Methods of making antibody-drug conjugates based on eribulin

Abstract

The present application relates to a method for preparing an antibody-drug conjugate based on eribulin. Provided is a method for preparing an antibody-drug conjugate represented by Formula (I), in the formula, Ab is an antibody or an antigen-binding fragment thereof, D is eribulin, m is an integer of 1 to 10, and p is an integer of 1 to 8, the method comprising: Step 1: obtaining a compound represented by Formula (B) by reaction of eribulin or a salt thereof with a compound represented by Formula (A), in the formula, m is an integer of 1 to 10, and X is phenoxy or nitrophenoxy, and in the formula, m is an integer of 1 to 10, and Step 2: obtaining the antibody-drug conjugate represented by Formula (I) by reaction of the compound represented by Formula (B) with Ab.

Description

[0001] This application is a divisional application of Chinese invention patent application No. 202080088854.3 (PCT application number PCT / JP2020 / 047706), filed on December 21, 2020, entitled "Method for preparing antibody-drug conjugates based on eribulin". Technical Field

[0002] This invention relates to a method for preparing eribulin-based antibody-drug conjugates. Background Technology

[0003] Cancer is a leading cause of disease and death, with approximately 14,000,000 new cancer cases and 8,200,000 cancer deaths reported in 2012. The most common causes of cancer death are lung cancer (1,590,000 deaths), liver cancer (745,000 deaths), stomach cancer (723,000 deaths), colorectal cancer (694,000 deaths), breast cancer (521,000 deaths), and esophageal cancer (400,000 deaths). The number of new cancer cases is projected to increase by approximately 70% over the next 20 years, with approximately 22,000,000 new cancer cases reported annually (Non-Patent Literature 1).

[0004] Microtubules are dynamic filamentous cytoskeletal proteins involved in a variety of cellular functions, including intracellular migration and transport, cell signaling, and cell morphology maintenance. Microtubules also play a crucial role in mitotic cell division by forming the mitotic spindle, which is required to divide chromosomes into two daughter cells. Most biological functions of microtubules in all cells are regulated by their polymerization kinetics, which occur through the reversible, non-covalent addition of α-tubulin dimers and β-tubulin dimers at the ends of the microtubules. This kinetic behavior and the resulting control over microtubule length are essential for the normal functioning of the spindle. Even small changes in microtubule kinetics can engage spindle checkpoints, halting cell cycle progression during mitosis and subsequently leading to cell death (Non-Patent Literature 2). Because cancer cells divide rapidly, they are generally more sensitive to compounds that bind to tubulin and disrupt its normal function compared to normal cells. Therefore, tubulin inhibitors and other agents targeting microtubules have emerged as a promising class of cancer therapeutics (Non-Patent Literature 3).

[0005] Folic acid receptor α (FRA) is a membrane protein linked to glycophosphatidylinositol (GPI), which binds to folate. While the role of FRA in the biology of normal and cancerous tissues is not fully understood, FRA is highly overexpressed in a high percentage of epithelial-derived ovarian cancer (Non-Patent Literature 4) and a certain percentage of non-small cell lung cancer (Non-Patent Literature 5). FRA expression in normal tissues is also limited. These properties make FRA an attractive target for cancer immunotherapy.

[0006] The proto-oncogene human epidermal growth factor receptor 2 (HER2) encodes a transmembrane tyrosine kinase receptor belonging to the human epidermal growth factor receptor (EGFR) family (Non-Patent Literature 6). Overexpression of HER2 enables constitutive activation of growth factor signaling pathways such as the PI3K-AKT-mTOR pathway, and thus acts as an oncogenic driver in several types of cancer, including approximately 20% of invasive breast cancers (Non-Patent Literatures 7 and 8). Due to the phenotype transformation mediated by HER2 amplification, HER2 is another promising target for cancer therapy.

[0007] List of cited references Patent documents [Patent Document 1] WO 2017 / 151979 Non-patent literature [Non-Patent Literature 1] World Cancer Report 2014 [Non-Patent Literature 2] Mukhtar et al. (2014) Mol. Cancer Ther. 13:275-84 [Non-Patent Literature 3] Dumontet and Jordan (2010) Nat. Rev. Drug Discov. 9:790-803 [Non-Patent Literature 4] O'Shannessy et al. (2013) Int. J. Gynecol. Pathol. 32(3):258-68 [Non-patent literature 5] Christoph et al. (2014) Clin. Lung Cancer 15(5):320-30 [Non-Patent Literature 6] King et al. (1985) Science 229:974-6 [Non-Patent Reference 7] Slamon et al. (1989) Science 244:707-12 [Non-Patent Literature 8] Gajria and Chandarlapaty (2011) Expert Rev. Anticancer Ther. 11:263-75 [Non-Patent Literature 9] O'Shannessy et al., (2011) Oncotarget 2:1227-43 Summary of the Invention

[0008] Technical issues The purpose of this invention is to provide a method for preparing antibody-drug conjugates in high yield and synthetic intermediates that can be used in this method.

[0009] Solution to the problem The present invention provides the following [1] to [8].

[0010] [1] A method for preparing the antibody-drug conjugate shown in formula (I), In this formula, Ab is an antibody or its antigen-binding fragment, D is eribulin, m is an integer from 1 to 10, and p is an integer from 1 to 8. The method includes: Step 1: The compound shown in formula (B) is obtained by reacting eribulin or its salt with the compound shown in formula (A). In this formula, m is an integer from 1 to 10, and X is phenoxy or nitrophenoxy. In this formula, m is an integer from 1 to 10, and Step 2: Obtain the antibody-drug conjugate shown in formula (I) by reacting the compound shown in formula (B) with Ab.

[0011] [2] According to the method of [1], wherein Ab comprises (i) a heavy chain domain comprising the amino acid sequence shown in SEQ ID NO: 1 and a light chain domain comprising the amino acid sequence shown in SEQ ID NO: 6, (ii) a heavy chain variable domain comprising the amino acid sequence shown in SEQ ID NO: 23 and a light chain variable domain comprising the amino acid sequence shown in SEQ ID NO: 24, (iii) a heavy chain variable domain comprising the amino acid sequence shown in SEQ ID NO: 27 and a light chain variable domain comprising the amino acid sequence shown in SEQ ID NO: 28, or (iv) a heavy chain domain comprising the amino acid sequence shown in SEQ ID NO: 347 and a light chain domain comprising the amino acid sequence shown in SEQ ID NO: 308.

