Anti-HER2 antibody-drug conjugates and uses thereof

Abstract

New anti-HER2 ADCs conjugated to camptothecin derivatives as therapeutic toxins are disclosed. The antitumor effects of ADCs of formula (PL-A) (e.g., compounds MB-2a and MB-3a (trastuzumab-meditecan)) make these compounds useful for the treatment of cell proliferative diseases such as cancer. JPEG2024509099000117.jpg3492

Description

[Background technology]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 63 / 153,530, filed February 25, 2021, the entirety of which is incorporated herein by reference. Sequence Listing

[0002] This application contains a Sequence Listing that has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. This ASCII copy, created on February 17, 2022, is entitled MDB-002WO_SLtxt and is 10,140 bytes in size.

[0003] HER2 (human epidermal growth factor receptor 2) has emerged as an important therapeutic target due to its involvement 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 in some gastric and gastroesophageal cancer cases. Because HER2 overexpression can be associated with a faster proliferation rate and poor prognosis, there remains a need for new effective anti-HER2 therapeutic agents. Summary of the Invention

[0004] Antibody drug conjugates (ADCs) have attracted great interest as a new class of therapeutics. For example, ADCs utilize monoclonal antibodies (mAbs) for targeted delivery of cytotoxic agents to tumor cells, thereby enabling the use of highly cytotoxic drugs that could not be used using traditional non-targeted modes. The design of ADCs, which typically feature the attachment of a cytotoxic agent to an antibody, typically via a linker, involves consideration of a variety of factors, including the presence of a conjugation handle on the agent for attachment to the linker, and linker technology to attach the agent to the antibody in a conditionally stable manner. Suboptimal design can result in reduced ADC potency, lack of immunological specificity of the conjugate, and increased toxicity due to non-specific release of the drug from the conjugate.

[0005] The present invention is based, at least in part, on the antitumor therapeutic effect of anti-HER2 ADCs conjugated to camptothecin derivatives as therapeutic toxins. The ADCs described herein are useful for the treatment of cell proliferative disorders such as cancer.

[0006] In an embodiment, the present invention provides a compound having the structure of formula PL-A: [ka] or a pharma- ceutically acceptable salt thereof, wherein E is a peptide comprising 2 to 8 amino acids, wherein E is optionally substituted with one or more polyols, and the N-terminus of the peptide is covalently linked to Z'; Z' is -C(=O)-L 1 -Y', [ka] In the formula, m represents an integer of 1 to 10, and * represents a site covalently bonded to the C, L 1 is -(C1-C 10 Alkylene)-, -CH2CH2(OCH2CH2) n N(R1)C(=O)-L 2 -*, or -CH2(OCH2CH2) n N(R1)C(=O)-L 2 -*, where n is an integer from 1 to 10, and * represents a site of covalent attachment to Y'; L 2 HA-(C1-C 10 alkylene)-, R1 is -H or -CH3, Y' is a group formed by reaction of an electrophilic group with a reactive nucleophilic group present on the cell-binding agent; p is the drug-to-antibody ratio (DAR), which is an average number between about 2 and 10; C is a HER2-targeting binding agent; a heavy chain comprising an amino acid sequence having at least 80% identity to SEQ ID NO:1; and a light chain comprising an amino acid sequence having at least 80% identity to SEQ ID NO:2.

[0007] In an embodiment, C is a heavy chain comprising an amino acid sequence having at least 85%, 90%, or 95% identity to SEQ ID NO:1; and a light chain comprising an amino acid sequence having at least 85%, 90%, 95% identity to SEQ ID NO:2.

[0008] In an embodiment, C is A heavy chain amino acid sequence of SEQ ID NO:1; and a 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) described herein.

[0010] In embodiments, C is a binding agent that is an antibody or an antigen-binding fragment thereof.

[0011] In embodiments, C is a binder that is trastuzumab (Herceptin), trastuzumab-dkst (Ogivri), trastuzumab-pkrb (Herzuma), trastuzumab-dttb (Ontruzant), trastuzumab-qyp (Trazimera), or trastuzumab-anns (Kanjinti).

[0012] In an embodiment, L 1 is -(C1-C 10 alkylene).

[0013] In an embodiment, L 1 is -CH2CH2(OCH2CH2) n N(R1)C(=O)-L 2 -* or -CH2(OCH2CH2) n N(R1)C(=O)-L 2-*, where n is an integer from 1 to 10, and where * represents the site of covalent bonding to Y'.

[0014] In an embodiment, L 1 is -CH2CH2CH2CH2CH2-, -CH2CH2-, -CH2-, -CH2CH2OCH2CH2OCH2CH2NHC(=O)CH2CH2-* or -CH2OCH2CH2OCH2CH2NHC(=O)CH2CH2-*, where * indicates the site of covalent attachment to Y'.

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

[0016] In embodiments, Y′ is [ka] is formed from In the formula, R 2 and R 3 are each independently -H or C1-C3 alkyl.

[0017] In embodiments, Y′ is [ka] and In the formula, R 2 and R 3 are each independently -H or C1-C3 alkyl, and * indicates the site of covalent bonding to the C.

[0018] In an embodiment, Z' is formed from: [ka]

[0019] In an embodiment, Z′ is [ka] and In the formula, * indicates the site of covalent attachment to C.

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

[0021] In an embodiment, Z' is [ka] where * indicates a covalent bond to C.

[0022] In an embodiment, Z' is [ka] where * indicates the site of covalent attachment to said C. In embodiments, m is 1. In embodiments, m is 2. In embodiments, m is 3. In embodiments, m is 4. In embodiments, m is 5. In embodiments, m is 6. In embodiments, m is 7. In embodiments, m is 8. In embodiments, m is 9. In embodiments, m is 10.

[0023] In an embodiment, Z' is [ka] where * indicates the site of covalent attachment to said C. In embodiments, m is 1. In embodiments, m is 2. In embodiments, m is 3. In embodiments, m is 4. In embodiments, m is 5. In embodiments, m is 6. In embodiments, m is 7. In embodiments, m is 8. In embodiments, m is 9. In embodiments, m is 10.

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

[0025] In an embodiment, E is a peptide of two, three, or four amino acids.

[0026] In 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 embodiments, E comprises one or more amino acids selected from glycine, alanine, valine, glutamine, glutamic acid, phenylalanine, and leucine, wherein the glutamine or glutamic acid is optionally substituted by a polyol.

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

[0029] In embodiments, E comprises an amino acid having the structure: [ka] In the formula, R 4 is -H or C1-C6 alkyl.

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

[0031] in embodiments、Eは、-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-Th r-*、-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-* * 、-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), - Z' is selected from the group consisting of 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 the N-terminus of the peptide covalently attached to Z'.

[0032] In embodiments, E is -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 the N-terminus of the peptide covalently attached to Z'.

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

[0034] In an embodiment, [ka] has one of the following structures, where * indicates the N-terminus of the peptide covalently attached to Z'. [ka]

[0035] In an embodiment, [ka] is formed from one of the following structures: [ka]

[0036] In an embodiment, [ka] is one of the following structures, where * indicates the point of attachment to C: [ka]

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

[0038] In embodiments, the compound has one of the following structures, or a pharma- ceutically acceptable salt thereof: [ka] In the formula, p is about 4 to 8, or 7 to 8.

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

[0040] In an embodiment, p is 8.

[0041] In another aspect, the invention features a pharmaceutical composition including any compound described herein, or a pharma- ceutically acceptable salt thereof.

[0042] In another aspect, the invention features a method of treating cancer, comprising administering to a subject in need thereof an effective amount of any compound described herein, or a pharma- ceutically acceptable salt thereof.

[0043] In an embodiment, the compound is [ka] or a pharma- ceutically acceptable salt thereof.

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

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

[0046] In an embodiment, p is 8.

[0047] In embodiments, the cancer is lung cancer, urothelial cancer, 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, colon cancer, rectal cancer, colorectal 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 an embodiment, the cancer is breast cancer.

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

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

[0051] In embodiments, the cancer is ovarian cancer.

[0052] In embodiments, the cancer is metastatic.

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

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

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

[0056] In embodiments, the cancer is trastuzumab-resistant. [Brief description of the drawings]

[0057] [Figure 1] Figures 1A-1F show exemplary results of in vitro cell growth inhibitory activity of ADC metabolites in NCI-N87 cell line (Figure 1A), JIMT-1 cell line (Figure 1B), MDA-MB-468 cell line (Figure 1C), SK-OV-3 cell line (Figure 1D), SK-Br-3 cell line (Figure 1E), and MCF-7 cell line (Figure 1F). DXd is an intracellular metabolite of DS-8201a (Enhertu), and MB-1 is a metabolite of certain ADCs described herein.

[0058] [Diagram 2] Figures 2A-2C show HIC-HPLC of ADCs MB-2a (Figure 2A), MB-3a (Figure 2B) and DS-8201a (Enhertu) (Figure 2C).

[0059] [Figure 3-1](FIG. 3A) Figures 3A-3D show exemplary results of in vitro cell growth inhibitory activity of ADCs in the Her2 high expressing cell line NCI-N87 (FIG. 3A), NCI-N87 with trastuzumab blockade (FIG. 3B), JIMT-1, a trastuzumab resistant cell line with intermediate Her2 expression (FIG. 3C), and the Her2 negative cell line MDA-MB-468 (FIG. 3D). (FIG. 3B) Ibid. [Figure 3-2] (Fig. 3C) Ibid. (Fig. 3D) Ibid.

[0060] [Figure 4] Figure 4 shows the tumor growth suppression effect of antibody drug conjugates (ADCs) in the NCI-N87 CDX model. MB-2a and MB-3a are ADCs encompassed by this formula and were studied along with vehicle control, trastuzumab, and the ADC DS-8201a (Enhertu). As shown in this figure, MB-2a (at doses 1 mg / kg and 4 mg / kg) and MB-3a (at doses 1 mg / kg and 4 mg / kg) demonstrated strong antitumor effects. DS-8201a (Enhertu) was included in the study for comparison.

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

[0062] [Figure 6] FIG. 6 shows a selection of data from FIG. 5 showing the antitumor efficacy of MB-2a and MB-3a using a single iv dose of 2.5 mg / kg.

[0063] [Figure 7] FIG. 7 shows a selection of data from FIG. 5 showing the antitumor efficacy of MB-2a and MB-3a using a single iv dose of 5 mg / kg.

[0064] [Figure 8] FIG. 8 shows a selection of data from FIG. 5 showing the antitumor efficacy of MB-2a and MB-3a using a single 10 mg / kg iv dose.

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

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

[0067] [Figure 11] Figure 11 shows the bystander effect of MB-3a, DS-8201a and T-DM1. For each test compound, the graph shows the Dan-G-Luc viability in co-cultures of SK-BR-3 and DAN-G-Luc (left) and in DAN-G-Luc cells alone (right). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0068] The present invention relates to an antibody drug conjugate comprising an anti-tumor agent conjugated to an anti-HER2 antibody via a linker structure moiety. Such compounds may be particularly useful for treating cancers (e.g., breast cancer and gastric cancer) that may be HER2 overexpressing and / or metastatic and / or trastuzumab resistant.

[0069] definition Unless otherwise stated, the following terms and phrases as used herein are intended to have the following meanings: When trade names are used herein, unless the context dictates otherwise, the trade names include formulations of the product, generic drugs, and active pharmaceutical ingredients of the trade name product.

[0070] As used herein, the term "antibody" refers to an agent that specifically binds to a particular antigen. In some embodiments, the term encompasses any polypeptide or polypeptide complex that contains sufficient immunoglobulin structural elements to confer specific binding. 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 sequence elements of humanized, primatized, chimeric, etc., as known in the art. In many embodiments, the term "antibody" is used to refer to one or more of the constructs or formats known or developed in the art to utilize the structural and functional features of antibodies in alternative presentations. For example, in embodiments, antibodies utilized in accordance with the present invention may be intact IgA, IgG, IgE or IgM antibodies, bi- or multispecific antibodies (such as, for example, Zybodies®), antibody fragments such as Fab fragments, Fab' fragments, F(ab')2 fragments, Fd' fragments, Fd fragments, and isolated CDRs or sets thereof, single chain Fv; polypeptide-Fc fusions, single domain antibodies (e.g., shark single domain antibodies, such as IgNAR or fragments thereof), cameloid antibodies, masked antibodies (e.g., Probodies®, Small Modular The formats are selected from, but are not limited to, ImmunoPharmaceuticals ("SMIPs™"), single chain or tandem diabodies (TandAb®), VHHs, Anticalins®, Nanobodies® minibodies, BiTEs®, ankyrin repeat proteins or DARPINs®, Avimers®, DARTs, TCR-like antibodies, Adnectins®, Affilins®, Trans-bodies®, Affibodies®, TrimerX®, MicroProteins, Fynomers®, Centyrins®, and KALBITORs®.In some embodiments, the antibody may lack covalent modifications (e.g., attachment of glycans) that it would have if produced naturally. In some embodiments, the antibody may contain covalent modifications (e.g., attachment of glycans, payloads (e.g., detectable moieties, therapeutic moieties, catalytic moieties, etc.), or other pendant groups (e.g., poly-ethylene glycol, etc.). In many embodiments, the antibody is or comprises a polypeptide whose amino acid sequence comprises one or more structural elements recognized by those skilled in the art as complementarity determining regions (CDRs), and in some embodiments, the antibody is or comprises a polypeptide whose amino acid sequence comprises at least one CDR (e.g., at least one heavy chain CDR and / or at least one light chain CDR) that is substantially identical to that found in a reference antibody. In some embodiments, the antibody agent is or comprises a polypeptide whose amino acid sequence comprises a structural element recognized by those skilled in the art as an immunoglobulin variable domain. In some embodiments, the antibody agent is a polypeptide protein having a binding domain that is homologous or largely homologous to an immunoglobulin binding domain.

[0071] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. The modifier "monoclonal" indicates the character of the antibody as it is obtained from a substantially homogeneous population of antibodies, but is not to be construed as requiring production of the antibody by any particular method.

[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 to be "human" notwithstanding that its amino acid sequence, e.g., in one or more CDRs, and particularly CDR3, includes residues or elements that are not encoded by human germline immunoglobulin sequences (e.g., including sequence mutations that may have been (initially) introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo).

[0073] As known in the art, the term "humanized" refers to a polypeptide whose amino acid sequence is derived from a reference antibody made in a non-human species (e.g., mouse). H and V LAlthough the term is generally used to refer to an antibody (or antibody component) that contains a region sequence, it also includes modifications of those sequences relative to a reference antibody that are intended to make the antibody more "human-like", i.e., more similar to human germline sequences. In some embodiments, a "humanized" antibody (or antibody component) is an antibody that immunospecifically binds to an antigen of interest and has framework (FR) regions with amino acid sequences substantially like a human antibody, and complementarity determining regions (CDRs) with amino acid sequences substantially like a non-human antibody. A humanized antibody comprises substantially all of at least one, and typically two, variable domains (Fab, Fab', F(ab')2, FabC, Fv), in which all or substantially all of the CDR regions correspond to the CDR regions of a non-human immunoglobulin (i.e., a donor immunoglobulin) and all or substantially all of the framework regions are framework regions of a human immunoglobulin consensus sequence. In some embodiments, a humanized antibody also comprises at least a portion of an immunoglobulin constant region (Fc), typically a portion of a human immunoglobulin constant region. In some embodiments, a humanized antibody contains both a light chain and at least the variable domain of a heavy chain. The antibody may also contain the CH1, hinge, CH2, CH3, and optionally CH4 regions of the heavy chain constant region. In some embodiments, a humanized antibody contains a humanized V L In some embodiments, the humanized antibody contains only a humanized V H In some particular embodiments, the humanized antibody contains only a humanized V H and V L Contains the region.

[0074] An "intact antibody" is one which comprises an antigen-binding variable region appropriate to the 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 native sequence constant domains (e.g., human native sequence constant domains) or amino acid sequence variants thereof.

[0075] "Antibody fragments" include a portion of an intact antibody, including its antigen-binding and / or variable regions. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments, diabodies, triabodies, tetrabodies, linear antibodies, single-chain antibody molecules, scFv, scFv-Fc, multispecific antibody fragments formed from antibody fragments, fragments produced by a Fab expression library, or epitope-binding fragments of any of the above that immunospecifically bind to a target antigen (e.g., a cancer cell antigen, a viral antigen, or a microbial antigen).

[0076] An "antigen" is an entity to which an antibody specifically binds.

[0077] As used herein, the term "binding" will be understood to typically refer to a non-covalent association between two or more entities. "Direct" binding involves physical contact between the entities or moieties, and indirect binding involves a physical interaction through physical contact with one or more intermediate entities. Binding between two or more entities can typically be assessed in any of a variety of contexts, including when the interacting entities or moieties are studied separately or in the context of a more complex system (e.g., while covalently or otherwise associated with a carrier entity and / or in a biological system or cell). In some embodiments, "binding" refers to the type of non-covalent interaction that occurs between an immunoglobulin molecule and an antigen for which the immunoglobulin is specific. The strength, or affinity, of an immunological binding interaction can be measured by the dissociation constant (K d ), K d A smaller K represents a higher affinity. The immunological binding properties of a selected polypeptide can be quantified using methods well known in the art. One such method involves measuring the rates of antigen-binding site / antigen complex formation and dissociation, which depend on the concentrations of the complex partners, the affinity of the interaction, and geometric parameters that affect the rates in both directions equally. Thus, the "on-rate constant" (K on ) and the "off-rate constant" (K offBoth the K and the KD can be determined by calculation of the concentration and the actual rates of association and dissociation. (See Nature 361:186-87 (1993)). off / K on The rate of affinity allows for the removal of all parameters not related to affinity and the dissociation constant K d (See generally, Davies et al. (1990) Annual Rev Biochem 59:439-473.)

[0078] The terms "specific binding" and "specifically bind" mean that an antibody or antibody derivative binds in a highly selective manner to its corresponding epitope of a target antigen and not to a multitude of other antigens. Typically, an antibody or antibody derivative binds to at least about 1×10 -7 M, and preferably 10 -8 M~10 -9 M, 10 -10 M, 10 -11 M, or 10 -12 and binds to a given antigen with an affinity of M that is at least two-fold higher than its affinity for binding to a non-specific antigen other than the given antigen or a closely related antigen (e.g., BSA, casein). The term "specificity" refers to the ability of a cell binding agent (e.g., as described herein, such as an antibody or fragment thereof) to specifically bind (e.g., immunoreact) with a given target antigen, e.g., a human target antigen.

[0079] Generally, a "protein" is a polypeptide (i.e., a chain of at least two amino acids linked together by peptide bonds). A protein may contain moieties other than amino acids (e.g., may be a glycoprotein) and / or may be otherwise processed or modified. One of skill in the art will understand that a "protein" may be an entire polypeptide chain produced by a cell (with or without a signal sequence), or a functional portion thereof. One of skill in the art will further understand that a protein may comprise two or more polypeptide chains, for example, linked by one or more disulfide bonds or associated by other means.

[0080] The term "inhibit" or "inhibition of" means to reduce by a measurable amount or to prevent completely.

[0081] The term "substantial" or "substantially" refers to the majority of a population of a mixture or sample, i.e., greater than 50%, preferably greater than 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the population.

[0082] The term "cytotoxic activity" refers to the cell-killing effect of a drug or a camptothecin conjugate or an intracellular metabolic product of a camptothecin conjugate. Cytotoxic activity is determined by the IC 50 It may be expressed as a value, which is the concentration (molar or mass) per unit volume at which half of the cells are viable.

[0083] The term "cytostatic activity" refers to the anti-proliferative effect of a drug or a camptothecin conjugate or an intracellular metabolic product of a camptothecin conjugate.

[0084] As used herein, the term "cytotoxic agent" refers to a substance that has cytotoxic activity and causes destruction of cells. The term is intended to include chemotherapeutic agents, as well as toxins, such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant, or animal origin, including synthetic analogs and derivatives thereof.

[0085] As used herein, the term "cytostatic agent" refers to a substance that inhibits the function of a cell, including the growth or proliferation of the cell. Cytostatic agents include inhibitors such as protein inhibitors, e.g., enzyme inhibitors. Cytostatic agents have cytostatic activity.

[0086] The terms "cancer" and "cancerous" refer to or describe the physiological condition or disorder in mammals that is typically characterized by unregulated cell growth. A "tumor" contains one or more cancerous cells.

[0087] As used herein, the term "patient" or "subject" refers to any organism to which a compound provided herein is administered in accordance with the present invention, for example, for experimental, diagnostic, prophylactic, and / or therapeutic purposes. Exemplary subjects include animals. The term "animal" refers to any member of the animal kingdom. In some embodiments, "animal" refers to humans at any stage of development. In some embodiments, "animal" refers to non-human animals at any stage of development. In certain 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, the animal includes, but is not limited to, mammals, birds, reptiles, amphibians, fish, insects, and / or parasites. In some embodiments, the animal may be a transgenic animal, a genetically engineered animal, and / or a clone. In embodiments, the animal is a mammal, insect, parasite, such as a mouse, rat, rabbit, non-human primate, and human. In embodiments, the subject is a human. In some embodiments, the subject may be suffering from and / or susceptible to a disease, disorder, and / or condition (e.g., cancer). As used herein, a "patient population" or a "population of subjects" refers to a plurality of patients or subjects.

[0088] As used herein, the term "normal", when used to modify the term "individual" or "subject", refers to an individual or group of individuals that do not have a particular disease or condition and are not carriers of that disease or condition. The term "normal" is also used herein to modify a biological specimen or sample isolated from a normal or wild-type individual or subject (e.g., a "normal biological sample").

[0089] An individual "suffering from" a disease, disorder, and / or condition (e.g., any of the cancers described herein) has been diagnosed with or exhibits one or more symptoms of that disease, disorder, and / or condition.

