibodies. Monoclonal antibodies are prepared by culturing fusion tumors.

[0226] Monoclonal antibodies can be effectively obtained by culturing the selected fusion tumors. However, it is preferable to screen the fusion tumors that produce the target monoclonal antibody before culturing.

[0227] In such screening, known methods can be used. The measurement of antibody titers in this invention can be performed, for example, by the ELISA method explained in paragraph (b) above. Fusomonas obtained by the above method can be stored frozen in liquid nitrogen or in a freezer at -80°C or below. After selection is completed, the culture medium is replaced from HT medium to normal medium, and the fusomonas are cultured.

[0228] Large-scale culture is carried out by rotation culture using large culture flasks or by rotary culture. Monoclonal antibodies that specifically bind to the proteins of the present invention can be purified from the supernatant obtained by large-scale culture using methods known to those skilled in the art (such as gel filtration).

[0229] In addition, the fusion tumor is injected into the peritoneal cavity of mice of the same strain as the fusion tumor (e.g., the above-mentioned BALB / c) or Nu / Nu mice to induce the proliferation of the fusion tumor, thereby obtaining ascites containing a large amount of the monoclonal antibody of the present invention.

[0230] In the case of intraperitoneal administration of fused tumors, a larger amount of ascites can be obtained if mineral oil such as 2,6,10,14-tetramethylpentadecane (squalane) is administered 3 to 7 days prior to administration.

[0231] For example, an immunosuppressant is injected into the peritoneal cavity of mice of the same strain as the fusion tumor to deactivate T cells. Twenty days later, 10⁶ to 10⁷ pure fusion tumor cells are suspended in serum-free culture medium (0.5 ml), and the suspension is injected into the peritoneal cavity of the mice. Generally, the ascites is collected from the mice when the abdomen is distended and filled with ascites. This method yields monoclonal antibodies at concentrations approximately 100 times or much higher than those in the culture medium.

[0232] The monoclonal antibody obtained by the above method can be purified by, for example, the method described in Weir, DM: Handbook of Experimental Immunology, Volumes I, II, III, Blackwell Scientific Publications, Oxford (1978).

[0233] The monoclonal antibody obtained thereby exhibits high antigen specificity for HER2. Examples of the monoclonal antibody of the present invention may include, but are not particularly limited to, mouse monoclonal antibody 4D5 (ATCC CRL 10463). Monoclonal antibody analysis

[0234] The isotypes and subtypes of the monoclonal antibodies obtained thereby can be determined as follows.

[0235] First, examples of identification methods include the Ouchterlony method, the ELISA method, and the RIA method.

[0236] The Ouchterlony method is simple, but when the concentration of single-strain antibodies is low, a concentration operation is required.

[0237] On the other hand, when using ELISA or RIA, the isotypes and subtypes of monoclonal antibodies can be identified by directly reacting the culture supernatant with the solid phase on which the antigen is adsorbed, and by using antibodies corresponding to the isotypes and subtypes of various types of immunoglobulins as secondary antibodies.

[0238] Alternatively, as a simpler method, commercially available identification kits (such as the Mouse Typer Kit manufactured by Bio-Rad Laboratories, Inc.) or similar products may be used.

[0239] In addition, the quantitative determination of protein can be performed by the Folin-Lowry method and the calculation method based on the absorbance at 280 nm (1.4 (OD 280) = 1 mg / ml of immunoglobulin).

[0240] Furthermore, even when monoclonal antibodies are obtained individually and independently by repeating steps (a) to (h) of (2), it is possible to obtain antibodies with equivalent cytotoxic activity to the HER2 antibody obtained in step (g). As an example of such antibodies, antibodies that bind to the same antigenic determinant as the HER2 antibody obtained in step (g) can be exemplified. If the newly generated monoclonal antibody binds to a portion of the peptide or a portion of the tertiary structure bound by the anti-HER2 antibody, it can be determined that the monoclonal antibody binds to the same antigenic determinant as the anti-HER2 antibody. Furthermore, by confirming that the monoclonal antibody and the anti-HER2 antibody compete for binding to HER2 (i.e., the monoclonal antibody inhibits the binding between the anti-HER2 antibody and HER2), it can be determined that the monoclonal antibody and the anti-HER2 antibody bind to the same antigenic determinant, even if the specific antigenic determinant sequence or structure has not yet been determined. When it is confirmed that the monoclonal antibody and the anti-HER2 antibody bind to the same antigenic determinant, it is strongly expected that the monoclonal antibody has antigen-binding affinity or biological activity equivalent to the anti-HER2 antibody. Other antibodies

[0241] The antibodies of the present invention include not only the monoclonal antibodies against HER2 described above, but also recombinant antibodies obtained by artificial modification for the purpose of reducing heteroantigenicity to humans, such as chimeric antibodies, humanized antibodies, and human antibodies. These antibodies can be produced using known methods.

[0242] Examples of chimeric antibodies include antibodies in which the variable and constant regions are derived from different species, such as chimeric antibodies in which the variable region of a mouse or rat-derived antibody is linked to the constant region of a human-derived antibody (see Proc. Natl. Acad. Sci. USA, 81, 6851-6855, (1984)). Examples of chimeric antibodies of the present invention may include, but are not particularly limited to, chimeric antibody 4D5 comprising the heavy chain constant region of human IgG1 or IgG2.

[0243] Examples of humanized antibodies include antibodies obtained by integrating only the complementarity-determining region (CDR) into a human-derived antibody (see Nature (1986) 321, pp. 522-525), antibodies obtained by transplanting a portion of the amino acid residues of the framework and the CDR sequence into a human antibody using a CDR transplantation method (WO 90 / 07861), and antibodies humanized using a gene conversion mutation induction strategy (US Patent No. 5,821,337).

[0244] As used herein, the term "several" means 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3 or 1 or 2.

[0245] As for the amino acid substitutions in this specification, conservative amino acid substitutions are preferred. Conservative amino acid substitution refers to substitutions that occur within a group of amino acids and are related to the amino acid side chain. Preferred amino acid groups are as follows: acidic groups (aspartic acid and glutamic acid); basic groups (lysine, arginine and histidine); nonpolar groups (alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine and tryptophan); and non-polar families (glycine, aspartic acid, glutamic acid, cysteine, serine, threonine and tyrosine). Preferred amino acid groups include: aliphatic hydroxyl groups (serine and threonine); acetylated groups (aspartic acid and glutamine); aliphatic groups (alanine, valine, leucine, and isoleucine); and aromatic groups (phenylalanine, tryptophan, and tyrosine). Such amino acid substitutions are preferably carried out within a range that does not impair the properties of the substance having the original amino acid sequence.

[0246] By combining a sequence having high homology with the above-described heavy chain amino acid sequence and a sequence having high homology with the above-described light chain amino acid sequence, it is possible to select antibodies with biological activities equivalent to each of the above-described antibodies. Such homology is generally 80% or higher, preferably 90% or higher, more preferably 95% or higher, and most preferably 99% or higher. Furthermore, by combining amino acid sequences in which one or more amino acid residues are substituted, deleted, or added in the heavy or light chain amino acid sequence, it is also possible to select antibodies with biological activities equivalent to each of the above-described antibodies. The term "homology" as used herein is used in the same sense as "identity".

[0247] Homology between two amino acid sequences can be determined using the default parameters of Blast algorithm version 2.2.2 (Altschul, Stephen F., Thomas L. Madden, Alejandro A. Schaeffer, Jinghui Zhang, Zheng Zhang, Webb Miller and David J. Lipman (1997), "Gapped BLAST and PSI-BLAST: a new generation of protein database search programs", Nucleic Acids Res. 25: 3389-3402). The Blast algorithm can also be used via the Internet by accessing the website www.ncbi.nlm.nih.gov / blast.

[0248] Furthermore, the antibodies of the present invention include human antibodies that bind to HER2. Anti-HER2 human antibody systems refer to human antibodies that possess only antibody sequences derived from human chromosomes. Anti-HER2 human antibodies can be obtained using mice that produce human antibodies and possess segments of human chromosomes containing the heavy and light chain genes of the human antibody (see Tomizuka, K. et al., Nature Genetics (1997) 16, pp. 133-143; Kuroiwa, Y. et al., Nucl. Acids Res. (1998) 26, pp. 3447-3448; Yoshida, H. et al., Animal Cell Technology: Basic and Applied Aspects, Vol. 10, pp. 69-73 (edited by Kitagawa, Y., Matuda, T. and Iijima, S.), Kluwer Academic Publishers, 1999; Tomizuka, K. et al., Proc. Natl. Acad. Sci. USA (2000) 97, pp. 722-727).

[0249] Such mice that produce human antibodies can be specifically produced as follows: Genetically modified animals in which the endogenous immunoglobulin heavy and light chain loci have been disrupted and, instead, introduced via a yeast artificial chromosome (YAC) vector or similar, the human immunoglobulin heavy and light chain loci, are produced by generating knockout animals and transgenic animals and pairing them with such animals.

[0250] In addition, according to recombinant DNA technology, eukaryotic cells are transformed and cultured to produce transformed somatic cells that produce recombinant human monoclonal antibodies by using cDNA encoding each of the heavy and light chains of such human antibodies, and preferably a vector containing such cDNA, thereby obtaining antibodies from the culture supernatant.

[0251] Here, as a host, eukaryotic cells, preferably mammalian cells, such as CHO cells, lymphocytes or myeloma cells, can be used.

[0252] In addition, a method for obtaining phage-presenting human antibodies selected from human antibody libraries is also known (see Wormstone, IM et al., Investigative Ophthalmology & Visual Science. (2002) 43 (7), pp. 2301-2308; Carmen, S. et al., Briefings in Functional Genomics and Proteomics (2002), 1 (2), pp. 189-203; Siriwardena, D. et al., Ophthalmology (2002) 109 (3), pp. 427-431, etc.).

[0253] For example, a phage presentation method can be used in which the variable region of a human antibody is presented as a single-chain antibody (scFv) on the surface of a phage and selectively binds to the antigen (Nature Biotechnology (2005), 23, (9), pp. 1105-1116).

[0254] By analyzing the genes of bacteriophages selected based on their binding to antigens, the DNA sequence encoding the variable region of human antibodies that bind to antigens can be determined.

[0255] If the DNA sequence of the scFv that binds to the antigen is determined, human antibodies can be obtained by preparing an expression vector containing the sequence and introducing the vector into an appropriate host to express the vector (WO 92 / 01047, WO 92 / 20791, WO 93 / 06213, WO 93 / 11236, WO 93 / 19172, WO 95 / 01438, WO 95 / 15388; Annu. Rev. Immunol. (1994) 12, pp. 433-455, Nature Biotechnology (2005) 23 (9), pp. 1105-1116).

[0256] As another example of an index for comparing antibody properties, antibody stability can be exemplified. Differential scanning calorimetry (DSC) is a device that can rapidly and accurately measure the midpoint temperature (Tm) of thermal denaturation, a favorable index for the relative conformational stability of proteins. By measuring Tm values ​​using DSC and comparing these values, differences in thermal stability can be compared. It is known that the storage stability of antibodies exhibits some correlation with their thermal stability (Lori Burton et al., Pharmaceutical Development and Technology (2007) 12, pp. 265-273), and thermal stability can be used as an index to select better antibodies. Examples of other indices for selecting antibodies include the following characteristics: higher yield in suitable host cells; and lower aggregation in aqueous solutions. For example, antibodies exhibiting the highest yield do not always exhibit the highest thermal stability, and therefore it is necessary to select the most suitable antibody for human administration by conducting a comprehensive evaluation based on the aforementioned indices.

[0257] In this invention, modified variants of antibodies are also included. A modified variant system refers to a variant obtained by subjecting the antibody of this invention to chemical or biological modification. Examples of chemically modified variants include variants chemically modified by linking the chemical portion to the amino acid backbone, and variants chemically modified by N-linking or O-linking the carbohydrate chain. Examples of biologically modified variants include variants obtained by post-translational modifications (such as N-linking or O-linking glycosylation, N- or C-terminal treatment, deacetylation, isomerization of aspartic acid, or oxidation of methionine), and variants in which methionine residues have been added to the N-terminus through expression in prokaryotic host cells. Furthermore, antibodies labeled to enable the detection or separation of the antibody or antigen of this invention (e.g., enzyme-labeled antibodies, fluorescently labeled antibodies, and affinity-labeled antibodies) are also included in the meaning of modified variants. The modified variants of the antibodies of this invention are suitable for improving antibody stability and blood retention, reducing their antigenicity, detecting or separating antibodies or antigens, etc.

[0258] Furthermore, by regulating the modification (glycosylation, defucosylation, etc.) of the glycans linked to the antibodies of the present invention, it is possible to enhance antibody-dependent cytotoxic activity. Techniques for regulating the modification of antibody glycans include WO 99 / 54342, WO 00 / 61739, and WO 02 / 31140. However, the techniques are not limited thereto. The antibodies of the present invention also include antibodies in which the glycan modification has been regulated.

[0259] In cases where antibodies are produced by first isolating an antibody gene and then introducing the gene into a suitable host, a combination of a suitable host and a suitable expression vector may be used. Specific examples of antibody genes include a combination of a gene encoding the heavy chain sequence of the antibody described in this specification and a gene encoding its light chain sequence. When host cells are transformed, it is possible for the heavy chain sequence gene and the light chain sequence gene to be inserted into the same expression vector, or they may be inserted into different expression vectors.

[0260] When eukaryotic cells are used as the host, animal cells, plant cells, and eukaryotic microorganisms may be used. Examples of animal cells include mammalian cells, such as monkey COS cells (Gluzman, Y, Cell, (1981) 23, pp. 175-182, ATCC CRL-1650), mouse fibroblast NIH3T3 (ATCC number CRL-1658), and a dihydrofolate reductase-deficient strain of Chinese hamster ovary cells (CHO cells; ATCC: CCL-61) (Urlaub, G. and Chasin, LA, Proc. Natl. Acad. Sci. USA (1980) 77, pp. 4126-4220). When prokaryotic cells are used, examples include Escherichia coli and Bacillus subtilis.

[0261] Antibodies can be obtained by introducing the desired antibody gene into these cells via transformation and culturing the transformed cells in vitro. In the above-described culture method, the yield can sometimes vary depending on the antibody sequence, and therefore, it is possible to select antibodies that are easy to produce as drugs from among antibodies with equivalent binding activity by using the yield as an indicator. Therefore, the antibodies of the present invention also include antibodies obtained by methods of antibody production, characterized by including the steps of culturing transformed host cells and collecting the desired antibody from the culture products obtained in the self-culture step.

[0262] It is known that the lysine residue at the C-terminus of the heavy chain of antibodies produced in cultured mammalian cells is deleted (Journal of Chromatography A, 705: 129-134 (1995)), and it is also known that two amino acid residues (glycine and lysine) at the C-terminus of the heavy chain of antibodies produced in mammalian cells are deleted and the proline residue newly located at the C-terminus is amided (Analytical Biochemistry, 360: 75-83 (2007)). However, such deletions and modifications of the heavy chain sequence do not affect the antigen-binding affinity and effector function of the antibody (complement activation, antibody-dependent cytotoxicity, etc.). Therefore, the antibodies according to the present invention also include antibodies and functional fragments of antibodies subjected to such modifications, as well as deletion variants in which one or two amino acids are missing at the carboxyl terminus of the heavy chain, variants obtained by acetamipation of the deletion variants (e.g., heavy chains in which the proline residue at the carboxyl terminus has been acetamipated), and the like. The types of deletion variants with a deletion at the carboxyl terminus of the heavy chain of the antibody according to the present invention are not limited to the above variants, as long as the antigen-binding affinity and effector function remain unchanged. The two heavy chains constituting the antibody of the present invention may belong to one type selected from the group consisting of the full-length heavy chain and the above-mentioned deletion variants, or may belong to a combination of two types selected therefrom. The ratio of the amounts of each deletion variant may be affected by the type and culture conditions of the cultured mammalian cells that produce the antibody according to the present invention; however, examples can be given of cases where one amino acid residue at the carboxyl terminus is missing from one of the two heavy chains contained as the main component of the antibody of the present invention.

[0263] For example, IgG (IgG1, IgG2, IgG3, IgG4) can be exemplified as the isotype of the antibody of the present invention, and IgG1 or IgG2 can be exemplified more preferably.

[0264] The biological activity of an antibody is generally antigen-binding activity, which can be exemplified by the activity of internalization in cells expressing the antigen by binding to the antigen, the activity of neutralizing antigen activity, the activity of enhancing antigen activity, antibody-dependent cytotoxicity (ADCC) activity, complement-dependent cytotoxicity (CDC) activity, and antibody-dependent cell-mediated phagocytosis (ADCP). The biological activity of the antibody of the present invention is HER2 binding activity, and preferably the activity of internalization in cells expressing HER2 by binding to HER2. In addition to the activity of internalization in cells, the antibody of the present invention may also have ADCC activity, CDC activity, and / or ADCP activity.

[0265] The obtained antibody can be purified to homogenization. The separation and purification of antibodies can be performed using conventional protein separation and purification methods. For example, antibodies can be separated and purified by appropriate selection and combination of column chromatography, filter filtration, ultrafiltration, salt precipitation, dialysis, preparative polyacrylamide gel electrophoresis, isoelectric focusing electrophoresis and the like (Strategies for Protein Purification and Characterization: A Laboratory Course Manual, Daniel R. Marshak et al., Cold Spring Harbor Laboratory Press (1996); Antibodies: A Laboratory Manual. Ed Harlow and David Lane, Cold Spring Harbor Laboratory (1988)), but the methods are not limited to these.