[0012] [3] The method described in [1] or [2], wherein p is 3 or 4.

[0013] [4] The method according to any one of [1] to [3], wherein eribulin or a salt thereof is eribulin methanesulfonate.

[0014] [5] The method according to any one of [1] to [4], wherein step 1 is carried out in the presence of a base.

[0015] [6] A method for preparing the compound shown in formula (B), In this formula, m is an integer from 1 to 10. The method includes: The steps for obtaining the compound shown in formula (B) by reacting eribulin or its salt with the compound shown in formula (A) are as follows: In this formula, m is an integer from 1 to 10, and X is phenoxy or nitrophenoxy.

[0016] [7] The method according to [6], wherein the step is carried out in the presence of a base.

[0017] [8] A compound of formula (A), In this formula, m is an integer from 1 to 10, and X is phenoxy or nitrophenoxy.

[0018] Beneficial effects of the present invention This invention provides a method for preparing antibody-drug conjugates in high yield. Furthermore, this invention also provides a synthetic intermediate that can be used to prepare antibody-drug conjugates in high yield. Detailed Implementation

[0019] The embodiments of the present invention will be described in detail below.

[0020] Embodiments of the present invention relate to a method for preparing the antibody-drug conjugate shown in formula (I).

[0021] First, the ADC shown in description (I) is used.

[0022] ADCs can bind to, internalize, and kill tumor cells (e.g., tumor cells expressing FRA). Furthermore, it is preferred that the antibody moiety (Ab) used in the ADC is an antibody or its antigen-binding fragment and targets tumor cells. The antibody or antigen-binding fragment includes, for example: (a) Three heavy chain CDRs and three light chain CDRs as defined by the Kabat numbering system (Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md., 1987 and 1991)), wherein the three heavy chain CDRs contain the amino acid sequences of the heavy chain complementarity-determining region (heavy chain CDR) 1 shown in SEQ ID NO: 2, heavy chain CDR 2 shown in SEQ ID NO: 3, and heavy chain CDR 3 shown in SEQ ID NO: 4, and the three light chain CDRs contain the amino acid sequences of the light chain complementarity-determining region (light chain CDR) 1 shown in SEQ ID NO: 7, light chain CDR 2 shown in SEQ ID NO: 8, and light chain CDR 3 shown in SEQ ID NO: 9; or (b) Three heavy chain CDRs and three light chain CDRs as defined by the IMGT numbering system (International Immunogenetic Information System (IMGT) (registered trade name)), wherein the three heavy chain CDRs contain the amino acid sequences of heavy chain CDR1 shown in SEQ ID NO: 13, heavy chain CDR2 shown in SEQ ID NO: 14, and heavy chain CDR3 shown in SEQ ID NO: 15, and the three light chain CDRs contain the amino acid sequences of light chain CDR1 shown in SEQ ID NO: 16, light chain CDR2 shown in SEQ ID NO: 17, and light chain CDR3 shown in SEQ ID NO: 18.

[0023] In this specification, the terms “antibody-drug conjugate,” “antibody conjugate,” “conjugate,” “immunoconjugate,” and “ADC” are used interchangeably to refer to a compound or derivative thereof linked to an antibody (e.g., an anti-FRA antibody) and defined by formula (I) [in which Ab is the antibody moiety (i.e., the antibody or its antigen-binding fragment), L is the linker moiety, D is eribulin, and p is the number of eribulin moieties on each antibody moiety].

[0024] The term "antibody" in its broadest sense refers to an immunoglobulin molecule that recognizes and specifically binds to a target such as a protein, polypeptide, carbohydrate, polynucleotide, lipid, or a combination of the foregoing via at least one antigen recognition site within the variable domain of an immunoglobulin molecule. The heavy chain of an antibody consists of a heavy chain variable domain (V... H ) and heavy chain constant structural domain (C H The antibody's light chain is composed of a light chain variable domain (V). L ) and light chain constant structural domain (C L The structure consists of a heavy chain variable domain and a light chain variable domain, each comprising three complementarity-determining regions (CDR1, CDR2, and CDR3) within four framework regions (FR1, FR2, FR3, and FR4) arranged from the N-terminus to the C-terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. An "antibody" may be naturally occurring or artificial, such as a monoclonal antibody produced by conventional hybridoma techniques. The term "antibody" includes full-length monoclonal antibodies, full-length polyclonal antibodies, and single-chain antibodies. Furthermore, examples of antigen-binding fragments of an antibody include Fab, Fab', F(ab')2, and Fv. An antibody may be any of the five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, or their subclasses (e.g., isotypes IgG1, IgG2, IgG3, and IgG4). The term further encompasses human antibodies, chimeric antibodies, humanized antibodies, and any modified immunoglobulin molecule containing an antigen recognition site, provided that it exhibits the desired biological activity.

[0025] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies constituting that population are identical except for the possible small number of naturally occurring mutations present. Monoclonal antibodies are highly specific for a single antigenic epitope. In contrast, conventional (polyclonal) antibody formulations typically comprise multiple antibodies that target or are specific to different epitopes. The modifier "monoclonal" indicates the characteristic of an antibody obtained from a substantially homogeneous population of antibodies and should not be construed as requiring the antibody to be prepared by any particular method. For example, monoclonal antibodies to be used according to this disclosure may be manufactured by a hybridoma method first described by Kohler et al. (1975) Nature [Nature] 256:495, or by a recombinant DNA method (e.g., see, U.S. Patent No. 4,816,567). Monoclonal antibodies can also be isolated from phage antibody libraries using techniques described, for example, those described in Clackson et al. (1991) Nature [Nature] 352:624-8 and Marks et al. (1991) J. Mol. Biol. [Journal of Molecular Biology] 222:581-97.

[0026] The monoclonal antibodies described herein specifically include “chimeric” antibodies (in which a portion of the heavy chain and / or light chain is identical or homologous to the corresponding sequence in an antibody derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain is identical or homologous to the corresponding sequence in an antibody derived from another species or belonging to another antibody class or subclass), as well as fragments of such antibodies; provided they specifically bind to the target antigen and / or exhibit the desired biological activity.