[0090] An individual who is "susceptible to" a disease, disorder, and / or condition may not have been diagnosed with and / or exhibit symptoms of the disease, disorder, and / or condition. In some embodiments, an individual who is susceptible to a disease, disorder, and / or condition (e.g., cancer) may be characterized by one of the following: (1) a genetic mutation associated with the development of the disease, disorder, and / or condition, (2) a genetic polymorphism associated with the development of the disease, disorder, and / or condition, (3) an increase and / or decrease in expression and / or activity of a protein associated with the disease, disorder, and / or condition, (4) a habit and / or lifestyle associated with the development of the disease, disorder, and / or condition, (5) a family history of the disease, disorder, and / or condition, (6) a response to a particular bacteria or virus, (7) exposure to a particular chemical. In some embodiments, an individual who is susceptible to a disease, disorder, and / or condition will develop the disease, disorder, and / or condition. In some embodiments, an individual who is susceptible to a disease, disorder, and / or condition will not develop the disease, disorder, and / or condition.

[0091] The term "treat" or "treatment", unless otherwise indicated by context, refers to any administration of a therapeutic molecule (e.g., any compound described herein) that partially or completely relieves, ameliorates, alleviates, inhibits, delays the onset of, delays the progression of, reduces the severity of, and / or reduces the occurrence of one or more symptoms or characteristics of a particular disease, disorder, and / or condition (e.g., cancer). Such treatment may be of subjects who do not exhibit symptoms of the relevant disease, disorder, and / or condition, and / or of subjects who exhibit only early signs of the disease, disorder, and / or condition. Alternatively or additionally, such treatment may be of subjects who exhibit one or more established signs of the relevant disease, disorder, and / or condition. Alternatively, the pharmacological and / or physiological effect may be prophylactic, i.e., an effect that completely or partially prevents a disease or its symptoms (e.g., delays the onset of or slows the progression of a disease or its symptoms). In this regard, the method of the present invention includes administering a "prophylactically effective amount" of a binding agent. "Prophylactically effective amount" refers to an amount effective at the dosage and duration required to achieve the desired prophylactic result. Thus, treatment (including prophylactic treatment) in which the purpose is to inhibit or slow down (reduce) undesirable physiological changes or disorders, such as the onset or spread of cancer. For the purposes of this invention, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, reduction in the extent of disease, stable (i.e., not worsening) state of disease, delay or slowing of disease progression, improvement or palliation of disease state, and remission (partial or complete), whether detectable or undetectable. Treatment can also include prolonging survival compared to expected survival in the absence of treatment. Those in need of treatment include those already with a disease condition or disorder, as well as those prone to having a disease condition or disorder.

[0092] In the context of cancer, the term "treating" includes any or all of 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 cancerous cells, and ameliorating one or more symptoms associated with the disease.

[0093] In the context of autoimmune disease, the term "treating" includes any or all of inhibiting the replication of cells associated with the autoimmune disease state, including, but not limited to, cells that produce autoimmune antibodies, reducing the autoimmune antibody load, and ameliorating one or more symptoms of the autoimmune disease.

[0094] The term "therapeutically effective amount" or "effective amount" refers to an amount of a conjugate effective to treat or prevent a disease or disorder (e.g., as described herein) in a mammal. In the case of cancer, a therapeutically effective amount of a conjugate can reduce the number of cancer cells, reduce tumor size, inhibit (i.e., slow to some extent, preferably stop) cancer cell invasion into peripheral organs, inhibit (i.e., slow to some extent, preferably stop) tumor metastasis, inhibit tumor growth to some extent, and / or alleviate to some extent one or more symptoms associated with cancer. To the extent that a drug can inhibit growth and / or kill existing cancer cells, the drug may be cytostatic and / or cytotoxic. For cancer therapy, efficacy can be measured, for example, by assessing the time to disease progression (TTP) and / or determining the response rate (RR).

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

[0096] In embodiments, the pharma- ceutically acceptable form is a pharma- ceutically acceptable salt. As used herein, the term "pharma- ceutically acceptable salt" refers to a pharma- ceutically acceptable organic or inorganic salt of a compound (e.g., camptothecin, camptothecin payload, or camptothecin conjugate). In some aspects, the compound can contain at least one amino group, and accordingly, an acid addition salt can be formed with the amino group. Exemplary salts include, but are not limited to, sulfate, trifluoroacetate, citrate, acetate, oxalate, chloride, bromide, iodide, nitrate, bisulfate, phosphate, acid phosphate, isonicotinate, lactate, salicylate, acid citrate, tartrate, oleate, tannate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate, gluconate, glucuronate, saccharate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, and pamoate (i.e., 1,1'-methylene-bis-(2-hydroxy-3-naphthoate)). Pharmaceutically acceptable salts may involve the inclusion of another molecule, such as an acetate ion, a succinate ion, or other counterion. Counterion may be any organic or inorganic moiety that stabilizes the charge on the parent compound. Furthermore, a pharmaceutically acceptable salt may have more than one charged atom in its structure. If multiple charged atoms are part of the pharmaceutically acceptable salt, it may have multiple counterions. Thus, a pharmaceutically acceptable salt may have one or more charged atoms and / or one or more counterions.

[0097] As used herein, the term "pharmaceutical composition" refers to a composition in which an active agent (e.g., a compound according to any of Formulas (I)-(III) described herein) is formulated together with one or more pharma- ceutically acceptable carriers. In some embodiments, the active agent is present in a unit dose suitable for administration in a treatment regimen that exhibits a statistically significant probability of achieving a predetermined therapeutic effect when administered to a relevant population. In some embodiments, the pharmaceutical compositions may be specially formulated for administration in solid or liquid form, including those adapted for: oral administration, e.g., drenches (aqueous or non-aqueous solutions or suspensions), tablets, e.g., buccal, sublingual, targeted for systemic absorption, boluses, powders, granules, pastes for application to the tongue, parenteral administration, e.g., by subcutaneous, intramuscular, intravenous, or epidural injection, e.g., as a sterile solution or suspension or sustained release formulation, topical application, e.g., as a cream, ointment, or controlled release patch or spray applied to the skin, lungs, or buccal cavity, intravaginally or rectally, sublingually, ophthalmically, transdermally, or intranasally, pulmonary, and other mucosal surfaces, e.g., as a pessary, cream, or foam.

[0098] As used herein, "carrier" or "pharmaceutical acceptable carrier" refers to a diluent, adjuvant, excipient, or vehicle with which the composition is administered. In some exemplary embodiments, the carrier may include sterile liquids, such as water and oils (oils of petroleum, animal, vegetable, or synthetic origin (e.g., peanut oil, soybean oil, mineral oil, sesame oil, etc.)). In some embodiments, the carrier is or includes one or more solid ingredients. In some embodiments, the carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerin, propylene glycol, and liquid polyethylene glycol, etc.), and suitable mixtures thereof. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by maintaining the required particle size in the case of dispersions, and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In some cases, it may be desirable to include isotonic agents, such as sugars, polyalcohols, such as mannitol, sorbitol, sodium chloride, in the composition. Prolonged absorption of the injectable composition can be brought about by including in the composition an agent that delays absorption, such as aluminum monostearate and gelatin.

[0099] As used herein, the term "kit" refers to any delivery system for delivering materials. Such delivery systems may include systems that allow for the storage, transport, or delivery of various diagnostic or therapeutic reagents (e.g., oligonucleotides, enzymes, etc. in appropriate containers) and / or supporting materials (e.g., buffers, written instructions for performing an assay, etc.) from one location to another. For example, a kit includes one or more enclosures (e.g., boxes, cartridges, bottles, ampoules, etc.) that contain the relevant reaction reagents and / or supporting materials. As used herein, the term "fragmented kit" refers to a delivery system that includes two or more separate containers, each containing a sub-portion of the overall kit components. The containers may be delivered to the intended recipient together or separately. For example, a first container may contain an enzyme for use in an assay, while a second container contains an oligonucleotide. The term "fragmented kit" is intended to encompass, but is not limited to, kits that contain analyte-specific reagents (ASRs) regulated under Section 520(e) of the Federal Food, Drug, and Cosmetic Act. Indeed, any delivery system that includes two or more separate containers, each containing a subportion of the total kit components, is included in the term "fragmented kit." In contrast, a "combination kit" refers to a delivery system that contains all of the components in a single container (e.g., in a single box that houses each of the desired components). The term "kit" includes both fragmented and combined kits.

[0100] As used herein, the term "administration" typically refers to administration of a composition to a subject or system to achieve delivery of the composition or an agent contained in the composition. A person skilled in the art will recognize the various routes that may be utilized for administration to a subject, e.g., a human, in the appropriate circumstances. Examples of routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (i.e., topical), transmucosal, and rectal administration. For example, in some embodiments, administration may be ocular, oral, parenteral, topical, etc. In embodiments, administration is parenteral (e.g., intravenous administration). In embodiments, intravenous administration is intravenous infusion. In some particular embodiments, administration may be or include one or more of bronchial (e.g., by bronchial instillation), oral, cutaneous (e.g., topical to the dermis, intradermal, interdermal, transdermal, etc.), intestinal, intra-arterial, intradermal, intragastric, intramedullary, intramuscular, intranasal, intraperitoneal, intrathecal, intravenous, intraventricular, within a particular organ (e.g., intrahepatic), mucosal, nasal, oral, rectal, subcutaneous, sublingual, topical, tracheal (e.g., by intratracheal instillation), vaginal, vitreous, etc.

[0101] As used herein, the term "nucleophile" refers to a reactive group that is electron-rich and has an unshared pair of electrons that act as a reactive site and react with positively charged or electron-deficient sites. Examples of nucleophilic groups suitable for use in the present invention include, but are not limited to, amino groups (e.g., primary amines, secondary amines, hydroxylamine, and / or hydrazine), thiols, phenols, and alcohols. In embodiments, the nucleophilic functional group includes amino, hydrazino, hydroxylamino, hydroxy, or thio. In embodiments, the nucleophilic functional group is carboxamide, N-hydroxycarboxamide, carboxylhydrazide, or guanidino. In embodiments, the nucleophilic group is or includes a thiol group. Certain nucleophilic groups must be activated with a base so that they can react with electrophilic groups. For example, if there are nucleophilic thiol and hydroxyl groups in a multifunctional compound, the compound must be mixed with aqueous base to remove a proton and provide a thiolate or hydroxylate anion to allow reaction with the electrophilic group. Non-nucleophilic bases are preferred unless it is desired that the base participate in the reaction, in some embodiments the base may be present as a component of a buffer solution.

[0102] As used herein, the term "electrophilic" refers to a reactive group that is susceptible to nucleophilic attack, i.e., susceptible to reaction with an incoming nucleophilic group. The electrophilic group can be selected to be capable of reacting with the nucleophilic group of a counter reactant. For example, if the nucleophilic reactive group is an amino group, the electrophilic group can be selected to react with an amino group. Similarly, if the nucleophilic reactive group is a thiol moiety, the corresponding electrophilic group can be a thiol reactive group, and the like. Examples of electrophilic groups suitable for use in the present invention include, but are not limited to, carboxylic acid esters, acid chloride groups, anhydrides, isocyanates, thioisocyanates, epoxides, activated hydroxyl groups, succinimidyl esters, sulfosuccinimidyl esters, maleimides, and ethenesulfonyl. In embodiments, the electrophilic group is an aldehyde, an α-haloketone, a maleimide, a succinimide, a hydroxysuccinimide, an isothiocyanate, an isocyanate, an acyl azide, a sulfonyl chloride, a tosylate ester, a glyoxal, an epoxide, an oxirane, a carbonate, an imido ester, an anhydride, a fluorophenyl ester, a hydroxymethylphosphine derivative, a carbonate, a haloacetyl, a chlorotriazine, a haloacetyl, an alkyl halide, an aziridine, an acryloyl derivative, a ketone, a carboxylic acid, an ester, an acetyl chloride, or an acetic anhydride. In embodiments, the electrophilic group is or includes a maleimide or succinimide group. The carboxylic acid group may be activated to react with a nucleophile, including reaction 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 be reacted with alkoxy-substituted N-hydroxysuccinimides or N-hydroxysulfosuccinimides in the presence of DCC to form reactive electrophilic groups, N-hydroxysuccinimide esters and N-hydroxysulfosuccinimide esters, respectively. Carboxylic acids may also be activated by reaction with acyl halides, such as acyl chlorides (e.g., acetyl chloride), to provide reactive anhydride groups.In a further example, a carboxylic acid may be converted to an acid chloride group using, for example, thionyl chloride or an acyl chloride capable of undergoing an exchange reaction.

[0103] Unless otherwise indicated, the term "alkyl," by itself or as part of another term, refers to a substituted or unsubstituted straight or branched chain, saturated or unsaturated hydrocarbon having the designated number of carbon atoms (e.g., "-C1-C8 alkyl" or "-C1-C8 alkyl"). 10 ("alkyl" refers to alkyl groups having 1 to 8 or 1 to 10 carbon atoms, respectively). If the number of carbon atoms is not specified, the alkyl group 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, -sec-butyl, -isobutyl, -tert-butyl, -isopentyl, and -2-methylbutyl, and unsaturated -C2-C8 alkyl groups include, but are not limited to, -isopropyl, -sec-butyl, -isobutyl, -tert-butyl, -isopentyl, and -2-methylbutyl. Alkyl includes, but is not limited to, -vinyl, -allyl, -1-butenyl, -2-butenyl, -isobutyrenyl, -1 pentenyl, -2 pentenyl, -3-methyl-1-butenyl, -2 methyl-2-butenyl, -2,3 dimethyl-2-butenyl, -1-hexyl, 2-hexyl, -3-hexyl, -acetylenyl, -propynyl, -1 butynyl, -2 butynyl, -1 pentynyl, -2 pentynyl, and -3 methyl 1 butynyl. Alkyl groups can be unsubstituted. Alkyl groups can be substituted with one or more groups. In other embodiments, the alkyl groups are saturated.

[0104] Unless otherwise indicated, "alkylene" by itself or as part of another term refers to a substituted or unsubstituted saturated, branched or straight chain, or cyclic hydrocarbon radical having a specified number of carbon atoms, typically 1 to 10 carbon atoms, and having two monovalent radical centers obtained by the removal of two hydrogen atoms from the same or two different carbon atoms of a parent alkane. Typical alkylene radicals include, but are not limited to, methylene (-CH2-), 1,2-ethylene (-CH2CH2-), 1,3-propylene (-CH2CH2CH2-), 1,4-butylene (-CH2CH2CH2CH2-), and the like. In preferred embodiments, the alkylene is a branched or straight chain hydrocarbon (i.e., not a cyclic hydrocarbon).

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

[0106] Unless otherwise indicated, an "arylene" by itself or as part of another term is an aryl group, as defined above, that has two covalent bonds (i.e., is divalent) and may be oriented ortho, meta, or para.

[0107] Unless otherwise indicated, "C3-C8 heterocycle" by itself or as part of another term refers to a monovalent substituted or unsubstituted, aromatic or non-aromatic, monocyclic or bicyclic ring system having 3 to 8 carbon atoms (also referred to as ring members) and one to four heteroatom ring members independently selected from N, O, P, or S, obtained by the removal of one hydrogen atom from a ring atom of a parent ring system. One or more N, C, or S atoms of a heterocycle may be oxidized. A ring containing a heteroatom may be aromatic or non-aromatic. A heterocycle in which all of the ring atoms participate in aromaticity is referred to as a heteroaryl, otherwise as a heterocarbocycle. Unless otherwise stated, a heterocycle is attached to its pendant group at any heteroatom or carbon atom that results in a stable structure. Thus, a heteroaryl may be attached through an aromatic carbon of its aromatic ring system, referred to as a C-linked heteroaryl, or through a non-double-bonded N atom (i.e., not =N-) of its aromatic ring system, referred to as an N-linked heteroaryl. Thus, nitrogen-containing heterocycles may be C-linked or N-linked and include pyrrole moieties such as pyrrol-l-yl (N-linked) and pyrrol-3-yl (C-linked), and imidazole moieties such as imidazol-l-yl and imidazol-3-yl (both N-linked), and imidazol-2-yl, imidazol-4-yl, and imidazol-5-yl moieties (all C-linked).

[0108] Unless otherwise indicated, "C3-C8 heteroaryl" refers to an aromatic C3-C8 heterocycle, with the subscript indicating the total number of carbons in the cyclic ring system of the heterocycle or the total number of aromatic carbons in the aromatic ring system of the heteroaryl, without implying the size of the ring system or whether the rings are fused. Representative examples of C3-C8 heterocycles include, but are not limited to, pyrrolidinyl, azetidinyl, piperidinyl, morpholinyl, tetrahydrofuranyl, tetrahydropyranyl, benzofuranyl, benzothiophene, indolyl, benzopyrazolyl, pyrrolyl, thiophenyl (thiophene), furanyl, thiazolyl, imidazolyl, pyrazolyl, pyrimidinyl, pyridinyl, pyrazinyl, pyridazinyl, isothiazolyl, and isoxazolyl. When explicitly indicated, the size of the heterocycle or heteroaryl ring system is indicated by the total number of atoms in the ring. For example, a designation as a 5- or 6-membered heteroaryl indicates the total number of aromatic atoms in the heteroaromatic ring system of the heteroaryl (i.e., 5 or 6), but does not imply the number of aromatic heteroatoms or aromatic carbons in the ring system. A fused heteroaryl is explicitly stated or implied by such context, and is typically indicated by the number of aromatic atoms in each aromatic ring that are fused together to form the fused heteroaromatic ring system. For example, a 5,6-membered heteroaryl is an aromatic 5-membered ring fused to an aromatic 6-membered ring, where one or both of the rings have an aromatic heteroatom or the heteroatom is shared between the two rings.

[0109] A heterocycle fused to an aryl or heteroaryl is an example of an optionally substituted heterocycle in which the heterocycle is replaced by ring fusion with an aryl or heteroaryl such that the heterocycle remains non-aromatic and is part of a larger structure through attachment to the non-aromatic portion of the fused ring system. Similarly, an aryl or heteroaryl fused to a heterocycle or carbocycle that is part of a larger structure through attachment to the aromatic portion of the fused ring system is an example of an optionally substituted aryl or heterocycle in which the aryl or heterocycle is replaced by ring fusion with a heterocycle or carbocycle.

[0110] Unless otherwise indicated, "C3-C8 heterocyclo" by itself or as part of another term refers to a C3-C8 heterocycle as defined above in which one of the heterocycle's hydrogen atoms is replaced with a bond (i.e., it is bivalent). Unless otherwise indicated, "C3-C8 heteroarylene" by itself or as part of another term refers to a C3-C8 heteroaryl group as defined above in which one of the heteroaryl group's hydrogen atoms is replaced with a bond (i.e., it is bivalent).

[0111] Unless otherwise indicated, a "C3-C8 carbocycle" by itself or as part of another term is a 3-, 4-, 5-, 6-, 7-, or 8-membered monovalent, substituted or unsubstituted, saturated or unsaturated, non-aromatic monocyclic or bicyclic carbocycle obtained by the removal of one hydrogen atom from a ring atom of a parent ring system. Representative -C3-C8 carbocycles include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentadienyl, cyclohexyl, cyclohexenyl, 1,3-cyclohexadienyl, 1,4-cyclo-hexadienyl, cycloheptyl, 1,3-cycloheptadienyl, 1,3,5-cyclo-heptatrienyl, cyclooctenyl, and cyclooctadienyl.

[0112] Unless otherwise indicated, "C3-C8 carbocyclo" by itself or as part of another term refers to a C3-C8 carbocyclic group, as defined above, in which another of the carbocyclic group's hydrogen atoms is replaced with a bond (i.e., is bivalent).

[0113] Unless otherwise indicated, the term "heteroalkyl", by itself or in combination with another term, means, unless otherwise stated, a stable straight or branched chain hydrocarbon, which is fully saturated or contains one to three degrees of unsaturation, and which consists of the 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, or a combination thereof, wherein the nitrogen and sulfur atoms may be optionally oxidized and the nitrogen heteroatom may be optionally quaternized. The heteroatom O, N, and S may be placed at any interior position of the heteroalkyl group or at the position at which the alkyl group is attached to the remainder of the molecule. The heteroatom Si may be placed at any position of the heteroalkyl group, including the position at which the alkyl group is attached to the remainder 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)-CH3, -CH=CH-O-CH3, -Si(CH3)3, -CH2-CH=NO-CH3, and -CH=CH-N(CH3)-CH3. Up to two heteroatoms may be consecutive, such as, for example, -CH2-NH-OCH3 and -CH2-O-Si(CH3)3. Typically, a C1-C4 heteroalkyl or heteroalkylene has 1 to 4 carbon atoms and 1 or 2 heteroatoms, and a C1-C3 heteroalkyl or heteroalkylene has 1 to 3 carbon atoms and 1 or 2 heteroatoms. In some embodiments, the heteroalkyl or heteroalkylene is saturated.

[0114] Unless otherwise indicated, the term "heteroalkylene," by itself or in combination with another term, means a divalent group derived from a heteroalkyl (as defined above), as exemplified by -CH-CH-S-CH-CH- and -CH-S-CH-CH-NH-CH-. For heteroalkylene groups, heteroatoms can also occupy either or both of the chain termini. Still further, for alkylene and heteroalkylene linking groups, no orientation of the linking group is implied.

[0115] Unless otherwise indicated, "aminoalkyl," by itself or in combination with another term, refers to a heteroalkyl in which the alkyl portion, as defined herein, is substituted with an amino, alkylamino, dialkylamino, or cycloalkylamino group. Exemplary non-limiting aminoalkyls are -CH2NH2, -CH2CH2NH2, -CH2CH2NHCH3, and -CH2CH2N(CH3)2, further including branched species such as -CH(CH3)NH2 and -C(CH3)CH2NH2 in the (R) or (S) configuration. Alternatively, an aminoalkyl may be a heteroalkyl group having a sp carbon other than the radical carbon. 3 The carbon is replaced with an amino or alkylamino moiety, and the sp 3 Nitrogen is alkyl sp 3 Replaces carbon with at least one sp 3 Provided that the carbon remains, the aminoalkyl moiety is an alkyl moiety, group, or substituent as defined herein. When the aminoalkyl moiety is referred to as a substituent on a larger structure or another moiety, the aminoalkyl is covalently attached to that structure or moiety via a carbon radical on the alkyl portion of the aminoalkyl.