[0266] Examples of this type of chromatography include affinity chromatography, ion exchange chromatography, hydrophobic chromatography, gel filtration chromatography, reverse phase chromatography, and adsorption chromatography. This type of chromatography can be performed using liquid chromatography methods such as HPLC or FPLC.

[0267] Examples of columns used for affinity chromatography include protein A columns and protein G columns. For example, examples of columns using protein A columns include Hyper D, POROS, Sepharose FF (Pharmacia Corporation), and their analogues.

[0268] Furthermore, antibodies can be purified by utilizing the antigen-binding properties of antibodies by using a carrier with antigens immobilized thereon. Antibody-drug conjugates containing anti-HER2 antibodies are an example.

[0269] In some embodiments, the present invention provides a conjugate comprising a cell binding agent (e.g., an anti-HER2 antibody as described herein) and a camptothecin derivative.

[0270] In the embodiments, compounds suitable for binding to anti-HER2 antibodies (e.g., trastuzumab) include those described in U.S. Provisional Application No. 62 / 981,197, filed February 25, 2020, which is incorporated herein by reference in its entirety. For example, suitable compounds include those of formulas (I), (II), and (III) as described in USSN 62 / 981,197.

[0271] In another embodiment, the present invention provides a compound of formula (PL-A'), {D—L1—L2—Q'—CH2—NH—E—Z'}p—C(PL-A'), or a pharmaceutically acceptable salt thereof, wherein: D is represented by the following structural formula: wherein R1 is independently -H, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, silyl, C3-C6 cycloalkyl, C1-C6 haloalkyl, C2-C6 haloalkenyl or C2-C6 haloalkynyl; R2 is independently -H, -F, -N(R4)2, -N(R4)(R5), -OR4, -SR4, -S(=O)R5, -SO2R5, C1-C6 alkyl or C1-C6 fluoroalkyl; and R3 is -H, -F, -CN, -OCH3, -CH3 or -CF3; or R2 and R3 together form a group of the formula -O(CH2)nO- or -O(CF2)nO-, where n is 1 or 2; R4 is independently -H or C1-C4 alkyl; R5 is independently C1-C4 alkyl; L1 is independently absent or is -(C1-C10 alkyl)-; L2 is independently absent or is -OCH2-L3-*, -SCH2-L3-*, -S(=O)-L3-*, -SO2-L3-*, -C(=O)-L3-*, -N(R6)CH2-L3-*, -N(R6)C(=O)-L3-*, -N(R6)C(=O)N(R7)-L3-*, -C(=O)N(R6)CH2-L3-*, -OC(=O)N(R6)CH2-L3-* or -N(R6)C(=O)OCH2-L3-*; where * indicates a site covalently linked to Q'; L3 is independently -(C1-C10 alkyl)-, -CH2OCH2CH2- or -CH2CH2OCH2CH2-; Each R6 and R7 is independently -H, C1-C6 alkyl, C1-C6 fluoroalkyl, C3-C6 cycloalkyl, aryl, heteroaryl, or benzyl; Q' is -O- or -S-; E is a peptide containing 2 to 10 amino acids, wherein E is substituted with one or more polyols as appropriate; and wherein the N-terminus of the peptide is covalently linked to Z'; Z' is -C(=O)-L4-Y', or; wherein m represents an integer from 1 to 10 and * indicates the site of covalent link to the C; L4 is -(C1-C10 alkyl)-, -CH2CH2(OCH2CH2)nN(R8)C(=O)-L5-*, or -CH2(OCH2CH2)nN(R8)C(=O)-L5-*; wherein n represents an integer from 1 to 10; and wherein * indicates the site of covalent link to Y'; L5 is -(C1-C10 alkyl)-; R8 represents -H or -CH3; C indicates a cell binder; Y' is a group formed by the reaction of an electrophilic group with a reactive nucleophilic group present on the cell binder;Furthermore, when R2 and R3 combine to form -OCH2O-, R1 is not -CH2CH2CH2CH3; and p has a value between 1 and 18.

[0272] Exemplary (PL-A') compounds include (PL-A) compounds, such as MB-2a and MB-3a (trastuzumab meditec) as described herein.

[0273] In the embodiments, L4 is -(C1-C10 alkyl)-.

[0274] In the embodiment, L4 is -CH2CH2(OCH2CH2)nN(R8)C(=O)-L5-* or -CH2(OCH2CH2)nN(R8)C(=O)-L5-*, where n represents an integer from 1 to 10; and * indicates a site covalently connected to Y'.

[0275] In the embodiments, L4 is -CH2CH2CH2CH2CH2-, -CH2CH2-, -CH2-, -CH2CH2OCH2CH2OCH2CH2NHC(=O)CH2CH2-* or -CH2OCH2CH2OCH2CH2NHC(=O)CH2CH2-*, where * indicates a site covalently linked to Y'.

[0276] In the embodiments, Y' is formed from a Michael acceptor group, succinimide, epoxide or halogen.

[0277] In the embodiments, Y' is formed from the following: R10, R11, R12, R13; wherein R10 and R11 are each independently -H or C1-C3 alkyl.

[0278] In the embodiments, Y' is , , , ; wherein R10 and R11 are each independently -H or C1-C3 alkyl, and * indicates the site covalently connected to the C.

[0279] In the embodiments, Z' is formed by the following: , , , or.

[0280] In the embodiment, Z' is: , , , , or; where * indicates a site covalently connected to C.

[0281] In an embodiment, Z' is, where * indicates a site covalently connected to the C. In an embodiment, m is 1. In an embodiment, m is 2. In an embodiment, m is 3. In an embodiment, m is 4. In an embodiment, m is 5. In an embodiment, m is 6. In an embodiment, m is 7. In an embodiment, m is 8. In an embodiment, m is 9. In an embodiment, m is 10.

[0282] In an embodiment, Z' is, where * indicates a site covalently connected to the C. In an embodiment, m is 1. In an embodiment, m is 2. In an embodiment, m is 3. In an embodiment, m is 4. In an embodiment, m is 5. In an embodiment, m is 6. In an embodiment, m is 7. In an embodiment, m is 8. In an embodiment, m is 9. In an embodiment, m is 10.

[0283] In the embodiments, E is a peptide of 2, 3, or 4 amino acids. Each amino acid in the peptide is an L-amino acid, or at least one amino acid in the peptide is a D-amino acid.

[0284] In the embodiments, E comprises one or more amino acids selected from glycine, alanine, valine, glutamic acid, glutamic acid, phenylalanine and leucine, wherein the glutamic acid or glutamic acid is substituted with a polyol as appropriate.

[0285] In the embodiments, E comprises an amino acid selected from glycine, alanine, valine, glutamic acid, glutamic acid, phenylalanine and leucine, wherein the glutamic acid or glutamic acid is substituted with a polyol as appropriate.

[0286] In the embodiments, E comprises an amino acid having the following structure: wherein R9 is -H or C1-C6 alkyl.

[0287] In the embodiments, E comprises an amino acid having the following structure:

[0288] In an embodiment, E is selected from the group consisting of: -Ala-Val-*, -Val-Ala-*, -Gly-Gly-*, -Val-Cit-*, -Cit-Val-*, -Leu-Ala-*, -Ala-Leu-*, -Leu-Cit-*, -Cit-Leu-*, -Leu-Ala-*, -Ala-Leu-*, -Lys-Lys-*, -Ala-Lys-*, -Lys-Ala-*, -Val-Lys-*, -Lys-Val-*, -Tyr-Arg-*, -Arg-Tyr-*, -Arg-Arg-*, -Ala-Ala-*, -Phe-Lys-*, -Lys-Phe-*, -Thr-Thr-*, -Thr-Met-*, -Met-Thr-*, -Met-Tyr-*, -Tyr-Met-*, -Phe-Gln-*, -Gln-Phe-*, -Gly-Ser-*, -Leu-Gln-*, -Gln-Leu-*, -Ser-Ala-*, -Ser-Gly-*, -Val-Thr-*, -Thr-Val-*, -Val-Gln-*, -Ser-Val-*, -Val-Ser-*, -Ala-Met-*, -Met-Ala-*, -Val-Arg-*, -Arg-Val-*, -Phe-Ala-*, -Ala-Phe-*, -Cit-Val-*, -Gln-Val-*, -Phe-Arg-*, -Arg-Phe-*, -Ala-Ala-Ala-*, -Gly-Gly-Gly-*, -Ala-Val-Ala-*, -Gly-Val-Gly-*, -Ala-Val-Gly-*, -Gly-Phe-Lys-*, -Lys-Phe-Gly-*, -Leu-Ala-Leu-*, -Val-Ala-Leu-*, -Leu-Ala-Val-*, -Val-Ala-Val-*, -Ala-Val-Ala-Gly-* (SEQ ID NO: 10), -Gly-Phe-Gly-Gly-* (SEQ ID NO: 11), -Gly-Gly-Phe-Gly-* (SEQ ID NO: 12), -Ala-Val-Gly-Gly-* (SEQ ID NO: 13), -Ala-Ala-Ala-Ala-* (SEQ ID NO: 14), -Ala-Val-Ala-Ala-* (SEQ ID NO: 15), -Ala-Leu-Ala-Leu-* (SEQ ID NO: 16),-Leu-Ala-Leu-Ala-* (SEQ ID NO: 17), -Gly-Phe-Leu-Gly-* (SEQ ID NO: 18), and -Gly-Leu-Phe-Gly-* (SEQ ID NO: 19), where * indicates covalently linked to the N-terminus of the Z' peptide.

[0289] In the embodiment, E is selected from the group consisting of: -L-Ala-D-Val-*, -L-Val-D-Ala-*, -L-Val-D-Lys-*, -L-Val-D-Arg-*, -L-Val-D-Cit-*, -L-Val-D-Arg-*, -L-Val-D-Cit-*, -L-Val-D-Lys-*, -L-Val-D-Arg-*, -L-Arg-D-Arg-*, -L-Ala-D-Ala-*, -L-Ala-D-Lys-*, -L-Ala-D-Arg-*, -L-Ala-D-Ala-L-Ala-*, -L-Ala-D-Val-L-Ala-*, -L-Ala-D-Ala-Gly-* and -L-Ala-D-Val-Gly-*, where * indicates covalently linked to the N-terminus of the Z' peptide.

[0290] In the embodiments, -E-NH-CH2- has one of the following structures, wherein * indicates covalently linked to the N-terminus of the peptide of Z': , , , or.

[0291] In the embodiments, Z'-E-NH-CH2 is formed by one of the following structures: , , , or.

[0292] In the embodiment, Z'-E-NH-CH2 is one of the following structures, where * indicates the connection point with C: , , , or.

[0293] In the embodiments, when R1 is -H or -CH2CH3, R2 is -OH or alkoxy, and R3 is -H, then -L1-L2-Q'- is not -CH(R')CH2O- or -CH(R')(CH2)2O-, wherein R' is -H or C1-C6 alkyl, alkoxy, substituted alkyl, phenyl, or PhCH2-. In the embodiments, when R1 is -H or -CH2CH3, R2 is -OH or alkoxy, and R3 is -H, then -L1-L2-Q'- is not -CH(R')CH2O- or -CH(R')(CH2)2O-, wherein R' is -H or C1-C6 alkyl, alkoxy, substituted alkyl, phenyl, or PhCH2-.

[0294] In the embodiment, at least one of L1 and L2 is present.

[0295] In the embodiment, at least one of R1, R2 and R3 is not -H.

[0296] In the examples, R1 is independently C1-C6 alkyl, C2-C6 alkenyl, C2-C6 ynyl, silyl, C3-C6 cycloalkyl, C1-C6 halogenated alkyl, olefin or ynyl.

[0297] In the embodiments, R1 is independently -H or C1-C6 alkyl.

[0298] In the embodiments, R2 is independently -H, -F, -N(R4)2, -N(R4)(R5), -OR4, -SR4, -S(=O)R5, -SO2R5, C1-C6 alkyl or C1-C6 fluoroalkyl; and R3 is independently -H, -F, -CN, -OCH3, -CH3 or -CF3.

[0299] In the examples, R2 is independently a C1-C6 alkyl, C1-C6 fluoroalkyl or -F.

[0300] In the embodiments, R3 is independently -H, -F, -CN or -CF3.

[0301] In the embodiments, R3 is independently -F, -CN, -OCH3, -CH3 or -CF3.

[0302] In the embodiment, R2 and R3 are combined to form -O(CH2)nO- or -O(CF2)nO-, where n is 1 or 2.

[0303] In the embodiments, D is represented by one of the following structures: (DI); (D-II); (D-III); (D-IV); (DV); (D-VI); (D-VII); or (D-VIII).

[0304] In the embodiment, D is... (D-II).

[0305] In the examples, R1 is -H or C1-C6 alkyl.

[0306] In the embodiments, D is represented by one of the following structures: (D1); (D2); (D3); (D4); (D5); (D6); (D7); (D8); (D9); (D10); (D11); (D12); (D13); (D14); (D15); or (D16)

[0307] In the embodiment, D is... (D3)

[0308] In the embodiments, L1 is -(C1-C10 alkyl)- and L2 is not present.

[0309] In the embodiments, L1 is -(C1-C10 alkyl)- and L2 is -N(R6)CH2-L3-* or -N(R6)C(=O)-L3-*, where * indicates a site covalently linked to Q'.

[0310] In the embodiment, L1 is absent and L2 is -N(R6)CH2-L3-* or -N(R6)C(=O)-L3-*, where * indicates a site covalently linked to Q'.

[0311] In the embodiments, L3 is -(C1-C10 alkyl)-.

[0312] In the embodiments, R6 is -H or -CH3.

[0313] In the embodiments, L1-L2 are -CH2-, -CH2CH2-, -CH2CH2CH2- or -CH2CH2CH2CH2-.

[0314] In the embodiments, L1-L2 are -OCH2CH2-*, -OCH2CH2OCH2CH2-*, -SCH2CH2-*, -SCH2CH2OCH2CH2-*, -S(=O)CH2-*, -SO2CH2-*, -C(=O)CH2-*, -NHCH2CH2-*, -N(CH3)CH2CH2-*, -N(CF3)CH2CH2-*, -NHC(=O)CH2-*, -CH2NHC(=O)CH2-*, -CH2CH2NHC(=O)CH2-*, -CH2N(CH3)C(=O)CH2-* -N(CH3)C(=O)CH2-*, -N(CH3)C(=O)CH2CH2-*, -C(=O)NHCH2CH2-*, -NHC(=O)NHCH2CH2-*, -NHC(=O)OCH2CH2-*, -CH2OC(=O)NHCH2CH2-* or -C(=O)N(CH3)CH2CH2-*, where * indicates a site covalently linked to Q'.

[0315] In embodiments, L1-L2-Q' is -CH2CH2CH2CH2O-, -CH2CH2CH2O-, -CH2CH2O-, -CH2CH2OCH2CH2O-, -CH2SCH2CH2O-, -CH2NHC(=O)CH2O-, -CH2CH2NHC(=O)CH2O-, - CH2N(CH3)C(=O)CH2O-, -OCH2CH2O-, -OCH2CH2CH2O-, -SCH2CH2CH2O-, -SCH2CH2O-, -NHCH2CH2O-, -NHCH2CH2CH2O-, -N(CH3)CH2CH2O-, -C(=O)NHCH2CH2O- , -NHC(=O)CH2O-, -CH2S(=O)CH2O-, -CH2SO2CH2O-, -CH2CH2CH2CH2S-, -CH2CH2CH2S-, -CH2CH2S-, -CH2CH2OCH2CH2S-, -CH2SCH2CH2S-, -CH2NHC(=O)CH2S- , -OCH2CH2CH2S-, -SCH2CH2CH2S-, -SCH2CH2S-, -NHCH2CH2CH2S-, -N(CH3)CH2CH2S-, -C(=O)NHCH2CH2S-, -NHC(=O)CH2S-, -CH2S(=O)CH2S- or -CH2SO2CH2S-.

[0316] In the embodiments, D-L1-L2 is represented by the following structure: (PI); (P-II); (P-III); (P-IV); or (PV).

[0317] In the embodiments, D-L1-L2 is represented by the following structure: (P-II).

[0318] In the examples, R1 is -H or C1-C6 alkyl.

[0319] In the embodiments, R1 is -H or -CH2CH3.

[0320] In the embodiment, Q' is -O-.

[0321] In the embodiment, Q' is -S-.

[0322] In the embodiment, D—L1—L2—Q'—has one of the following structures: (P1'); (P2'); (P3'); (P4'); (P5'); or (P6')

[0323] In the embodiment, D—L1—L2—Q'— is: (P1').

[0324] In the embodiment, D—L1—L2—Q'—CH2—NH—E—Z'—is formed by one of the following structures: (PL1), (PL2), (PL3), (PL4), (PL5), (PL6), (PL7), (PL8), (PL9), (PL10), (PL11), (PL12), (PL13), or (PL14).

[0325] In the embodiment, D—L1—L2—Q'—CH2—NH—E—Z'— is formed from MB-2, (PL1).

[0326] In the embodiment, D—L1—L2—Q'—CH2—NH—E—Z'— is formed from MB-3 (medotecan), (PL3).