[0027] The term "homology" refers to a molecule that exhibits homology with another molecule, for example, by having the same or similar sequence of chemical residues at corresponding positions.

[0028] As used herein, the term "human antibody" refers to an antibody produced by humans or an antibody having the amino acid sequence of an antibody produced by humans.

[0029] As used herein, the term "chimeric antibody" refers to an antibody in which the amino acid sequence of the immunoglobulin molecule is derived from two or more species. In some cases, the variable domains of both the heavy and light chains correspond to the variable domains of an antibody derived from one species with the desired specificity, affinity, and activity, while the constant region is homologous to an antibody derived from another species (e.g., human) to minimize the immune response in the latter species.

[0030] As used herein, the term "humanized antibody" refers to an antibody form containing sequences derived from non-human (e.g., mouse) antibodies as well as human antibodies. Such antibodies are chimeric antibodies containing minimal sequences derived from non-human immunoglobulins. Generally, humanized antibodies contain substantially all of at least one, and typically two, variable domains, wherein all or substantially all of the hypervariable loops correspond to the hypervariable loops of the non-human immunoglobulin and all or substantially all of the frame (FR) regions are frame regions of the human immunoglobulin sequence. Humanized antibodies optionally also contain at least a portion of the immunoglobulin constant region (Fc), typically the constant region (Fc) of human immunoglobulins. Humanized antibodies can be further modified by neutralization and / or substitution of residues within the Fv frame regions to improve and optimize antibody specificity, affinity, and / or activity.

[0031] As used herein, the term "antigen-binding fragment" of an antibody refers to one or more antibody fragments that retain the ability to specifically bind to an antigen (e.g., FRA). The antigen-binding fragment preferably also retains the ability to be internalized into cells expressing the antigen. In some embodiments, the antigen-binding fragment also retains immune effector activity. Fragments of full-length antibodies have been shown to perform the antigen-binding function of full-length antibodies. Examples of binding fragments covered within the term "antigen-binding fragment" of an antibody include: (i) Fab fragments, which are composed of V... L V H C L and C H1 (ii) A monovalent segment composed of structural domains; (iii) A divalent segment consisting of two Fab segments connected by a disulfide bridge in the hinge region; (iv) A Fd segment consisting of V H Domain and C H1 (iv) The binding domain consists of the Fv fragment, which is composed of the V arm of the antibody. L Domain and V H Domain composition; (v) dAb fragment, which contains a single variable domain, for example, V H Structural domains (e.g., see Ward et al. (1989) Nature 341:544-6; and Winter et al. or WO 90 / 05144); and (vi) separated complementarity-determining regions (CDRs). Furthermore, although the two structural domains V of the Fv segment... L and V H Encoded by individual genes, but which can be conjugated using recombination methods by synthetic linkers that enable them to be made in the form of single protein chains, where V L Domain and V HDomains pair to form a monovalent molecule (called a single-chain Fv (scFv)). See, for example, Bird et al. (1988) Science 242:423-6; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-83. Such single-chain antibodies are also intended to be covered within the term "antigen-binding fragment" of antibody and are known in the art as exemplary types of binding fragments that can be internalized into cells after binding. See, for example, Zhu et al. (2010) 9:2131-41; He et al. (2010) J Nucl. Med. 51:427-32; and Fitting et al. (2015) MAbs 7:390-402. In some embodiments, the scFv molecule may be incorporated into a fusion protein. Other forms of single-chain antibodies, such as biantibodies, are also covered. Biantibodies are bispecific biantibodies, where V H and V L The domains are expressed on a single polypeptide chain, but a linker too short to allow pairing between two regions on the same chain is used, thereby forcing these domains to pair with complementary regions of another chain and creating two antigen-binding sites (see, for example, Holliger et al. (1993) Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences] 90:6444-8; and Poljak et al. (1994) Structure [Structure] 2:1121-3). Antigen-binding fragments are obtained using conventional techniques known to those skilled in the art, and are screened for utility (e.g., binding affinity, internalization) in the same manner as intact antibodies. Antigen-binding fragments can be prepared by cleaving intact proteins, for example by protease or chemical cleavage.

[0032] In this specification, an antibody or its antigen-binding fragment is an internalizing antibody or its internalizing antigen-binding fragment. As used herein, "internalization" of an antibody or antigen-binding fragment means that, after binding to a cell, the antibody or antigen-binding fragment can cross the cell's lipid bilayer membrane and enter the internal compartments (i.e., "be internalized"), preferably into degradation compartments within the cell such as lysosomes. For example, an internalizing anti-FRA antibody is an antibody that can enter the cell after binding to FRA on the cell membrane.

[0033] As used herein, the term "folate receptor α" or "FRA" refers to any naturally occurring form of human FRA. This term encompasses full-length FRA (e.g., NCBI reference sequence: NP_000793; SEQ ID NO: 19) and any form of human FRA derived from cellular processing. The term also encompasses variants of naturally occurring FRA, including but not limited to splice variants, allele variants, and isotypes. FRAs can be isolated from humans or can be produced recombinantly or synthetically.

[0034] The term "anti-FRA antibody" or "antibody that specifically binds to FRA" refers to any form of antibody that specifically binds to FRA or its antigen-binding fragment, and encompasses monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, and biologically functional antibody fragments, provided they specifically bind to FRA. Preferably, the anti-FRA antibody used in the ADCs disclosed herein is an antibody or its antigen-binding fragment. MORAb-003 is an exemplary anti-human FRA antibody. As used herein, the terms "specificity" and "specific binding" refer to the selective binding of an antibody to a target antigen epitope. The binding specificity of an antibody can be tested by comparing binding to an appropriate antigen and binding to unrelated antigens or mixtures of antigens under a given set of conditions. An antibody is considered specific if its affinity for binding to an appropriate antigen is at least 2, 5, 7, and preferably 10 times that for binding to unrelated antigens or mixtures of antigens. In one embodiment, a specific antibody is an antibody that binds only to the FRA antigen but not to other antigens (or exhibits minimal binding).