[0116] Unless otherwise indicated, "alkylamino" and "cycloalkylamino", by themselves or in combination with another term, refer to an alkyl or cycloalkyl radical in which the radical carbon is replaced with a nitrogen radical, provided that at least one sp 3means an alkyl or cycloalkyl radical as described herein, provided that the carbon remains. When an alkylamino is substituted at its nitrogen with another alkyl moiety, the resulting substituted radical is referred to as a dialkylamino moiety, group, or substituent, and the alkyl moieties substituting the nitrogen are independently selected. Exemplary and non-limiting amino, alkylamino, and dialkylamino substituents include those having the structure -N(R')2, where R' in these examples is independently selected from hydrogen or C1-6 alkyl, typically hydrogen or methyl, while in cycloalkylamines contained within heterocycloalkyls, both R', together with the nitrogen to which they are attached, define a heterocycle. When both R' are hydrogen or alkyl, the moieties may be described as primary amino and tertiary amine groups, respectively. When one R' is hydrogen and the other is alkyl, the moiety is described as a secondary amino group. Primary and secondary alkylamino moieties are more reactive as nucleophiles toward carbonyl-containing electrophilic centers, while tertiary amines are more basic.

[0117] "Substituted alkyl" and "substituted aryl" refer to alkyl and aryl, respectively, in which one or more hydrogen atoms, typically one, are each independently replaced with a substituent. Exemplary substituents include -X, -R', -OH, -OR', -SR', -N(R'), -N(R'), =NR', -CX, -CN, -NO, -NR'C(=O)R', -C(=O)R', -C(=O)N(R'), -S(=O)R', -S(=O)NR, -S(=O)R', -OP(=O)(OR'), -P(=O)(OR'), -PO=, POH, -C(=O)R and -C(=NR)N(R')2, where each X is independently selected from the group consisting of a halogen: -F, -CI, -Br, and -I, and where each R' is independently selected from the group consisting of -H, -C1-C 20 Alkyl, -C6-C20 Aryl, -C3-C 14 It is selected from the group consisting of a heterocycle, a protecting group, and a prodrug moiety.

[0118] More typically, the substituents are selected 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, where each X is independently -F and and -CI, or selected 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 groups, and prodrug moieties, wherein each X is -F and each R' is independently hydrogen, -C1-C 20 Alkyl, -C6-C 20 Aryl, -C3-C 14 In some embodiments, the alkyl substituent is selected from the group consisting of -N(R')2, -N(R')3, and -C(=NR)N(R')2, where R is selected from the group consisting of hydrogen and -C1-C 20 In another embodiment, the alkyl is substituted with a series of ethyleneoxy moieties to define the PEG unit. The alkylene, carbocycle, carbocyclo, arylene, heteroalkyl, heteroalkylene, heterocycle, heterocyclo, heteroaryl, and heteroarylene groups described above may also be similarly substituted.

[0119] As used herein, a "protecting group" refers to a moiety that prevents or reduces the ability of an atom or functional group to which it is attached to participate in an undesired reaction. Exemplary protecting groups for atoms or functional groups are described in Greene (1999), "Protective Groups In Organic Synthesis, 3 RD " Ed.", Wiley Interscience. Protecting groups for heteroatoms such as oxygen, sulfur, and nitrogen are used in some cases to minimize or avoid their undesired reactions with electrophilic compounds. In other cases, protecting groups are used to reduce or eliminate the nucleophilic and / or basic nature of the unprotected heteroatom. A non-limiting example of a protected oxygen is -OR. PR where R PR is a protecting group for hydroxyl, which is typically protected as an ester (e.g., acetate, propionate, or benzoate). Other protecting groups for hydroxyl avoid interfering with the nucleophilicity of organometallic or other highly basic reagents, and hydroxyl is typically protected as an ether, including alkyl or heterocycloalkyl ethers (e.g., methyl or tetrahydropyranyl ethers), alkoxymethyl ethers (e.g., methoxymethyl or ethoxymethyl ethers), optionally substituted aryl ethers, and silyl ethers (e.g., trimethylsilyl (TMS), triethylsilyl (TES), tert-butyldiphenylsilyl (TBDPS), tert-butyldimethylsilyl (TBS / TBDMS), triisopropylsilyl (TIPS), and [2-(trimethylsilyl)ethoxy]-methylsilyl (SEM)). Nitrogen protecting groups include -NHR PR or -N(R PR ) 2- for primary or secondary amines, in which R PR At least one of R is a nitrogen atom protecting group or PR together contain a protecting group.

[0120] A protecting group is suitable if it can prevent or avoid undesirable side reactions or premature loss of the protecting group under the reaction conditions required to effect the desired chemical transformation elsewhere in the molecule, and if necessary, during purification of the newly formed molecule, and can be removed under conditions that do not adversely affect the structure or stereochemical integrity of the newly formed molecule. By way of example and not limitation, suitable protecting groups can include those previously described for protecting functional groups. Suitable protecting groups can also be those used in peptide coupling reactions.

[0121] "Aromatic alcohol" refers to an aromatic ring system substituted with a hydroxyl functional group -OH, either by itself or as part of a larger structure. Thus, aromatic alcohol refers to any aryl, heteroaryl, arylene, and heteroarylene moiety described herein that has a hydroxyl functional group attached to an aromatic carbon of its aromatic ring system. The aromatic alcohol may be part of a larger moiety, such as when the aromatic ring system is a substituent of the moiety, or may be embedded in the larger moiety by ring fusion, and may be optionally substituted with one or more other hydroxyl substituents as described herein. Phenolic alcohol is an aromatic alcohol that has a phenol group as the aromatic ring.

[0122] "Aliphatic alcohol" refers to a moiety that, by itself or as part of a larger structure, has a non-aromatic carbon bonded to a hydroxyl functional group -OH. The hydroxy-containing carbon may be unsubstituted (i.e., methyl alcohol) or may have one, two, or three optionally substituted branched or unbranched alkyl substituents to define a primary alcohol, or a secondary or tertiary aliphatic alcohol within a linear or cyclic structure. When part of a larger structure, the alcohol may be a substituent of the structure by bonding through the hydroxy-containing carbon, through a carbon of an alkyl or other moiety described herein to the hydroxy-containing carbon, or through a substituent of the alkyl or other moiety. Aliphatic alcohol contemplates non-aromatic cyclic structures (i.e., optionally substituted carbocyclic and hetero-carbocyclic rings) in which a hydroxy functional group is bonded to a non-aromatic carbon of the cyclic ring system.

[0123] As used herein, "arylalkyl" or "heteroarylalkyl" refers to a substituent, moiety, or group in which an aryl moiety is linked to an alkyl moiety, i.e., aryl-alkyl-, where the alkyl and aryl groups are as described above, e.g., CH-CH- or CH-CH(CH)CH-. An arylalkyl or heteroarylalkyl is a group in which the sp 3 Related to a larger structure or moiety through carbon.

[0124] As used herein, "succinimide moiety" refers to an organic moiety comprised of a succinimide ring system, which is typically present in one type of Y' in a compound of formula (PL-A) further comprised of an alkylene-containing moiety attached to the imide nitrogen of the ring system. The succinimide moiety typically results from the Michael addition of a thiol group of a cell-binding agent to the maleimide ring system of a camptothecin payload compound (formula II). Thus, the succinimide moiety is comprised of a thio-substituted succinimide ring system, the imide nitrogen of which, when present in a camptothecin conjugate, is replaced by the remaining moiety of the cell-binding agent of the camptothecin conjugate, and optionally replaced by a substituent that was present on the maleimide ring system of the compound of formula II.

[0125] As used herein, "acid-amide moiety" refers to a succinic acid having an amide substituent resulting from the thio-substituted succinimide ring system of the succinimide moiety that has undergone cleavage of one of its carbonyl-nitrogen bonds by hydrolysis. Hydrolysis resulting in a succinic acid-amide moiety provides a linker that is less likely to suffer premature loss of the antibody-linked linker via elimination of the thio-substituent. Hydrolysis of the succinimide ring system of the thio-substituted succinimide moiety is expected to result in regiochemical isomers of the acid-amide moiety due to differences in reactivity of the two carbonyl carbons of the succinimide ring system, at least in part due to any substituents present in the maleimide ring system of the compound of formula II and the thio-substituents introduced by the targeting ligand.

[0126] As used herein, the term "prodrug" refers to a less biologically active or inactive compound that is transformed into a more biologically active compound in the body through a chemical or biological process (i.e., chemical reaction or enzymatic biotransformation). Typically, a biologically active compound is rendered less biologically active (i.e., converted into a prodrug) by chemically modifying the compound with a prodrug moiety. In some embodiments, the prodrug is a type II prodrug, which is bioactivated outside the cell, e.g., in digestive fluids, or in the circulatory system of the body, e.g., in blood. Exemplary prodrugs are esters and (β-D-glucopyranosides.

[0127] In many cases, the conjugates, linkers, and component assemblies described herein refer to reactive groups. A "reactive group" or RG is a group that contains a reactive site (RS) that can form a bond with either a camptothecin payload or a linker component of a camptothecin conjugate, or camptothecin. RS is a reactive site within the reactive group (RG). Reactive groups include thiol groups that form disulfide or thioether bonds, aldehydes, ketones, or hydrazine groups that form hydrazone bonds, carboxylic acids or amino groups that form peptide bonds, carboxylic acids or hydroxy groups that form ester bonds, sulfonic acids that form sulfonamide bonds, alcohols that form carbamate bonds, and amines that form sulfonamide or carbamate bonds. The following table illustrates exemplary functional groups that can be formed after the reaction of reactive groups, reactive sites, and reactive sites. This table is not intended to be limiting. One of ordinary skill in the art will appreciate that the R' and R'' moieties depicted in the table are effectively any organic moiety (e.g., an alkyl, aryl, heteroaryl, or substituted alkyl, aryl, or heteroaryl group) compatible with the bond formation provided upon conversion of RG to one of the exemplary functional groups. It will also be appreciated that, as applied to embodiments of the present invention, R' may optionally represent one or more components of a self-stabilizing linker or an optional secondary linker, and R' may optionally represent one or more components of an optional secondary linker, camptothecin, stabilizing unit, or detection unit. [Table 8]

[0128] Combinations of substituents and variables envisioned by the present invention are only those that result in the formation of stable compounds. As used herein, the term "stable" refers to compounds that have sufficient stability to permit manufacture and maintain compound integrity for a sufficient period of time to be useful for the purposes detailed herein (e.g., therapeutic or prophylactic administration to a subject).

[0129] After their preparation, the compounds of the invention are preferably isolated and purified to obtain compositions containing 95% or more by weight ("substantially pure"), which can then be used or formulated as described herein.

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

[0131] As used herein, the term "linkable to a cell binding agent" refers to a compound or derivative thereof that includes at least one linking group or precursor thereof suitable for linking the compound or derivative thereof described herein to a cell binding agent.

[0132] The term "precursor" of a given group refers to any group that can be connected to that group by any deprotection, chemical modification, or coupling reaction.

[0133] The term "linked to a cell binding agent" refers to a conjugate molecule comprising at least one of the compounds described herein, or a derivative thereof, attached to a cell binding agent via a suitable linking group or precursor thereof.

[0134] The terms "abnormal cell growth" and "proliferative disorder" are used interchangeably in this application. As used herein, unless otherwise indicated, "abnormal cell growth" refers to cell growth that is independent of normal regulatory mechanisms (e.g., loss of contact inhibition). This includes, for example, the abnormal growth of (1) tumor cells (tumors) that grow due to expression of mutant tyrosine kinases or overexpression of receptor tyrosine kinases, (2) benign and malignant cells of other proliferative diseases in which abnormal tyrosine kinase activation occurs, (3) any tumor that grows due to receptor tyrosine kinases, (4) any tumor that grows due to abnormal serine / threonine kinase activation, and (5) benign and malignant cells of other proliferative diseases in which abnormal serine / threonine kinase activation occurs.

[0135] The terms "cancer" and "cancerous" refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth. A "tumor" contains one or more cancerous cells and / or benign or pre-cancerous cells.

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

[0137] A "chemotherapeutic agent" is a chemical compound useful in the treatment of cancer.

[0138] A "metabolite" is a product produced through metabolism in the body of a particular compound, its derivative, or its conjugate, or its salt. Metabolites of a compound, its derivative, or its conjugate may be identified using routine techniques known in the art, and their activity may be determined using tests such as those described herein. Such products may result, for example, from oxidation, hydroxylation, reduction, hydrolysis, amidation, deamidation, esterification, deesterification, enzymatic cleavage, and the like, of an administered compound. Thus, the present invention includes metabolites of a compound, its derivative, or its conjugate of the present invention, including compounds, its derivative, or its conjugate produced by a process comprising contacting a compound, its derivative, or its conjugate of the present invention with a mammal for a period of time sufficient to obtain a metabolic product thereof.

[0139] As defined herein, a "linker," "linker moiety," or "linking group" refers to a moiety that connects two groups together, such as a cell-binding agent and a cytotoxic compound. Typically, a linker is substantially inert under the conditions under which the two groups it connects are linked. A bifunctional crosslinker may contain two reactive groups, one at each end of the linker moiety, such that one reactive group can react first with a cytotoxic compound to provide a compound having a linker moiety and a second reactive group, and the second reactive group can then react with a cell-binding agent. Alternatively, one end of the bifunctional crosslinker may react first with a cell-binding agent to provide a cell-binding agent having a linker moiety and a second reactive group, and the second reactive group can then react with a cytotoxic compound. The linking moiety may contain a chemical bond that allows for release of the cytotoxic moiety at a specific site. Suitable chemical bonds are well known in the art and include disulfide bonds, thioether bonds, acid labile bonds, photolabile bonds, peptidase labile bonds, and esterase labile bonds (see, for example, U.S. Patent Nos. 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, thioethers, and peptidase labile bonds are preferred. Other linkers that may be used in the present invention include non-cleavable linkers, such as those described and detailed in U.S. Publication No. 2005 / 0169933, or charged or hydrophilic linkers, and are 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 mimetics that function in a manner similar to naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those that are later modified, such as hydroxyproline, γ-carboxyglutamic acid, selinocystiene, and O-phosphoserine. Amino acid analogs refer to compounds that have the same basic chemical structure as naturally occurring amino acids, i.e., carbons bound to hydrogen, carboxyl groups, amino groups, and R groups, such as homoserine, norleucine, methionine sulfoxide, and methionine methylsulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as naturally occurring amino acids. One amino acid that can be used in particular is citrulline, which is a derivative of arginine and is involved in the formation of urea in the liver. Amino acid mimetics refer to compounds that have a structure that is different from the general chemical structure of amino acids, but function in a manner similar to naturally occurring amino acids. The term "unnatural amino acid" is intended to refer to the "D" stereochemical forms of the twenty naturally occurring amino acids mentioned above. It is further understood that the term unnatural amino acid includes the homologues 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 with side chains shortened or lengthened by up to two carbon atoms, amino acids containing optionally substituted aryl groups, amino acids containing halogenated groups, preferably halogenated alkyl groups and aryl groups, and N-substituted amino acids, such as N-methyl-alanine. The amino acid or peptide can be attached to the linker / spacer or cell-binding agent through the terminal amine or terminal carboxylic acid of the amino acid or peptide. The amino acid can also be attached to the linker / spacer or cell-binding agent through a side chain reactive group, such as, but not limited to, the thiol group of cysteine, the epsilon amine of lysine, or the side chain hydroxyl of serine or threonine.

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

[0142] As used herein, amino acids can be L or D isomers. Unless otherwise specified, when an amino acid is referred to, it can be L or D isomer or a mixture thereof. In embodiments, when a peptide is referred to by its amino acid sequence, each of the amino acids can be L or D isomers unless otherwise specified. When one of the amino acids in a peptide is specified as D isomer, the other amino acid is L isomer unless otherwise specified. For example, 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 moieties that protect the N-terminus of amino acids or peptides from undesired reactions and can be used during the synthesis of drug-ligand conjugates. They should remain attached to the N-terminus throughout the entire synthesis and can be removed after the completion of the synthesis of drug conjugates by chemical or other conditions that selectively achieve their removal. Blocking groups suitable for N-terminus protection are well known in the art of peptide chemistry. Exemplary blocking groups include, but are not limited to, methyl ester, tert-butyl ester, 9-fluorenylmethylcarbamate (Fmoc), and carbobenzoxy (Cbz).

[0144] The term "protease-cleavable peptide" refers to a peptide that contains a protease cleavage recognition sequence. As used herein, a protease is an enzyme that can cleave a peptide bond. A protease cleavage recognition sequence is a specific amino acid sequence that is recognized by a protease during proteolytic cleavage. Many protease cleavage sites are known in the art, and these and other cleavage sites can be included in the linker moiety. 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. 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, ed. Masters et al. al. 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 cathepsin 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 the art.

[0146] In another embodiment, peptide sequence is selected based on its ability to be cut by lysosomal proteases.Lysosomal proteases are proteases that are mainly located in lysosomes, but can also be located in endosomes.Examples of lysosomal proteases include, but are not limited to, cathepsin B, C, D, H, L and S, and furin.

[0147] In another embodiment, the peptide sequence is selected based on its ability to be cleaved by tumor-associated proteases, such as proteases found extracellularly on the surface of cancerous cells or in the vicinity of tumor cells, non-limiting examples of which include thimeto oligopeptidase (TOP), CD10 (neprilysin), matrix metalloproteases (such as MMP2 or MMP9), type II transmembrane serine proteases (hepsin, testisin, TMPRSS4, or matriptase / MT-SP1), legumain, and enzymes described in the following references (Current Topics in Developmental Biology: Cell Surface Proteases, vol. 54 Zucker S. 2003, Boston, MA). The ability of the peptide to be cleaved by tumor-associated proteases can be tested using in vitro protease cleavage assays known in the art.

[0148] The term "cation" refers to an ion having a positive charge. Cations can be monovalent (e.g., Na+, K+, etc.), divalent (e.g., Ca2+, Mg2+, etc.), or multivalent (e.g., Al3+, etc.). In embodiments, the cation is monovalent.

[0149] HER2 HER2 is one of the oncogene products of typical growth factor receptor oncogenes identified as human epidermal growth factor receptor 2-related oncogenes, 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), and is known to be activated by autophosphorylation at intracellular tyrosine residues through its homodimerization or heterodimerization with another EGFR receptor, HER1, HER3, or HER4, thereby playing an important role in cell proliferation, differentiation, and survival in normal and tumor cells.

[0150] The HER2 protein used in the present invention can be directly purified from HER2-expressing cells of human or non-human mammals (such as rats or mice), or can be prepared and used as a cell membrane fraction of the above-mentioned cells. In addition, HER2 can be obtained by in vitro synthesis or production in a host cell by genetic engineering. In genetic engineering, specifically, HER2 protein can be obtained by synthesizing HER2 protein in a solution containing the enzymes, substrates and energy materials required for transcription and translation after inserting HER2 cDNA into an expression vector, or by expressing HER2 in other prokaryotic or eukaryotic transformed host cells. Alternatively, the above-mentioned genetically engineered HER2-expressing cells or cell lines expressing HER2 can be used as the HER2 protein.

[0151] The DNA sequence and amino acid sequence of HER2 are disclosed in public databases, and can be found under, for example, accession numbers M11730 (GenBank) and NP_004439.2 (NCBI).

[0152] Furthermore, HER2 also includes proteins that have an amino acid sequence in which one or more amino acids have been substituted, deleted and / or added in any of the above amino acid sequences of HER2, and have biological activity equivalent to that of the protein.

[0153] The human HER2 protein consists of an N-terminal signal sequence consisting of 22 amino acid residues, 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 to be a poor prognostic factor in breast cancer.

[0155] Anti-HER2 antibody An anti-HER2 antibody is an antibody that can target tumor cells, i.e., has the property of recognizing tumor cells, binding to tumor cells, being internalized into tumor cells, cytocidal activity against tumor cells, etc., and can be conjugated to a drug having anti-tumor activity via a linker to form an antibody-drug conjugate. Non-limiting anti-HER2 antibodies suitable for use in the compounds, compositions, and methods described herein include those having any of the following exemplary properties, or any combination thereof: (1)Anti-HER2 antibody having the following characteristics: (a) specifically binds to HER2; (b) It has the activity of being internalized into HER2-expressing cells by binding to HER2. (2) The antibody described in (1) above binds to the extracellular domain of HER2. (3) The antibody according to (1) or (2) above, wherein the antibody is a monoclonal antibody. (4) The antibody according to any one of the above (1) to (3), wherein the antibody has antibody-dependent cellular cytotoxicity (ADCC) activity and / or complement-dependent cytotoxicity (CDC) activity. (5) The 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) The 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 of the murine anti-HER2 antibody 4D5, named recombinant humanized anti-HER2 monoclonal antibody (huMAb4D5-8, rhuMAb HER2). Trastuzumab specifically binds to the extracellular domain IV of HER2 and exerts an anti-cancer effect by inducing antibody-dependent cellular cytotoxicity (ADCC) or inhibiting signal transduction from HER2. Trastuzumab is highly effective against tumors that overexpress HER2, and 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] Trastuzumab has therapeutic effect in treating breast cancer, but some breast cancer patients who overexpress HER2 show no or only weak response to trastuzumab treatment. The anti-HER2 antibody used in the anti-HER2 antibody drug conjugate of the present invention can be derived from any species, and preferred examples of species can include human, rat, mouse, and rabbit. If the antibody is derived from a species other than human, it is preferably chimerized 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 the amino acid sequence represented by SEQ ID NO:1 and a light chain consisting of the amino acid sequence represented by SEQ ID NO:2.

[0159] Trastuzumab heavy chain amino acid sequence: EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGT LVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT CPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 1)

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

[0161] The CDR sequences of trastuzumab according to Kabat are: [Table 9]

[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.

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

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

[0165] In embodiments, the antibody lacks a lysine residue at the carboxyl terminus of the heavy chain defined by the sequence of SEQ ID NO: 1. In embodiments, the antibody comprises a heavy chain consisting of an amino acid sequence consisting of amino acid residues 1-449 of SEQ ID NO: 1, and a light chain consisting of an amino acid sequence consisting of amino acid residues 1-214 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 ADCs of the present invention are also effective against antibody constructs that are commercially available or highly similar to or biosimilar to "innovator" antibody constructs. Biosimilar ADCs are expected to perform similarly to immunoconjugates of innovator products (e.g., trastuzumab). As used herein, the term "biosimilar" in reference to a biological product means that the biological product is very similar to the reference product, with minor differences in clinically inactive components, and there are no clinically meaningful differences between the biological product and the reference product in terms of safety, purity, and potency of the product.