[0327] In the embodiments, {D—L1—L2—Q'—CH2—NH—E—Z'}p—C is one of the following structures, wherein C is a monoclonal antibody and p is the drug-to-antibody ratio (DAR), and p is an average number in the range of about 2-10, 4-8 or 7-8 (e.g., 3.2 to 8.0), (PL1'), (PL2'), (PL3'), (PL4'), (PL5'), (PL6'), (PL7'), (PL8'), (PL9'), (PL10'), (PL11'), (PL12'), (PL13'), or (PL14').

[0328] In the embodiments, {D—L1—L2—Q'—CH2—NH—E—Z'}p—C is (PL1'), where C is a monoclonal antibody and p is the drug-to-antibody ratio (DAR). In the embodiments, p is an average number in the range of about 2-10, 4-8, or 7-8 (e.g., 3.2 to 8.0).

[0329] In the embodiments, {D—L1—L2—Q'—CH2—NH—E—Z'}p—C is (PL3'), where C is a monoclonal antibody and p is the drug-to-antibody ratio (DAR). In the embodiments, p is an average number in the range of about 2-10, 4-8, or 7-8 (e.g., 3.2 to 8.0).

[0330] In the examples, C is an anti-HER2 antibody.

[0331] In the examples, C is trastuzumab (Herceptin).

[0332] In the examples, C is trastuzumab-dkst (Ogivri).

[0333] In the examples, C is trastuzumab-pkrb (Herzuma).

[0334] In the examples, C is trastuzumab-dttb (Ontruzant).

[0335] In the examples, C is trastuzumab-qyyp (Trazimera).

[0336] In the examples, C is trastuzumab-anns (Kanjinti).

[0337] In the embodiments, {D—L1—L2—Q'—CH2—NH—E—Z'}p—C is MB-2a, where p is the drug-to-antibody ratio (DAR). In the embodiments, p is an average number in the range of about 4-8 or 7-8 (e.g., 7.9).

[0338] In the embodiments, {D—L1—L2—Q'—CH2—NH—E—Z'}p—C is MB-3a (trastuzumab meditec), where p is the drug-to-antibody ratio (DAR). In the embodiments, p is an average in the range of about 4-8 or 7-8, or p is 8.

[0339] The conjugates described herein (e.g., any compound according to formula PL-A' or formula PL-A, such as MB-2a and MB-3a) may contain at least a camptothecin derivative covalently linked.

[0340] In the embodiments, the subscript p indicates the number of camptothecin effective loading portions on the cell binder and has a value of 1 to 18, 1 to 12, or 1 to 8. Individual camptothecin conjugates may also be referred to as camptothecin conjugate compounds. In the embodiments herein, there may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 camptothecin effective loading portions bound to individual camptothecin conjugates.

[0341] In the embodiments, except for the number of camptothecin effective loading portions bound to each cell binder (i.e., the camptothecin conjugate composition), the group of individual camptothecin conjugates is substantially identical, such that p represents the average number of camptothecin effective loading portions bound to the cell binder of the camptothecin conjugate composition. In this group of embodiments, p is an average number ranging from 1 to about 18, 1 to about 10, or 1 to about 8, 2 to about 6, 3 to about 5, or 6 to about 8. In the embodiments, p is an average number ranging from about 2 to 10, 4 to 8, or 7 to 8 (e.g., 3.2 to 8.0). In the embodiments, p is about 2. In the embodiments, p is about 4. In the embodiments, p is about 6. In the embodiments, p is about 8. In the embodiments, p is about 10. In the embodiments, p is about 12. In the embodiments, p is 2. In the embodiments, p is 4. In the embodiments, p is 8. In the embodiments, the value of p is 3 to 4. In the embodiments, the value of p is 4 to 5. In the embodiments, the value of p is 5 to 6. In the examples, the value of p is 6 to 7. In the examples, the value of p is 7 to 8. In the examples, the value of p is 7.4 to 8. In the examples, the p value refers to the average drug load in the composition and the drug load of the main ADC.

[0342] In the embodiments, the binding (e.g., as found in any compound according to formula PL-A' or formula PL-A, such as MB-2a or MB-3a as described herein) will be via a reduced interchain disulfide bond, and there may be about 1 to about 8, or 3 to 5, or 6 to 8 camptothecin-loaded compounds that bind to the cell binder.

[0343] In the embodiments, camptothecin-loaded compounds (e.g., as found in any compound according to formula PL-A' or formula PL-A, such as MB-2a or MB-3a as described herein) are incorporated via introduced cysteine ​​residues and reduced interchain disulfide bonds, and may be present in 1 to 8, or 1 to 10, or 1 to 12, or 1 to 18, of the cell binder.

[0344] In the embodiments, the binding (e.g., as found in any compound according to formula PL-A' or formula PL-A, such as MB-2a or MB-3a as described herein) will be via the introduced cysteine ​​residues, and there will be 2, or 3, or 4, or 5, or 6, or 7, or 8 camptothecin-loaded compounds that bind to the cell binder.

[0345] In embodiments, the camptothecin-loaded compound (e.g., as found in any compound according to formula PL-A' or formula PL-A, such as MB-2a or MB-3a as described herein) will be present via lysine residues, and may be 1 to 10, or 1 to 12, or 1 to 14, or 1 to 18, of the reactive groups on the cell binder for covalent linkage.

[0346] In embodiments, the cell binder binds to the peptide to release a linker to form a conjugate, such as according to formula PL-A' or its derivative (e.g., MB-2a or MB-3a). As noted above, other linking components may be present in the conjugate described herein to provide additional space between the camptothecin compound and the cell binder. In embodiments, the cell binder is linked to the linker unit via heteroatom bonds of the cell binder.

[0347] Heteratoms that may be present on the cell binder used for the bond include sulfur (in one embodiment, from a thiol group of the targeting ligand), oxygen (in one embodiment, from a carboxyl or hydroxyl group of the targeting ligand), and, where appropriate, substituted nitrogen (in one embodiment, from a primary or secondary amine functional group of the targeting ligand, or in another embodiment, from a substituted amide nitrogen, where appropriate). These heteroatoms may be present on the targeting ligand in their native state as a cell binder, for example, in naturally occurring antibodies, or may be introduced into the targeting ligand via chemical modification or bioengineering.

[0348] In one embodiment, the cell binder has a thiol functional group, such that the cell binder binds to the camptothecin-loaded compound via the sulfur atom of the thiol functional group.

[0349] In another embodiment, the cell binder has one or more lysine residues capable of reacting with activated esters of camptothecin-loaded compounds (such esters include, but are not limited to, N-hydroxybutyrylimide, pentafluorophenyl and p-nitrophenyl esters), and thus provides acetamide bonds consisting of the nitrogen atom of the cell binder and the C=O group of the compound.

[0350] In another embodiment, the cell binder has one or more lysine residues that can be chemically modified to introduce one or more thiol groups. In those embodiments, the cell binder is covalently linked to the camptothecin-supported compound via the sulfur atom of the thiol functional group. Reagents that can be used to modify lysine in this manner include, but are not limited to, S-acetylosylacetic acid N-butadiene imide (SATA) and 2-iminothiocyclopentane hydrochloride (Traut's Reagent).

[0351] In another embodiment, the cell binder has one or more carbohydrate groups that can be modified to provide one or more thiol functional groups. The chemically modified cell binder in the camptothecin conjugate is bound to the camptothecin effective loading compound via the sulfur atom of the thiol functional group.

[0352] In another embodiment, the cell binder has one or more carbohydrate groups that can be oxidized to provide an aldehyde (-CHO) functional group (see, for example, Laguzza et al., 1989, J. Med. Chem. 32(3):548-55). In these embodiments, the corresponding aldehyde interacts with reactive sites on the camptothecin-supported compound to form a bond between the camptothecin-supported compound and the cell binder. Reactive sites on the camptothecin-supported compound that can interact with reactive carbonyl functional groups on the target ligand include, but are not limited to, hydrazine and hydroxylamine.

[0353] In some embodiments, the cell binder can form a covalent bond between Y' in formula PL-A' and the cell binder corresponding to the target ligand by interacting with a reactive functional group Y. The functional group Y capable of interacting with the target ligand will depend on the nature of the cell binder. In an embodiment, the reactive group is a maleic anhydride present on the camptothecin support compound prior to its attachment to form the cell binder. The covalent attachment of the cell binder to the camptothecin support compound is achieved by interacting with the maleic anhydride functional group Y of the support compound to form a thiol functional group of the thiosubstituted succinyl anhydride cell binder. The thiol functional group may exist on the cell binder in its native state, for example, in naturally occurring residues, or may be introduced into the cell binder through chemical modification or bioengineering.

[0354] In another embodiment, the cell binder is an antibody and the thiol group is generated by reducing the interchain disulfide bond of the antibody. Therefore, in the embodiment, the camptothecin-loaded compound binds to cysteine ​​residues from the reduced interchain disulfide bond.

[0355] In another embodiment, the cell binder is an antibody, and the thiol functional group is chemically introduced into the antibody, for example, by introducing cysteine ​​residues. Therefore, in this embodiment, the camptothecin-loaded compound binds to the cell binder via the cysteine ​​residues introduced by the cell binder.

[0356] For bioconjugates, drug binding sites have been observed to affect multiple parameters, including binding ease, drug-linker stability, effects on the biophysical properties of the resulting bioconjugate, and in vitro cytotoxicity. Regarding drug-linker stability, the binding site of the drug-linker moiety to the cell binder can affect the ability of the bound drug-linker moiety to undergo elimination reactions in some cases, leading to premature release of the free drug. Binding sites on the targeting ligand include, for example, reduced inter-chain disulfide bonds and selected cysteine ​​residues at engineered sites. In examples, the binding method for forming camptothecin conjugates as described herein uses thiol residues at genetically engineered sites (e.g., position 239 according to the EU index as shown in Kabat) that are less susceptible to elimination reactions compared to binding methods using thiol residues from reduced disulfide bonds. In other examples, the binding method for forming camptothecin conjugates as described herein uses thiol residues at sites more susceptible to elimination reactions (e.g., generated by reduction of inter-chain disulfide bonds). Synthetic Methods

[0357] Exemplary synthesis methods are described herein. Other exemplary synthesis methods include those described in U.S. Provisional Application No. 62 / 981,197 and methods known in the art, which are incorporated herein by reference in their entirety.

[0358] In the embodiments, process 1 provides an exemplary method for the synthesis of the described compound MB-1 (herotecan) (P1). Process 1

[0359] In the embodiment, process 2 provides an exemplary synthesis method for preparing the described connectable payloads (PL1), (PL2), (PL4), and (PL7): Process 2

[0360] In the embodiments, process 3 provides an exemplary synthetic method for the described compound MB-2 (PL1): Process 3

[0361] In the embodiments, process 4 provides an exemplary method for the synthesis of the described compound MB-3 (medotecan) (PL3): Process 4

[0362] In the embodiments, process 5 provides an exemplary general method for preparing the conjugate (PL'): Process 5

[0363] In the examples, an exemplary general experimental procedure for preparing conjugates (PL') with a drug-to-antibody ratio (DAR) of 7-8 or 8 is as follows: Antibody C is treated at 25°C with 50 mM pH 7.4 phosphate buffer containing 8 equivalents (2 equivalents / disulfide bond) of tris(2-carboxyethyl)phosphonic acid hydrochloride (TCEP) and 10 mM DTPA (diethylenetriaminepentaacetic acid) for 2 hours, followed by the addition of DMSO containing 12 equivalents of effective load (PL) (the volume of DMSO is approximately 12-15% of the volume of phosphate buffer). The resulting reaction solution is vortexed at 25°C on a tube vortex mixer for 1 hour. The reaction mixture is immediately purified for several cycles using an ultrafiltration tube (30 KD) with formulation buffer. The resulting conjugate (PL') typically has a drug-to-antibody ratio (DAR) of 7-8 or 8 and is >95% monomeric as measured by size exclusion chromatography.

[0364] In the embodiments, the antibody-drug conjugate is MB-2a. In the embodiments, process 6 provides an exemplary method for the synthesis of the described antibody-drug conjugate MB-2a: Process 6

[0365] In the examples, the antibody-drug conjugate is MB-3a (trastuzumab-medotecan). In the examples, process 7 provides an exemplary method for the synthesis of the described antibody-drug conjugate MB-3a (trastuzumab-medotecan): Process 7 Treatment Method

[0366] In another embodiment, the present invention provides a method for treating a proliferative disease or condition or inhibiting abnormal cell growth, the method comprising administering a compound of any formula (PL-A) as described herein, such as MB-2a or MB-3a (trastuzumab-medotecan) or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising a compound of any formula (PL-A) as described herein (e.g., MB-2a or MB-3a (trastuzumab-medotecan)) or a pharmaceutically acceptable salt thereof.

[0367] In an embodiment, the method is used to treat cancer. In an embodiment, the cancer is characterized by HER2 overexpression and / or metastatic and / or trastuzumab resistance.

[0368] In the embodiments, the cancer is adenocarcinoma, brain cancer, bladder cancer, breast cancer, cervical cancer, choriocarcinoma, CNS tumor, colorectal or rectal cancer, diffuse endogenous pontine glioma (DIPG), endometrial cancer, esophageal cancer, Ewing's sarcoma, fallopian tube cancer, gallbladder cancer, gastric cancer, glioblastoma, head and neck cancer, hematologic cancer, Hodgkin's lymphoma, kidney cancer, laryngeal cancer, leukemia, liver cancer, lung cancer, lymphoma, melanoma, Merkel cell carcinoma, mesothelioma, multiple myeloma, myelodysplastic syndrome (MDS), neuroblastoma, non-Hodgkin's lymphoma, osteosarcoma, pancreatic cancer, peritoneal cancer, prostate cancer, ovarian cancer, kidney cancer, rhabdomyosarcoma, salivary gland cancer, sarcoma, skin cancer, small intestine cancer, squamous cell carcinoma, testicular cancer, thyroid cancer, uterine cancer, or Wilms' tumor.

[0369] In the embodiments, the cancer is lung cancer, urothelial carcinoma, colorectal cancer, prostate cancer, ovarian cancer, pancreatic cancer, breast cancer, bladder cancer, gastric cancer, gastrointestinal stromal tumor, cervical cancer, esophageal cancer, squamous cell carcinoma, peritoneal cancer, liver cancer, hepatocellular carcinoma, colorectal cancer, rectal cancer, endometrial cancer, uterine cancer, salivary gland cancer, kidney cancer, vulvar cancer, thyroid cancer, penile cancer, leukemia, malignant lymphoma, plasmacytoma, myeloma, or sarcoma.

[0370] In the embodiments, the cancer is breast cancer, gastric cancer, lung cancer (e.g., non-small cell lung cancer) and ovarian cancer, and has been reported as a negative prognostic factor for breast cancer.

[0371] In the embodiments, the cancer is characterized by low HER2 expression.

[0372] In the embodiments, the cancer is characterized by moderate HER2 expression.

[0373] In the embodiments, the cancer is characterized by high HER2 expression.

[0374] In the embodiments, the cancer is characterized by HER2 overexpression.

[0375] In the embodiments, the cancer is trastuzumab resistant.

[0376] In this embodiment, the cancer is breast cancer. In this embodiment, the breast cancer is metastatic. In this embodiment, the breast cancer is characterized by low Her2 expression. In this embodiment, the breast cancer is characterized by intermediate Her2 expression. In this embodiment, the breast cancer is characterized by high Her2 expression. In this embodiment, the breast cancer is trastuzumab resistant.

[0377] In this embodiment, the cancer is gastric cancer. In this embodiment, the gastric cancer is metastatic. In this embodiment, the gastric cancer is characterized by low Her2 expression. In this embodiment, the gastric cancer is characterized by intermediate Her2 expression. In this embodiment, the gastric cancer is characterized by high Her2 expression. In this embodiment, the gastric cancer is trastuzumab resistant.

[0378] In an embodiment, the cancer is lung cancer (e.g., non-small cell lung cancer). In an embodiment, the lung cancer (e.g., non-small cell lung cancer) is metastatic. In an embodiment, the lung cancer (e.g., non-small cell lung cancer) is characterized by low Her2 expression. In an embodiment, the lung cancer (e.g., non-small cell lung cancer) is characterized by intermediate Her2 expression. In an embodiment, the lung cancer (e.g., non-small cell lung cancer) is characterized by high Her2 expression. In an embodiment, the lung cancer (e.g., non-small cell lung cancer) is trastuzumab resistant.

[0379] In this embodiment, the cancer is ovarian cancer. In this embodiment, the ovarian cancer is metastatic. In this embodiment, the ovarian cancer is characterized by low Her2 expression. In this embodiment, the ovarian cancer is characterized by intermediate Her2 expression. In this embodiment, the ovarian cancer is characterized by high Her2 expression. In this embodiment, the ovarian cancer is trastuzumab resistant.

[0380] Cancer, including but not limited to tumors, metastases, or other diseases or conditions characterized by uncontrolled cell growth, can be treated or inhibited by administration of camptothecin conjugates.

[0381] In other embodiments, a method of treating cancer is provided, comprising administering an effective amount of a camptothecin conjugate and a chemotherapeutic agent to a patient in need. In one embodiment, the chemotherapeutic agent is another therapeutic agent that has not yet been found to be difficult to use for treating cancer. In another embodiment, the chemotherapeutic agent is another therapeutic agent that has been found to be difficult to use for treating cancer. The camptothecin conjugate may be administered to a patient who has also undergone surgery as cancer treatment.