[0035] As used herein, the terms “human epidermal growth factor receptor 2,” “her2,” or “her2 / neu” refer to any naturally occurring form of human her2. This term encompasses full-length her2 (e.g., NCBI reference sequence: NP_004439.2; SEQ ID NO: 21) and any form of human her2 derived from cellular processing. The term also encompasses variants of naturally occurring her2, including but not limited to splice variants, allele variants, and isotypes. Her2 can be isolated from humans or produced recombinantly or synthetically.

[0036] The term "anti-HER2 antibody" or "antibody that specifically binds to HER2" refers to any form of antibody that specifically binds to HER2 or its antigen-binding fragment, and encompasses monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, and biologically functional antibody fragments, provided that they specifically bind to HER2. U.S. Patent No. 5,821,337 (incorporated herein by reference) provides exemplary HER2 binding sequences, including exemplary anti-HER2 antibody sequences. Preferably, the anti-HER2 antibody used in the ADCs disclosed herein is an antibody or its antigen-binding fragment. Trastuzumab is an exemplary anti-human HER2 antibody.

[0037] The term "epitope" refers to a portion of an antigen that can be recognized and specifically bound by an antibody. When the antigen is a polypeptide, the epitope can be formed from consecutive amino acids or discontinuous amino acids adjacent to each other via the tertiary folding of the polypeptide. Epitopes bound by antibodies can be identified using any epitope mapping technique known in the art. Examples of epitope mapping techniques include X-ray crystallography for epitope identification (which is performed by direct visual inspection of the antigen-antibody complex), monitoring the binding of antibodies to fragments or mutant variants of antigens, and monitoring solvent accessibility of different portions of antibodies and antigens. Exemplary strategies for epitope mapping include, but are not limited to, array-based oligopeptide scanning, restriction proteolysis, site-directed mutagenesis, high-throughput mutagenesis mapping, hydrogen-deuterium exchange, and mass spectrometry (e.g., see Gershoni et al. (2007) 21:145-56; and Hager-Braun and Tomer (2005) Expert Rev. Proteomics 2:745-56).

[0038] Competitive binding and epitope clustering can also be used to identify antibodies that share the same or overlapping epitopes. Competitive binding can be assessed using cross-blocking analyses, such as those described in "Antibodies, A Laboratory Manual," Cold Spring Harbor Laboratory, Harlow and Lane (1st edition 1988, 2nd edition 2014). In some embodiments, the binding of the test antibody or its antigen-binding fragment to a target antigen such as FRA or her2 (e.g., binding proteins containing CDR and / or variable domains selected from those identified in Tables 2, 4, and 6) is evaluated as competitive when, in a cross-blocking analysis, the binding of the test antibody or its antigen-binding fragment to a reference antibody or its antigen-binding fragment is reduced by at least about 50% (e.g., 50%, 60%, 70%, 80%, 90%, 95%, 99%, 99.5%, or higher, or any percentage between thereof). In some embodiments, competitive binding may be attributed to identical or similar (e.g., partially overlapping) epitopes, or to steric hindrance, where an antibody or its antigen-binding fragment binds to a neighboring epitope. See, for example, Tzartos, Methods in Molecular Biology (Morris, ed., (1998) Vol. 66, pp. 55–66). In some embodiments, competitive binding can be used to classify groups of binding proteins sharing similar epitopes; for example, those binding proteins that bind competitively may be “boxed” into groups of binding proteins with overlapping or neighboring epitopes, while those binding proteins that do not compete are classified into separate groups of binding proteins without overlapping or neighboring epitopes.

[0039] The term "k" on "or "k a "" refers to the association rate constant of antibodies and antigens to form antibody / antigen complexes. This rate can be determined using standard analyses such as Biacore or ELISA.

[0040] The term "k" off "or "k d "" refers to the dissociation rate constant of the antibody from the antibody / antigen complex. This rate can be determined using standard analyses such as Biacore or ELISA.

[0041] Term "K" D "K" refers to the equilibrium dissociation constant of a specific antibody-antigen interaction. D via k a / k dThis rate can be calculated using standard analyses such as Biacore or ELISA.

[0042] The term "p" or "antibody:drug ratio" refers to the number of structural units (i.e., drug loading) containing a linker portion and eribulin on each antibody moiety (Ab). In some embodiments, p is an integer of 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 or 2, and preferably an integer of 3 or 4. In compositions containing multiple copies of the ADC shown in Formula I, "p" refers to the average number (also known as average drug loading) of structural units containing a linker portion and eribulin on each antibody moiety (Ab). When p is expressed as average drug loading, p can be 3 to 4, 3.2 to 3.8, 3.5 to 4.5, 3.6 to 4.4, or 4.

[0043] In some embodiments, p is an integer from 1 to 6, 2 to 5, or 3 or 4. When p is a larger number, the number of eribulin portions on each antibody moiety increases. Therefore, a larger amount of eribulin can be delivered to target cells with a single antibody, and its pharmacological effect can be further enhanced.

[0044] When an ADC is present outside the cell, it remains intact. When an ADC is internalized into a cell (e.g., a cancer cell), the ADC's linker portion cleaves, and eribulin is released into the cell. Outside the cell, the linker portion is typically stable. That is, the ADC recognizes and enters a cell (e.g., a cancer cell) that expresses an antigen specific to the antibody portion (Ab). For ADCs within a cell, the linker portion connecting eribulin and the antibody portion (Ab) cleaves, thereby releasing eribulin and exhibiting pharmacological effects.

[0045] 1. Antibody portion (Ab) The antibody portion (Ab) in an ADC is an antibody or its antigen-binding fragment (in particular, an anti-folate receptor α (FRA) antibody or its antigen-binding fragment) and can bind to tumor cells expressing FRA.