[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-qyp (Trazimera), and trastuzumab-anns (Kanjinti).

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

[0170] In other embodiments, the anti-HER2 antibody is a biobetter of trastuzumab. In embodiments, a "biobetter" biologic is in the same class as an FDA approved biologic, is directed to the same target protein as the reference biologic but is not identical, and has improvements over the reference formulation in terms of safety, efficacy, stability, route of administration, etc.

[0171] Antibody Binding and Antitumor Activity The binding activity of the antibody to tumor cells can be confirmed using flow cytometry. The internalization of the antibody into tumor cells can be confirmed using (1) an assay in which the antibody taken up into the cell is visualized under a fluorescent microscope using a secondary antibody (fluorescently labeled) that binds to the therapeutic antibody (Cell Death and Differentiation (2008) 15,751-761), (2) an assay in which the intensity of fluorescence taken up into the cell is measured using a therapeutic antibody (fluorescently labeled) (Molecular Biology of the Cell, Vol. 15,5268-5282, December 2004), or (3) a Mab-ZAP assay using an immunotoxin that binds to the therapeutic antibody, which is released when taken up into the cell and inhibits cell proliferation (Bio Techniques 28: 162-165, January 2000). A recombinant complex protein of the diphtheria toxin catalytic domain and protein G can be used as the immunotoxin.

[0172] Antitumor activity of an antibody can be confirmed in vitro by determining the inhibitory activity against cell proliferation.For example, a cancer cell line that overexpresses the antibody's target protein is cultured, and the antibody is added to the culture system at various concentrations to determine the inhibitory activity against foci formation, colony formation, and spheroid growth.Antitumor activity can be confirmed in vivo, for example, by administering the antibody to nude mice implanted with tumor cell lines that highly express the target protein, and measuring the changes in cancer cells.

[0173] Since the compound conjugated to the antibody-drug conjugate exerts an antitumor effect, it is preferable, but not essential, that the antibody itself has an antitumor effect. In order to specifically and selectively exert the cytotoxic activity of the antitumor compound against tumor cells, it is important and preferable that the antibody has the property of being internalized and translocated into tumor cells.

[0174] Anti-HER2 antibody production The antibody against HER2 of the present invention can be obtained by a method commonly practiced by those skilled in the art, for example, immunizing an animal with HER2 or any polypeptide selected from the amino acid sequence of HER2, and collecting and purifying the antibody produced in vivo. The biological species of HER2 used as an antigen is not limited to human, and animals can be immunized with HER2 derived from animals other than humans, such as mice and rats, or rat p185neu. In this case, antibodies applicable to human diseases can be selected by examining the cross-reaction between the obtained antibody that binds to heterologous HER2 and human HER2.

[0175] Furthermore, monoclonal antibodies can be obtained from hybridomas 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 antigen can be obtained by expressing HER2 gene in host cell using genetic engineering.Specifically, a vector capable of expressing HER2 gene is produced, the obtained vector is transfected into host cell to express gene, and then the expressed HER2 is purified.

[0177] Alternatively, the above-mentioned genetically engineered HER2-expressing cells or cell lines expressing HER2 may be used as HER2 protein. Anti-HER2 antibodies can be obtained by procedures known to those skilled in the art. The method of obtaining antibodies against HER2 is specifically described below.

[0178] Antigen preparation Examples of antigens used to generate anti-HER2 antibodies include HER2, or a polypeptide consisting of a partial amino acid sequence containing at least 6 consecutive amino acids of HER2, or a derivative obtained by adding a given amino acid sequence or its carrier.

[0179] HER2 can be directly purified and used from human tumor tissue or tumor cells. In addition, HER2 can be obtained by synthesizing it in vitro or by producing it in a host cell by genetic engineering.

[0180] With regard to genetic engineering, specifically, HER2 can be obtained by incorporating HER2 cDNA into an expression vector and then synthesizing HER2 in a solution containing the enzymes, substrates and energy materials required for transcription and translation, or by expressing HER2 in other prokaryotic or eukaryotic transformed host cells.

[0181] Furthermore, the antigen can be obtained as a secreted protein by expressing a fusion protein obtained by ligating the extracellular domain of HER2, a membrane protein, to the constant region of an antibody in an appropriate host vector system.

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

[0183] An example of an in vitro synthesis of a polypeptide is the Rapid Translation System (RTS) manufactured by Roche Diagnostics, Inc., but is not limited thereto.

[0184] Examples of prokaryotic host cells include Escherichia coli and Bacillus subtilis. To transform a host cell with a target gene, the host cell is transformed with a replicon, i.e., a plasmid vector consisting of an origin of replication and regulatory sequences derived from a species compatible with the host. In addition, the vector preferably contains sequences capable of conferring phenotypic selection on the transformed cell.

[0185] Examples of eukaryotic host cells include vertebrate cells, insect cells, and yeast cells. Vertebrate cells are often used, such as simian COS cells (Gluzman, Y., Cell, (1981) 23, pp. 175-182, ATCC CRL-1650; ATCC: American Type Culture Collection), mouse fibroblasts NIH3T3' ATCC No. CRL-1658), and Chinese hamster ovary cells (CHO cells; ATCC: CCL-61) with dihydrofolate reductase deficiency (Urlaub, G. and Chasin, LA, Proc. Natl. Acad. Sci. USA (1980) 77, pp. 4126-4220), but are not limited thereto.

[0186] The transformant thus obtained can be cultured according to a method commonly used by those skilled in the art, and the target polypeptide is produced intracellularly or extracellularly by culturing the transformant.

[0187] The appropriate medium used for the culture can be selected from various commonly used culture media depending on the host cell used. When E. coli is used, for example, LB medium supplemented with antibiotics such as ampicillin or IPMG as necessary can be used.

[0188] The recombinant protein produced intracellularly or extracellularly by the transformant through such culture can be separated and purified by any of a variety of known separation methods utilizing the physical or chemical properties of the protein.

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

[0190] Additionally, by attaching a tag of six histidine residues (SEQ ID NO: 9) to the expressed recombinant protein, the protein can be efficiently purified on a nickel affinity column, or by attaching an IgG Fc region to the expressed recombinant protein, the protein can be efficiently purified on a protein A column.

[0191] By combining the above-mentioned methods, the target polypeptide can be easily produced in large quantities with high yield and purity.

[0192] The above-mentioned transformant itself can be used as an antigen. 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 lines NCI-N87, and human ovarian cancer lines SK-OV-3. The cell lines of the present invention are not limited to these cell lines as long as they express HER2.

[0193] Generation of anti-HER2 monoclonal antibodies 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 generally requires the following steps: (a) purifying a biopolymer to be used as an antigen or preparing an antigen-expressing cell; (b) preparing antibody-producing cells by immunizing the animal by injection of the antigen, collecting blood and assaying the blood for 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 a myeloma; (e) screening the group of hybridomas that produce the desired antibodies; (f) dividing the hybridomas into single cell clones (cloning); (g) optionally culturing the hybridomas or breeding the animals engrafted with the hybridomas to produce large amounts of monoclonal antibodies; (h) examining the biological activity and binding specificity of the monoclonal antibodies thus produced, or similarly assaying their properties as labeling reagents, etc.

[0195] The method for producing the monoclonal antibody of the present invention will be described in detail below in accordance with the above steps, but the method of the present invention is not limited thereto, and for example, antibody-producing cells other than spleen cells and myelomas can be used.

[0196] Antigen purification As an antigen, HER2 or a partial peptide thereof prepared by the above-mentioned method can be used.

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

[0198] In addition, HER2 expressing cell lines can also be used as antigens.

[0199] Preparation of antibody-producing cells The antigen obtained in step (a) is mixed with an adjuvant such as Freund's complete or incomplete adjuvant, or an adjuvant such as aluminum potassium sulfate, and the mixture obtained is used as an immunogen to immunize an experimental animal. Another method involves immunizing an experimental animal using the antigen-expressing cells as an immunogen. As the experimental animal, any animal used in a known hybridoma production method can be used without any problems. Specifically, for example, mice, rats, goats, sheep, cows, horses, etc. can be used. However, from the viewpoint of easy availability of myeloma cells to be fused with the extracted antibody-producing cells, it is preferable to use mice or rats as the animal to be immunized.

[0200] Furthermore, the mouse or rat strain used is not particularly limited. For example, 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, 129, etc. can be used, and in the case of rats, Wistar, Low, Lewis, Sprague, Dawley, ACI, BN, Fischer, etc. can be used.

[0201] These mice and rats are commercially available from laboratory animal breeders / distributors such as CLEA Japan and Charles River Animal Hospital Japan.

[0202] As animals to be immunized, taking into consideration the fusion with myeloma cells described below, the BALB / c strain of mice and the Wistar and Low strains of rats are particularly preferred.

[0203] Furthermore, taking into consideration the antigenic homology between humans and mice, it is also preferable to use mice with a reduced biological function for removing autoantibodies, that is, mice with an autoimmune disease.

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

[0205] To immunize animals with HER2 or its recombinants, known methods can be used, as described in detail in, for example, Weir, DM, Handbook of Experimental Immunology Vol. I.II.III., Blackwell Scientific Publications, Oxford (1987); Kabat, EA and Mayer, MM, Experimental Immunochemistry, Charles C Thomas Publisher Springfield, Ill. (1964).

[0206] Among these immunization methods, specific preferred methods in the present invention are, for example, as follows.

[0207] That is, first, either or both of the membrane protein fraction functioning as an antigen or the cells expressing the antigen are administered intradermally or intraperitoneally to an animal. However, in order to increase the immune efficiency, it is preferable to use both administration routes in combination, and the immune 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 final administration.

[0208] The administration schedule of the antigen varies depending on the type of animal to be immunized, individual differences, etc. However, in general, an administration schedule in which the antigen is administered 3 to 6 times at an administration interval of 2 to 6 weeks is preferable, and an administration schedule in which the antigen is administered 3 to 4 times at an administration interval of 2 to 4 weeks is more preferable.

[0209] Furthermore, the dose of the antigen varies depending on the type of animal, individual differences, etc., but is generally set to 0.05 to 5 mg, preferably about 0.1 to 0.5 mg.

[0210] The booster immunization is carried out 1 to 6 weeks, preferably 1 to 4 weeks, more preferably 1 to 3 weeks after the above-mentioned antigen administration. When the immunogen is a cell, the booster immunization is carried out at a dose of 1×10 6 ~1×10 7 Using cells.

[0211] The dose of antigen in booster immunization varies depending on the type and size of the animal. For example, in the case of mice, the dose is generally set to 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, the dose is set to 1×10 6 ~1×10 7 Using cells.

[0212] Spleen cells or lymphocytes containing antibody-producing cells are aseptically removed from the immunized animal 1 to 10 days, preferably 2 to 5 days, more preferably 2 to 3 days after booster immunization. At this time, the antibody titer is measured, and if an animal with a sufficiently increased antibody titer is used as a source of antibody-producing cells, the subsequent procedures can be carried out more efficiently.

[0213] Examples of the antibody titer measurement method used herein include, but are not limited to, RIA and ELISA.For example, when using ELISA, the antibody titer measurement in the present invention can be carried out according to the procedure described below.

[0214] First, a purified or partially purified antigen is adsorbed onto a solid-phase surface such as a 96-well ELISA plate, and the solid-phase surface to which the antigen is not adsorbed is covered with a protein unrelated to the antigen, such as bovine serum albumin (BSA). After washing the surface, it is contacted with a serially diluted sample (e.g. mouse serum) as the primary antibody, and the antibody in the sample is allowed to bind to the antigen.

[0215] Furthermore, an antibody labeled with an enzyme is added as a secondary antibody to the mouse antibody, and allowed to bind to the mouse antibody. After washing, a substrate for the enzyme is added, and the change in absorbance caused by color development due to the decomposition of the substrate is measured, and the antibody titer is calculated based on the measured value.

[0216] Isolation of antibody-producing cells from spleen cells or lymphocytes of an immunized animal can be carried out according to 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 general method can be adopted in which the spleen is homogenized and filtered through a stainless steel mesh to obtain cells, which are then suspended in Eagle's minimum essential medium (MEM) to isolate antibody-producing cells.

[0217] Preparation of myeloma cells (hereinafter referred to as "myeloma cells") The myeloma cells used for cell fusion are not particularly limited, and appropriate cells can be selected from known cell lines. However, in consideration of the convenience of selecting hybridomas from fused cells, it is preferable to use HGPRT (hypoxanthine guanine phosphoribosyltransferase)-deficient strains for which selection procedures have been established.

[0218] More specifically, examples of HGPRT-deficient strains include 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 from mouse, 210.RSY3.Ag.1.2.3 (Y3) from rat, and U266AR (SKO-007), GM1500.GTG-A12 (GM1500), UC729-6,LICR-LOW-HMy2 (HMy2), and 8226AR / NIP4-1 (NP41) from human. These HGPRT-deficient strains are available from, for example, ATCC.

[0219] These cell lines are subcultured in appropriate media such as 8-azaguanine medium (a medium obtained by adding 8-azaguanine to RPMI1640 medium supplemented with glutamine, 2-mercaptoethanol, gentamicin, and fetal calf serum (hereinafter referred to as "FCS")), Iscove's Modified Dulbecco's Medium (hereinafter referred to as "IMDM"), or Dulbecco's Modified Medium (hereinafter referred to as "DMEM"). In this case, 3 to 4 days before cell fusion, 2 × 10 7 The cells are subcultured in a normal medium (for example, ASF104 medium (manufactured by Ajinomoto Co., Inc.) containing 10% FCS) to obtain a cell number of 100 or more.

[0220] cell fusion Fusion of antibody-producing cells and myeloma cells can be carried out according to known methods (Weir, D. M. Handbook of Experimental Immunology Vol. I.II.III., Blackwell Scientific Publications, Oxford (1987); Kabat, E. A. and Mayer, M. M., Experimental Immunochemistry, Charles C. Thomas Publisher, Springfield, Ill. (1964), etc.) under conditions that do not excessively reduce the viability of the cells.

[0221] Examples of such methods include a chemical method in which antibody-producing cells and myeloma cells are mixed in a solution containing a high concentration of a polymer such as polyethylene glycol, a physical method using electrical stimulation, etc. Among these methods, specific examples of the chemical method are described below.

[0222] That is, when polyethylene glycol is used in a solution containing a polymer at a high concentration, antibody-producing cells and myeloma cells are mixed in a solution of polyethylene glycol having 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.

[0223] Selection of Hybridoma Populations The method for selecting hybridomas obtained by the above-mentioned cell fusion is not particularly limited. Usually, the HAT (hypoxanthine, aminopterin, thymidine) selection method (Kohler et al., Nature (1975), 256, p. 495; Milstein et al., Nature (1977), 266, p. 550) is used.

[0224] This method is effective when obtaining hybridomas using HGPRT-deficient myeloma cells that cannot survive in the presence of aminopterin. In other words, by culturing unfused cells and hybridomas in HAT medium, only hybridomas resistant to aminopterin are selectively allowed to survive and grow.

[0225] Division into single cell clones (cloning) As a method for cloning hybridomas, known methods such as the methylcellulose method, the soft agarose method, or the limiting dilution method can be used (see, for example, Barbara, BM and Stanley, MS: Selected Methods in Cellular Immunology, WH Freeman and Company, San Francisco (1980)). Among these methods, three-dimensional culture methods such as the methylcellulose method are particularly preferred. For example, a group of hybridomas produced by cell fusion is suspended in a methylcellulose medium such as ClonaCell-HY Selection Medium D (manufactured by StemCell Technologies, Inc., #03804) and cultured. The formed hybridoma colonies are then collected, thereby obtaining monoclonal hybridomas. Each of the collected hybridoma colonies is cultured, and hybridomas confirmed to have a stable antibody titer in the obtained hybridoma culture supernatant are selected as HER2 monoclonal antibody-producing hybridoma strains.

[0226] Preparation of monoclonal antibodies by hybridoma culture By culturing the hybridomas selected in this manner, monoclonal antibodies can be obtained efficiently. However, it is preferable to screen hybridomas that produce the target monoclonal antibody prior to culturing.

[0227] In such screening, a known method can be adopted. The antibody titer in the present invention can be measured, for example, by the ELISA method described in the above item (b). The hybridoma obtained by the above method can be stored in a frozen state in liquid nitrogen or in a freezer at -80°C or lower. After the cloning is completed, the medium is changed from HT medium to a normal medium, and the hybridoma is cultured.

[0228] Large-scale culture is carried out by rotary culture or spinner culture using a large culture bottle. From the supernatant obtained by large-scale culture, a monoclonal antibody that specifically binds to the protein of the present invention can be obtained by purification using a method known to those skilled in the art, such as gel filtration.

[0229] Furthermore, the hybridomas can be injected into the peritoneal cavity of mice of the same strain as the hybridomas (e.g., the BALB / c described above) or Nu / Nu mice to proliferate the hybridomas, thereby obtaining ascites fluid containing large amounts of the monoclonal antibodies of the present invention.

[0230] When hybridomas are administered intraperitoneally, a large amount of ascites can be obtained by administering a mineral oil such as 2,6,10,14-tetramethylpentadecane (pristane) 3 to 7 days prior to administration.

[0231] For example, an immunosuppressant is injected into the abdominal cavity of a mouse of the same strain as the hybridoma to inactivate T cells. 6 ~10 7The hybridoma clone cells are suspended in serum-free medium (0.5 ml) and the suspension is administered intraperitoneally to a mouse. In general, when the abdomen is expanded and filled with ascites, the ascites fluid is collected from the mouse. By this method, monoclonal antibodies can be obtained at a concentration of about 100 times or more that of the culture fluid.

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

[0233] The monoclonal antibody thus obtained has high antigen specificity to HER2. Examples of the monoclonal antibody of the present invention can include, but are not limited to, mouse monoclonal antibody 4D5 (ATCC CRL 10463).

[0234] Monoclonal antibody assays The isotype and subclass of the monoclonal antibody thus obtained can be determined as follows.

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

[0236] The Ouchterlony method is simple, but when the concentration of the monoclonal antibody is low, a concentration procedure is necessary.

[0237] On the other hand, when using the ELISA or RIA method, the culture supernatant is directly reacted with an antigen-adsorbed solid phase, and antibodies corresponding to various immunoglobulin isotypes and subclasses are used as secondary antibodies, thereby enabling the isotype and subclass of the monoclonal antibody to be identified.

[0238] Furthermore, as a simpler method, a commercially available identification kit (eg, Mouse Typer Kit manufactured by Bio-Rad Laboratories, Inc.) can also be used.

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

[0240] Furthermore, by repeating steps (a) to (h) in (2), even if a monoclonal antibody is obtained separately and independently, it is possible to obtain an antibody having a cytotoxic activity equivalent to that of the HER2 antibody obtained in step (g). An example of such an antibody is an antibody that binds to the same epitope as the HER2 antibody obtained in step (g). When a newly produced monoclonal antibody binds to a partial peptide or partial tertiary structure to which an anti-HER2 antibody binds, it can be determined that the monoclonal antibody binds to the same epitope as the anti-HER2 antibody. Furthermore, by confirming that the monoclonal antibody competes with the binding of the anti-HER2 antibody to HER2 (i.e., that the monoclonal antibody inhibits the binding of the anti-HER2 antibody to HER2), it can be determined that the monoclonal antibody binds to the same epitope as the anti-HER2 antibody even if a specific epitope sequence or structure has not been determined. When it is confirmed that the monoclonal antibody binds to the same epitope as the anti-HER2 antibody, it is highly expected that the monoclonal antibody has an antigen-binding affinity or biological activity equivalent to that of the anti-HER2 antibody.

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

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

[0243] Examples of humanized antibodies include antibodies obtained by incorporating only the complementarity determining region (CDR) into a human-derived antibody (see Nature (1986) 321, pp. 522-525), antibodies obtained by grafting not only the CDR sequences but also some of the amino acid residues in the framework onto a human antibody using the CDR grafting method (WO90 / 07861), and antibodies humanized using a gene conversion mutagenesis strategy (US at. No. 5,821,337).

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

[0245] As the amino acid substitution herein, conservative amino acid substitution is preferred. Conservative amino acid substitution refers to substitution occurring within the amino acid group related to the amino acid side chain. Preferred amino acid groups are as follows: acidic group (aspartic acid and glutamic acid), basic group (lysine, arginine, and histidine), non-polar group (alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan), and uncharged polar family (glycine, asparagine, glutamine, cysteine, serine, threonine, and tyrosine). More preferred amino acid groups are as follows: aliphatic hydroxy group (serine and threonine), amide-containing group (asparagine and glutamine), aliphatic group (alanine, valine, leucine, and isoleucine), and aromatic group (phenylalanine, tryptophan, and tyrosine). Such amino acid substitution is preferably performed 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 to the above heavy chain amino acid sequence with a sequence having high homology to the above light chain amino acid sequence, an antibody having biological activity equivalent to each of the above antibodies can be selected. Such homology is generally 80% or more homology, preferably 90% or more homology, more preferably 95% or more homology, and most preferably 99% or more homology. Furthermore, by combining amino acid sequences in which one to several amino acid residues are substituted, deleted or added in the heavy or light chain amino acid sequence, it is also possible to select an antibody having biological activity equivalent to each of the above antibodies. As used herein, the term "homology" is used synonymously with "identity."

[0247] The homology between two amino acid sequences can be determined using the default parameters of the Blast algorithm version 2.2.2. (Ltschul, 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 visiting the site: www.ncbi.nlm.nih.gov / blast.

[0248] Furthermore, the antibodies of the present invention include human antibodies that bind to HER2. An anti-HER2 human antibody refers to a human antibody that has only antibody sequences derived from human chromosomes. Anti-HER2 human antibodies can be obtained by a method using a human antibody-producing mouse having a human chromosomal fragment containing heavy and light chain genes of a 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 (Kitagawa, Y., Matuda, T. and Iijima, S. eds.), Kluwer Academic Publishers, 1999; Tomizuka, K. et al., Proc. Natl. Acad. Sci. USA (2000) 97, pp. 722-727, etc.).