[0382] In embodiments, the patient also receives additional treatment, such as radiation therapy. In one particular embodiment, the camptothecin conjugate is administered concurrently with a chemotherapy agent or with radiation therapy. In another particular embodiment, the chemotherapy agent or radiation therapy is administered before or after the administration of the camptothecin conjugate (e.g., MB-2a or MB-3a (trastuzumab-medotecan)).

[0383] Chemotherapy agents may be administered in a series of stages. Any single chemotherapy agent or combination thereof may be administered, such as one or more standard care chemotherapy agents.

[0384] Additionally, the use of camptothecin conjugates to treat cancer is offered as an alternative to chemotherapy or radiation therapy, where chemotherapy or radiation therapy has been proven or can be proven to be excessively toxic to the individual being treated, for example, producing unacceptable or intolerable side effects. The treated patient may be treated with another cancer therapy, such as surgery, radiation therapy, or chemotherapy, depending on which treatment is found to be acceptable or tolerable.

[0385] In embodiments, the treatment methods described herein include administration of MB-2a or a medically acceptable salt thereof.

[0386] In the embodiments, the treatment methods described herein comprise administration of MB-3a (trastuzumab / medotecan). Composition and method of administration.

[0387] In another embodiment, the present invention provides a pharmaceutical composition comprising any compound described herein (e.g., any (PL-A) compound, such as MB-2a and MB-3a (trastuzumab-medotecan) as described herein), or a pharmaceutically acceptable salt thereof. In embodiments, the pharmaceutical composition comprises a pharmaceutically acceptable carrier.

[0388] In the embodiments, the pharmaceutical composition comprises a conjugate according to formula (PL-A), such as MB-2a or MB-3a (trastuzumab-medotecan) or a pharmaceutically acceptable salt thereof.

[0389] In the embodiments, the pharmaceutical composition comprises MB-2a or a pharmaceutically acceptable salt thereof.

[0390] In the embodiments, the pharmaceutical composition comprises MB-3a (trastuzumab meditec) or a pharmaceutically acceptable salt thereof.

[0391] In embodiments, the present invention provides pharmaceutical compositions comprising a camptothecin conjugate as described herein (e.g., any (PL-A) compound, such as MB-2a or MB-3a (trastuzumab-medotecan)) and a pharmaceutically acceptable carrier. The camptothecin conjugate may be in any form that allows administration of the compound to a patient for the treatment of a condition associated with the expression of an antigen bound to a cell binder. For example, the conjugate may be in liquid or solid form. A preferred route of administration is non-enteral. Non-enteral administration includes subcutaneous injection, intravenous, intramuscular, intrasternal injection, or infusion techniques. In one embodiment, the composition is non-enterally administered. In another embodiment, the conjugate is administered intravenously. Administration may be by any suitable route, such as by infusion or bolus injection.

[0392] The pharmaceutical composition may be formulated such that the compound is biologically usable after administration to a patient. The composition may be in the form of one or more dosage units.

[0393] The materials used to prepare pharmaceutical compositions may be non-toxic at the amounts used. It will be apparent to those skilled in the art that the optimal dosage of one or more active ingredients in a pharmaceutical composition will depend on a variety of factors. These factors include, but are not limited to, the type of animal (e.g., human), the specific form of the compound, the method of administration, and the composition used.

[0394] The composition may be, for example, in liquid form. The liquid may be suitable for delivery by injection. In the composition intended for injection, one or more of surfactants, preservatives, wetting agents, dispersants, suspending agents, buffers, stabilizers and isotonics may also be included.

[0395] Liquid compositions (whether in the form of solutions, suspensions, or other similar forms) may also include one or more of the following: sterile diluents, such as water for injection, saline solution (preferably physiological saline), Ringer's solution, isotonic sodium chloride, non-volatile oils (such as synthetic mono- or diglycerides of glycerol that can act as solvents or suspension media), polyethylene glycol, glycerol, cyclodextrin, propylene glycol, or other solvents; antibacterial agents, such as benzyl alcohol or methylparaben; antioxidants, such as ascorbic acid or sodium bisulfite; chelating agents, such as ethylenediaminetetraacetic acid; buffers, such as amino acids, acetates, citrates, or phosphates; detergents, such as nonionic surfactants, polyols; and agents for regulating tension, such as sodium chloride or dextran. Non-enteral compositions may be encapsulated in ampoules, disposable syringes, or multi-dose vials made of glass, plastic, or other materials. Physiological saline is used as an illustrative adjuvant. Injectable compositions are preferably sterile.

[0396] The amount of the compound that is effective in treating a specific condition or symptom will depend on the nature of the condition or symptom and can be determined using standard clinical techniques. Additionally, in vitro or in vivo analysis may be used, as appropriate, to help identify the optimal dosage range. The precise dosage to be used in the composition will also depend on the route of administration and the severity of the disease or symptom, and should be determined based on the physician's judgment and the individual patient's condition.

[0397] The composition contains an effective amount of the compound to obtain a suitable dosage. Typically, this amount is at least about 0.01% of the compound by weight of the composition.

[0398] For intravenous administration, the composition may include about 0.01 to about 100 mg camptothecin conjugate / kg animal body weight. In one formulation, the composition may include about 1 to about 100 mg camptothecin conjugate / kg animal body weight. In another formulation, the dosage will be in the range of about 0.1 to about 25 mg of compound per kg body weight. Depending on the drug used, the dosage may be even lower, for example, from 1.0 µg / kg to 5.0 mg / kg, 4.0 mg / kg, 3.0 mg / kg, 2.0 mg / kg, or 1.0 mg / kg, or from 1.0 µg / kg to 500.0 µg / kg individual body weight.

[0399] Generally, the dose of the conjugate administered to a patient is typically from about 0.01 mg / kg to about 100 mg / kg body weight or from 1.0 µg / kg to 5.0 mg / kg body weight. In examples, the dose administered to a patient is between about 0.01 mg / kg and about 15 mg / kg body weight. In examples, the dose administered to a patient is between about 0.1 mg / kg and about 15 mg / kg body weight. In examples, the dose administered to a patient is between about 0.1 mg / kg and about 20 mg / kg body weight. In examples, the dose administered is between about 0.1 mg / kg and about 5 mg / kg or between about 0.1 mg / kg and about 10 mg / kg body weight. In examples, the dose administered is between about 1 mg / kg and about 15 mg / kg body weight. In examples, the dose administered is between about 1 mg / kg and about 10 mg / kg body weight. In the embodiments, the dose administered during the treatment period is between about 0.1 to 4 mg / kg, even more preferably 0.1 to 3.2 mg / kg or even more preferably 0.1 to 2.7 mg / kg of individual body weight.

[0400] The term "carrier" refers to a diluent, adjuvant, or excipient administered together with a compound. Such pharmaceutical carriers can be liquids, such as water and oils, including petroleum, animal, plant, or synthetic oils, such as peanut oil, soybean oil, mineral oil, and sesame oil. Carriers can be saline, gum arabic, gelatin, starch paste, talc, keratin, colloidal silica, and urea. Additionally, adjuvants, stabilizers, thickeners, lubricants, and colorants may be used. In one embodiment, the compound or composition and the pharmaceutically acceptable carrier are sterile when administered to a patient.

[0401] When the compound is administered intravenously, water is used as an illustrative carrier. Saline solutions and aqueous solutions of dextran and glycerol can also be used as liquid carriers, especially for injectable solutions. Suitable pharmaceutical carriers also include excipients such as starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silicone, sodium stearate, glyceryl monostearate, talc, sodium chloride, dried skim milk powder, glycerol, propylene, ethylene glycol, water, and ethanol. Where necessary, the compositions of the present invention may also contain small amounts of wetting agents, emulsifiers, or pH buffers.

[0402] In one embodiment, the conjugate is formulated according to conventional procedures to suit the intravenous administration of the pharmaceutical composition to animals, particularly humans. Typically, the carrier or mediator used for intravenous administration is a sterile isotonic buffered aqueous solution. If necessary, the composition may also include a solubilizer. The composition for intravenous administration may, where appropriate, contain a local anesthetic, such as lidocaine, to relieve pain at the injection site. Typically, these components are provided individually or in unit dosage forms, for example, as dry lyophilized powders or anhydrous concentrates in hermetically sealed containers (such as ampoules or capsules) indicating the active dose. When the conjugate is administered by infusion, it may be prepared, for example, using an infusion bottle containing sterile pharmaceutical-grade water or saline. When the conjugate is administered by injection, ampoules of sterile water or saline for injection may be provided so that the components can be mixed prior to administration.

[0403] Pharmaceutical compositions are generally formulated to be sterile, substantially isotonic, and fully compliant with all Good Manufacturing Practices (GMP) regulations of the U.S. Food and Drug Administration. Kits for medical use.

[0404] In some forms, kits are provided for the treatment of cancer and autoimmune diseases. Such kits may include pharmaceutical compositions comprising the camptothecin conjugates described herein.

[0405] In embodiments, the kit may include instructions for use with any of the treatments described herein. The included instructions provide a description of administering the pharmaceutical composition to an individual to achieve the intended efficacy in that individual, such as treating a disease or condition (e.g., cancer). In embodiments, the instructions relating to the use of the pharmaceutical composition described herein may include information regarding the dosage, timing of administration, and route of administration for the intended treatment. The container may be a unit dose, bulk (e.g., multi-dose packaging), or subunit dose. The instructions provided in the kit of the present invention are typically written instructions on a label or packaging insert. The label or packaging insert indicates that the pharmaceutical composition is used to treat, delay the onset of, and / or alleviate an individual's disease or condition.

[0406] In embodiments, the kits provided herein are in suitable packaging. Suitable packaging includes, but is not limited to, vials, bottles, wide-mouth flasks, flexible packaging, and the like. Packaging for use with a specific device, such as an inhaler, nasal delivery device, or infusion device, is also included. In embodiments, the kit may have a sterile access port (e.g., the container may be an intravenous solution bag or a vial with a stopper that can be punctured by a hypodermic needle).

[0407] In embodiments, the kits provided herein include additional therapeutic agents suitable for treating cancers with autoimmune diseases as described herein. Multimodal therapy for cancer.

[0408] Cancer, including but not limited to tumors, metastases, or other diseases or conditions characterized by uncontrolled cell growth, can be treated or inhibited by administration of camptothecin conjugates.

[0409] In other embodiments, a method of treating cancer is provided, comprising administering an effective amount of a camptothecin conjugate and a chemotherapeutic agent to a patient in need. In one embodiment, the chemotherapeutic agent is another therapeutic agent that has not yet been found to be difficult to use for treating cancer. In another embodiment, the chemotherapeutic agent is another therapeutic agent that has been found to be difficult to use for treating cancer. The camptothecin conjugate may be administered to a patient who has also undergone surgery as cancer treatment.

[0410] In embodiments, the patient also receives additional treatment, such as radiation therapy. In one particular embodiment, the camptothecin conjugate is administered simultaneously with a chemotherapy agent or with radiation therapy. In another particular embodiment, the chemotherapy agent or radiation therapy is administered before or after the administration of the camptothecin conjugate.

[0411] Chemotherapy agents can be administered in a series of stages. Any single chemotherapy agent or combination thereof can be administered, such as one or more standard care chemotherapy agents.

[0412] Additionally, the use of camptothecin conjugates to treat cancer is offered as an alternative to chemotherapy or radiation therapy, where chemotherapy or radiation therapy has been proven or can be proven to be excessively toxic to the individual being treated, for example, producing unacceptable or intolerable side effects. The treated patient may be treated with another cancer therapy, such as surgery, radiation therapy, or chemotherapy, depending on which treatment is found to be acceptable or tolerable.

[0413] The present invention is further illustrated by the following examples, which should not be construed as limiting the scope or spirit of the invention to the specific procedures described herein. It should be understood that these examples are merely illustrative of certain embodiments and are therefore not intended to limit the scope of the invention. It should also be understood that various other embodiments, modifications, and equivalents may be used to suggest to those skilled in the art that they do not depart from the spirit of the invention and / or the scope of the appended claims. Examples

[0414] The invention is specifically described with reference to the examples shown below. However, the invention is not limited to these examples. Furthermore, it is in no way intended to be limited. In addition, unless otherwise specifically described, the reagents, solvents, and starting materials described in this specification are readily available from commercial suppliers.

[0415] The following abbreviations are used for the following terms: ADCs Antibody-Drug Conjugates ACN Acetonitrile DAR Drug-to-Antibody Ratio DCC N,N'-Dicyclohexylcarbodiimide DCM Dichloromethane DIPA Diisopropylamine DIPEA Diisopropylethylamine DMF Dimethylformamide DMSO Dimethylimide DMTMM 4-(4,6-dimethoxy-1,3,5-tri-2-yl)-4-methylmorpholinium chloride DMTMMT 4-(4,6-dimethoxy-1,3,5-tri-2-yl)-4-methyllinonium tetrafluoroborate DTPA Diethylenetriaminepentaacetic acid EDCI 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide HOPO 2-Hydroxypyridine-1-oxide IV Intravenous M Morphine nM Nemor NMM N-Methyllinonium PPTS Pyridinium p-Toluenesulfonic acid PTSA 4-Methylbenzenesulfonic acid (SEC) Size exclusion chromatography (TBS) Tributyldimethylsilyl TCEP 3,3',3''-phosphine trimethyltripropionate salt (TEA) Triethylamine (TFA) Trifluoroacetic acid (THF) Tetrahydrofuran (TLC) Thin-layer chromatography (p-TsOH) p-Toluenesulfonic acid Example 1. Exemplary synthesis of compound MB-1 (P1)

[0416] General preparation procedure for 2,6-dibromo-4-fluoro-3-methyl-aniline (1-2)

[0417] Br2 (223.48 g, 1.40 mol, 72.09 mL, 2.5 equivalents) in CH2Cl2 / methanol (1:1, 1.2 L) was added dropwise to a stirred solution of compound 1-1 (70 g, 559.36 mmol, 1 equivalent) in CH2Cl2 / methanol (1:1, 200 mL) in 1.5 h at 15 °C using a feeding funnel. The reaction mixture was stirred at 25 °C for 4 h, and TLC (petroleum ether / ethyl acetate = 6 / 1, Rf = 0.6) showed depletion of the starting material. Three additional vials were prepared as described above, and mixtures from the four reactions were combined and concentrated. 1 N Na2S2O3 (1.5 L) and ethyl acetate (1.5 L) were added to the resulting residue. The solution was stirred for 10 min and then carefully alkalized with 1 N Na2CO3 (150 mL). It was transferred to a separatory funnel and the organic layer was separated. The aqueous layer was extracted with ethyl acetate (2 × 1 L). The combined organic layers were washed with 1 N Na₂S₂O₃ (1 L), followed by washing with brine (1 L) and drying over Na₂SO₄. The residue was filtered and concentrated under reduced pressure to give the residue. The residue was wet-milled with petroleum ether (1 L) and filtered to give product 1-2 (574 g, 1.93 mol, yield 86%, purity 95%) as a pale purple solid. ¹H NMR (400 MHz, chloroform-d) δ 7.18 (d, J = 8.6 Hz, 1H), 4.52 - 4.30 (m, 2H), 2.29 (d, J = 2.4 Hz, 3H).

[0418] General preparation procedure for 1,3-dibromo-5-fluoro-2-iodo-4-methylbenzene (1-3)

[0419] Compounds 1-2 (49.25 g, 174.07 mmol, 1 equivalent) were added to a solution of p-TsOH (90 g, 522.2 mmol, 3 equivalents) in acetonitrile (700 mL). The resulting white suspension was cooled to 10-15 °C and then NaNO2 (24.02 g, 348.14 mmol, 2 equivalents) and KI (73.22 g, 435.13 mmol, 2.5 equivalents) in water (105 mL) were gradually added. The suspension turned dark brown and released gas. The thickened mixture was stirred at 10 °C for 10 min, followed by stirring at 20 °C for 1 h. TLC (petroleum ether / ethyl acetate = 6 / 1, Rf = 0.6) showed depletion of the starting material. The reaction mixture was poured into water (400 mL). 1 N sodium bicarbonate solution (200 mL) was added to adjust the pH to 9-10, followed by 2 N sodium thiosulfate solution (200 mL). The resulting mixture was extracted with ethyl acetate (3 × 500 mL). Eleven additional vials were prepared as described above. The combined organic layers from the 12 reactions were combined, dried over Na₂SO₄, and concentrated under reduced pressure. The resulting residue was purified by silica gel chromatography and dissolved in petroleum ether to give products 1-3 as yellow solids (504 g, 1.09 mol, yield 56%, purity 85%). ¹H NMR (400 MHz, chloroform-d) δ 7.41 (d, J = 8.8 Hz, 1H), 2.43 (d, J = 2.4 Hz, 3H).