[0046] In some embodiments, the antibody or its antigen-binding fragment binds to folate receptor α (FRA) and can target tumor cells expressing FRA. Furthermore, in some embodiments, the antibody or its antigen-binding fragment comprises: (a) three heavy chain CDRs (heavy chain CDR1 shown in SEQ ID NO: 2, heavy chain CDR2 shown in SEQ ID NO: 3, and heavy chain CDR3 shown in SEQ ID NO: 4) and three light chain CDRs (light chain CDR1 shown in SEQ ID NO: 7, light chain CDR2 shown in SEQ ID NO: 8, and light chain CDR3 shown in SEQ ID NO: 9) as defined by the Kabat numbering system; or (b) three heavy chain CDRs (heavy chain CDR1 shown in SEQ ID NO: 13, heavy chain CDR2 shown in SEQ ID NO: 14, and heavy chain CDR3 shown in SEQ ID NO: 15) and three light chain CDRs (light chain CDR1 shown in SEQ ID NO: 16, light chain CDR2 shown in SEQ ID NO: 17, and light chain CDR3 shown in SEQ ID NO: 18) as defined by the IMGT numbering system. Additionally, in some embodiments, the antibody or its antigen-binding fragment comprises a human frame sequence. In some embodiments, the antibody or its antigen-binding fragment comprises the heavy chain variable domain shown in SEQ ID NO: 23 and the light chain variable domain shown in SEQ ID NO: 24. In some embodiments, the antibody or antigen-binding fragment comprises the human IgG1 heavy chain constant domain and the Igκ light chain constant domain. In some embodiments, the antibody or antigen-binding fragment competitively binds to and / or binds to the same epitope as an antibody comprising the heavy chain variable domain shown in SEQ ID NO: 23 and the light chain variable domain shown in SEQ ID NO: 24. In some embodiments, the antibody or its antigen-binding fragment binds to an epitope comprising alanine-histidine-lysine-aspartic acid (Ala-His-Lys-Asp, SEQ ID NO: 345) (Non-Patent Document 9). In some embodiments, the antibody or antigen-binding fragment binds to an epitope comprising NTSQEAHKDVSYL (asn-Thr-Ser-Gln-Glu-Ala-His-Lys-Asp-Val-Ser-Tyr-Leu, SEQ ID NO: 346).

[0047] In other embodiments, the antibody or its antigen-binding fragment binds to human epidermal growth factor receptor 2 (her2) and can target tumor cells expressing her2. In some embodiments, the antibody or its antigen-binding fragment may comprise: (a) three heavy chain CDRs (heavy chain CDR1 shown in SEQ ID NO: 71, heavy chain CDR2 shown in SEQ ID NO: 72, and heavy chain CDR3 shown in SEQ ID NO: 73) and three light chain CDRs (light chain CDR1 shown in SEQ ID NO: 74, light chain CDR2 shown in SEQ ID NO: 75, and light chain CDR3 shown in SEQ ID NO: 76) defined by the Kabat numbering system; or (b) three heavy chain CDRs (heavy chain CDR1 shown in SEQ ID NO: 191, heavy chain CDR2 shown in SEQ ID NO: 192, and heavy chain CDR3 shown in SEQ ID NO: 193) and three light chain CDRs (light chain CDR1 shown in SEQ ID NO: 194, light chain CDR2 shown in SEQ ID NO: 195, and light chain CDR3 shown in SEQ ID NO: 196). Furthermore, in some embodiments, the antibody or its antigen-binding fragment comprises a human frame sequence. In some embodiments, the antibody or its antigen-binding fragment comprises the heavy chain variable domain shown in SEQ ID NO: 27 and the light chain variable domain shown in SEQ ID NO: 28. In some embodiments, the antibody or antigen-binding fragment comprises the human IgG1 heavy chain constant domain and the Igκ light chain constant domain. In some embodiments, the antibody or antigen-binding fragment competitively binds to and / or binds to the same epitopes as an antibody comprising the heavy chain variable domain shown in SEQ ID NO: 27 and the light chain variable domain shown in SEQ ID NO: 28.

[0048] In other embodiments, the antibody or its antigen-binding fragment binds to mesothelin (MSLN) and can target tumor cells expressing MSLN. In some embodiments, the antibody or its antigen-binding fragment may comprise: (a) three heavy chain CDRs (heavy chain CDR1 shown in SEQ ID NO: 65, heavy chain CDR2 shown in SEQ ID NO: 66, and heavy chain CDR3 shown in SEQ ID NO: 67) and three light chain CDRs (light chain CDR1 shown in SEQ ID NO: 68, light chain CDR2 shown in SEQ ID NO: 69, and light chain CDR3 shown in SEQ ID NO: 70) as defined by the Kabat numbering system; or (b) three heavy chain CDRs (heavy chain CDR1 shown in SEQ ID NO: 185, heavy chain CDR2 shown in SEQ ID NO: 186, and heavy chain CDR3 shown in SEQ ID NO: 187) and three light chain CDRs (light chain CDR1 shown in SEQ ID NO: 188, light chain CDR2 shown in SEQ ID NO: 189, and light chain CDR3 shown in SEQ ID NO: 190) as defined by the IMGT numbering system. In some embodiments, the antibody or antigen-binding fragment comprises the heavy chain variable domain shown in SEQ ID NO: 25 and the light chain variable domain shown in SEQ ID NO: 26. In some embodiments, the antibody or antigen-binding fragment comprises the human IgG1 heavy chain constant domain and the Igκ light chain constant domain. In some embodiments, the antibody or antigen-binding fragment competitively binds to and / or binds to the same epitope as an antibody comprising the heavy chain variable domain shown in SEQ ID NO: 25 and the light chain variable domain shown in SEQ ID NO: 26.