[0249] Specifically, such a human antibody-producing mouse can be produced as follows: A knockout animal and a transgenic animal are produced and then crossed to produce a genetically modified animal in which the endogenous immunoglobulin heavy and light chain loci are destroyed and instead human immunoglobulin heavy and light chain loci are introduced via a yeast artificial chromosome (YAC) vector or the like.

[0250] Furthermore, according to recombinant DNA technology, the antibody can also be obtained from the culture supernatant by transforming eukaryotic cells with cDNA encoding each of the heavy and light chains of such a human antibody, preferably with a vector containing such cDNA, and culturing the transformed cells that produce recombinant human monoclonal antibody.

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

[0252] Furthermore, methods for obtaining phage display-derived human antibodies selected from a human antibody library (see Wormstone, I M 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.) are also known.

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

[0254] By analyzing the genes of phages selected based on antigen binding, the DNA sequence encoding the variable region of the antigen-binding human antibody can be determined.

[0255] If the DNA sequence of an antigen-binding scFv has been determined, an expression vector consisting of that sequence can be prepared and introduced into an appropriate host for expression to obtain a human antibody (see WO92 / 01047, WO92 / 20791, WO93 / 06213, WO93 / 11236, WO93 / 19172, WO95 / 01438, WO95 / 15388; Annu. Rev. Immunol. (1994) 12, pp. 433-455, Nature Biotechnology (2005) 23(9), pp. 1105-1116).

[0256] Another example of an index for use in comparing antibody properties is antibody stability. Differential scanning calorimetry (DSC) is an instrument that can rapidly and accurately measure the thermal denaturation midpoint temperature (Tm), which is used as a preferred index of the relative structural stability of proteins. By measuring the Tm value using DSC and comparing the values, differences in thermal stability can be compared. It is known that the storage stability of an antibody shows some correlation with its thermal stability (Lori Burton, et al., Pharmaceutical Development and Technology (2007) 12, pp. 265-273), and a preferred antibody can be selected by using thermal stability as an index. Examples of other indices for selecting an antibody include high yield in a suitable host cell and low aggregation in an aqueous solution. For example, an antibody that shows the highest yield does not always show the highest thermal stability, so it is necessary to select an antibody that is optimal for administration to humans by performing a comprehensive evaluation based on the above-mentioned indices.

[0257] The present invention also includes modified variants of antibodies. Modified variants refer to variants obtained by subjecting the antibody of the present invention to chemical or biological modification. Examples of chemically modified variants include variants chemically modified by linking a chemical moiety to the amino acid backbone, variants chemically modified with N-linked or O-linked carbohydrate chains, etc. Examples of biologically modified variants include variants obtained by post-translational modification (such as N-linked or O-linked glycosylation, N- or C-terminal processing, deamidation, isomerization of aspartic acid, or oxidation of methionine), and variants in which a methionine residue has been added to the N-terminus by expression in a prokaryotic host cell. Furthermore, antibodies labeled to allow detection or isolation of the antibody or antigen of the present invention, such as enzyme-labeled antibodies, fluorescent-labeled antibodies, and affinity-labeled antibodies, are also included in the meaning of modified variants. Such modified variants of the antibody of the present invention are useful for improving the stability and blood retention of the antibody, reducing antigenicity, detecting or isolating the antibody or antigen, etc.

[0258] Furthermore, antibody-dependent cellular cytotoxicity can be enhanced by controlling the modification of glycans linked to the antibody of the present invention (glycosylation, defucosylation, etc.). Techniques for controlling the modification of antibody sugar chains are known, such as WO99 / 54342, WO00 / 61739, and WO02 / 31140. However, the techniques are not limited thereto. The antibody of the present invention also includes an antibody in which the modification of the sugar chain is regulated.

[0259] When an antibody gene is first isolated and then introduced into a suitable host to produce an antibody, a combination of a suitable host and a suitable expression vector can be used. A specific example of an antibody gene includes a combination of a gene encoding the heavy chain sequence of an antibody described herein and a gene encoding its light chain sequence. When transforming a host cell, the heavy chain sequence gene and the light chain sequence gene can be inserted separately into the same expression vector or into different expression vectors.

[0260] When eukaryotic cells are used as hosts, animal cells, plant cells, and eukaryotic microorganisms can be used. Examples of animal cells include mammalian cells, such as simian COS cells (Gluzman, Y, Cell, (1981) 23, pp. 175-182, ATCC CRL-1650), mouse fibroblast cells NIH3T3 (ATCC No. CRL-1658), and Chinese hamster ovary cells (CHO cells; ATCC: CCL-61) with dihydrofolate reductase deficiency (Urlaub, G. and Chasin, LA, Proc. Natl. Acad. Sci. USA (1980) 77, pp. 4126-4220). Examples of prokaryotic cells include Escherichia coli and Bacillus subtilis.

[0261] The desired antibody gene is introduced into these cells via transformation, and the transformed cells are cultured in vitro to obtain the antibody. In the above culture method, the yield may vary depending on the nucleotide sequence of the antibody, so an antibody that is easy to manufacture as a pharmaceutical product can be selected from antibodies with equivalent binding activity using the yield as an indicator. Therefore, the antibody of the present invention also includes an antibody obtained by a method for producing an antibody, which is characterized by including a step of culturing a transformed host cell and a step of collecting the desired antibody from the culture obtained in the culture step.

[0262] It is known that the lysine residue at the carboxyl terminus of the heavy chain of an antibody produced in cultured mammalian cells is deleted (Journal of Chromatography A, 705: 129-134 (1995)), and it is also known that two amino acid residues (glycine and lysine) are deleted at the carboxyl terminus of the heavy chain of an antibody produced in cultured mammalian cells, and a proline residue newly positioned at the carboxyl terminus is amidated (Analytical Biochemistry, 360: 75-83 (2007)). However, such deletion and modification of the heavy chain sequence does not affect the antigen-binding affinity and effector functions (complement activation, antibody-dependent cellular cytotoxicity, etc.) of the antibody. Therefore, the antibody according to the present invention also includes antibodies and functional fragments thereof that have been subjected to such modifications, and also includes deletion variants in which one or two amino acids are deleted at the carboxyl terminus of the heavy chain, and variants obtained by amidating the deletion variants (for example, heavy chains in which the carboxyl-terminal proline residue is amidated). The types of deletion variants having deletions at the carboxyl termini of the heavy chains of the antibody according to the present invention are not limited to the above variants, so long as the antigen-binding affinity and effector function are preserved. The two heavy chains constituting the antibody according to the present invention may be one type selected from the group consisting of full-length heavy chains and the above deletion variants, or may be a combination of two types selected therefrom. The ratio of the amounts of each deletion variant may be influenced by the type of cultured mammalian cells producing the antibody according to the present invention and the culture conditions, but an example can be one in which one amino acid residue is deleted at the carboxyl terminus in both of the two heavy chains contained as major components of the antibody according to the present invention.

[0263] Examples of the isotype of the antibody of the present invention include IgG (IgG1, IgG2, IgG3, IgG4), and preferably IgG1 or IgG2.

[0264] Examples of the biological activity of an antibody generally include antigen-binding activity, antigen-binding and internalization activity in cells expressing the antigen, neutralizing activity of the antigen, enhancing activity of the antigen, antibody-dependent cellular 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 binding activity to HER2, and preferably binding to HER2 and internalization activity in HER2-expressing cells. Furthermore, the antibody of the present invention may have ADCC activity, CDC activity, and / or ADCP activity in addition to the activity of internalization into cells.

[0265] The obtained antibody can be purified to homogeneity. The antibody can be separated and purified by using a conventional protein separation and purification method. For example, the antibody can be separated and purified by appropriately selecting and combining column chromatography, filter filtration, ultrafiltration, salt precipitation, dialysis, preparative polyacrylamide gel electrophoresis, isoelectric focusing, etc. (Strategies for Protein Purification and Characterization: A Laboratory Course Manual, Daniel R. Marshak et al. eds., Cold Spring Harbor Laboratory Press (1996); Antibodies: A Laboratory Manual. Ed Harlow and David Lane, Cold Spring Harbor Laboratory (1988)), but the method is not limited thereto.

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

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

[0268] Furthermore, by using a carrier on which an antigen is immobilized, the antibody can be purified by utilizing the binding properties of the antibody to the antigen.

[0269] Antibody-drug conjugates including anti-HER2 antibodies In some aspects, the invention features a conjugate that includes a cell binding agent (eg, an anti-HER2 antibody described herein) and a camptothecin derivative.

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

[0271] In yet a further aspect, the present invention provides a compound of formula (PL-A'): {D-L1-L2-Q'-CH2-NH-E-Z'} p -C(PL-A'), or a pharma- ceutically acceptable salt thereof, wherein D is represented by the following structural formula: [ka] During the ceremony, R 1are independently -H, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, silyl, C3-C6 cycloalkyl, C1-C6 halogenated alkyl, C2-C6 halogenated alkenyl, or C2-C6 halogenated alkynyl; R 2 are independently -H, -F, -N(R 4 )2, -N(R 4 )(R 5 ), -OR 4 , -SR 4 , -S(=O)R 5 , -SO2R 5 , C1-C6 alkyl, or C1-C6 fluoroalkyl; R 3 is -H, -F, -CN, -OCH3, -CH3, or -CF3, or R 2 and R 3 together, the formula -O(CH2) n O- or -O(CF2) n O- group, where n is 1 or 2, R 4 are independently -H or C1-C4 alkyl; R 5 are independently C1-C4 alkyl; L1 is independently absent or -(C1-C 10 alkylene)-, L2 is independently absent or -OCH2-L3-*, -SCH2-L3-*, -S(=O)-L3-*, -SO2-L3-*, -C(=O)-L3-*, -N(R 6 )CH2-L3-*, -N(R 6 )C(=O)-L3-*, -N(R 6 )C(=O)N(R 7 )-L3-*, -C(=O)N(R 6 )CH2-L3-*, -OC(=O)N(R 6 )CH2-L3-* or -N(R 6 )C(=O)OCH2-L3-*, where * indicates the site of covalent attachment to Q'; L3 is independent, -(C1-C 10alkylene)-, -CH2OCH2CH2-, or -CH2CH2OCH2CH2-; Each R 6 and R 7 are independently -H, C1-C6 alkyl, C1-C6 fluoroalkyl, C3-C6 cycloalkyl, aryl, heteroaryl, or benzyl; Q' is -O- or -S-; E is a peptide comprising 2 to 10 amino acids, wherein E is optionally substituted with one or more polyols, and the N-terminus of the peptide is covalently linked to Z'; Z' is -C(=O)-L4-Y', [ka] In the formula, m represents an integer of 1 to 10, and * represents a site covalently bonded to the C, L4 is -(C1-C 10 Alkylene)-, -CH2CH2(OCH2CH2) n N(R 8 )C(=O)-L5-*, or -CH2(OCH2CH2) n N(R 8 )C(═O)-L5-*, where n is an integer from 1 to 10, and * represents a site of covalent bonding to Y′; L5 is -(C1-C 10 alkylene)-, R 8 is -H or -CH3, C represents a cell binding agent; Y' is a group formed by reaction of an electrophilic group with a reactive nucleophilic group present on the cell-binding agent; In the formula, R 2 and R 3 When they combine to form -OCHO-, R 1 is not -CH2CH2CH2CH3, p has a value of 1 to 18.

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

[0273] In embodiments, L4 is -(C1-C 10 alkylene).

[0274] In an embodiment, L4 is -CH2CH2(OCH2CH2) n N(R 8 )C(=O)-L5-* or -CH2(OCH2CH2) n N(R 8 )C(═O)-L5-*, where n represents an integer of 1 to 10, and where * represents the site of covalent bonding to Y′.

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

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

[0277] In an embodiment, Y' is formed from: [ka] In the formula, R 10 and R 11 are each independently -H or C1-C3 alkyl.

[0278] In embodiments, Y′ is [ka] where R 10 and R 11are each independently -H or C1-C3 alkyl, and * indicates the site of covalent bonding to the C.

[0279] In an embodiment, Z' is formed from: [ka]

[0280] In an embodiment, Z′ is [ka] and In the formula, * indicates the site of covalent attachment to C.

[0281] In an embodiment, Z' is [ka] where * indicates the site of covalent attachment to said C. In embodiments, m is 1. In embodiments, m is 2. In embodiments, m is 3. In embodiments, m is 4. In embodiments, m is 5. In embodiments, m is 6. In embodiments, m is 7. In embodiments, m is 8. In embodiments, m is 9. In embodiments, m is 10.

[0282] In an embodiment, Z' is [ka] where * indicates the site of covalent attachment to said C. In embodiments, m is 1. In embodiments, m is 2. In embodiments, m is 3. In embodiments, m is 4. In embodiments, m is 5. In embodiments, m is 6. In embodiments, m is 7. In embodiments, m is 8. In embodiments, m is 9. In embodiments, m is 10.

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

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

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

[0286] In embodiments, E comprises an amino acid having the structure: [ka] In the formula, R 9 is -H or C1-C6 alkyl.

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

[0288] in embodiments、Eは、-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-Th r-*、-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-* * 、-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), -A Z' is selected from the group consisting of La-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 the N-terminus of the peptide covalently attached to Z'.

[0289] In embodiments, E is -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 the N-terminus of the peptide covalently attached to Z'.

[0290] In embodiments, -E-NH-CH 2- has one of the following structures, where * indicates the N-terminus of the peptide covalently attached to Z'. [ka]

[0291] In an embodiment, Z'-E-NH-CH2 is formed from one of the following structures: [ka]

[0292] In embodiments, Z'-E-NH-CH2 is one of the following structures, where * indicates the point of attachment to C: [ka]

[0293] In embodiments, R 1 is -H or -CH2CH3, and R 2 is -OH or alkoxy, and R 3 is -H, then -L1-L2-Q'- is not -CH(R')CHO- or -CH(R')(CH2)2O-, where R' is -H, or C1-C6 alkyl, alkoxy, substituted alkyl, phenyl, or PhCH2-. In embodiments, R 1 is -H or -CH2CH3, and R 2 is -OH or alkoxy, and R 3 is -H, then -L1-L2-Q'- is not -CH(R')CHO- or -CH(R')(CH2)2O-, where R' is -H, or C1-C6 alkyl, alkoxy, substituted alkyl, phenyl, or PhCH2-.

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

[0295] In embodiments, R 1 , R 2 , and R 3 At least one of is not -H.

[0296] In embodiments, R 1is independently a C1-C6 alkyl, a C2-C6 alkenyl, a C2-C6 alkynyl, a silyl, a C3-C6 cycloalkyl, a C1-C6 alkyl halide, an alkene, or an alkyne.

[0297] In embodiments, R 1 is independently -H or C1-C6 alkyl.

[0298] In embodiments, R 2 are independently -H, -F, -N(R 4 )2, -N(R 4 )(R 5 ), -OR 4 , -SR 4 , -S(=O)R 5 , -SO2R 5 , C1-C6 alkyl, or C1-C6 fluoroalkyl; R 3 are independently -H, -F, -CN, -OCH3, -CH3, or -CF3.

[0299] In embodiments, R 2 is independently C1-C6 alkyl, C1-C6 fluoroalkyl, or -F.

[0300] In embodiments, R 3 are independently -H, -F, -CN, or -CF3.

[0301] In embodiments, R 3 are independently -F, -CN, -OCH3, -CH3, or -CF3.

[0302] In embodiments, R 2 and R 3 is bonded to -O(CH2) n O- or -O(CF2) n O-, where n is 1 or 2.

[0303] In embodiments, D is represented by one of the following structures: [ka] [ka]

[0304] In an embodiment, D is: [ka]

[0305] In embodiments, R 1 is -H or C1-C6 alkyl.

[0306] In embodiments, D is represented by one of the following structures: [ka]

[0307] In an embodiment, D is: [ka]

[0308] In an embodiment, L is -(C 10 alkylene)- and L2 is absent.

[0309] In an embodiment, L is -(C 10 alkylene)- and L2 is -N(R 6 )CH2-L3-* or -N(R 6 )C(=O)-L3-*, where * indicates the site of covalent attachment to Q'.

[0310] In an embodiment, L 1 does not exist, L 2 is -N(R 6 )CH2-L3-* or -N(R 6 )C(=O)-L3-*, where * indicates the site of covalent attachment to Q'.

[0311] In embodiments, L3 is -(C1-C 10 alkylene).

[0312] In embodiments, R 6 is -H or -CH3.

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

[0314] In embodiments, L1-L2 is -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 the site of covalent attachment 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-, -OCH2 CH2CH2S-, -SCH2CH2CH2S-, -SCH2CH2S-, -NHCH2CH2CH2S-, -N(CH3)CH2CH2S-, -C(=O)NHCH2CH2S-, -NHC(=O)CH2S-, -CH2S(=O)CH2S-, or -CH2SO2CH2S-.

[0316] In an embodiment, D-L1-L2 is represented by the structure: [ka]

[0317] In an embodiment, D-L1-L2 is represented by the structure: [ka]

[0318] In embodiments, R 1 is -H or C1-C6 alkyl.

[0319] In embodiments, R 1 is -H or -CH2CH3.

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

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

[0322] In embodiments, D-L1-L2-Q'- has one of the following structures: [ka]

[0323] In an embodiment, D-L1-L2-Q'- is: [ka]

[0324] In an embodiment, D-L1-L2-Q'-CH2-NH-E-Z'- is formed from one of the following structures: [ka] [ka]

[0325] In an embodiment, D-L1-L2-Q'-CH2-NH-E-Z'- is formed from MB-2. [ka]

[0326] In an embodiment, D-L1-L2-Q'-CH2-NH-E-Z'- is formed from MB-3 (meditecan). [ka]

[0327] In an embodiment, {D-L1-L2-Q'-CH2-NH-E-Z'} p-C is one of the following structures, where C is a monoclonal antibody and p is the drug-to-antibody ratio (DAR), where 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). [ka] [ka] [ka]

[0328] In an embodiment, {D-L1-L2-Q'-CH2-NH-E-Z'} p -C is [ka] where C is a monoclonal antibody and p is the drug-to-antibody ratio (DAR). In 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).

[0329] In an embodiment, {D-L1-L2-Q'-CH2-NH-E-Z'} p -C is [ka] where C is a monoclonal antibody and p is the drug-to-antibody ratio (DAR). In 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).

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

[0331] In an embodiment, C is trastuzumab (Herceptin).

[0332] In an embodiment, C is trastuzumab-dkst (Ogivri).

[0333] In an embodiment, C is trastuzumab-pkrb (Herzuma).

[0334] In an embodiment, C is trastuzumab-dttb (Ontruzant).

[0335] In an embodiment, C is trastuzumab-qyp (Trazimera).

[0336] In an embodiment, C is trastuzumab-an (Kanjinti).

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

[0338] In an embodiment, {D-L1-L2-Q'-CH2-NH-E-Z'} p -C is MB-3a (trastuzumab meditecan), [ka] where p is the drug-to-antibody ratio (DAR). In embodiments, p is an average number ranging from about 4 to 8 or 7 to 8, or p is 8.

[0339] The conjugates described herein (eg, any compound according to formula PL-A' or formula PL-A, such as MB-2a and MB-3a) can include at least the covalent attachment of a camptothecin derivative.

[0340] In embodiments, subscript p represents the number of camptothecin payload moieties on the cell-binding agent and has a value of 1 to 18, 1 to 12, or 1 to 8. An individual camptothecin conjugate may also be referred to as a camptothecin conjugate compound. In 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 payload moieties conjugated to the cell-binding agent of an individual camptothecin conjugate.

[0341] In embodiments, the populations of individual camptothecin conjugates are substantially identical except for the number of camptothecin payload moieties attached to each cell-binding agent (i.e., camptothecin conjugate composition), such that p represents the average number of camptothecin payload moieties attached to the cell-binding agents of the camptothecin conjugate composition. In that 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 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 embodiments, p is about 2. In embodiments, p is about 4. In embodiments, p is about 6. In embodiments, p is about 8. In embodiments, p is about 10. In embodiments, p is about 12. In embodiments, p is 2. In embodiments, p is 4. In embodiments, p is 8. In embodiments, p has a value of 3 to 4. In embodiments, p has a value of 4 to 5. In embodiments, p has a value of 5 to 6. In embodiments, p has a value of 6 to 7. In embodiments, p has a value of 7 to 8. In embodiments, p has a value of 7.4 to 8. In embodiments, the p value refers to the average drug loading as well as the drug loading of the major ADC in the composition.

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

[0343] In embodiments, conjugation (e.g., as found in any compound according to formula PL-A' or formula PL-A, such as MB-2a or MB-3a described herein) is via an introduced cysteine ​​residue, as well as a reduced interchain disulfide, and there may be 1-8, or 1-10, or 1-12, or 1-18 camptothecin payload compounds conjugated to a cell-binding agent.

[0344] In embodiments, conjugation (e.g., as found in any compound according to formula PL-A' or formula PL-A, such as MB-2a or MB-3a described herein) is via an introduced cysteine ​​residue, and there may be 2, or 3, or 4, or 5, or 6, or 7, or 8 camptothecin payload compounds conjugated to the cell-binding agent.

[0345] In embodiments, conjugation (e.g., as found in any compound according to formula PL-A' or formula PL-A, such as MB-2a or MB-3a described herein) is via a lysine residue, and there may be about 1-10, or 1-12, or 1-14, or 1-18 camptothecin payload compounds conjugated to the cell-binding agent.

[0346] Reactive groups on cell-binding agents for covalent attachment In embodiments, the cell-binding agent is linked to a peptide releasable linker to form a conjugate such as one according to formula PL-A' or formula PL-A (e.g., MB-2a or MB-3a). As mentioned above, further linking moieties can be present in the conjugates described herein to serve the purpose of providing additional space between the camptothecin compound and the cell-binding agent. In embodiments, the cell-binding agent is linked to the linker unit via a heteroatom of the cell-binding agent.

[0347] Heteroatoms that may be present on the cell-binding agent for attachment 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 optionally substituted nitrogen (in one embodiment, from a primary or secondary amine functional group of the targeting ligand, or in another embodiment, from an optionally substituted amide nitrogen). These heteroatoms may be present on the native targeting ligand of the cell-binding agent, for example, in a naturally occurring antibody, or may be introduced into the targeting ligand via chemical modification or biological engineering.