[0420] General preparation procedure for 2,6-dibromo-4-fluoro-3-methylbenzaldehyde (1-4)

[0421] A solution of magnesium chloride (isopropyl) (2 M in tetrahydrofuran, 76.80 mL, 1.2 equivalents) was added over a 10-minute interval to a solution of compounds 1-3 (50.4 g, 127.98 mmol, 1 equivalent) in anhydrous toluene (300 mL), while maintaining the internal temperature below -25°C. A clear brown solution was obtained, and the mixture was stirred for 1.5 h. Then, N,N-dimethylformamide (30.86 g, 422.33 mmol, 3.3 equivalents) was added over 10 minutes. After the addition, the temperature of the reaction mixture was increased to -19°C. The reaction mixture was heated to 20°C over 0.5 h and stirred for 1.5 h. TLC (petroleum ether / ethyl acetate = 10 / 1, Rf = 0.45) showed that the reaction was complete. The reaction mixture was quenched with 50 mL of saturated aqueous NH4Cl solution. Ten additional vials were prepared as described above, and all eleven reaction mixtures were combined. The combined mixture was filtered, and the filtrate was evaporated under reduced pressure to obtain a residue. The residue was purified by silica gel column chromatography and dissolved in petroleum ether to give product 1-4 as a yellow solid (253 g, 812.18 mmol, yield 60%, purity 95%). ¹H NMR (400 MHz, chloroform-d) δ 10.22 (s, 1H), 7.40 (d, J=8.6 Hz, 1H), 2.37 (d, J=2.4 Hz, 3H).

[0422] General preparation procedure for 2-(2,6-dibromo-4-fluoro-3-methyl-phenyl)-1,3-dioxane (4).

[0423] Ethylene glycol (53.06 g, 878.58 mmol, 47.80 mL, 5 equivalents), triethyl orthoformate (25.34 g, 170.99 mmol, 28.44 mL, 1 equivalent), and p-toluenesulfonic acid (1.47 g, 8.55 mmol, 0.05 equivalents) were added to a solution of compounds 1-4 (50.6 g, 170.99 mmol, 28.44 mL, 1 equivalent) in 1,2-dichloroethane (430 mL). The reaction mixture was stirred at 80 °C for 3 h and TLC (petroleum ether / ethyl acetate = 10 / 1, Rf = 0.59) showed that the reaction was complete. Four additional vials were prepared as described above, and the reaction mixtures from the five reactions were combined. The combined reaction mixture was then washed with saturated aqueous solution of Na₂CO₃ (1 L), saturated aqueous solution of NH₄Cl (1 L), and water (1 L). The organic layer was dried with Na₂SO₄, filtered, and evaporated under reduced pressure to obtain the crude product. The crude product was wet-milled with petroleum ether at 20 °C for 15 minutes and filtered to obtain product 4 (280 g, 741.21 mmol, yield 84%, purity 90%), a pale yellow solid. ¹H NMR (400 MHz, chloroform-d) δ 7.34 (d, J=8.6 Hz, 1H), 6.44 (s, 1H), 4.37–4.31 (m, 2H), 4.11–4.06 (m, 2H), 2.34 (d, J=2.4 Hz, 3H).

[0424] General preparation procedure for N-[3-bromo-2-(1,3-dioxolane-2-yl)-5-fluoro-4-methyl-phenyl]-1,1-diphenyl-methylimine (5)

[0425] Compound 4a (29.67 g, 163.69 mmol, 27.46 mL, 1.05 equivalents), sodium tributoxide (29.97 g, 311.78 mmol, 2 equivalents), palladium(II) acetate (3.5 g, 15.59 mmol, 0.1 equivalents), and 4,5-bis(diphenylphosphino)-9,9-dimethyldibenzopiperanan (9.02 g, 15.59 mmol, 0.1 equivalents) were added to a solution of compound 4 (53 g, 155.89 mmol, 1 equivalent) in toluene (100 mL) under N2 protection. The reaction mixture was stirred at 100 °C under N2 protection for 12 h, and TLC (petroleum ether / ethyl acetate = 10 / 1, Rf = 0.32) showed that the reaction was complete. Two additional vials were assembled as described above, and all three reaction mixtures were combined and filtered through a diatomaceous earth mat. The filter cake was washed with ethyl acetate (500 mL). The combined filtrates were concentrated under reduced pressure to obtain a residue. The residue was purified by column chromatography and dissociated with petroleum ether / ethyl acetate = 10 / 1 to give product 5 (105 g, 214.62 mmol, yield 45.61%, purity 80%) as a yellow solid. The product was used in the next step without further purification. 1H NMR (400MHz, chloroform-d) δ 7.77 (br d, J=7.3 Hz, 2H), 7.54 - 7.37 (m, 4H), 7.31 (br d, J=4.5 Hz, 3H), 7.26 - 7.22 (m, 1H), 6.48 (s, 1H), 5.93 (d, J=10.3 Hz, 1H), 4.11 - 4.05 (m, 2H), 3.96 - 3.91 (m, 2H), 2.23 (d, J=2.3 Hz, 3H).

[0426] General preparation procedure for N-[3-[4-[tri-butyl(dimethyl)silyl]oxybutyl]-2-(1,3-dioxolane-2-yl)-5-fluoro-4-methyl-phenyl]-1,1-diphenyl-methylimine (1-6)

[0427] 9-BBN (0.5 M in tetrahydrofuran, 68.13 mL, 1.2 equivalents) was added to a stirred mixture of compound 5a (5.3 g, 28.44 mmol, 1 equivalent) in toluene (80 mL) at 10 °C under a nitrogen atmosphere. The resulting mixture was stirred at 80 °C under nitrogen protection for 20 min, and TLC (petroleum ether / ethyl acetate = 1 / 1, product Rf = 0.2, I2) showed that the reaction was complete. A solution of NaOH (2.27 g, 56.78 mmol, 2 equivalents) in water (20 mL) was added to the above mixture at 10 °C under a nitrogen atmosphere. The resulting mixture was stirred at 10°C under a nitrogen atmosphere for 10 minutes, followed by the addition of compound 5 (10.00 g, 22.71 mmol, 0.8 equivalents), tetrabutylammonium iodide (524.31 mg, 1.42 mmol, 0.05 equivalents), and [1,1-bis(diphenyl-phosphino)ferrocene]palladium(II) dichloromethane adduct (463.7 mg, 568.8 µmol, 0.02 equivalents). The resulting mixture was stirred at 80°C under a nitrogen atmosphere for 15 hours, and LCMS (retention time = 3.620) showed the reaction was complete. Seven additional vials were prepared as described above, and all eight reaction mixtures were combined. The combined reaction mixtures were washed with water (500 mL × 3), dried over Na₂SO₄, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 10 / 1 to 5 / 1) to obtain a crude product, which was further purified by reverse-phase HPLC to obtain a yellow gel-like product 1-6 (45 g, 82.15 mmol, yield 50%, purity 80%). 1H NMR (400MHz, DMSO-d6) δ 7.72 - 7.61 (m, 2H), 7.59 - 7.51 (m, 1H), 7.50 - 7.42 (m, 2H), 7.37 - 7.22 (m, 4H), 7.19 (br d, J=3.5 Hz, 1H), 6.03 (s, 1H), 5.89 (s, 1H), 4.07 - 3.99 (m, 2H), 3.93 - 3.78 (m, 2H), 3.65 - 3.56 (m, 2H), 2.75 - 2.64 (m, 2H), 2.01 (s, 2H), 1.64 (s, 1H), 1.59 - 1.44 (m, 4H), 0.87 (s, 9H), 0.03 (s, 6H).

[0428] General preparation procedure for (19S)-19-ethyl-6-fluoro-19-hydroxy-8-(4-hydroxybutyl)-7-methyl-17-oxa-3,13-diazapentacyclo[11.8.0.02,11.04,9.015,20] icosico-1(21),2,4,6,8,10,15(20)-hepten-14,18-dione (MB-1, helotecone)

[0429] 35% HCl (5.7 g, 6 equivalents) was added to a solution of compound 7 (2.4 g, 1 equivalent) in ethanol (100 mL), and the solution was heated to 50-60°C. Then, ethanol (100 mL) containing compounds 1-6 (10 g, 2 equivalents) was added dropwise over 2 hours. The mixture was stirred at 50-60°C for 1.5 hours. The heating bath was removed, and the reaction mixture was cooled to room temperature. Methyl-tertiary butyl ether (2000 mL) was added to precipitate the product. The product was filtered and the solid was washed with methyl-tertiary butyl ether to give MB-1 (herotecan) as a pale yellow solid (2.45 g, 59% yield and 98% purity). 1H NMR (400MHz, DMSO-d6) δ 8.89 (s, 1H), 7.77 (d, J=11.0 Hz, 1H), 7.31 (s, 1H), 6.54 (br s, 1H), 5.53 - 5.33 (m, 2H), 5.26 (s, 2H), 4.80 - 4.06 (m, 1H), 3.49 (br t, J=5.9 Hz, 2H), 3.19 - 3.11 (m, 2H), 2.43 (d, J=1.8 Hz, 3H), 1.86 (tt, J=7.2, 14.5 Hz, 2H), 1.62 (br s, 4H), 0.88 (t, J=7.3 Hz, 3H). Example 2. Exemplary synthesis of compound MB-2 (PL1).

[0430] General preparation procedure for C17H14NO4(S2).

[0431] A column containing 1500 mL of dichloromethane containing 2-(9H-furo-9-ylmethoxycarbonylamino)acetic acid (74.92 g, 252.10 mmol, 2 equivalents), Trt-resin S1 (120.00 g, 126.05 mmol, 1 equivalent), and N,N-diisopropylethylamine (162.85 g, 1.26 mol, 219.47 mL, 10 equivalents) was bubbled with nitrogen at 20 °C for 12 hours. After filtration, the residue was washed with dichloromethane (3 × 300 mL), dimethylformamide dichloromethane / methanol = 1 / 1 (3 × 300 mL), and dimethylformamide (3 × 300 mL). The residue was further dried under high vacuum to give a yellow solid resin-C17H14NO4 (150 g, 123.66 mmol, 98.10% yield, crude purity). The product was used directly in the next step without purification. Piperidine (105.30 g, 1.24 mol, 122.13 mL, 10 equivalents) was added to a column packed with DMF containing resin-C17H14NO4 (150 g, 123.66 mmol, 1 equivalent). The mixture was bubbled with N2 at 20 °C for 1 hour. The resulting resin was filtered off and subsequently washed with dimethylformamide (2 × 500 mL) and dichloromethane (2 × 500 mL). The resin was dried to obtain a yellow solid resin - C2H4NO2(S2) (120 g, 121.21 mmol, 98.02% yield) which was used directly in the next step.

[0432] General preparation procedure for resin-C5H9N2O3(S3)

[0433] HCTU (O-(6-chloro-1-hydrobenzotriazol-1-yl)-1,1,3,3-tetramethylglucuronide hexafluorophosphate) (100.29 g, 242.42 mmol, 2 equivalents) and N,N-diisopropylethylamine (78.33 g, 606.06 mmol, 105.56 mL, 5 equivalents) was added to a column containing dimethylformamide (1200 mL) containing (2S)-2-(9H-en-9-ylmethoxycarbonylamino)-( ... The obtained resin was filtered and washed successively with dimethylformamide (2 × 500 mL) and dichloromethane (2 × 500 mL). After drying, a yellow solid resin-C20H19N2O5 (150 g, crude material) was obtained, which was used directly in the next step. Piperidine (99.55 g, 1.17 mol, 115.46 mL, 10 equivalents) was added to a column containing dimethylformamide (1200 mL) containing resin-C20H19N2O5 (150 g, 116.91 mmol, 1 equivalent). The mixture was bubbled with N2 at 20°C for 1 hour. The obtained resin was filtered and washed successively with dimethylformamide (2 × 500 mL) and dichloromethane (2 × 500 mL). The resin was dried to obtain a yellow solid resin C5H9N2O3(S3) (120 g, crude material), which was then used directly in the next step. General preparation procedure for 2-[[(2S)-2-[[(2S)-2-(9H-furo-9-ylmethoxy-carbonylamino)-3-methyl-butyryl]amino]propionic]amino]acetic acid (1-7)

[0435] O-(6-chloro-1-hydrobenzotriazol-1-yl)-1,1,3,3-tetramethylaurocyanurate hexafluorophosphate (93.49 g, 225.99 mmol, 2 equivalents) and N,N-diisopropylethylamine (73.02 g, 564.97 mmol, 98.41 mL, 5 equivalents) were added to a column containing dimethylformamide (200 mL) containing resin C5H9N2O3(S3) (120 g, 112.99 mmol, 1 equivalent) and (2S)-2-(9H-en-9-ylmethoxycarbonylamino)-3-methyl-butyric acid (76.70 g, 225.99 mmol, 2 equivalents). The mixture was bubbled with N2 at 20 °C for 12 hours. The obtained resin was filtered off and washed successively with dimethylformamide (2 × 500 mL) and dichloromethane (2 × 500 mL). The resin was quenched with trifluoroacetic acid / dichloromethane (10%, 3 × 500 mL). The organic layer was combined and concentrated under reduced pressure to obtain the residue. The residue was wet-milled with n-hexane at 20 °C for 12 hours. It was filtered to give product 1-7 as a white solid (60 g, 39.05 mmol, yield 34.56%, purity 90%). 1H NMR (400MHz, DMSO-d6) δ 8.17 (br t, J=5.7 Hz, 1H), 7.99 (d, J=7.5 Hz, 1H), 7.89 (d, J=7.3 Hz, 2H), 7.74 (t, J=6.6 Hz, 2H), 7.45 - 7.38 (m, 3H), 7.37 - 7.29 (m, 2H), 4.39 - 4.19 (m, 4H), 3.93 - 3.66 (m, 3H), 2.03 - 1.92 (m, 1H), 1.22 (d, J=7.1 Hz, 3H), 0.85 (dd, J=6.9, 9.8 Hz, 6H).

[0436] General preparation procedure for [[(2S)-2-[[(2S)-2-(9H-furo-9-ylmethoxycarbonylamino)-3-methyl-butyryl]amino]propionic]amino]methyl acetate (1-8).

[0437] A solution of compounds 1-7 (90.0 g), pyridine (30.6 g, 2 equivalents), lead(IV) acetate (189 g, 2 equivalents), and Cu(OAc)₂ (3.6 g, 0.1 equivalents) in THF (1000 mL) and toluene (250 mL) was stirred at 50–60 °C for 2 hours. The reaction mixture was cooled to room temperature and filtered to remove insoluble salts. The filtrate was diluted with THF / MTBE (1:4) to precipitate the desired product. The precipitate was filtered and the solid was washed with THF / MTBE (1:4) to give product 1-8 (84.2 g, yield = 90.8%, purity 91.9%) as a pale yellow solid. 1H NMR (400MHz, DMSO-d6) δ 8.03 - 7.93 (m, 1H), 7.89 (d, J=7.5 Hz, 2H), 7.74 (br t, J=6.3 Hz, 2H), 7.47 - 7.36 (m, 3H), 7.36 - 7.28 (m, 2H), 5.16 - 5.02 (m, 1H), 4.39 - 4.26 (m, 2H), 4.22 (br d, J=3.9 Hz, 2H), 3.93 - 3.82 (m, 1H), 1.98 (s, 2H), 1.78 (s, 1H), 1.27 - 1.13 (m, 3H), 0.92 - 0.75 (m, 6H).

[0438] General preparation procedure for (2S)-2-amino-N-[(1S)-2-[4-[(19S)-19-ethyl-6-fluoro-19-hydroxy-7-methyl-14,18-disideoxy-17-oxa-3,13-diazapentacyclo[11.8.0.02,11.04,9.015,20] icosico-1(21),2,4,6,8,10,15(20)-heptane-8-yl]butoxymethylamino]-1-methyl-2-sideoxy-ethyl]-3-methyl-butyramine (1-10)

[0439] At room temperature, a mixture of BF3 ether (12.1 g, 0.0852 mol, 3.6 equivalents) was added to a stirred solution of MB-1 (herotecan) (10.7 g, 0.0236 mol) and compounds 1-8 (14.7 g, 0.0306 mol, 1.3 equivalents) in anhydrous DMSO (90 mL). The reaction solution was stirred at room temperature for 13 hours. LCMS analysis of the reaction sample showed approximately 75% formation of product 1-9 and 6% remaining of starting material MB-1. A porphyrin (20.5 g, 0.236 mol) was added to the reaction solution, and the reaction solution was stirred at room temperature for 2 hours. The product 1-10 (9.1 g, y=59%, purity 96.1%) was purified directly by preparative HPLC (C18 column, mobile phase CH3CN / H2O) and lyophilized to obtain a grayish-white solid. 1H NMR (400MHz, DMSO-d6) δ 8.87 (s, 1H), 8.68 (br s, 1H), 8.09 (br s, 1H), 7.77 (br d, J=10.6 Hz, 1H), 7.32 (s, 1H), 6.51 (s, 1H), 5.43 (s, 2H), 5.29 (br s, 2H), 4.55 (br s, 2H), 4.28 (br s, 1H), 3.46 (br s, 1H), 3.45 - 3.42 (m, 1H), 3.14 (br s, 2H), 3.02 (br s, 1H), 2.43 (br s, 3H), 1.88 (br dd, J=7.9, 14.8 Hz, 3H), 1.70 (br s, 2H), 1.58 (br s, 2H), 1.19 (br d, J=6.8 Hz, 3H), 0.88 (br t, J=7.2 Hz, 3H), 0.82 (br d, J=6.6 Hz, 3H), 0.73 (br d, J=6.6 Hz, 3H).