[0049] In embodiments of the present invention, the preferred antibody moiety (Ab) is an anti-folate receptor α antibody or its antigen-binding fragment, comprising: (a) three heavy chain CDRs comprising the amino acid sequences shown in SEQ ID NO: 2 (heavy chain CDR1), SEQ ID NO: 3 (heavy chain CDR2), and SEQ ID NO: 4 (heavy chain CDR3) as defined by the Kabat numbering system, or three light chain CDRs comprising the amino acid sequences shown in SEQ ID NO: 7 (light chain CDR1), SEQ ID NO: 8 (light chain CDR2), and SEQ ID NO: 9 (light chain CDR3); or (b) three heavy chain CDRs comprising the amino acid sequences shown in SEQ ID NO: 13 (heavy chain CDR1), SEQ ID NO: 14 (heavy chain CDR2), and SEQ ID NO: 15 (heavy chain CDR3) as defined by the IMGT numbering system, or three light chain CDRs comprising SEQ ID NO: 16 (light chain CDR1), SEQ ID NO: 17 (light chain CDR2), and SEQ ID NO: 18 (light chain CDR3). The amino acid sequence shown in 18 (light chain CDR3) consists of three light chain CDRs. Another preferred antibody moiety (Ab) is an anti-human epidermal growth factor receptor 2 (her2) antibody or its antigen-binding fragment, comprising: (c) three heavy chain CDRs comprising the amino acid sequences shown in SEQ ID NO: 71 (heavy chain CDR1), SEQ ID NO: 72 (heavy chain CDR2), and SEQ ID NO: 73 (heavy chain CDR3) as defined by the Kabat numbering system, or three light chain CDRs comprising the amino acid sequences shown in SEQ ID NO: 74 (light chain CDR1), SEQ ID NO: 75 (light chain CDR2), and SEQ ID NO: 76 (light chain CDR3); or (d) three heavy chain CDRs comprising the amino acid sequences shown in SEQ ID NO: 191 (heavy chain CDR1), SEQ ID NO: 192 (heavy chain CDR2), and SEQ ID NO: 193 (heavy chain CDR3) as defined by the IMGT numbering system, or three light chain CDRs comprising SEQ ID NO: 194 (light chain CDR1), SEQ ID NO: The three light chain CDRs of the amino acid sequence shown in SEQ ID NO: 195 (light chain CDR2) and SEQ ID NO: 196 (light chain CDR3). More preferably, the antibody moiety (Ab) is an anti-folate receptor α antibody or its antigen-binding fragment.

[0050] The antibody portion (Ab) includes any antibody or antigen-binding fragment that specifically binds to a target antigen on a range of cancer cells. The antibody or antigen-binding fragment can bind to the target antigen, wherein, when measured by, for example, BIAcore (registered trade name) analysis, the dissociation constant (Ki) is... DK ≤ 1 mM, ≤ 100 nM, or ≤ 10 nM, or any amount in between. In some embodiments, K D The range is from 1 pM to 500 pM. In some embodiments, K D The range is 500 pM to 1 µM, 1 µM to 100 nm, or 100 mM to 10 nM.

[0051] In some embodiments, the antibody portion is a four-chain antibody (also known as an immunoglobulin) comprising two heavy chains and two light chains. In some embodiments, the antibody portion is a double-chain half-antibody of an immunoglobulin (one light chain and one heavy chain) or an antigen-binding fragment thereof.

[0052] Tables 1-9 clearly show the amino acid and nucleic acid sequences of exemplary antibodies according to this disclosure.

[0053] [Table 1]

[0054] Abbreviation: xi stands for chimeric antibody, and is the most humanized antibody.

[0055] The amino acid sequence of the mAb variable domain [Table 2]

[0056] Nucleic acid sequences encoding the variable domains of mAb [Table 3]

[0057] amino acid sequence of mAb Kabat CDR [Table 4]

[0058] [Table 4 (continued)]

[0059] Nucleic acid sequence encoding mAb Kabat CDR [Table 5]

[0060] [Table 5 (continued)]

[0061] amino acid sequence of mAb IMGT CDR [Table 6]

[0062] [Table 6 (continued)]

[0063] Nucleic acid sequence encoding mAb IMGT CDR [Table 7]

[0064] [Table 7 (continued)]

[0065] Amino acid sequence of the full-length mAb Ig chain [Table 8]

[0066] Nucleic acid sequence encoding the full-length mAb Ig chain [Table 9]

[0067] The exemplary sequence does not include a leader sequence.

[0068] An ADC may comprise any set of heavy and light chain variable domains listed in the table above (e.g., the MORAb-003 heavy and light chain variable domains, or the trastuzumab heavy and light chain variable domains), or a set of six CDR sequences from the heavy and light chain groups. In some embodiments, an ADC comprises human heavy and light chain constant domains or fragments thereof. For example, an ADC may comprise a human IgG heavy chain constant domain (e.g., IgG1) and a human κ or λ light chain constant domain. The antibody portion comprises the human immunoglobulin G subtype 1 (IgG1) heavy chain constant region together with the human Igκ light chain constant region.

[0069] The target cancer antigen of ADCs can be folate receptor α (FRA).

[0070] Anti-FRA antibodies or their antigen-binding fragments may contain three heavy chain CDRs (heavy chain CDR1 shown in SEQ ID NO: 2, heavy chain CDR2 shown in SEQ ID NO: 3, and heavy chain CDR3 shown in SEQ ID NO: 4) and three light chain CDRs (light chain CDR1 shown in SEQ ID NO: 7, light chain CDR2 shown in SEQ ID NO: 8, and light chain CDR3 shown in SEQ ID NO: 9) as defined by the Kabat numbering system.

[0071] In addition, the anti-FRA antibody or its antigen-binding fragment may contain three heavy chain CDRs (heavy chain CDR1 shown in SEQ ID NO: 13, heavy chain CDR2 shown in SEQ ID NO: 14, and heavy chain CDR3 shown in SEQ ID NO: 15) and three light chain CDRs (light chain CDR1 shown in SEQ ID NO: 16, light chain CDR2 shown in SEQ ID NO: 17, and light chain CDR3 shown in SEQ ID NO: 18) as defined by the IMGT numbering system.

[0072] In various embodiments, the anti-FRA antibody or its antigen-binding fragment comprises: a heavy chain variable domain comprising the amino acid sequence shown in SEQ ID NO: 23 and a light chain variable domain comprising the amino acid sequence shown in SEQ ID NO: 24; or a heavy chain variable domain comprising the amino acid sequence shown in SEQ ID NO: 1 and a light chain variable domain comprising the amino acid sequence shown in SEQ ID NO: 6. In some embodiments, the anti-FRA antibody or its antigen-binding fragment comprises the heavy chain variable domain amino acid sequence shown in SEQ ID NO: 23 and the light chain variable domain amino acid sequence shown in SEQ ID NO: 24, or comprises a sequence having at least 95% identity with the above sequences. In some embodiments, the anti-FRA antibody or its antigen-binding fragment comprises a heavy chain variable domain amino acid sequence having at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO: 23 and a light chain variable domain amino acid sequence having at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO: 24.

[0073] Anti-FRA antibodies may contain a human IgG1 heavy chain constant domain along with a human Igκ light chain constant domain.