[0348] In one embodiment, the cell-binding agent has a thiol functional group such that the cell-binding agent is attached to the camptothecin payload compound via the sulfur atom of the thiol functional group.

[0349] In another embodiment, the cell-binding agent has one or more lysine residues that can react with an activated ester of a camptothecin payload compound (e.g., any compound according to formula (II) described herein or formed from a compound of formula (I) described herein), thus providing an amide bond consisting of a nitrogen atom of the cell-binding agent and a C=O group of the compound.

[0350] In yet another aspect, the cell-binding agent has one or more lysine residues that can be chemically modified to introduce one or more thiol groups. In these embodiments, the cell-binding agent is covalently linked to the camptothecin payload compound via the sulfur atom of the thiol functional group. Reagents that can be used to modify lysines in such a manner include, but are not limited to, N-succinimidyl S-acetylthioacetate (SATA) and 2-iminothiolane hydrochloride (Traut's reagent).

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

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

[0353] In some aspects, the cell-binding agent can form a bond by interacting with a reactive functional group Y to form a covalent bond between Y' in formula PL-A' and the cell-binding agent corresponding to the targeting ligand. The functional group Y capable of interacting with the targeting ligand depends on the nature of the cell-binding agent. In an embodiment, the reactive group is a maleimide present on the camptothecin payload compound prior to its attachment to form the cell-binding agent. The covalent attachment of the cell-binding agent to the camptothecin payload compound is achieved through a thiol functional group of the cell-binding agent, which interacts with the maleimide functional group Y of the payload compound to form a thio-substituted succinimide. The thiol functional group can be present on the cell-binding agent in its native state, for example in a naturally occurring residue, or can be introduced into the cell-binding agent via chemical modification or by biological engineering.

[0354] In yet another embodiment, the cell-binding agent is an antibody and the thiol group is generated by reduction of an interchain disulfide of the antibody. Thus, in an embodiment, a camptothecin payload compound is conjugated to a cysteine ​​residue from the reduced interchain disulfide.

[0355] In yet another embodiment, the cell-binding agent is an antibody and the thiol functional group is chemically introduced into the antibody, for example, by introduction of a cysteine ​​residue. Thus, in an embodiment, the camptothecin payload compound is conjugated to the cell-binding agent via the introduced cysteine ​​residue of the cell-binding agent.

[0356] For bioconjugation, it has been observed that the site of drug conjugation can affect many parameters, including ease of conjugation, drug-linker stability, impact on the biophysical properties of the resulting bioconjugation, and in vitro cytotoxicity. With regard to drug-linker stability, the site of conjugation of the drug-linker moiety to the cell-binding agent can affect the ability of the conjugated drug-linker moiety to undergo elimination reactions, in some cases, to cause early release of free drug. Sites of conjugation on targeting ligands include, for example, reduced interchain disulfides as well as selected cysteine ​​residues at engineered sites. In embodiments, the conjugation method for forming camptothecin conjugates described herein uses a thiol residue at engineered sites (e.g., at position 239 according to the EU index as described in Kabat) that is less susceptible to elimination reactions compared to conjugation methods that use thiol residues from reduced disulfide bonds. In other embodiments, the conjugation methods for forming camptothecin conjugates described herein use thiol residues at sites that are more susceptible to elimination reactions (e.g., resulting from interchain disulfide reduction).

[0357] Synthesis method Exemplary synthetic methods are described herein. Still other exemplary synthetic methods include those described in U.S. Provisional Application No. 62 / 981,197, which is incorporated by reference in its entirety, and methods known in the art.

[0358] In an embodiment, Scheme 1 provides an exemplary method for the synthesis of the described compound MB-1 (hydrotecan) (P1). Scheme 1 [ka]

[0359] In embodiments, Scheme 2 provides exemplary synthetic methods for preparing the linkable payloads (PL1), (PL2), (PL4), and (PL7) described. Scheme 2 [ka]

[0360] In an embodiment, Scheme 3 provides an exemplary method for the synthesis of the described compound MB-2 (PL1). Scheme 3 [ka]

[0361] In an embodiment, Scheme 4 provides an exemplary method for the synthesis of the described compound MB-3 (Meditecan, Medi-Tekan) (PL3). Scheme 4 [ka]

[0362] In an embodiment, Scheme 5 provides an exemplary general method for preparing the conjugate (PL'). Scheme 5 [ka]

[0363] In embodiments, an exemplary experimental procedure for preparing a conjugate (PL') with a drug-to-antibody ratio (DAR) of 7-8 or 8: Antibody C is treated with 8 equivalents (2 equivalents per disulfide bond) of tris(2-carboxyethyl)phosphine hydrochloride (TCEP) in 50 mM pH 7.4 phosphate buffer and 10 mM DTPA (diethylenetriaminepentaacetic acid) for 2 h at 25 °C, followed by the addition of 12 equivalents of payload (PL) in DMSO (the volume of DMSO is approximately 12-15% of the volume of phosphate buffer). The resulting reaction solution is rotated on a tube rotator for 1 h at 25 °C. The reaction mixture is immediately purified using ultrafiltration tubes (30 KD) for several cycles with formulation buffer. The resulting conjugate (PL') typically has a drug-to-antibody ratio (DAR) of 7-8 or 8 and is greater than 95% monomeric as measured by size exclusion chromatography.

[0364] In an embodiment, the antibody drug conjugate is MB-2a. In an embodiment, Scheme 6 provides an exemplary method for the synthesis of the described antibody drug conjugate MB-2a. Scheme 6 [ka]

[0365] In an embodiment, the antibody drug conjugate is MB-3a (trastuzumab meditecan). In an embodiment, scheme 7 provides an exemplary method for the synthesis of the antibody drug conjugate MB-3a (trastuzumab meditecan) described. Scheme 7 [ka]

[0366] Treatment method In another aspect, the invention features a method of treating a cell proliferative disease or disorder or inhibiting abnormal cell proliferation, the method includes administering any compound of formula (PL-A), such as MB-2a or MB-3a (trastuzumab meditecan), or a pharma- ceutically acceptable salt thereof, or a pharmaceutical composition including any compound of formula (PL-A) (e.g., MB-2a or MB-3a (trastuzumab meditecan)), or a pharma- ceutically acceptable salt thereof, as described herein.

[0367] In an embodiment, the method is for treating cancer. In an embodiment, the cancer is characterized by HER2 overexpression and / or is metastatic and / or is trastuzumab resistant.

[0368] In embodiments, the cancer is adenocarcinoma, brain cancer, bladder cancer, breast cancer, cervical cancer, choriocarcinoma, CNS tumors, colon or colorectal cancer, diffuse pontine glioma (DIPG), endometrial cancer, esophageal cancer, Ewing's sarcoma, fallopian tube cancer, gallbladder cancer, gastric cancer, glioblastoma, head and neck cancer, blood 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, renal 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 embodiments, the cancer is lung cancer, urothelial cancer, 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, colon cancer, rectal cancer, colorectal 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 embodiments, the cancer is breast cancer, gastric cancer, lung cancer (e.g., non-small cell lung cancer), and ovarian cancer, which has been reported to be a poor prognostic factor for breast cancer.

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

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

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

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

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

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

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

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

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

[0380] Cancer, including but not limited to tumors, metastases, or other diseases or disorders characterized by uncontrolled cell growth, may be treated or inhibited by administration of a camptothecin conjugate.

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

[0382] In embodiments, the patient also receives additional treatment, such as radiation therapy. In certain embodiments, the camptothecin conjugate is administered simultaneously with a chemotherapy agent or radiation therapy. In another specific 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-meditecan)).

[0383] The chemotherapy agents may be administered over a series of sessions. Any one or a combination of chemotherapy agents, such as standard of care chemotherapy agents, may be administered.

[0384] Furthermore, the method of treating cancer with camptothecin conjugates is provided as an alternative to chemotherapy or radiation therapy, which, for example, have proven or can prove to be excessively toxic to the subject being treated, for example resulting in unacceptable or intolerable side effects. The patient being treated can optionally be treated with another cancer treatment, such as surgery, radiation therapy, or chemotherapy, depending on which treatment is acceptable or tolerable.

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

[0386] In embodiments, the methods of treatment described herein include administration of MB-3a (trastuzumab meditecan).

[0387] Compositions and Methods of Administration In another aspect, the invention features a pharmaceutical composition comprising any compound described herein (e.g., any compound of formula (PL-A), such as MB-2a and MB-3a (trastuzumab meditecan)) or a pharma- ceutically acceptable salt thereof described herein. In an embodiment, the pharmaceutical composition comprises a pharma- ceutically acceptable carrier.

[0388] In an embodiment, the pharmaceutical composition comprises a conjugate according to formula (PL-A), such as MB-2a or MB-3a (trastuzumab meditecan), or a pharma- ceutically acceptable salt thereof.

[0389] In an embodiment, the pharmaceutical composition comprises MB-2a, or a pharma- ceutically acceptable salt thereof.

[0390] In an embodiment, the pharmaceutical composition comprises MB-3a (trastuzumab meditecan), or a pharma- ceutically acceptable salt thereof.

[0391] In an embodiment, the present invention provides a pharmaceutical composition comprising a camptothecin conjugate described herein (e.g., any compound of formula (PL-A), such as MB-2a or MB-3a (trastuzumab meditecan)) and a pharma- ceutically acceptable carrier. The camptothecin conjugate may be in any form that allows the compound to be administered to a patient for the treatment of a disorder associated with the expression of an antigen to which the cell-binding agent binds. For example, the conjugate may be in liquid or solid form. The preferred route of administration is parenteral. Parenteral administration includes subcutaneous injection, intravenous, intramuscular, intrasternal injection or infusion techniques. In one aspect, the composition is administered parenterally. In one aspect, the conjugate is administered intravenously. Administration may be by any convenient route, for example, by infusion or bolus injection.

[0392] The pharmaceutical composition can be formulated to allow the compound to be bioavailable upon administration of the composition to a patient. The composition can be in the form of one or more dosage units.

[0393] The materials used in the preparation of pharmaceutical compositions can be non-toxic in the amounts used.The optimal dosage of active ingredients in pharmaceutical compositions will be clear to those skilled in the art, depending on a variety of factors.Relevant factors include, but are not limited to, the type of animal (e.g., human), the specific form of the compound, the method of administration, and the composition used.

[0394] Composition can be, for example, in liquid form.Liquid can be useful for delivery by injection.In the composition for administration by injection, one or more of surfactant, preservative, wetting agent, dispersing agent, suspending agent, buffer, stabilizer and isotonic agent can also be included.

[0395] Liquid compositions, whether in solution, suspension, or other similar form, may also contain one or more of the following: sterile diluents such as water for injection, saline solution, preferably saline, Ringer's solution, isotonic sodium chloride, fixed oils such as synthetic mono- or di-silsesquioxanes that can serve as solvents or suspending media, polyethylene glycol, glycerin, 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 non-ionic surfactants, polyols; and tonicity adjusters such as sodium chloride or dextrose. Parenteral compositions may be enclosed in ampoules, disposable syringes, or multiple dose vials made of glass, plastic, or other materials. Saline is an exemplary adjuvant. Injectable compositions are preferably sterile.

[0396] The amount of the conjugate that is effective in treating a particular disorder or condition depends on the nature of the disorder or condition and can be determined by standard clinical techniques.In addition, in vitro or in vivo assays can be optionally used to help identify optimal dosage ranges.The exact dose to be used in the composition also depends on the route of administration and the severity of the disease or disorder, and should be determined according to the judgment of the practitioner and each patient's circumstances.

[0397] The compositions contain an effective amount of the compound such that a suitable dosage will be obtained, typically this amount being at least about 0.01% of the compound by weight of the composition.

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

[0399] In general, the dosage of the conjugate administered to a patient is typically about 0.01 mg / kg to about 100 mg / kg of subject body weight, or 1.0 μg / kg to 5.0 mg / kg of subject body weight. In an embodiment, the dosage administered to a patient is about 0.01 mg / kg to about 15 mg / kg of subject body weight. In an embodiment, the dosage administered to a patient is about 0.1 mg / kg to about 15 mg / kg of subject body weight. In an embodiment, the dosage administered to a patient is about 0.1 mg / kg to about 20 mg / kg of subject body weight. In an embodiment, the dosage administered is about 0.1 mg / kg to about 5 mg / kg or about 0.1 mg / kg to about 10 mg / kg of subject body weight. In an embodiment, the dosage administered is about 1 mg / kg to about 15 mg / kg of subject body weight. In an embodiment, the dosage administered is about 1 mg / kg to about 10 mg / kg of subject body weight. In embodiments, the dosage administered is about 0.1-4 mg / kg, even more preferably 0.1-3.2 mg / kg, or even more preferably 0.1-2.7 mg / kg of the subject's body weight over a treatment cycle.

[0400] The term "carrier" refers to a diluent, adjuvant, or excipient that is administered with the compound. Such pharmaceutical carriers can be liquids such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, for example, peanut oil, soybean oil, mineral oil, sesame oil. Carriers can be saline, gum acacia, gelatin, starch paste, talc, keratin, colloidal silica, urea. In addition, auxiliary agents, stabilizers, thickeners, lubricants, and colorants can be used. In one embodiment, when administered to a patient, the compound or composition and pharma-ceutically acceptable carriers are sterile.

[0401] Water is an exemplary carrier when compound is administered intravenously.Saline solution and aqueous dextrose and glycerol solution can also be used as liquid carrier, especially for injectable solution.Suitable pharmaceutical carriers also include excipients such as starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, skimmed milk powder, glycerol, propylene, glycol, water, ethanol, etc.If desired, the composition can also contain small amounts of wetting or emulsifying agents, or pH buffering agents.

[0402] In one embodiment, the conjugate is formulated according to routine procedures as a pharmaceutical composition adapted for intravenous administration to animals, particularly humans. Typically, the carrier or vehicle for intravenous administration is a sterile isotonic aqueous buffer solution. If necessary, the composition can also include a solubilizing agent. Compositions for intravenous administration can optionally include a local anesthetic, such as lignocaine, to ease pain at the site of injection. Generally, the ingredients are supplied separately or mixed together in unit dosage form, for example as a lyophilized powder or water-free concentrate in a sealed container, such as an ampoule or sachet indicating the quantity of active agent. When the conjugate is administered by injection, it can be dispensed, for example, with an infusion bottle containing sterile pharmaceutical grade water or saline. When the composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients can be mixed prior to administration.

[0403] Pharmaceutical compositions are generally formulated as sterile, substantially isotonic, and in full compliance with all Good Manufacturing Practice (GMP) regulations of the US Food and Drug Administration.

[0404] Kits for therapeutic use In some embodiments, kits for use in the treatment of cancer and autoimmune diseases are provided. Such kits can include pharmaceutical compositions comprising the camptothecin conjugates described herein.

[0405] In embodiments, the kit may include instructions for use in any of the therapeutic methods described herein. The included instructions may provide instructions for administration of the pharmaceutical composition to a subject to achieve the intended activity in the subject, e.g., treatment of a disease or condition, such as cancer. In embodiments, the instructions for use of the pharmaceutical composition described herein may include information regarding dosage, administration schedule, and route of administration for the intended treatment. The container may be a unit dose, bulk package (e.g., multi-dose package), or partial unit dose. The instructions provided with the kit of the present disclosure are typically written instructions on a label or package insert. The label or package insert indicates that the pharmaceutical composition is used to treat, delay the onset of, and / or alleviate a disease or disorder in a subject.

[0406] In an embodiment, the kit provided herein is suitable packaging.Suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packaging, etc.Packaging for use in combination with a specific device, such as an inhaler, a nasal administration device, or an injection device, is also contemplated.In an embodiment, the kit can have a sterile access port (for example, the container can be an intravenous solution bag or a vial with a stopper that can be pierced by a hypodermic needle).

[0407] In embodiments, the kits provided herein include additional therapeutic agents useful for treating the autoimmune cancer diseases described herein.

[0408] Multidisciplinary cancer therapy Cancer, including but not limited to tumors, metastases, or other diseases or disorders characterized by uncontrolled cell growth, may be treated or inhibited by administration of a camptothecin conjugate.

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

[0410] In embodiments, the patient also receives an additional treatment, such as radiation therapy. In certain embodiments, the camptothecin conjugate is administered simultaneously with the chemotherapeutic agent or radiation therapy. In another specific embodiment, the chemotherapeutic agent or radiation therapy is administered before or after administration of the camptothecin conjugate.

[0411] The chemotherapy agents may be administered over a series of sessions. Any one or a combination of chemotherapy agents, such as standard of care chemotherapy agents, may be administered.

[0412] Furthermore, the method of treating cancer with camptothecin conjugates is provided as an alternative to chemotherapy or radiation therapy, which, for example, have proven or can prove to be excessively toxic to the subject being treated, for example resulting in unacceptable or intolerable side effects. The patient being treated can optionally be treated with another cancer treatment, such as surgery, radiation therapy, or chemotherapy, depending on which treatment is acceptable or tolerable.

[0413] The present disclosure is further illustrated by the following examples, which should not be construed as limiting the scope or spirit of the present disclosure to the specific procedures described herein. It should be understood that the examples are provided to illustrate specific embodiments, and no limitation to the scope of the present disclosure is intended thereby. It should be further understood that, as would be obvious to one skilled in the art, various other embodiments, modifications, and their equivalents may be resorted to without departing from the spirit of the present disclosure and / or the scope of the appended claims. EXAMPLES

[0414] The present invention will be specifically described based on the following examples. However, the present invention is not limited thereto. Moreover, it is not to be construed in any way as being limiting. Moreover, unless otherwise specified, the reagents, solvents and starting materials described herein are readily available from commercial suppliers.

[0415] The following abbreviations are used in the following terms: ADC Antibody-drug conjugates ACN Acetonitrile DAR Drug to Antibody Ratio DCC N,N'-dicyclohexylcarbodiimide DCM Dichloromethane DIPA Diisopropylamine DIPEA Diisopropylethylamine DMF Dimethylformamide DMSO Dimethyl sulfoxide DMTMM 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride DMTMMT 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium tetrafluoroborate DTPA Diethylenetriaminepentaacetic acid EDCI 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide HOPO 2-Hydroxypyridine-1-oxide iv M mole nM nanomolar NMM N-Methylmorpholine PPTS Pyridinium p-toluenesulfonate PTSA 4-Methylbenzenesulfonic acid SEC Size Exclusion Chromatography TBS tert-Butyldimethylsilyl TCEP 3,3',3''-Phosphinetriyltripropanoic acid hydrochloride TEA Triethylamine TFA Trifluoroacetic acid THF Tetrahydrofuran TLC Thin Layer Chromatography p-TsOH p-Toluenesulfonic acid

[0416] Example 1. Exemplary synthesis of compound MB-1 (P1) General procedure for the preparation of 2,6-dibromo-4-fluoro-3-methyl-aniline (1-2) [ka]

[0417] To a stirred solution of compound 1-1 (70 g, 559.36 mmol, 1 equiv) in CHCl / methanol (1:1, 1.2 L) was added a solution of Br2 (223.48 g, 1.40 mol, 72.09 mL, 2.5 equiv) in CHCl / methanol (1:1, 200 mL) dropwise over 1.5 h at 15 °C using an addition funnel. The reaction mixture was stirred at 25 °C for 4 h and TLC (petroleum ether / ethyl acetate = 6 / 1, Rf = 0.6) showed that the starting material was consumed. Three additional vials were set up as above and the mixtures from the four reactions were combined and concentrated. To the resulting residue was added 1N Na2S2O3 (1.5 L) and ethyl acetate (1.5 L). The solution was stirred for 10 min and then carefully basified with 1N Na2CO3 (150 mL). It was transferred to a separatory funnel and the organic layer was isolated. The aqueous layer was extracted with ethyl acetate (2×1 L). The combined organic layers were washed with 1N Na2S2O3 (1 L), followed by brine (1 L), and then dried over Na2SO4. It was filtered and concentrated under reduced pressure to give a residue. The residue was triturated with petroleum ether (1 L) and filtered to give the product 1-2 (574 g, 1.93 mol, 86% yield, 95% purity) as a pale purple solid. 1 H 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 procedure for the preparation of 1,3-dibromo-5-fluoro-2-iodo-4-methyl-benzene (1-3) [ka]

[0419] To a solution of p-TsOH (90 g, 522.2 mmol, 3 equiv.) in acetonitrile (700 mL) was added compound 1-2 (49.25 g, 174.07 mmol, 1 equiv.). The resulting white suspension was cooled to 10-15 °C, then a solution of NaNO2 (24.02 g, 348.14 mmol, 2 equiv.) and KI (73.22 g, 435.13 mmol, 2.5 equiv.) in water (105 mL) was added slowly. The suspension became dark brown and gas was released. The thick mixture was stirred at 10 °C for 10 min and then at 20 °C for an additional 1 h. TLC (petroleum ether / ethyl acetate = 6 / 1, Rf = 0.6) showed that the starting material was consumed. The reaction mixture was poured into water (400 mL). A 1N solution of sodium bicarbonate (200 mL) was added to adjust the pH to 9-10, followed by the addition of a 2N solution of sodium thiosulfate (200 mL). The resulting mixture was extracted with ethyl acetate (3 x 500 mL). Eleven additional vials were installed 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 eluting with petroleum ether to give the product 1-3 (504 g, 1.09 mol, 56% yield, 85% purity) as a yellow solid. 1 H NMR (400 MHz, chloroform-d) δ 7.41 (d, J = 8.8 Hz, 1H), 2.43 (d, J = 2.4 Hz, 3H).