[0440] General preparation procedure for 6-(2,5-disideroxypyrrolo-1-yl)-N-[(1S)-1-[[(1S)-2-[4-[(19S)-19-ethyl-6-fluoro-19-hydroxy-7-methyl-14,18-disideroxy-17-oxa-3,13-diazapentacyclo[11.8.0.02,11.04,9.015,20] icosico-1(21),2,4,6,8,10,15(20)-heptane-8-yl]butoxymethylamino]-1-methyl-2-sideroxy-ethyl]aminomethyl]-2-methyl-propyl]hexamylamine (MB-2)

[0441] Compound 10A (283.8 mg, 920.6 µmol, 1.5 equivalents) was added to a solution of compound 1-10 (400 mg, 613.8 µmol, 1 equivalent) in N,N-dimethylformamide (10 mL). The reaction mixture was stirred at 15 °C for 12 h. LCMS (product retention time = 2.080) showed that all compounds 1-10 were depleted and new peaks with the desired MS were detected. The reaction mixture was filtered and the filtrate was purified by preparative HPLC using acetonitrile and deionized water as the mobile phase to give product MB-2 as a white solid (173 mg, 203.9 µmol, yield 33.36%, purity 95.74%). 1H NMR (400MHz, DMSO-d6) δ 8.87 (s, 1H), 8.57 (t, J=6.4 Hz, 1H), 7.97 (d, J=7.2 Hz, 1H), 7.81 - 7.71 (m, 2H), 7.32 (s, 1H), 6.99 (s, 2H), 6.52 (s, 1H), 5.43 (s, 1H), 5.49 - 5.37 (m, 1H), 5.30 (s, 2H), 4.54 (dq, J=6.6, 10.1 Hz, 2H), 4.21 (quin, J=7.1 Hz, 1H), 4.10 (dd, J=6.8, 8.4 Hz, 1H), 3.48 - 3.41 (m, 2H), 3.37 - 3.34 (m, 2H), 3.20 - 3.08 (m, 2H), 2.43 (d, J=2.0 Hz, 3H), 2.18 - 2.01 (m, 2H), 1.95 - 1.79 (m, 3H), 1.68 (br d, J=7.0 Hz, 2H), 1.58 (br s, 2H), 1.51 - 1.38 (m, 4H), 1.20 - 1.10 (m, 5H), 0.88 (t, J=7.3 Hz, 3H), 0.76 (dd, J=6.8, 9.3 Hz, 6H).13C NMR (101MHz, DMSO-d6) δ 173.09, 172.54, 172.26, 171.09, 170.85, 160.54, 156.86, 152.36, 150.00, 147.85, 145.38, 140.13, 134.46, 129.33, 128.33, 125.02, 124.04, 119.05, 110.14, 96.73, 72.40, 69.18, 66.72, 65.27 (br s, 1C), 57.44, 50.49 (br s, 1C), 48.29, 37.02, 34.88, 30.31 (br s, 1C), 29.00, 27.78, 27.62 (br s, 1C), 26.73 (br s, 1C), 25.78, 24.89, 19.18, 18.03 (br d, J=5.8 Hz, 1C), 11.46, 7.80. HRMS (ESI-TOF) m / z: [M + H]+ Calculated value 845.39; Experimental value 845.3859.

[0442] Preparative HPLC method: Gilson 281 semi-preparative HPLC system and Phenomenex Gemini C18 column (75 × 40 mm × 3 μm); mobile phase: acetonitrile and water; flow rate: 25 mL / min; monitoring wavelength: 220 & 254 nm. Gradient: 30% to 50% acetonitrile over 8 min, 50% to 100% acetonitrile over 0.2 min, 100% acetonitrile for 2 min, 100% to 30% acetonitrile over 0.1 min, followed by 30% acetonitrile for 1.2 min. Example 3. Exemplary synthesis of MB-3 (medotecan) (PL3).

[0443] General preparation procedure for (2S)-2-(benzyloxycarbonylamino)-5-sideoxy-5-[[(2S,3R,4R,5R)-2,3,4,5,6-pentylhydroxyhexyl]amino]benzyl valerate (1-12).

[0444] 4-(4,6-dimethoxy-1,3,5-tris-2-yl)-4-methylmorpholinium tetrafluoroborate (DMTMMT) (9.7 g, 29.59 mmol, 2 equivalents) and triethylamine (2.24 g, 22.19 mmol, 1.5 equivalents) were added sequentially to a solution of compound 11A (6.03 g, 16.27 mmol, 1.1 equivalents) in N,N-dimethylformamide (27 mL). After stirring at 25 °C for 0.5 hours, compound 1-11 (2.68 g, 14.79 mmol, 1 equivalent) was added, and the reaction mixture was stirred at 25 °C for 12 hours. LCMS (product retention time = 0.253) showed depletion of the starting material and detection of a new peak with the desired MS. The reaction mixture was diluted with water (50 mL) and extracted with dichloromethane (6 × 50 mL). The combined organic layers were washed with brine (3 × 130 mL), dried over Na₂SO₄, and filtered. The filtrate was concentrated under reduced pressure, and the residue was treated under high vacuum to obtain an oily substance. The oily substance was purified by reverse-phase HPLC (3 kg Agela C18 column, CH₃CN / H₂O, 300 mL / min, gradient: 30% CH₃CN for 10 min, 30% to 45% CH₃CN over 30 min, 45% CH₃CN for 35 min; approximately 15 g of crude product was dissolved in 70 mL DMF and loaded onto the column) to give product 1-12 (4 g, 6.74 mmol, yield 46.1%, purity 99%) as a white solid. 1H NMR (400 MHz, DMSO-d6) δ 1.72 - 1.85 (m, 1 H) 1.91 - 2.03 (m, 1 H) 2.18 (br t, J=7.44 Hz, 2 H) 2.96 - 3.03 (m, 1 H) 3.24 (dt, J=13.16, 5.17 Hz, 2 H) 3.37 - 3.40 (m, 2 H) 3.44 (br s, 2 H) 4.04 - 4.11 (m, 1 H) 4.29 (d, J=6.38 Hz, 1 H) 4.39 - 4.45 (m, 2 H) 4.51 (d, J=5.63 Hz, 1 H) 4.75 (d, J=4.63 Hz, 1 H) 4.96 - 5.14 (m, 4 H) 7.19 - 7.46 (m, 10 H) 7.68 - 7.85 (m, 2 H).

[0445] General preparation procedure for (2S)-2-amino-5-sideoxy-5-[[(2S,3R,4R,5R)-2,3,4,5,6-pentylhydroxyhexyl]amino]valeric acid (1-13).

[0446] Pd / C (15.86 g, 14.96 mmol, 10 wt%, 2 equivalents) was added to a solution of compound 1-12 (4 g, 7.48 mmol, 1 equivalent) in water (192 mL) and tetrahydrofuran (48 mL). The mixture was stirred at 25 °C under H2 (15 psi) for 12 h. LCMS (product retention time = 0.137) showed depletion of the starting material and detection of the desired product. The mixture was filtered through a diatomaceous earth mat and the filtrate was concentrated to give product 1-13 (2 g, 6.26 mmol, yield 83.6%, purity 97.1%) as a white solid. ¹H NMR (400MHz, deuterium oxide) δ 3.84 (dt, J=7.76, 4.74 Hz, ¹H) 3.70 - 3.80 (m, 4H) 3.58 - 3.64 (m, 2H) 3.41 (dd, J=14.06, 4.03 Hz, ¹H) 3.25 (dd, J=14.06, 7.83 Hz, ¹H) 2.41 (br s, 2H) 2.10 (br s, 2H).

[0447] General preparation procedure for (2S)-2-[6-(2,5-di-side-oxypyrrolo-1-yl)hexamido]-5-side-oxy-5-[[(2S,3R,4R,5R)-2,3,4,5,6-pentylhydroxyhexyl]amino]valeric acid (1-14).

[0448] TEA (7.3 g, 0.0722 mol) was added to a stirred solution of compound 1-13 (containing water and 0.0361 mol or 11.2 g of 1-13) and 10A (11.13 g, 0.0361 mol) in acetonitrile (100 mL) and water (100 mL) at 10-20 °C. The reaction solution was stirred at 10-20 °C for 18 hours. It was purified by preparative HPLC (C18 column, mobile phase: CH3CN and water) and after lyophilization, product 1-14 (9.7 g, y=53%, purity 95.7%) was obtained as a pale red solid. ¹H NMR (400MHz, deuterium oxide) δ 6.78 (s, ¹H) 4.30 (dd, J=9.11, 5.07 Hz, ¹H) 3.83 (dt, J=7.89, 4.74 Hz, ¹H) 3.79 - 3.74 (m, ¹H) 3.74 - 3.68 (m, ²H) 3.65 - 3.57 (m, ²H) 3.46 (t, J=6.91 Hz, ²H) 3.40 (dd, J=14.06, 4.16 Hz, ¹H) 3.23 (dd, J=14.00, 7.89 Hz, ¹H) 2.39 - 2.30 (m, ²H) 2.24 (t,J=7.27 Hz, 2 H) 2.20 - 2.08 (m, 1 H) 2.04 - 1.89 (m, 1 H) 1.55 (dquin,J=14.04, 7.19, 7.19, 7.19, 7.19 Hz, 4 H) 1.28 - 1.17 (m, 2 H).

[0449] (2S)-2-[6-(2,5-disideroxypyrrolo-1-yl)hexamyl]-N-[(1S)-1-[[(1S)-2-[4-[(19S)-19-ethyl-6-fluoro-19-hydroxy-7-methyl-14,18-disideroxy-17-oxa-3,13-diazapentacyclo[11.8.0.02,11.04,9.01] General preparation procedure for [5,20] 1-(21),2,4,6,8,10,15(20)-heptane-8-yl]butoxymethylamino]-1-methyl-2-sideoxy-ethyl]aminomethoxy]-2-methyl-propyl]-N'-[(2S,3R,4R,5R)-2,3,4,5,6-pentylhydroxyhexyl]pentanediamine (MB-3, Meditec)

[0450] A mixture of EDCI (8.04 g, 0.0518 mol) and HOPO (5.75 g, 0.0518 mol) was added to a stirred solution of compounds 1-10 (15 g, purity: 90.1%, 0.0207 mol) and 1-14 (8.7 g, 0.0173 mol) in anhydrous DMF (400 mL) at 5-15 °C. The mixture was stirred at 5-15 °C for 13.5 h and purified by preparative HPLC (C18 column, mobile phase: CH3CN / H2O) to give MB-3 (meditecan) (7.1 g, y=30.2%, purity 98.8%) as a grayish-white solid.1H NMR (400MHz, DMSO-d6) δ 8.87 (s, 1H), 8.60 (br s, 1H), 8.08 (br d,J=6.2 Hz, 1H), 8.00 (br d, J=7.7 Hz, 1H), 7.77 (br d, J=11.5 Hz, 1H), 7.72 (br s, 1H), 7.63 (br d, J=9.3 Hz, 1H), 7.32 (s, 1H), 6.99 (s, 2H), 6.53 (s, 1H), 5.43 (br s, 2H), 5.29 (br s, 2H), 4.74 (br s, 1H), 4.57 (br s, 1H), 4.52 (br d, J=6.6 Hz, 1H), 4.47 (br d, J=5.5 Hz, 1H), 4.38 (br d, J=5.1 Hz, 1H), 4.33 (br s, 1H), 4.26 (br d, J=6.4 Hz, 1H), 4.23 (br d, J=6.6 Hz, 2H), 4.13 (br s, 1H), 3.55 (br d, J=4.4 Hz, 3H), 3.45 (br s, 7H), 3.14 (br s, 3H), 3.00 (br s, 1H), 2.42 (br s, 3H), 2.08 (br d, J=5.7 Hz, 4H), 1.96 - 1.78 (m, 1H), 1.96 - 1.78 (m, 4H), 1.69 (br s, 3H), 1.58 (br s, 2H), 1.45 (br s, 4H), 1.18 (br d, J=6.4 Hz, 5H), 0.88 (br t, J=7.1 Hz, 3H), 0.76 - 0.76 (m, 1H), 0.76 (br dd, J=6.9, 11.4 Hz, 5H)。13C NMR (101MHz, DMSO-d6) δ 173.14, 172.49 (d, J=10.3 Hz, 1C), 172.08, 171.53, 171.14, 170.58, 163.06, 160.59, 156.93, 152.35, 150.07, 147.96 (d, J=13.9 Hz, 1C), 145.41, 140.24, 134.48, 129.34, 128.39, 125.08, 124.09, 119.08, 110.24 (br d, J=22.7 Hz, 1C), 96.83, 72.44, 72.10, 71.78, 71.55, 69.67, 69.25, 66.80, 65.33, 63.38, 57.25, 52.34, 50.52, 48.41, 42.09, 37.02, 34.99, 31.98, 30.69, 30.36, 29.01, 27.81, 27.65 (br s, 1C), 26.75, 25.82, 24.80, 19.12, 17.88 (d, J=11.7 Hz, 1C), 11.44 (d, J=5.9 Hz, 1C), 7.81. HRMS (ESI-TOF) m / z: [M + H]+ calculated value 1137.52; experimental value 1137.5140. Example 4. Illustrative synthesis of antibody-drug conjugates MB-2a and MB-3a (trastuzumab-medotecan).

[0451] General preparation procedure for trastuzumab-drug conjugate MB-2a

[0452] 50 mM binding buffer (pH 7.4): One liter contains 6.86 g Na2HPO4·2H2O and 1.58 g NaH2PO4·H2O.

[0453] 10 mM DTPA (vaponectin) solution: One liter contains 3.90 g DTPA and 1.20 g NaOH.

[0454] 25 mM His / His-HCl buffer (pH 5.5): One liter contains 0.90 g L-histamine and 4.04 g L-histamine hydrochloride monohydrate.

[0455] Antibody preparation: 452 mg of lyophilized trastuzumab powder was dissolved in 22 mL of purified water. The obtained antibody solution was dialyzed for 4 cycles using an ultrafiltration tube (30KD) with 50 mM binding buffer to obtain an antibody concentration of 8.63 mg / mL (extinction coefficient ε280 = 213380 M-1cm-1 for trastuzumab).

[0456] Antibody reduction: Add 6.2 mL of 50 mM binding buffer to a tube containing 12.2 mL (105 mg, 0.000724 mmol trastuzumab) of the trastuzumab solution prepared above, followed by 579.2 μl of TCEP (10 mM) and 2.1 mL of 10 mM DTPA. Place the tube in a hot mixer and incubate the reduction reaction at 25°C for 2 hours.

[0457] Binding between antibody and payload: Add MB-2 (7.45 mg, 0.00882 mmol) in DMSO (1.76 mL) to the above trastuzumab reduction solution. Place the tube in a hot mixer and run the binding reaction at 25°C for 1 hour.

[0458] Purification: The above binding reaction solution was purified for 6 cycles using an ultrafiltration tube (30KD) with 25 mM His / His-HCl formulation buffer to obtain 5.5 mL (15.1 mg / mL, antibody yield = 83 mg, yield % = 79%) of MB-2a in formulation buffer.

[0459] Physicochemical characterization of MB-2a (using extinction coefficients ε280 = 4546 M⁻¹cm⁻¹ and ε360 = 17513 M⁻¹cm⁻¹ of the effective load) (Table 1): Table 1 Analysis Project method result monomer content SEC-HPLC 99.4% DAR HIC-HPLC DAR = 7.9 D6 = 5.9% D8 = 94.1% concentration UV-Vis mass concentration 15.1 mg / ml molar concentration Antibody 98.9 μmol / L Payload 797.3 μmol / L

[0460] General preparation procedure for trastuzumab-drug conjugate MB-3a

[0461] 50 mM binding buffer (pH 7.4): One liter contains 6.86 g Na2HPO4·2H2O and 1.58 g NaH2PO4·H2O.

[0462] 10 mM DTPA (pentetic acid) solution: One liter contains 3.90 g DTPA and 1.20 g NaOH.

[0463] 25 mM His / His-HCl buffer (pH 5.5): One liter contains 0.90 g L-histidine and 4.04 g L-histidine monohydrate.

[0464] Antibody preparation: 452 mg of lyophilized trastuzumab powder was dissolved in 22 mL of purified water. The obtained antibody solution was dialyzed for 4 cycles using an ultrafiltration tube (30KD) with 50 mM binding buffer to obtain an antibody concentration of 8.63 mg / mL (extinction coefficient ε280 = 213380 M-1cm-1 for trastuzumab).

[0465] Antibody reduction: Add 6.2 mL of 50 mM binding buffer to a tube containing 12.2 mL (105 mg, 0.000724 mmol trastuzumab) of the trastuzumab solution prepared above, followed by 579.2 μl of TCEP (10 mM) and 2.1 mL of 10 mM DTPA. Place the tube in a hot mixer and incubate the reduction reaction at 25°C for 2 hours.

[0466] Binding between antibody and payload: Add MB-3 (10.02 mg, 0.00886 mmol) in DMSO (1.77 mL) to the above trastuzumab reduction solution. Place the tube in a hot mixer and run the binding reaction at 25°C for 1 hour.

[0467] Purification: The above binding reaction solution was purified for 6 cycles using an ultrafiltration tube (30KD) with 25 mM His / His-HCl formulation buffer to obtain 6.2 mL (14.6 mg / mL, antibody yield = 90.5 mg, yield % = 86%) of MB-3a in formulation buffer.