[0074] The anti-FRA antibody comprises the heavy chain amino acid sequence shown in SEQ ID NO: 1 or a sequence having at least 95% identity with SEQ ID NO: 1, and the light chain amino acid sequence shown in SEQ ID NO: 6 or a sequence having at least 95% identity with SEQ ID NO: 6. In some embodiments, the antibody comprises the heavy chain amino acid sequence shown in SEQ ID NO: 1 and the light chain amino acid sequence shown in SEQ ID NO: 6, or comprises a sequence having at least 95% identity with the above sequences. In some embodiments, the anti-FRA antibody comprises a heavy chain amino acid sequence having at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO: 1 and / or a light chain amino acid sequence having at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO: 6. In some embodiments, the anti-FRA antibody comprises a heavy chain encoded by the nucleotide sequence shown in SEQ ID NO: 11 (containing a nucleotide encoding a leader sequence) or SEQ ID NO: 325 (not containing a nucleotide encoding a leader sequence) and a light chain encoded by the nucleotide sequence shown in SEQ ID NO: 12 (containing a nucleotide encoding a leader sequence) or SEQ ID NO: 326 (not containing a nucleotide encoding a leader sequence). In some embodiments, the heavy chain amino acid sequence lacks a C-terminal lysine. In various embodiments, the anti-FRA antibody has the amino acid sequence of an antibody produced by a cell line that lacks such a sequence as the C-terminal lysine of the heavy chain, and is deposited in accordance with the provisions of the Budapest Treaty on April 24, 2006 at the American Type Culture Collection (ATCC, 10801 University Blvd., Manassas, Va., 20110-2209), accession number PTA-7552. In various embodiments, the anti-FRA antibody is MORAb-003 (USAN name: farletuzumab) (Ebel et al. (2007) Cancer Immunity 7:6), or its antigen-binding fragment.

[0075] The target cancer antigen of ADCs can be human epidermal growth factor receptor 2 (her2).

[0076] In addition, the anti-her2 antibody or its antigen-binding fragment may contain three heavy chain CDRs (heavy chain CDR1 shown in SEQ ID NO: 71, heavy chain CDR2 shown in SEQ ID NO: 72 and heavy chain CDR3 shown in SEQ ID NO: 73) and three light chain CDRs (light chain CDR1 shown in SEQ ID NO: 74, light chain CDR2 shown in SEQ ID NO: 75 and light chain CDR3 shown in SEQ ID NO: 76) as defined by the Kabat numbering system.

[0077] In addition, the anti-her2 antibody or its antigen-binding fragment may contain three heavy chain CDRs (heavy chain CDR1 shown in SEQ ID NO: 191, heavy chain CDR2 shown in SEQ ID NO: 192 and heavy chain CDR3 shown in SEQ ID NO: 193) and three light chain CDRs (light chain CDR1 shown in SEQ ID NO: 194, light chain CDR2 shown in SEQ ID NO: 195 and light chain CDR3 shown in SEQ ID NO: 196) as defined by the IMGT numbering system.

[0078] In various embodiments, the anti-her2 antibody or its antigen-binding fragment comprises: a heavy chain variable domain comprising the amino acid sequence shown in SEQ ID NO: 27 and a light chain variable domain comprising the amino acid sequence shown in SEQ ID NO: 28; or a heavy chain domain comprising the amino acid sequence shown in SEQ ID NO: 347 and a light chain domain comprising the amino acid sequence shown in SEQ ID NO: 308. In some embodiments, the anti-her2 antibody or its antigen-binding fragment comprises the heavy chain variable domain amino acid sequence shown in SEQ ID NO: 27 and the light chain variable domain amino acid sequence shown in SEQ ID NO: 28, or comprises a sequence having at least 95% identity with the above sequences. In some embodiments, the anti-her2 antibody or its antigen-binding fragment comprises a heavy chain variable domain amino acid sequence having at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO: 27 and / or a light chain variable domain amino acid sequence having at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO: 28.

[0079] Antiher2 antibodies may contain a human IgG1 heavy chain constant domain and a human Igκ light chain constant domain.

[0080] In various embodiments, the anti-her2 antibody comprises the heavy chain amino acid sequence shown in SEQ ID NO: 307 or a sequence having at least 95% identity with SEQ ID NO: 307, and the light chain amino acid sequence shown in SEQ ID NO: 308 or a sequence having at least 95% identity with SEQ ID NO: 308. In some embodiments, the antibody comprises the heavy chain amino acid sequence shown in SEQ ID NO: 307 and the light chain amino acid sequence shown in SEQ ID NO: 308, or comprises a sequence having at least 95% identity with the above sequences. In some embodiments, the anti-her2 antibody comprises a heavy chain amino acid sequence having at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO: 307, and a light chain amino acid sequence having at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO: 308. In various embodiments, the anti-her2 antibody is trastuzumab or its antigen-binding fragment.

[0081] In various embodiments, the anti-FRA antibody or its antigen-binding fragment comprises three heavy chain CDRs and three light chain CDRs of MORAb-003, or comprises an amino acid sequence obtained by adding, deleting, or substituting one, two, three, four, five, or six amino acids on heavy chain CDR1 (SEQ ID NO: 2 or SEQ ID NO: 13 as defined in the Kabat system or SEQ ID NO: 13 as defined in the IMGT system), heavy chain CDR2 (SEQ ID NO: 3 or SEQ ID NO: 14 as defined in the Kabat system or SEQ ID NO: 14 as defined in the IMGT system), heavy chain CDR3 (SEQ ID NO: 4 or SEQ ID NO: 15 as defined in the Kabat system or SEQ ID NO: 16 as defined in the IMGT system), light chain CDR1 (SEQ ID NO: 8 or SEQ ID NO: 17 as defined in the Kabat system or SEQ ID NO: 18 as defined in the IMGT system), and light chain CDR3 (SEQ ID NO: 9 or SEQ ID NO: 18 as defined in the Kabat system).

[0082] In various other embodiments, the anti-her2 antibody or its antigen-binding fragment comprises three heavy chain CDRs and three light chain CDRs of trastuzumab, or comprises CDRs formed by the following heavy chain CDRs: SEQ ID NO: 71 (defined in the Kabat system or SEQ ID NO: 191) , SEQ ID NO: 72 (defined in the Kabat system or SEQ ID NO: 192) , SEQ ID NO: 73 (defined in the Kabat system or SEQ ID NO: 193) , SEQ ID NO: 74 (defined in the Kabat system or SEQ ID NO: 194) , SEQ ID NO: 75 (defined in the Kabat system or SEQ ID NO: 195) , and SEQ ID NO: 76 (defined in the Kabat system or SEQ ID NO: 193) . The amino acid sequence obtained by adding, deleting, or substituting one, two, three, four, five, or six amino acids in 196).