[0420] General procedure for the preparation of 2,6-dibromo-4-fluoro-3-methyl-benzaldehyde (1-4) [ka]

[0421] To a solution of compound 1-3 (50.4 g, 127.98 mmol, 1 equiv.) in anhydrous toluene (300 mL) was added a solution of chloro(isopropyl)magnesium (2 M in tetrahydrofuran, 76.80 mL, 1.2 equiv.) over 10 min while maintaining the internal temperature below -25 °C. A clear brown solution was obtained and the mixture was stirred for 1.5 h, followed by the addition of N,N-dimethylformamide (30.86 g, 422.33 mmol, 3.3 equiv.) over 10 min. The temperature of the reaction mixture rose to -19 °C after the addition. The reaction mixture was warmed to 20 °C over 0.5 h and stirred for 1.5 h. TLC (petroleum ether / ethyl acetate = 10 / 1, Rf = 0.45) indicated the reaction was complete. The reaction mixture was quenched with saturated aqueous NH4Cl (50 mL). Ten additional vials were set up as above and all eleven reaction mixtures were combined. The combined mixture was filtered, and the filtrate was evaporated under reduced pressure to give a residue. The residue was purified by silica gel column chromatography, eluted with petroleum ether to give the product 1-4 (253 g, 812.18 mmol, yield 60%, purity 95%) as a yellow solid. 1 H 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 procedure for the preparation of 2-(2,6-dibromo-4-fluoro-3-methyl-phenyl)-1,3-dioxolane (4). [ka]

[0423] To a solution of compound 1-4 (50.6 g, 170.99 mmol, 1 equiv.) in 1,2-dichloroethane (430 mL) was added ethylene glycol (53.06 g, 878.58 mmol, 47.80 mL, 5 equiv.), triethyl orthoformate (25.34 g, 170.99 mmol, 28.44 mL, 1 equiv.), and p-toluenesulfonic acid (1.47 g, 8.55 mmol, 0.05 equiv.). The reaction mixture was stirred at 80 °C for 3 h, and TLC (petroleum ether / ethyl acetate = 10 / 1, Rf = 0.59) indicated the reaction was complete. Four additional vials were set up as described above and the reaction mixtures from the five reactions were combined. The combined reaction mixture was then washed with saturated aqueous Na2CO3 (1 L), saturated aqueous NH4Cl (1 L), and water (1 L). The organic layer was dried over Na2SO4, filtered, and evaporated under reduced pressure to give the crude product. The crude product was triturated with petroleum ether at 20°C for 15 min and filtered to give product 4 (280 g, 741.21 mmol, 84% yield, 90% purity) as a pale yellow solid. 1 H 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 procedure for the preparation of N-[3-bromo-2-(1,3-dioxolan-2-yl)-5-fluoro-4-methyl-phenyl]-1,1-diphenyl-methanimine (5) [ka] To a solution of compound 4 (53 g, 155.89 mmol, 1 equiv.) in toluene (100 mL), compound 4a (29.67 g, 163.69 mmol, 27.46 mL, 1.05 equiv.), sodium tert-butoxide (29.97 g, 311.78 mmol, 2 equiv.), palladium(II) acetate (3.5 g, 15.59 mmol, 0.1 equiv.), and 4,5-bis(diphenylphosphino)-9,9-dimethylxanthene (9.02 g, 15.59 mmol, 0.1 equiv.) were added under N2 protection. The reaction mixture was stirred at 100 °C for 12 h under N2 protection, and TLC (petroleum ether / ethyl acetate = 10 / 1, Rf = 0.32) showed the reaction was complete. Two additional vials were set up as above, and all three reaction mixtures were combined and filtered through a Celite pad. The filter cake was washed with ethyl acetate (500 mL). The combined filtrate was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography, eluted with petroleum ether / ethyl acetate=10 / 1 to give product 5 (105 g, 214.62 mmol, 45.61% yield, 80% purity) as a yellow solid. The product was used in the next step without further purification. 1 H NMR(400MHz,chloroform-d)δ 7.77(br d,J=7.3Hz,2H),7.54-7.37(m,4H),7.31(br d,J=4.5Hz,3H),7.26-7.22(m,1H),6.48(s,1H),5.93(d,J=10.3Hz,1H),4.11-4.05(m,2H),3.96-3.91(m,2H),2.23(d,J=2.3Hz,3H).

[0425] General procedure for the preparation of N-[3-[4-[tert-butyl(dimethyl)silyl]oxybutyl]-2-(1,3-dioxolan-2-yl)-5-fluoro-4-methyl-phenyl]-1,1-diphenyl-methanimine (1-6) [ka]

[0426] To a stirred mixture of compound 5a (5.3 g, 28.44 mmol, 1 equiv.) in toluene (80 mL) was added 9-BBN (0.5 M in tetrahydrofuran, 68.13 mL, 1.2 equiv.) under nitrogen atmosphere at 10° C. The resulting mixture was stirred at 80° C. for 20 min under nitrogen protection, and TLC (petroleum ether / ethyl acetate=1 / 1, product Rf=0.2, I2) showed the reaction was complete. A solution of NaOH (2.27 g, 56.78 mmol, 2 equiv.) in water (20 mL) was added to the above mixture under nitrogen atmosphere at 10° C. The resulting mixture was stirred at 10° C. for 10 min, followed by the addition of compound 5 (10.00 g, 22.71 mmol, 0.8 equiv), tetrabutylammonium iodide (524.31 mg, 1.42 mmol, 0.05 equiv), and [1,1-bis(diphenyl-phosphino)ferrocene]palladium(II) dichloride dichloromethane adduct (463.7 mg, 568.8 μmol, 0.02 equiv) at 10° C. under nitrogen atmosphere. The resulting mixture was stirred at 80° C. under nitrogen atmosphere for 15 h, and LCMS (retention time=3.620) indicated the reaction was complete. Seven additional vials were installed as above, and all eight reaction mixtures were combined. The combined organic layers were washed with water (500 mL×3), dried over Na2SO4, filtered, and concentrated under reduced pressure. The resulting residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 10 / 1 to 5 / 1) to give the crude product, which was further purified by reverse-phase HPLC to give the product 1-6 (45 g, 82.15 mmol, 50% yield, 80% purity) as a yellow gum. 1 H 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=3.5Hz,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).

[0427] (19S)-19-Ethyl-6-fluoro-19-hydroxy-8-(4-hydroxybutyl)-7-methyl-17-oxa-3,13-diazapentacyclo[11.8.0.0 2,11 .0 4,9 .0 15,20 General procedure for the preparation of heptane-1(21),2,4,6,8,10,15(20)-heptaene-14,18-dione (MB-1, hydrotecen) [ka]

[0428] To a solution of compound 7 (2.4 g, 1 equiv.) in ethanol (100 mL), 35% HCl (5.7 g, 6 equiv.) was added, the solution was heated to 50-60 °C, and then compound 1-6 (10 g, 2 equiv.) in ethanol (100 mL) was added dropwise over 2 h. The mixture was stirred at 50-60 °C for 1.5 h. The heating bath was removed and the reaction mixture was cooled to room temperature. Methyl-tert-butyl ether (2000 mL) was added to precipitate the product. It was filtered and the solid was washed with methyl-tert-butyl ether to give the product MB-1 (hydrotecan, Hai Luo Pei Kang) as a pale yellow solid (2.45 g, 59% yield, 98% purity). 1 H NMR(400MHz,DMSO-d6)δ 8.89(s,1H),7.77(d,J=11.0Hz,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=5.9Hz,2H),3.19-3.11(m,2H),2.43(d,J=1.8Hz,3H),1.86(tt,J=7.2,14.5Hz,2H),1.62(br s,4H),0.88(t,J=7.3Hz,3H).

[0429] Example 2. Exemplary synthesis of compound MB-2 (PL1) C 17 H 14 General procedure for the preparation of NO4(S2). [ka]

[0430] A column packed with 2-(9H-fluoren-9-ylmethoxycarbonylamino)acetic acid (74.92 g, 252.10 mmol, 2 equiv.), Trt-Resin S1 (120.00 g, 126.05 mmol, 1 equiv.), and N,N-diisopropylethylamine (162.85 g, 1.26 mol, 219.47 mL, 10 equiv.) in dichloromethane (1500 mL) was bubbled with nitrogen for 12 h at 20 °C. After filtration, the residue was washed successively with dichloromethane (3 x 300 mL), dimethylformamide dichloromethane / methanol = 1 / 1 (3 x 300 mL), and dimethylformamide (3 x 300 mL). The residue was further dried under high vacuum to give crude resin-C. 17 H 14 NO4 (150 g, 123.66 mmol, 98.10% yield, crude purity) was obtained as a yellow solid. The product was used directly in the next step without purification. Resin-C in DMF (1200 mL) 17 H 14 To a column packed with NO4 (150 g, 123.66 mmol, 1 equiv.) was added piperidine (105.30 g, 1.24 mol, 122.13 mL, 10 equiv.). The mixture was bubbled with N2 at 20 °C for 1 h. The resulting resin was filtered and subsequently washed with dimethylformamide (2 x 500 mL) and dichloromethane (2 x 500 mL). The resin was dried to give Resin-C2H4NO2(S2) (120 g, 121.21 mmol, 98.02% yield) as a yellow solid, which was used directly in the next step.

[0431] General Procedure for Preparation of Resin-C5H9N2O3 (S3) [ka]

[0432] (2S)-2-(9H-fluoren-9-ylmethoxycarbonylamino)propanoic acid (75.47 g, 242.42 mmol, 2 equiv.) in dimethylformamide (1200 mL) and Resin-C 17 H 14 To a column packed with NO4(S2) (120 g, 121.21 mmol, 1 equiv.) was added HCTU (O-(6-chloro-1-hydroxybenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate) (100.29 g, 242.42 mmol, 2 equiv.) and N,N-diisopropylethylamine (78.33 g, 606.06 mmol, 105.56 mL, 5 equiv.). The mixture was bubbled with N2 at 20 °C for 1 h. The resulting resin was filtered and washed successively with dimethylformamide (2 × 500 mL) and dichloromethane (2 × 500 mL). It was dried to give resin-C. 20 H 19 N2O5 (150 g, crude) was obtained as a yellow solid, which was used directly in the next step. Resin-C in dimethylformamide (1200 mL) 20 H 19 Piperidine (99.55 g, 1.17 mol, 115.46 mL, 10 eq.) was added to a column packed with N2O5 (150 g, 116.91 mmol, 1 eq.). The mixture was bubbled with N2 at 20 °C for 1 h. The resulting resin was filtered and washed successively with dimethylformamide (2 x 500 mL) and dichloromethane (2 x 500 mL). It was dried to give the resin C5H9N2O3 (S3) (120 g, crude) as a yellow solid, which was used directly in the next step.

[0433] General procedure for the preparation of 2-[[(2S)-2-[[(2S)-2-(9H-fluoren-9-ylmethoxy-carbonylamino)-3-methyl-butanoyl]amino]propanoyl]amino]acetic acid (1-7) [ka]

[0434] To a column packed with dimethylformamide (200 mL) resin CHNO (S3) (120 g, 112.99 mmol, 1 equiv.) and (2S)-2-(9H-fluoren-9-ylmethoxycarbonylamino)-3-methyl-butanoic acid (76.70 g, 225.99 mmol, 2 equiv.), O-(6-chloro-1-hydroxybenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (93.49 g, 225.99 mmol, 2 equiv.) and N,N-diisopropyl-ethylamine (73.02 g, 564.97 mmol, 98.41 mL, 5 equiv.) were added. The mixture was bubbled with N at 20 °C for 12 h. The resulting resin was filtered 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 layers were combined and concentrated under reduced pressure to give a residue. The residue was triturated with n-hexane at 20° C. for 12 h. It was filtered to give the product 1-7 (60 g, 39.05 mmol, 34.56% yield, 90% purity) as a white solid. 1 H NMR(400MHz,DMSO-d6)δ 8.17(br t,J=5.7Hz,1H),7.99(d,J=7.5Hz,1H),7.89(d,J=7.3Hz,2H),7.74(t,J=6.6Hz,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=7.1Hz,3H),0.85(dd,J=6.9,9.8Hz,6H).

[0435] General procedure for the preparation of methyl [[(2S)-2-[[(2S)-2-(9H-fluoren-9-ylmethoxycarbonylamino)-3-methyl-butanoyl]amino]propanoyl]amino]acetate (1-8). [ka]

[0436] A solution of compounds 1-7 (90.0 g), pyridine (30.6 g, 2 equiv.), lead(IV) acetate (189 g, 2 equiv.), and Cu(OAc)2 (3.6 g, 0.1 equiv.) in THF (1000 mL) and toluene (250 mL) was stirred at 50-60 °C for 2 h. 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. After filtration and washing the solid with THF / MTBE (1:4), the product 1-8 was obtained as a pale yellow solid (84.2 g, yield = 90.8%, purity 91.9%). 1 H NMR(400MHz,DMSO-d6)δ 8.03-7.93(m,1H),7.89(d,J=7.5Hz,2H),7.74(br t,J=6.3Hz,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=3.9Hz,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).

[0437] (2S)-2-amino-N-[(1S)-2-[4-[(19S)-19-ethyl-6-fluoro-19-hydroxy-7-methyl-14,18-dioxo-17-oxa-3,13-diazapentacyclo[11.8.0.0 2,11 .0 4,9 .0 15,20 General procedure for the preparation of ]henicosa-1(21),2,4,6,8,10,15(20)-heptaen-8-yl]butoxymethylamino]-1-methyl-2-oxoethyl]-3-methyl-butanamide (1-10) [ka]

[0438] To a stirred solution of MB-1 (hydrotecan) (10.7 g, 0.0236 mol) and compound 1-8 (14.7 g, 0.0306 mol, 1.3 equiv.) in anhydrous DMSO (90 mL), BF3 etherate (12.1 g, 0.0852 mol, 3.6 equiv.) was added at room temperature. The reaction solution was kept stirring at room temperature for 13 h. LCMS of the reaction sample showed that about 75% of product 1-9 was produced and 6% of starting material MB-1 remained. Morpholine (20.5 g, 0.236 mol) was added to the reaction solution and the reaction solution was kept stirring at room temperature for 2 h. This was directly purified by preparative HPLC (C18 column, mobile phase CH3CN / H2O) to give product 1-10 (9.1 g, y=59%, purity 96.1%) as an off-white solid after lyophilization. 1 H NMR(400MHz,DMSO-d6)δ 8.87(s,1H),8.68(br s,1H),8.09(br s,1H),7.77(br d,J=10.6Hz,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=7.9,14.8Hz,3H),1.70(br s,2H),1.58(br s,2H),1.19(br d,J=6.8Hz,3H),0.88(br t,J=7.2Hz,3H),0.82(br d,J=6.6Hz,3H),0.73(br d,J=6.6Hz,3H).

[0439] 6-(2.5-dioxopyrrol-1-yl)-N-[(1S)-1-[[(1S)-2-[4-[(19S)-19-ethyl-6-fluoro-19-hydroxy-7-methyl-14,18-dioxo-17-oxa-3,13-diazapentacyclo[11.8.0.0 2,11 .0 4,9 .0 15,20General procedure for the preparation of]henicosa-1(21),2,4,6,8,10,15(20)-heptan-8-yl]butoxymethylamino]-1-methyl-2-oxo-ethyl]carbamoyl]-2-methyl-propyl]hexanamide (MB-2) [ka]

[0440] To a solution of compound 1-10 (400 mg, 613.8 μmol, 1 equiv.) in N,N-dimethylformamide (10 mL) was added compound 10A (283.8 mg, 920.6 μmol, 1.5 equiv.). The reaction mixture was stirred at 15° C. for 12 h. LCMS (product retention time=2.080) showed that all of compound 1-10 was consumed and a new peak with the desired MS was 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 the product MB-2 (173 mg, 203.9 μmol, 33.36% yield, 95.74% purity) as a white solid. 1 H NMR(400MHz,DMSO-d6)δ 8.87(s,1H),8.57(t,J=6.4Hz,1H),7.97(d,J=7.2Hz,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=6.6,10.1Hz ,2H),4.21(quin,J=7.1Hz,1H),4.10(dd,J=6.8,8.4Hz,1H),3.48-3.41(m,2H),3.37-3.34(m ,2H),3.20-3.08(m,2H),2.43(d,J=2.0Hz,3H),2.18-2.01(m,2H),1.95-1.79(m,3H),1.68(br d,J=7.0Hz,2H),1.58(br s,2H),1.51-1.38(m,4H),1.20-1.10(m,5H),0.88(t,J=7.3Hz,3H),0.76(dd,J=6.8,9.3Hz,6H). 13C NMR(101MHz,DMSO-d6)δ 173.09,172.54,172.26,171.09,170.85,160.54,156.86,152.36,150.00,147.85,145.38,140.1 3,134.46,129.33,128.33,125.02,124.04,119.05,110.14,96.73,72.40,69.18,66.72,65.27(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=5.8Hz,1C),11.46,7.80. HRMS(ESI-TOF)m / z:[M+H] + Calculated value 845.39; measured value 845.3859.

[0441] Preparative HPLC method: Gilson 281 semi-preparative HPLC system and Phenomenex Gemini C18 column (75 x 40 mm x 3 um); Mobile phase: acetonitrile and water; Flow rate: 25 mL / min; Monitor wavelengths: 220 and 254 nm. Gradient: 30% to 50% acetonitrile in 8 min, 50% to 100% acetonitrile in 0.2 min, 100% acetonitrile for 2 min, 100% to 30% acetonitrile in 0.1 min, then 30% acetonitrile for 1.2 min.

[0442] Example 3. Synthesis of MB-3 (Meditecan) (PL3) General procedure for the preparation of benzyl (2S)-2-(benzyloxycarbonylamino)-5-oxo-5-[[(2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl]amino]pentanoate (1-12). [ka]

[0443] To a solution of compound 11A (6.03 g, 16.27 mmol, 1.1 equiv) in N,N-dimethylformamide (27 mL) were added 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium tetrafluoroborate (DMTMMT) (9.7 g, 29.59 mmol, 2 equiv) and triethylamine (2.24 g, 22.19 mmol, 1.5 equiv) successively. After stirring at 25 °C for 0.5 h, compound 1-11 (2.68 g, 14.79 mmol, 1 equiv) was added and the reaction mixture was stirred at 25 °C for 12 h. LCMS (product retention time = 0.253) showed that the starting material was consumed and a new peak with the desired MS was detected. The reaction mixture was diluted with water (50 mL) and extracted with dichloromethane (6 x 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 placed on high vacuum to give an oil. The oil 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 in 30 min, 45% CH3CN for 35 min; approximately 15 grams of crude product was dissolved in 70 mL of DMF and loaded onto the column) to give the product 1-12 (4 g, 6.74 mmol, 46.1% yield, 99% purity) as a white color. 1 H NMR(400MHz,DMSO-d6)δ 1.72-1.85(m,1H)1.91-2.03(m,1H)2.18(br t,J=7.44Hz,2H)2.96-3.03(m,1H)3.24(dt,J=13.16,5.17Hz,2H)3.37-3.40(m,2H)3.44(br s,2H)4.04-4.11(m,1H)4.29(d,J=6.38Hz,1H)4.39-4.45(m,2H)4.51(d,J=5.63Hz ,1H)4.75(d,J=4.63Hz,1H)4.96-5.14(m,4H)7.19-7.46(m,10H)7.68-7.85(m,2H).

[0444] General procedure for the preparation of (2S)-2-amino-5-oxo-5-[[(2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl]amino]pentanoic acid (1-13). [ka]

[0445] To a solution of compound 1-12 (4 g, 7.48 mmol, 1 equiv.) in water (192 mL) and tetrahydrofuran (48 mL), Pd / C (15.86 g, 14.96 mmol, 10 wt.%, 2 equiv.) was added. The mixture was stirred under H2 (15 psi) at 25°C for 12 h. LCMS (product retention time = 0.137) showed that the starting material was consumed and the desired product was detected. The mixture was filtered through a celite pad and the filtrate was concentrated to give product 1-13 (2 g, 6.26 mmol, 83.6% yield, 97.1% purity) as a white solid. 1 H NMR(400MHz, heavy water)δ 3.84(dt,J=7.76,4.74Hz,1H)3.70-3.80(m,4H)3.58-3.64(m,2H)3.41(dd,J=14.06,4.03Hz,1H)3.25(dd,J=14.06,7.83Hz,1H)2.41(br s,2H)2.10(br s,2H).

[0446] General procedure for the preparation of (2S)-2-[6-(2,5-dioxopyrrol-1-yl)hexanoylamino]-5-oxo-5-[[(2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl]amino]pentanoic acid (1-14). [ka]

[0447] 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) was added TEA (7.3 g, 0.0722 mol) at 10-20 °C. The reaction solution was kept stirring at 10-20 °C for 18 h. It was purified by preparative HPLC (C18 column, mobile phase: CH3CN and water) to give the product 1-14 as a pale red solid after lyophilization (9.7 g, y=53%, purity 95.7%). 1 H NMR(400MHz, heavy water)δ 6.78(s,1H)4.30(dd,J=9.11,5.07Hz,1H)3.83(dt,J=7.89,4.74Hz,1H)3.79-3.74(m,1H) 3.74-3.68(m,2H)3.65-3.57(m,2H)3.46(t,J=6.91Hz,2H)3.40(dd,J=14.06,4.16Hz,1H) 3.23(dd,J=14.00,7.89Hz,1H)2.39-2.30(m,2H)2.24(t,J=7.27Hz,2H)2.20-2.08(m,1H) 2.04-1.89(m,1H)1.55(dquin,J=14.04,7.19,7.19,7.19,7.19Hz,4H)1.28-1.17(m,2H).

[0448] (2S)-2-[6-(2.5-dioxopyrrol-1-yl)hexanoylamino]-N-[(1S)-1-[[(1S)-2-[4-[(19S)-19-ethyl-6-fluoro-19-hydroxy-7-methyl-14,18-dioxo-17-oxa-3,13-diazapentacyclo[11.8.0.02,11.04,9.015,20]henicosa- General procedure for the preparation of 1(21),2,4,6,8,10,15(20)-heptan-8-yl]butoxymethylamino]-1-methyl-2-oxo-ethyl]carbamoyl]-2-methyl-propyl]-N'-[(2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl]pentanediamide (MB-3, Meditechan, Meditechan) [ka]

[0449] To a stirred solution of Compound 1-10 (15 g, purity: 90.1%, 0.0207 mol) and Compound 1-14 (8.7 g, 0.0173 mol) in anhydrous DMF (400 mL), a mixture of EDCI (8.04 g, 0.0518 mol) and HOPO (5.75 g, 0.0518 mol) was added at 5 - 15 °C. Stirring was continued at 5 - 15 °C for 13.5 h and the product MB-3 (Meditcan, Mediticon) was obtained as an off-white solid (7.1 g, y = 30.2%, purity 98.8%) by preparative HPLC (C18 column, mobile phase: CH3CN / H2O). 1 H NMR (400 MHz, 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=10.3Hz,1C),172.08,171.53,171.14,170.58,163.06,160.59,156.93,152.35,150. 07,147.96(d,J=13.9Hz,1C),145.41,140.24,134.48,129.34,128.39,125.08,124.09,119.08,110.24(br d,J=22.7Hz,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=11.7Hz,1C),11.44(d,J=5.9Hz,1C),7.81. HRMS(ESI-TOF)m / z:[M+H] + Calculated value 1137.52; measured value 1137.5140.