[0468] Physicochemical characterization of MB-3a (using extinction coefficients ε280 = 4546 M⁻¹cm⁻¹ and ε360 = 17513 M⁻¹cm⁻¹ of the effective load) (Table 2): Table 2 Analysis Project method result monomer content SEC-HPLC 98.4% DAR HIC-HPLC DAR = 8 D8 = 100% concentration UV-Vis mass concentration 14.6 mg / ml molar concentration Antibody 94.8 μmol / L Payload 772.5 μmol / L Example 5. In vitro analysis of toxins and ADCs

[0469] 175 µL of cell suspension was dispensed into 96-well plates at a concentration of 1500 cells / well and incubated for 24 hours in a humidified incubator (37°C, 5% CO2). For antibody blockade, cells were cultured (15000 cells / mL) with 2×10⁻⁶ M trastuzumab (final concentration 1 µM). 25 µL of different concentrations of the compound were added to the cell culture medium (fetal bovine serum, Invitrogen) in the plates as a 5× solution and incubated for 120 hours. CCK-8 was thawed on the workbench or in a 37°C water bath, and 10 µL of CCK-8 was added to each well of the culture plate (avoiding air bubbles as they would interfere with OD readings), followed by further incubation for 1–4 hours. The absorbance at 450 nm was measured using a SpectraMax i3x microplate reader, and the cell inhibition rate was calculated. IC50 curves and IC50 values ​​were generated using GraphPad Prism software.

[0470] The results of the in vitro cytotoxicity analysis of the toxin (the expected metabolite of the ADC) are summarized in Table 3 and Figures 1A-1F below. The cytotoxicity of metabolite MB-1 in multiple cell lines was comparable to that of DXd, a metabolite of DS-8201a (Enhertu). However, in the intermediate Her-2 expression and trastuzumab-resistant cell line JIMT-1, MB-1 was ten times more effective than DXd. Table 3 compound Cell line (IC) 50 (nM) SK-BR-3 MCF-7 NCI-N87 SK-OV-3 MDA-MB-468 JIMT-1 OVCAR-3 0.77 0.37 1.3 1.0 0.42 0.78 0.10 >30 >30 >30 >30 1.9 0.57 3.6 4.7 0.57 8.7 0.20

[0471] Due to the presence of a polyol moiety in the linker, the active load MB-3 (medotecan) is more hydrophilic than the active loads of MB-2 and Daiichi (deruxtecan). Therefore, MB-3 is more reactive with disulfide-reduced trastuzumab, yielding MB-3a (trastuzumab-medotecan), a homogeneous ADC with a DAR value of 8. In contrast, MB-2a and DS-8201a (trastuzumab-deruxtecan) both contain a small percentage of DAR6 species, resulting in lower reactivity of their active loads to the reducing antibody. The HIC-HPLC spectra of the ADCs are shown in Figures 2A-2C. The hydrophilic active load MB-3 is more reactive in the binding reaction, yielding MB-3a as a homogeneous ADC. The active loads MB-2MB-3 incorporated into ADCs MB-2a and MB-3a, respectively, were designed to give the ADCs better bystander activity than DS-8201a (Enhertu).

[0472] The results of the in vitro cytotoxicity analysis of ADCs are summarized in Table 4 and Figures 3A-3D below. In addition to illustrative Formula III compounds such as MB-2a and MB-3a, the activities of trastuzumab and the ADC trastuzumab derutecan (DS-8201a) were evaluated for comparison. As shown in Table 4 and Figures 3A-3D, the trastuzumab ADCs MB-2a and MB-3a exhibited the same potency as DS-8201a in the Her2-high expression cell line NCI-N87 (Figure 3A). However, when the Her2 antigen was blocked with trastuzumab, the cell growth inhibitory ability of the ADCs decreased (Figure 3B). Furthermore, the ADCs were ineffective in the Her2-negative cell line MDA-MB-468 (Figure 3D), indicating the specificity of the ADCs for Her2-expressing cells. Although the ADCs were not sensitive to JIMT-1 cells with moderate Her2 expression levels in in vitro analyses, MB-2a and MB-3a were still relatively more effective than DS-8201a in this cell line (Figure 3C). Table 4 compound Cell line (IC) 50 (nM) NCI-N87 NCI-N87 Trastuzumab blocking JIMT-1 MDA-MB-468 SK-Br-3 Trastuzumab 495 0.56 MB-2a 11.5 37.8 3773 > 1000 MB-3a 11.1 36.4 1815 > 1000 0.12 DS-8201a 11.3 29 6496 > 1000 0.09

[0473] In the stability study of the ADC in the formulation buffer, MB-2a, MB-3a, and DS-8201a were all stable at room temperature. The DAR value remained unchanged at 25°C and 37°C, indicating no effective load detachment from the antibody. MB-2a and DS-8201a formulations showed an increase in both high molecular weight (HMW) and low molecular weight (LMW) species over time at 37°C. Only MB-3a showed a slight increase in LMW species after 6 days at 37°C. MB-3a was more stable than MB-2a and DS-8201a at higher temperatures due to its more hydrophilic effective load. Stability data are shown in Table 5. Table 5 ADC temperature time (sky) SEC(%) HIC DAR HMW monomer LMW MB-2a 25℃ 0 0.6 99.4 / 7.55 1 0.7 99.3 / 7.52 3 1.0 98.4 0.6 7.53 37℃ 3 0.7 91.3 8 7.56 6 3.9 86.0 9.1 7.51 MB-3a 25℃ 0 0.7 99.3 / 8 1 0.7 99.1 0.2 8 3 0.7 99.1 0.2 8 37℃ 3 0.8 95.0 4.2 8 6 0.9 94.2 4.9 8 DS-8201a 25℃ 0 0.9 99.1 / 7.55 1 1.0 99.0 / 7.55 3 0.8 99.2 / 7.57 37℃ 3 0.8 94.9 4.3 7.58 6 3.7 86.6 9.7 7.58 Example 6. In vivo efficacy of ADC in the NCI-N87 CDX model

[0474] NCI-N87 tumor cells (5 × 10⁶) were subcutaneously inoculated into the right ventral region of female Balb / c-Nude mice from Vital Rivers. These cells were mixed with Matrigel (50:50) in 0.2 mL PBS for tumor development. Animals were randomly assigned to groups on day 6 post-inoculation, when the average tumor volume reached approximately 160 mm³, and then treatment was initiated for efficacy studies. Each group contained 8 mice. Test and control drugs were administered to tumor-bearing mice via the tail vein at a volume of 5 mL / kg.

[0475] The size of the tumor was measured twice a week in two dimensions using calipers, and the volume was expressed in mm3 using the following formula: V = 0.5 a × b2, where a and b are the long and short dimensions of the tumor, respectively. The results are expressed as mean and standard error (mean ± SEM).

[0476] Statistical analysis: Two-way ANOVA was performed to compare tumor volume between the two groups. All data were analyzed using GraphPad Prism 6.0, and P < 0.05 was considered statistically significant. Statistical analysis and biological observations were both considered.

[0477] Tumor growth inhibition: The T / C value was calculated using tumor size. The relative tumor proliferation rate T / C (%) was calculated using the following formula: T / C (%) = (Ti / T0) / (Vi / V0) × 100%. Relative tumor growth inhibition was calculated using the following formula: TGI (%) = [1 - (Ti / T0) / (Vi / V0)] × 100%. Ti refers to the mean tumor volume of the treatment group measured at each indicated time point after treatment; T0 refers to the tumor volume of the treatment group at the time of grouping; Vi refers to the mean tumor volume of the mediator control group measured at each indicated time point after treatment; V0 refers to the tumor volume of the mediator control group at the time of grouping. If T / C > 40%, there is no efficacy; if T / C < 40% and p < 0.05, there is tumor inhibition.

[0478] The antitumor effects of ADCs in the NCI-N87 CDX model are illustrated in Figure 4 and Table 6. As shown in Figure 4, both MB-2a (1 mg / kg and 4 mg / kg doses) and MB-3a (1 mg / kg and 4 mg / kg doses) exhibited strong antitumor effects and were more effective than DS-8201a (Enhertu). Table 6 Model ADC dose (mg / Kg, single IV) fade TGI (%) (Day 23) Commentary part completely NCI-N87 Mediator N / A --- --- --- --- Trastuzumab 4 0 / 8 0 / 8 16.8 Inactive MB-2a 0.25 0 / 8 0 / 8 12.0 Inactive MB-3a 0.25 0 / 8 0 / 8 28.1 Inactive DS-8201a 0.25 0 / 8 0 / 8 24.7 Inactive MB-2a 1 2 / 8 0 / 8 75.5 active MB-3a 1 3 / 8 0 / 8 82.4 Highly active DS-8201a 1 0 / 8 0 / 8 52.2 active MB-2a 4 5 / 8 3 / 8 97.1 Highly active MB-3a 4 5 / 8 3 / 8 97.6 Highly active DS-8201a 4 8 / 8 0 / 8 97.3 Highly active Example 7. In vivo efficacy of ADC in the JIMT-1 CDX model

[0479] JIMT-1 tumor cells (1 × 10⁷) were subcutaneously inoculated into the right ventral region of each Scid-Beige mouse (from Shanghai Lingchang Biotech). These cells were mixed with Matrigel (50:50) in 0.2 mL PBS for tumor development. Animals were randomly assigned to groups on day 6 post-inoculation, when the average tumor volume reached approximately 175 mm³, and then treatment was initiated for efficacy studies. Each group contained 8 mice. Test and control drugs were administered to tumor-bearing mice via the tail vein at a volume of 5 mL / kg.

[0480] The size of the tumor was measured twice a week in two dimensions using calipers, and the volume was expressed in mm3 using the following formula: V = 0.5 a × b2, where a and b are the long and short dimensions of the tumor, respectively. The results are expressed as mean and standard error (mean ± SEM).

[0481] Statistical analysis: Two-way ANOVA was performed to compare tumor volume between the two groups. All data were analyzed using GraphPad Prism 6.0 and P < 0.05 was considered statistically significant. Statistical analysis and biological observations were both considered.

[0482] Tumor growth inhibition: The T / C value was calculated using tumor size. The relative tumor proliferation rate T / C (%) was calculated using the following formula: T / C (%) = (Ti / T0) / (Vi / V0) × 100%. Relative tumor growth inhibition was calculated using the following formula: TGI (%) = [1 - (Ti / T0) / (Vi / V0)] × 100%. Ti refers to the mean tumor volume of the treatment group measured at each indicated time point after treatment; T0 refers to the tumor volume of the treatment group at the time of grouping; Vi refers to the mean tumor volume of the mediator control group measured at each indicated time point after treatment; V0 refers to the tumor volume of the mediator control group at the time of grouping. If T / C > 40%, there is no efficacy; if T / C < 40% and p < 0.05, there is tumor inhibition.

[0483] The antitumor effects of ADCs in the JIMT-1 CDX model are illustrated in Figures 5-8 and Table 7. Figure 5 illustrates the effects of antibody-drug conjugates (ADCs) at three different doses in the JIMT-1 CDX model. In this study, all three doses of MB-2a and MB-3a studied showed significant antitumor effects. The different doses studied in these experiments are also illustrated in Figure 6 (2.5 mg / kg IV single dose), Figure 7 (5 mg / kb IV single dose), and Figure 8 (10 mg / kg IV single dose). Table 7 Model ADC dose (mg / Kg, single IV) fade TGI (%) (Day 27) Commentary part completely JIMT-1 Mediator N / A --- --- --- --- Trastuzumab 10 0 / 8 0 / 8 22.5 Inactive MB-2a 2.5 5 / 8 0 / 8 83.9 Highly active MB-3a 2.5 3 / 8 0 / 8 76.2 active DS-8201a 2.5 1 / 8 0 / 8 66.3 active MB-2a 5 6 / 8 0 / 8 85.1 Highly active MB-3a 5 7 / 8 0 / 8 88.4 Highly active DS-8201a 5 4 / 8 0 / 8 78.4 active MB-2a 10 8 / 8 0 / 8 90.9 Highly active MB-3a 10 6 / 8 1 / 8 90.9 Highly active DS-8201a 10 7 / 8 0 / 8 87.5 Highly active

[0484] All publications and patent applications cited in this specification are incorporated herein by reference in their entirety for all purposes, as if specifically and individually indicated that each individual publication or patent application is incorporated by reference, and as if each reference were fully described in its entirety. Although the invention has been described in considerable detail by way of illustration and example for purposes of clarity, it will be apparent to those skilled in the art, based on the teachings of this invention, that certain changes and modifications may be made without departing from the spirit or scope of the appended claims. [Simplified Explanation of the Diagram]

[0057] Figures 1A-1F illustrate exemplary results of the in vitro cell growth inhibitory activity of ADC metabolites in the following cell lines: NCI-N87 (Figure 1A); JIMT-1 (Figure 1B); MDA-MB-468 (Figure 1C); SK-OV-3 (Figure 1D); SK-Br-3 (Figure 1E); and MCF-7 (Figure 1F). DXd is a metabolite of DS-8201a (Enhertu) in the cells, and MB-1 is a metabolite of some of the ADCs described herein.

[0058] Figures 2A-2C depict the HIC-HPLC of ADC MB-2a (Figure 2A); MB-3a (Figure 2B) and DS-8201a (Enhertu) (Figure 2C).

[0059] Figures 3A-3D show exemplary results of the in vitro cell growth inhibition activity of the ADC in the following: Her2 highly expressed cell line NCI-N87 (Figure 3A); NCI-N87 with trastuzumab blockade (Figure 3B); JIMT-1, which has moderate Her2 expression and is a trastuzumab resistant cell line (Figure 3C); and Her2 negative cell line MDA-MB-468 (Figure 3D).

[0060] Figure 4 illustrates the tumor growth inhibition effect of antibody-drug conjugates (ADCs) in the NCI-N87 CDX model. MB-2a and MB-3a are ADCs covered by this invention and were studied together with a mediator control, trastuzumab, and ADC DS-8201a (Enhertu). As shown in this figure, MB-2a (1 mg / kg and 4 mg / kg doses) and MB-3a (1 mg / kg and 4 mg / kg doses) exhibited strong antitumor activity. For comparison, DS-8201a (Enhertu) was included in the study.

[0061] Figure 5 illustrates the tumor growth inhibition effect of antibody-drug conjugates (ADCs) in the JIMT-1 CDX model. In this study, all three doses of MB-2a and MB-3a (trastuzumab-medotecan) studied showed significant antitumor effects. For comparison, DS-8201a (Enhertu) was included in the study.

[0062] Figure 6 illustrates a series of data from Figure 5, showing the antitumor effects of a single dose of MB-2a and MB-3a at 2.5 mg / kg IV.

[0063] Figure 7 illustrates a series of data from Figure 5, showing the antitumor effects of a single dose of 5 mg / kg IV of MB-2a and MB-3a.

[0064] Figure 8 illustrates a series of data from Figure 5, showing the antitumor effects of a single dose of MB-2a and MB-3a at 10 mg / kg IV.

[0065] Figure 9 illustrates the comparison of the hydrophilicity of MB-3a, DS-8201a and trastuzumab.

[0066] Figure 10 illustrates the antigen-binding affinity of MB-3a, DS-8201a and trastuzumab.