[0450] Example 4. Exemplary synthesis of antibody-drug conjugates MB-2a and MB-3a (trastuzumab meditecan) General procedure for the preparation of trastuzumab-drug conjugate MB-2a [ka]

[0451] 50 mM conjugation buffer (pH 7.4): 1 liter contains 6.86 g Na2HPO4·2H2O and 1.58 g NaH2PO4·H2O.

[0452] 10 mM DTPA (pentetic acid) solution: 1 liter contains 3.90 g DTPA and 1.20 g NaOH.

[0453] 25 mM His / His-HCl formulation buffer (pH 5.5): 1 liter contains 0.90 g L-histidine and 4.04 g L-histidine hydrochloride monohydrate.

[0454] Antibody preparation: 452 mg of lyophilized trastuzumab powder was dissolved in 22 mL of purified water. The resulting antibody solution was dialyzed for 4 cycles with 50 mM conjugation buffer using an ultrafiltration tube (30 KD) to obtain an antibody concentration of 8.63 mg / ml (the extinction coefficient of trastuzumab, ε 280 =213380M -1 cm -1 was used).

[0455] Reduction of antibody: To a tube containing 12.2 mL (105 mg, 0.000724 mmol of trastuzumab) of the above prepared trastuzumab solution, 6.2 mL of 50 mM conjugation buffer was added, followed by 579.2 μl of TCEP (10 mM) and 2.1 mL of 10 mM DTPA. The tube was placed in a thermomixer and the reduction reaction was carried out at 25° C. for 2 hours.

[0456] Conjugation between antibody and payload: To the above trastuzumab reduced solution, a solution of MB-2 (7.45 mg, 0.00882 mmol) in DMSO (1.76 mL) was added. The tube was placed in a thermomixer and the conjugation reaction was carried out at 25° C. for 1 hour.

[0457] Purification: The above conjugation reaction solution was subjected to purification using ultrafiltration tubes (30 KD) for 6 cycles 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.

[0458] Physicochemical characterization of MB-2a (payload extinction coefficient ε 280 =4546M -1 cm -1 and ε 360 =17513M -1 cm-1 (Table 1): [Table 1-1] [Table 1-2]

[0459] General procedure for the preparation of trastuzumab-drug conjugate MB-3a [ka]

[0460] 50 mM conjugation buffer (pH 7.4): 1 liter contains 6.86 g Na2HPO4·2H2O and 1.58 g NaH2PO4·H2O.

[0461] 10 mM DTPA (pentetic acid) solution: 1 liter contains 3.90 g DTPA and 1.20 g NaOH.

[0462] 25 mM His / His-HCl formulation buffer (pH 5.5): 1 liter contains 0.90 g L-histidine and 4.04 g L-histidine hydrochloride monohydrate.

[0463] Antibody preparation: 452 mg of lyophilized trastuzumab powder was dissolved in 22 mL of purified water. The resulting antibody solution was dialyzed for 4 cycles with 50 mM conjugation buffer using an ultrafiltration tube (30 KD) to obtain an antibody concentration of 8.63 mg / ml (the extinction coefficient of trastuzumab, ε 280 =213380M -1 cm -1 was used).

[0464] Reduction of antibody: To a tube containing 12.2 mL (105 mg, 0.000724 mmol of trastuzumab) of the above prepared trastuzumab solution, 6.2 mL of 50 mM conjugation buffer was added, followed by 579.2 μl of TCEP (10 mM) and 2.1 mL of 10 mM DTPA. The tube was placed in a thermomixer and the reduction reaction was carried out at 25° C. for 2 hours.

[0465] Conjugation between antibody and payload: To the above trastuzumab reduced solution, a solution of MB-3 (10.02 mg, 0.00886 mmol) in DMSO (1.77 mL) was added. The tube was placed in a thermomixer and the conjugation reaction was carried out at 25° C. for 1 h.

[0466] Purification: The above conjugation reaction solution was subjected to purification using ultrafiltration tubes (30 KD) for 6 cycles 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.

[0467] Physicochemical characterization of MB-3a (payload extinction coefficient ε 280 =4546M -1 cm -1 and ε 360 =17513M -1 cm -1 (Table 2): [Table 2]

[0468] Example 5. In vitro assays of toxins and ADCs 175 μL of cell suspension was dispensed into 96-well plates at 1500 cells per well and incubated for 24 h in a humidified incubator (37 °C, 5% CO2). For antibody blocking, 2 × 10 -6Cells (15000 cells / mL) were incubated with 1000 M trastuzumab (final concentration 1 μM). 25 μL of various concentrations of compounds as 5× solutions were added to cell culture medium (fetal bovine serum, Invitrogen) in the plates and incubated for 120 h in an incubator. CCK-8 was thawed on the benchtop or in a 37°C water bath and 10 μL of CCK-8 was added to each well of the incubated plate (careful not to introduce air bubbles into the wells as this will interfere with the OD reading) and then further incubated for 1–4 h in the incubator. Absorbance was measured at 450 nm using a SpectraMax i3x Microplate Reader and the percentage of cell inhibition was calculated. IC 50 Curves were analyzed using GraphPad Prism software to determine IC 50 Generated with the value.

[0469] The results of in vitro cytotoxicity assays of the toxins (predicted metabolites of the ADCs) are summarized in Table 3 below and Figures 1A-1F. The cytotoxicity of metabolite MB-1 is comparable to DXd, a metabolite of DS-8201a (Enhertu), in multiple cell lines, except for the moderately Her-2 expressing and trastuzumab resistant cell line JIMT-1, where MB-1 is ten-fold more potent than DXd. [Table 3-1] [Table 3-2]

[0470] The payload MB-3 (Meditecan) is more hydrophilic than MB-2 and Daiichi Pharmaceutical's payload, deruxtecan, due to the presence of a polyol moiety in the linker. Thus, MB-3 has a higher reactivity with disulfide-reduced trastuzumab, resulting in a homogenous ADC, MB-3a (trastuzumab meditecan), with a DAR value of 8. In contrast, both MB-2a and DS-8201a (trastuzumab deruxtecan) have a lower content of DAR6 species due to the lower reactivity of the payload with the reduced antibody. The HIC-HPLC spectra of the ADCs are shown in Figures 2A-2C. The hydrophilic payload MB-3 is more reactive in the conjugation reaction, resulting in MB-3a as a homogenous ADC. The payloads MB-2a and MB-3a incorporated into the ADCs MB-2a and MB-3a, respectively, are designed to enable ADCs with better bystander activity than DS-8201a (Enhertu).

[0471] The results of the in vitro cytotoxicity assays of the ADCs are summarized in Table 4 below and Figures 3A-3D. In addition to the exemplary compounds of formula III, such as MB-2a and MB-3a, the activity of trastuzumab and the ADC trastuzumab deruxtecan (DS-8201a) was also evaluated for comparison. As shown in Table 4 and Figures 3A-3D, the trastuzumab ADCs MB-2a and MB-3a showed the same potency as DS-8201a in the Her2-high expressing cell line, NCI-N87 (Figure 3A). However, blocking the Her2 antigen with trastuzumab reduced the cell growth inhibition ability of the ADCs (Figure 3B). Furthermore, the ADCs were not as potent in the Her2-negative cell line MDA-MB-468 (Figure 3D), demonstrating the specificity of the ADCs for Her2-expressing cells. Although the ADCs were not sensitive in in vitro assays in JIMT-1 cells, which have moderate levels of Her2 expression, MB-2a and MB-3a were still relatively more potent than DS-8201a in this cell line (Figure 3C). [Table 4] [ka]

[0472] In stability studies of the ADCs in formulation buffer, MB-2a, MB-3a, and DS-8201a were all stable at room temperature. The DAR values ​​did not change at 25°C or 37°C, indicating that the payload was not shed from the antibody. MB-2a and DS-8201a formulations showed an increase in high molecular weight (HMW) and low molecular weight (LMW) species over time at 37°C. For MB-3a, only the LMW species increased slightly after 6 days at 37°C. MB-3a is more stable at higher temperatures than MB-2a and DS-8201a due to the incorporation of a more hydrophilic payload. Stability data are shown in Table 5. [Table 5]

[0473] Example 6. In vivo efficacy of ADCs in the NCI-N87 CDX model The right flank of each mouse (female Balb / c-Nude from Vital Rivers) was inoculated with NCI-N87 tumor cells (5 × 10 cells) mixed with Matrigel in 0.2 mL of PBS for tumor development. 6 ) (50:50) were subcutaneously inoculated. Animals were randomized into groups 6 days after tumor inoculation, and the mean tumor volume was approximately 160 mm 3 Treatment for efficacy studies was initiated when the tumor-bearing mice reached 100%. Each group contained 8 mice. Test and control articles were administered to tumor-bearing mice via the tail vein at a volume of 5 mL / kg.

[0474] Tumor size was measured twice weekly in two dimensions using calipers and volume was calculated in mm using the following formula: 3 Expressed as: V = 0.5a × b 2 (where a and b were the major and minor dimensions of the tumor, respectively.) Results were expressed as the mean and standard error (mean±SEM).

[0475] Statistical analysis: Two-way ANOVA was performed to compare the tumor volume between the two groups. All data were analyzed using Graphpad Prism 6.0, and P less than 0.05 was considered statistically significant. Both statistical analysis and biological observations were taken into account.

[0476] Tumor growth inhibition: Tumor size was used to calculate T / C value. Relative tumor growth rate T / C (%) was calculated using the formula: T / C (%) = (Ti / T0) / (Vi / V0) x 100%. Relative tumor growth inhibition was calculated by the formula: TGI (%) = [1-(Ti / T0) / (Vi / V0)] x 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 when divided, Vi refers to the mean tumor volume of the vehicle control group measured at each indicated time point after treatment, and V0 refers to the tumor volume of the vehicle control group when divided. If T / C is more than 40%, there is no efficacy, and if T / C is less than 40% and p-value is less than 0.05, there is tumor inhibition.

[0477] The antitumor efficacy of the ADCs in the NCI-N87 CDX model is shown in Figure 4 and Table 6. As shown in Figure 4, both MB-2a (at doses of 1 mg / kg and 4 mg / kg) and MB-3a (at doses of 1 mg / kg and 4 mg / kg) demonstrated potent antitumor efficacy and were more effective than DS-8201a (Enhertu). [Table 6]

[0478] Example 7. In vivo efficacy of ADCs in the JIMT-1 CDX model The right flank of each mouse (Scid-Beige, Shanghai Lingchang Biotech) was inoculated with JIMT-1 tumor cells (1 × 10 ) mixed with Matrigel in 0.2 mL of PBS for tumor development. 7 ) (50:50) subcutaneously. Animals were randomized into groups 6 days after tumor inoculation to obtain a mean tumor volume of approximately 175 mm 3Treatment for efficacy studies was initiated when the tumor-bearing mice reached 100%. Each group contained 8 mice. Test and control articles were administered to tumor-bearing mice via the tail vein at a volume of 5 mL / kg.

[0479] Tumor size was measured twice weekly in two dimensions using calipers and volume was calculated in mm using the following formula: 3 Expressed as: V = 0.5a × b 2 (where a and b were the major and minor dimensions of the tumor, respectively.) Results were expressed as the mean and standard error (mean±SEM).

[0480] Statistical analysis: Two-way ANOVA was performed to compare the tumor volume between the two groups. All data were analyzed using Graphpad Prism 6.0, and P less than 0.05 was considered statistically significant. Both statistical analysis and biological observations were taken into account.

[0481] Tumor growth inhibition: Tumor size was used to calculate T / C value. Relative tumor growth rate T / C (%) was calculated using the formula: T / C (%) = (Ti / T0) / (Vi / V0) x 100%. Relative tumor growth inhibition was calculated by the formula: TGI (%) = [1-(Ti / T0) / (Vi / V0)] x 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 when divided, Vi refers to the mean tumor volume of the vehicle control group measured at each indicated time point after treatment, and V0 refers to the tumor volume of the vehicle control group when divided. If T / C is more than 40%, there is no efficacy, and if T / C is less than 40% and p-value is less than 0.05, there is tumor inhibition.

[0482] The antitumor effects of ADC in the JIMT-1 CDX model are shown in Figures 5-8 and Table 7. Figure 5 shows the effect of antibody drug conjugates (ADC) in the JIMT-1 CDX model at three different doses. In this study, all three doses of MB-2a and MB-3a showed significant antitumor effects. The different doses studied in these experiments are also shown separately in Figure 6 (2.5 mg / kg, single intravenous dose), Figure 7 (5 mg / kg, single intravenous dose), and Figure 8 (10 mg / kg, single intravenous dose). [Table 7]

[0483] All publications and patent applications cited herein are incorporated by reference in their entirety and for all purposes as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference and as if each reference was set forth in its entirety. Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be apparent to those skilled in the art in light of the teachings of the present invention that certain changes and modifications can be made thereto without departing from the spirit or scope of the appended claims.

Claims

1. A compound having the structure of formula PL-A, 【Chemistry 83】 or a pharma- ceutically acceptable salt thereof, E is a peptide comprising 2-8 amino acids, where E is optionally substituted with one or more polyols, and the N-terminus of the peptide is covalently linked to Z'; Z'は、-C(=O)-L １ -Y'、 【Chemistry 84】 In the formula, m represents an integer of 1 to 10, and * represents a site covalently bonded to the C; L １ is -(C １ -C １０ alkylene)-, -CH ２ CH ２ (O.C.H. ２ CH ２ ) ｎ N (R １ ) C(=O)-L ２ -* or -CH ２ (O.C.H. ２ CH ２ ) ｎ N (R １ ) C(=O)-L ２ -*, where n is an integer from 1 to 10, and * represents a site covalently bonded to Y'; L ２ Ha-(C １ -C １０ alkylene)-, and R １ is -H or -CH ３ and Y' is a group formed by reaction of an electrophilic group with a reactive nucleophilic group present on the cell-binding agent; p is the drug-to-antibody ratio (DAR), which is an average number between about 2 and 10; C is a binding agent that targets HER2; a heavy chain comprising an amino acid sequence having at least 80% identity to SEQ ID NO:1; and a light chain comprising an amino acid sequence having at least 80% identity to SEQ ID NO:2, or a pharma- ceutically acceptable salt thereof.

2. C, a heavy chain comprising an amino acid sequence having at least 85%, 90%, or 95% identity to SEQ ID NO:1; and a light chain comprising an amino acid sequence having at least 85%, 90%, 95% identity to SEQ ID NO:

2.

3. C, A heavy chain amino acid sequence of SEQ ID NO:1; The compound of claim 1 or 2, which is a binding agent comprising the light chain amino acid sequence of SEQ ID NO:

2.

4. The compound of any one of claims 1 to 3, wherein C is a binding agent that is an antibody or an antigen-binding fragment thereof.

5. 5. The compound of any one of claims 1 to 4, wherein C is a binder that is trastuzumab (Herceptin), trastuzumab-dkst (Ogivri), trastuzumab-pkrb (Herzuma), trastuzumab-dttb (Ontruzant), trastuzumab-qyp (Trazimera), or trastuzumab-anns (Kanjinti).

6. L １ But -(C １ -C １０ The compound according to any one of claims 1 to 5, wherein the aryl group is aryl, ...

7. L １ But -CH ２ CH ２ (O.C.H. ２ CH ２ ) ｎ N (R １ ) C(=O)-L ２ -* or -CH ２ (O.C.H. ２ CH ２ ) ｎ N (R １ ) C(=O)-L ２ A compound according to any one of claims 1 to 5, wherein n represents an integer from 1 to 10, and wherein * represents the site of covalent attachment to Y'.

8. L １ But -CH ２ CH ２ CH ２ CH ２ CH ２ --, --CH ２ CH ２ --, --CH ２ --, --CH ２ CH ２ OCH ２ CH ２ OCH ２ CH ２ NHC(=O)CH ２ CH ２ -* or -CH ２ OCH ２ CH ２ OCH ２ CH ２ NHC(=O)CH ２ CH ２ The compound of any one of claims 1 to 7, wherein * represents the site of covalent attachment to Y'.

9. The compound of any one of claims 1 to 8, wherein Y' is formed from a Michael acceptor group, a succinimide, an epoxide, or a halogen.

10. Y' is 【Chemistry 85】 is formed from In the formula, R ２ and R ３ are each independently -H or C １ -C ３ The compound according to any one of claims 1 to 9, which is alkyl.

11. Y' is 【Chemistry 86】 and In the formula, R ２ and R ３ are each independently -H or C １ -C ３ The compound according to any one of claims 1 to 9, wherein: R is alkyl and * indicates the site of covalent attachment to said C.

12. A compound according to any one of claims 1 to 11, wherein Z' is formed from 【Chemistry 87】 。

13. Z' is 【Chemistry 88】 and A compound according to any one of claims 1 to 12, wherein * indicates the site of covalent attachment to C.

14. Z' is 【Chemistry 89】 2. The compound according to claim 1, wherein * represents a covalent bond to C and n is an integer from 2 to 10.

15. Z' is 【Chemistry 90】 15. The compound according to claim 14, wherein * indicates a covalent bond to C.

16. The compound of any one of claims 1 to 15, wherein E is a peptide of 2, 3, 4, or 5 amino acids.

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

18. 18. The compound of claim 16 or 17, wherein 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.

19. 19. The compound of any one of claims 1 to 18, wherein E comprises one or more amino acids selected from glycine, alanine, valine, glutamine, glutamic acid, phenylalanine, and leucine, wherein said glutamine or glutamic acid is optionally substituted by a polyol.

20. 20. The compound of claim 19, wherein E comprises an amino acid selected from glycine, alanine, valine, glutamine, glutamic acid, phenylalanine, and leucine, wherein the glutamine or glutamic acid is optionally replaced by a polyol.

21. E comprises an amino acid having the structure: 【Chemistry 91】 In the formula, R ４ is -H or C １ -C ６ The compound according to any one of claims 1 to 20, which is alkyl.

22. 22. The compound of claim 21 , wherein E comprises an amino acid having the structure: 【Chemistry 92】 。

23. 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); 21. The compound according to any one of claims 1 to 20, wherein * denotes the N-terminus of the peptide covalently attached to Z'.

24. E is -L-Ala-D-Val-*, -L-Val-D-Ala-*, -L-Val-D-Lys-*, -L-Val-D-Arg-*, -L-Val-D-C it-*, -L-Val-D-Arg-*, -L-Val-D-Cit-*, -L-Val-D-Lys-*, -L-Val-D-Arg-*, -L-Arg-D selected from the group consisting of -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-*; 24. The compound of claim 23, wherein * indicates the N-terminus of the peptide covalently attached to Z'.

25. 21. The compound of any one of claims 1 to 20, wherein E comprises -(L-Val)-(L-Ala)-.

26. 26. The compound of claim 25, wherein E comprises an amino acid having the structure: 【Chemistry 93】 。

27. 【Catalogue 94】 The compound according to any one of claims 1 to 15, having one of the following structures, where * denotes the N-terminus of the peptide covalently attached to Z': 【Chemistry 95】 。

28. 【Chemical 96】 The compound according to any one of claims 1 to 5, wherein is formed from one of the following structures: 【Chemistry 97】 。

29. 【Catalysis 98】 is one of the following structures, where * indicates the point of attachment to C: 【Chemistry 99】 。

30. The compound has one of the following structures, or a pharma- ceutically acceptable salt thereof: 【Chemistry 100】 【Chemistry 101】 2. The compound of claim 1, wherein p is about 2-10, 4-8, or 7-8.

31. 31. The compound of claim 30, wherein the compound has one of the following structures: 【Chemistry 102】 。

32. The compound has one of the following structures, or a pharma- ceutically acceptable salt thereof: 【Chemistry 103】 32. The compound of any one of claims 1 and 30-31, wherein p is about 4-8, or 7-8.

33. 33. The compound of any one of claims 30 to 32, wherein p is about 7 to 8.

34. The compound according to any one of claims 30 to 33, wherein p is 8.

35. A pharmaceutical composition comprising a compound according to any one of claims 1 to 34, or a pharma- ceutically acceptable salt thereof.

36. A composition for treating cancer in a subject in need thereof, comprising a compound according to any one of claims 1 to 34, or a pharma- ceutically acceptable salt thereof.

37. The compound is 【Chemistry 104】 or a pharma- ceutically acceptable salt thereof.

38. 38. The composition of claim 37, wherein p is about 4 to 8, or 7 to 8.

39. 39. The composition of claim 38, wherein p is about 7 to 8.

40. 40. The composition of claim 39, wherein p is 8.

41. The composition of any one of claims 36 to 40, wherein the cancer is lung cancer, urothelial cancer, 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, colon cancer, rectal cancer, colorectal cancer, endometrial cancer, uterine cancer, salivary gland cancer, kidney cancer, vulvar cancer, thyroid cancer, penile cancer, leukemia, malignant lymphoma, plasmacytoma, myeloma, or sarcoma.

42. The composition of any one of claims 36 to 41, wherein the cancer is breast cancer.

43. The composition of any one of claims 36 to 41, wherein the cancer is gastric cancer.

44. The composition of any one of claims 36 to 41, wherein the cancer is lung cancer.

45. 45. The composition of claim 44, wherein the lung cancer is non-small cell lung cancer.

46. The composition of any one of claims 36 to 41, wherein the cancer is ovarian cancer.

47. The composition of any one of claims 36 to 46, wherein the cancer is metastatic.

48. The composition of any one of claims 36 to 46, wherein the cancer is characterized by low Her2 expression.

49. The composition of any one of claims 36 to 46, wherein the cancer is characterized by intermediate Her2 expression.

50. The composition of any one of claims 36 to 46, wherein the cancer is characterized by high Her2 expression.

51. The composition of any one of claims 36 to 50, wherein the cancer is trastuzumab resistant.

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