[0067] Figure 11 illustrates the bystander effect of MB-3a, DS-8201a and T-DM1. For each tested compound, the figure shows the Dan-G-Luc activity in the SK-BR-3 and DAN-G-Luc co-culture (left) and the Dan-G-Luc activity in the DAN-G-Luc cells alone (right). [Sequence List]

[0485] <![CDATA[ <110> Hong Kong-based MEDIBOSTON LIMITED <![CDATA[ <120> Anti-HER2 antibody-drug conjugates and their uses <![CDATA[ <130> MDB-002TW1]]> <![CDATA[ <140> 111107091]]> <![CDATA[ <141> 2022-02-25 <![CDATA[ <150> 63 / 153,530]]> <![CDATA[ <151> 2021-02-25 <![CDATA[ <160> 19 ]]> <![CDATA[ <170> PatentIn version 3.5]]> <![CDATA[ <210> 1]]> <![CDATA[ <211> 450]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial sequence <![CDATA[ <220> ]]> <![CDATA[ <223> Description of artificial sequences: synthetic peptides]]> <![CDATA[ <400> 1]]> Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr 20 25 30 Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val SerSer Ala Ser Thr Lys Gly Pro Ser Val 115 120 125 Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala 130 135 140 Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser 145 150 155 160 Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val 165 170 175 Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro 180 185 190 Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys 195 200 205 Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp 210 215 220 Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly 225 230 235 240 Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 245 250 255 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu 260 265 270 Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 275 280 285 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 290 295 300 Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys 305 310 315 320 Glu Tyr Lys Cys Lys Val SerAsn Lys Ala Leu Pro Ala Pro Ile Glu 325 330 335 Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr 340 345 350 Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu 355 360 365 Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 370 375 380 Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 385 390 395 400 Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp 405 410 415 Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His 420 425 430 Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 445 Gly Lys 450 <![CDATA[<210> 2]]> <![CDATA[<211> 214]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Artificial Sequence]]> <![CDATA[<220>]]> <![CDATA[<223> Description of Artificial Sequence: Synthetic polypeptide]]> <![CDATA[<400> 2]]> Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Asn Thr Ala 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ser Ala Ser Phe Leu TyrSer Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Pro 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 <![CDATA[<210> 3]]> <![CDATA[<211> 10]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Artificial Sequence]]> <![CDATA[<220>]]> <![CDATA[<223> Description of Artificial Sequence: Synthetic Peptide]]> <![CDATA[<400> 3]]> Gly Phe Asn Ile Lys Asp Thr Tyr Ile His 1 5 10 <![CDATA[<210> 4]]> <![CDATA[<211> 17]]><![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial sequence <![CDATA[ <220> ]]> <![CDATA[ <223> Description of artificial sequences: synthetic peptides]]> <![CDATA[ <400> 4]]> Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly <![CDATA[ <210> 5]]> <![CDATA[ <211> 11]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial sequence <![CDATA[ <220> ]]> <![CDATA[ <223> Description of artificial sequences: synthetic peptides]]> <![CDATA[ <400> 5]]> Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr 1 5 10 <![CDATA[ <210> 6]]> <![CDATA[ <211> 11]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial sequence <![CDATA[ <220> ]]> <![CDATA[ <223> Description of artificial sequences: synthetic peptides]]> <![CDATA[ <400> 6]]> Arg Ala Ser Gln Asp Val Asn Thr Ala Val Ala 1 5 10 <![CDATA[ <210> 7]]> <![CDATA[ <211> 7]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial sequence <![CDATA[ <220> ]]> <![CDATA[ <223> Description of artificial sequences: synthetic peptides]]> <![CDATA[ <400> 7]]> Ser Ala Ser Phe Leu Tyr Ser 1 5 <![CDATA[ <210> 8]]> <![CDATA[ <211> 9]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial sequence <![CDATA[ <220> ]]> <![CDATA[ <223> Description of artificial sequences: synthetic peptides]]> <![CDATA[ <400> 8]]> Gln Gln His Tyr Thr Thr Pro Pro Thr 1 5 <![CDATA[ <210> 9]] ><![CDATA[ <211> 6]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial sequence <![CDATA[ <220> ]]> <![CDATA[ <223> Description of artificial sequences: Synthetic 6xHis tags]]> <![CDATA[ <400> 9]]> His His His His His 1 5 <![CDATA[ <210> 10]]> <![CDATA[ <211> 4]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial sequence <![CDATA[ <220> ]]> <![CDATA[ <223> Description of artificial sequences: synthetic peptides]]> <![CDATA[ <400> 10]]> Ala Val Ala Gly 1 <![CDATA[ <210> 11]]> <![CDATA[ <211> 4]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial sequence <![CDATA[ <220> ]]> <![CDATA[ <223> Description of artificial sequences: synthetic peptides]]> <![CDATA[ <400> 11]]> Gly Phe Gly Gly 1 <![CDATA[ <210> 12]]> <![CDATA[ <211> 4]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial sequence <![CDATA[ <220> ]]> <![CDATA[ <223> Description of artificial sequences: synthetic peptides]]> <![CDATA[ <400> 12]]> Gly Gly Phe Gly 1 <![CDATA[ <210> 13]]> <![CDATA[ <211> 4]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial sequence <![CDATA[ <220> ]]> <![CDATA[ <223> Description of artificial sequences: synthetic peptides]]> <![CDATA[ <400> 13]]> Ala Val Gly Gly 1 <![CDATA[ <210> 14]]> <![CDATA[ <211> 4]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial sequence <![CDATA[ <220> ]]> <![CDATA[ <223> Description of artificial sequences: synthetic peptides ><![CDATA[ <400> 14]]> Ala Ala Ala Ala 1 <![CDATA[ <210> 15]]> <![CDATA[ <211> 4]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial sequence <![CDATA[ <220> ]]> <![CDATA[ <223> Description of artificial sequences: synthetic peptides]]> <![CDATA[ <400> 15]]> Ala Val Ala Ala 1 <![CDATA[ <210> 16]]> <![CDATA[ <211> 4]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial sequence <![CDATA[ <220> ]]> <![CDATA[ <223> Description of artificial sequences: synthetic peptides]]> <![CDATA[ <400> 16]]> Ala Leu Ala Leu 1 <![CDATA[ <210> 17]]> <![CDATA[ <211> 4]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial sequence <![CDATA[ <220> ]]> <![CDATA[ <223> Description of artificial sequences: synthetic peptides]]> <![CDATA[ <400> 17]]> Leu Ala Leu Ala 1 <![CDATA[ <210> 18]]> <![CDATA[ <211> 4]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial sequence <![CDATA[ <220> ]]> <![CDATA[ <223> Description of artificial sequences: synthetic peptides]]> <![CDATA[ <400> 18]]> Gly Phe Leu Gly 1 <![CDATA[ <210> 19]]> <![CDATA[ <211> 4]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial sequence <![CDATA[ <220> ]]> <![CDATA[ <223> Description of artificial sequences: synthetic peptides]]> <![CDATA[ <400> 19]]> Gly Leu Phe Gly 1

Claims

1. A compound having the structure of formula PL-A or a pharmaceutically acceptable salt thereof, (PL-A), wherein E is a peptide comprising 2 to 8 amino acids; wherein E is substituted with one or more polyols as appropriate; and wherein the N-terminus of the peptide is covalently linked to Z'; Z' is -C(=O)-L1-Y', or; wherein m represents an integer from 1 to 10 and * indicates a site covalently linked to the C; L1 is -(C1-C10 alkyl)-, -CH2CH2(OCH2CH2)nN(R1)C(=O)-L2-* or -CH2(OCH2CH2)nN(R1)C(=O)-L2-*; wherein n represents an integer from 1 to 10; and wherein * indicates a site covalently linked to the Y'; L2 is -(C1-C10 alkyl)-; R1 is -H or -CH3; Y' is a group formed by the reaction of an electrophilic group with a reactive nucleophilic group present on the cell binder C; and p is the drug-to-antibody ratio (DAR), which is an average of 2-10; and C is an anti-HER2 antibody or its antigen-binding fragment, which includes the following CDRs: HCDR1, which has the sequence GFNIKDTYIH (SEQ ID NO:3); HCDR2, which has the sequence RIYPTNGYTRYADSVKG (SEQ ID NO:4); HCDR3, which has the sequence WGGDGFYAMDY (SEQ ID NO:5); LCDR1, which has the sequence RASQDVNTAVA (SEQ ID NO:6); LCDR2, which has the sequence SASFLYS (SEQ ID NO:7); and LCDR3, which has the sequence QQHYTTPPT (SEQ ID NO:8).

2. The compound of claim 1 or a pharmaceutically acceptable salt thereof, wherein C is a binder comprising a heavy chain comprising an amino acid sequence having at least 85%, 90%, or 95% similarity to SEQ ID NO:1; and a light chain comprising an amino acid sequence having at least 85%, 90%, or 95% similarity to SEQ ID NO:

2.

3. The compound of claim 1 or 2 or a pharmaceutically acceptable salt thereof, wherein C is a binder comprising the heavy chain amino acid sequence of SEQ ID NO:1 and the light chain amino acid sequence of SEQ ID NO:

2.

4. A compound of claim 1 or 2 or a pharmaceutically acceptable salt thereof, wherein C is a binder, which is an antibody or an antigen-binding fragment thereof.

5. The compound of claim 1 or 2 or a pharmaceutically acceptable salt thereof, wherein C is a conjugate, which is trastuzumab (Herceptin), trastuzumab-dkst (Ogivri), trastuzumab-pkrb (Herzuma), trastuzumab-dttb (Ontruzant), trastuzumab-qyyp (Trazimera), or trastuzumab-anns (Kanjinti).

6. A compound of claim 1 or 2 or a pharmaceutically acceptable salt thereof, wherein L1 is -(C1-C10 alkyl)-.

7. The compound of claim 1 or 2 or a pharmaceutically acceptable salt thereof, wherein L1 is -CH2CH2(OCH2CH2)nN(R1)C(=O)-L2-* or -CH2(OCH2CH2)nN(R1)C(=O)-L2-*; wherein n represents an integer from 1 to 10; and wherein * indicates a site covalently linked to Y'.

8. A compound of claim 1 or 2 or a pharmaceutically acceptable salt thereof, wherein L1 is -CH2CH2CH2CH2CH2-, -CH2CH2-, -CH2-, -CH2CH2OCH2CH2OCH2CH2NHC(=O)CH2CH2-* or -CH2OCH2CH2OCH2CH2NHC(=O)CH2CH2-*; wherein * indicates a site covalently linked to Y'.

9. A compound of claim 1 or 2 or a pharmaceutically acceptable salt thereof, wherein Y' is formed from a Michael acceptor group, a succinimide, an epoxide, or a halogen.

10. A compound of claim 1 or 2 or a pharmaceutically acceptable salt thereof, wherein Y' is formed of: , , or; wherein R2 and R3 are each independently -H or C1-C3 alkyl.

11. A compound of claim 1 or 2 or a pharmaceutically acceptable salt thereof, wherein Y' is , , or ; wherein R2 and R3 are each independently -H or C1-C3 alkyl, and * indicates a site covalently linked to the C.

12. The compound of claim 1 or 2 or its pharmaceutically acceptable salt, wherein Z' is formed from: , , , or.

13. A compound of claim 1 or 2 or a pharmaceutically acceptable salt thereof, wherein Z' is: , , , , , or; wherein * indicates a site covalently linked to C.

14. The compound of claim 1 or a pharmaceutically acceptable salt thereof, wherein Z' is; wherein * indicates a covalent connection with C, and n is an integer from 2 to 10.

15. A compound of claim 14 or a pharmaceutically acceptable salt thereof, wherein Z' is; wherein * indicates a covalent connection with C.

16. A compound of claim 1 or 2 or a pharmaceutically acceptable salt thereof, wherein E is a peptide of 2, 3, 4 or 5 amino acids.

17. The compound of claim 16 or a pharmaceutically acceptable salt thereof, wherein E is a peptide of 2, 3 or 4 amino acids.

18. The compound of claim 16 or a pharmaceutically acceptable salt thereof, wherein each amino acid in the peptide is an L-amino acid, or wherein at least one amino acid in the peptide is a D-amino acid.

19. The compound of claim 1 or 2 or a pharmaceutically acceptable salt thereof, wherein E comprises one or more amino acids selected from glycine, alanine, valine, glutamic acid, glutamic acid, phenylalanine and leucine, and wherein the glutamic acid or glutamic acid is, where appropriate, substituted with a polyol.

20. The compound of claim 19 or a pharmaceutically acceptable salt thereof, wherein E comprises an amino acid selected from glycine, alanine, valine, glutamic acid, glutamic acid, phenylalanine and leucine, and wherein the glutamic acid or glutamic acid is, where appropriate, substituted with a polyol.

21. A compound of claim 1 or 2 or a pharmaceutically acceptable salt thereof, wherein E comprises an amino acid having the following structure: , wherein R4 is -H or C1-C6 alkyl.

22. The compound of claim 21 or a pharmaceutically acceptable salt thereof, wherein E comprises an amino acid having the following structure:

23. The compound of claim 1 or 2 or a pharmaceutically acceptable salt thereof, wherein E is selected from the group consisting of: -Ala-Val-*, -Val-Ala-*, -Gly-Gly-*, -Val-Cit-*, -Cit-Val-*, -Leu-Ala-*, -Ala-Leu-*, -Leu-Cit-*, -Cit-Leu-*, -Leu-Ala-*, -Ala-Leu-*, -Lys-Lys-*, -Ala-Lys-*, -Lys-Ala-*, -Val-Lys-*, -Lys-Val-*, -Tyr-Arg-*, -Arg-Tyr-*, -Arg-Arg-*, -Ala-Ala-*, -Phe-Lys-*, -Lys-Phe-*, -Thr- Thr-*, -Thr-Met-*, -Met-Thr-*, -Met-Tyr-*, -Tyr-Met-*, -Phe-Gln-*, -Gln-Phe-*, -Gly-Se r-*, -Leu-Gln-*, -Gln-Leu-*, -Ser-Ala-*, -Ser-Gly-*, -Val-Thr-*, -Thr-Val-*, -Val-Gln- *, -Ser-Val-*, -Val-Ser-*, -Ala-Met-*, -Met-Ala-*, -Val-Arg-*, -Arg-Val-*, -Phe-Ala-*, -Ala-Phe-*, -Cit-Val-*, -Gln-Val-*, -Phe-Arg-*, -Arg-Phe-*, -Ala-Ala-Ala-*, -Gly-Gly- Gly-*, -Ala-Val-Ala-*, -Gly-Val-Gly-*, -Ala-Val-Gly-*, -Gly-Phe-Lys-*, -Lys-Phe-Gly- *, -Leu-Ala-Leu-*, -Val-Ala-Leu-*, -Leu-Ala-Val-*, -Val-Ala-Val-*, -Ala-Val-Ala-Gly-* (SEQ ID NO: 10), -Gly-Phe-Gly-Gly-* (SEQ ID NO: 11), -Gly-Gly-Phe-Gly-* (SEQ ID NO: 12), -Ala-Val-Gly-Gly-* (SEQ ID NO: 13), -Ala-Ala-Ala-Ala-* (SEQ ID NO: 14), -Ala-Val-Ala-Ala-* (SEQ ID NO: 15), -Ala-Leu-Ala-Leu-* (SEQ ID NO: 16),-Leu-Ala-Leu-Ala-* (SEQ ID NO: 17), -Gly-Phe-Leu-Gly-* (SEQ ID NO: 18), and -Gly-Leu-Phe-Gly-* (SEQ ID NO: 19), wherein * indicates covalently linked to the N-terminus of the Z' peptide.

24. The compound of claim 23 or a pharmaceutically acceptable salt thereof, wherein E is selected from the group consisting of: -L-Ala-D-Val-*, -L-Val-D-Ala-*, -L-Val-D-Lys-*, -L-Val-D-Arg-*, -L-Val-D-Cit-*, -L-Val-D-Arg-*, -L-Val-D-Cit-*, -L-Val-D-Lys-*, -L-Val-D -Arg-*, -L-Arg-D-Arg-*, -L-Ala-D-Ala-*, -L-Ala-D-Lys-*, -L-Ala-D-Arg-*, -L-Ala-D-Ala-L-Ala-*, -L-Ala-D-Val-L-Ala-*, -L-Ala-D-Ala-Gly-* and -L-Ala-D-Val-Gly-*, where * indicates covalently linked to the N-terminus of the peptide of Z'.

25. A compound of claim 1 or 2 or a pharmaceutically acceptable salt thereof, wherein E comprises -(L-Val)-(L-Ala)-.

26. The compound of claim 25 or a pharmaceutically acceptable salt thereof, wherein E further comprises an amino acid having the following structure:

27. A compound of claim 1 or 2 or a pharmaceutically acceptable salt thereof, having one of the following structures, wherein * indicates covalently linked to the N-terminus of a Z' peptide: , , , or.

28. A compound of claim 1 or 2 or a pharmaceutically acceptable salt thereof, wherein it is formed of one of the following structures: , , , or.

29. A compound of claim 1 or 2 or a pharmaceutically acceptable salt thereof, wherein it is one of the following structures, wherein * indicates the connection point with C: , , , , or.

30. The compound of claim 1 or a pharmaceutically acceptable salt thereof, wherein the compound is one of the following structures, or a pharmaceutically acceptable salt thereof: , , , , , and wherein p is 2-10, 4-8 or 7-8.

31. The compound of claim 30 or a pharmaceutically acceptable salt thereof, wherein the compound is one of the following structures, or a pharmaceutically acceptable salt thereof.

32. A compound or a pharmaceutically acceptable salt thereof of any one of claims 1, 30 and 31, wherein the compound has one of the following structures, or a pharmaceutically acceptable salt thereof: , and wherein p is 4-8 or 7-8.

33. The compound of claim 32 or a pharmaceutically acceptable salt thereof, wherein p is 7-8.

34. The compound of claim 33 or a pharmaceutically acceptable salt thereof, wherein p is 8.

35. A pharmaceutical composition comprising a compound of any one of claims 1 to 34 or a pharmaceutically acceptable salt thereof.

36. Use of any compound of claims 1 to 34 or a pharmaceutically acceptable salt thereof for the preparation of a medicament for treating cancer.

37. As claimed in claim 36, wherein the compound is or, or a pharmaceutically acceptable salt thereof.

38. As requested in item 37, where p is 4-8 or 7-8.

39. As requested in item 38, where p is 7-8.

40. As requested in item 39, where p is 8.

41. The use of any of claims 36 to 40, wherein the cancer is lung cancer, ovarian cancer, breast cancer or stomach cancer.

42. The use of any of claims 36 to 40, wherein the cancer is breast cancer.

43. The use of any of claims 36 to 40, wherein the cancer is stomach cancer.

44. The use of any of claims 36 to 40, wherein the cancer is lung cancer.

45. As used in claim 44, wherein the lung cancer is non-small cell lung cancer.

46. ​​The use of any of claims 36 to 40, wherein the cancer is ovarian cancer.

47. The use of any of claims 36 to 40, wherein the cancer is metastatic.

48. As requested in any of items 36 to 40, wherein the cancer is characterized by low Her2 expression.

49. As requested in any of items 36 to 40, wherein the cancer is characterized by intermediate Her2 manifestation.

50. Use of any one of claims 36 to 40, wherein the cancer is characterized by high Her2 expression.

51. The use of any of claims 36 to 40, wherein the cancer is trastuzumab resistant.

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