[0083] In various embodiments, amino acid substitutions are single-residue substitutions. Insertions are typically from about 1 to about 20 amino acid residues, but significantly larger insertions are permissible as long as biological function (e.g., binding to FRA or her2) is preserved. Deletions are typically from about 1 to about 20 amino acid residues, but in some cases, deletions may be much larger. Substitutions, deletions, insertions, or any combination thereof can be used to obtain the final derivative or variant. Generally, these changes are made to several amino acids to minimize alterations to the molecule, particularly to minimize alterations to the immunogenicity and specificity of the antigen-binding protein. However, in some cases, more changes are permissible. Conserved substitutions are generally performed according to the diagram shown in Table 10 below.

[0084] [Table 10]

[0085] Substantial changes in functional or immunological properties can be made by selecting substitutions with lower conservation than those shown in Table 10. For example, substitutions can more significantly affect: the structure of the polypeptide backbone in a region, such as α-helical or β-sheet structures; the charge or hydrophobicity of the molecule at the target site; or the volume of the side chains. Substitutions that typically produce the most significant changes in polypeptide properties are those shown in (a) through (d): (a) Hydrophilic amino acid residues (e.g., Ser or Thr) are replaced by hydrophobic amino acid residues (e.g., Leu, Ile, Phe, Val or Ala); (b) Cys or Pro is substituted by any other residue; (c) Amino acid residues with positively charged side chains (e.g., Lys, Arg, or His) are replaced by negatively charged amino acid residues (e.g., Gln or Asn); and (d) Residues with large side chains (e.g., Phe) are replaced by amino acids without side chains (e.g., Gly).

[0086] In the various embodiments in which variant antibody sequences are used in ADCs, the variants typically exhibit the same qualitative biological activity and elicit the same immune response, but variants may also be selected as needed to alter the characteristics of the antigen-binding protein. Alternatively, variants may be designed to alter the biological activity of the antigen-binding protein. For example, as discussed herein, glycosylation sites may be modified or removed.

[0087] In the ADCs according to embodiments of the present invention, various antibodies can be used to target cancer cells. Suitable antigens expressed on tumor cells rather than healthy cells, or expressed on tumor cells at higher levels than on healthy cells, and antibodies against such antigens are known in the art. These antibodies can be used in conjunction with the adapters and eribulin disclosed herein.

[0088] The antibody portion of the ADC may be an antibody portion targeting FRA, such as MORAb-003. In some embodiments, the linker and eribulin according to this disclosure can surprisingly be effective on several different tumor-targeting antibodies. When the antibody portion of the ADC is an antibody portion targeting FRA, such as MORAb-003, the ADC can deliver significant improvements in antibody ratio, tumor targeting, bystander effect, and therapeutic efficacy, as well as a reduction in off-target killing. Improvements in therapeutic efficacy can be measured in vitro or in vivo and may include a reduction in tumor growth rate and / or tumor volume.

[0089] When the antibody moiety in an ADC is a HER2-targeting antibody moiety, such as trastuzumab, some or all of its beneficial functional properties are observed. Furthermore, the antibody moiety in an ADC can be a HER2-targeting antibody moiety, such as trastuzumab. When the antibody moiety in an ADC is an MSLN-targeting antibody moiety, such as MORAb-009, some or all of its beneficial functional properties are observed.

[0090] In some embodiments, free cysteine ​​residues are introduced into the amino acid sequence of the antibody moiety. For example, antibodies in which one or more amino acids in the amino acid sequence of the parent antibody are replaced by cysteine ​​(cysteine-modified antibodies) can be prepared. For example, a cysteine-modified Fab antibody (also known as “ThioFab”) can be formed by introducing cysteine ​​into a fragment of a parent FAb antibody. Similarly, a cysteine-modified monoclonal antibody (also known as “ThioMab”) can be formed by introducing cysteine ​​into a parent monoclonal antibody. A single point mutation in ThioFab produces a single modified cysteine ​​residue, while a single point mutation in ThioMab produces two modified cysteine ​​residues due to the dimer nature of IgG antibodies. The DNA encoding the amino acid sequence variant of the parent polypeptide can be prepared by various methods known in the art (see, for example, the method described in WO 2006 / 034488). These methods include, but are not limited to, preparation by site-directed (or oligonucleotide-mediated) mutagenesis, PCR mutagenesis, and cassette mutagenesis of DNA encoding the polypeptide previously prepared. Variants of recombinant antibodies can also be constructed via restriction fragment manipulation or by using overlapping extension PCR with synthetic oligonucleotides. The ADCs shown in Formula I include, but are not limited to, antibodies having one, two, three, or four modified cysteine ​​residues (Lyon et al. (2012) Methods Enzymol. [Enzymological Methods] 502:123-38). In some embodiments, when one or more free cysteine ​​residues are already present in the antibody moiety, the existing free cysteine ​​residues can be used to conjugate the antibody moiety with eribulin instead of modifying it.

[0091] 2. Connector section The linker portion of this invention has the following chemical structure and is composed of five units, which, starting from the side adjacent to the antibody portion, include a maleimide unit (Mal), an ethylene oxide unit (PEG), a propionic acid unit (PA), an amino acid unit (Val-Cit), and a self-ablating unit (pAB: p-aminobenzyloxycarbonyl).

[0092] The linker portion is stable outside the cells to ensure adequate therapeutic efficacy. In some embodiments, the linker portion is stable outside the cells such that the ADC remains intact when present in extracellular conditions (e.g., prior to transport or delivery to cells). The term “intact” as used in the context of ADC means that the antibody portion remains bound to eribulin. As used herein, the term “stable” in the context of ADC means that less than 20%, less than about 15%, less than about 10%, less than about 5%, less than about 3%, or less than about 1% (or any percentage between) of the linker in the ADC sample is cleaved when the ADC is present in extracellular conditions (or in cases where the overall ADC is otherwise incomplete).

[0093] Extracellular stability of the linker moiety can be determined, for example, by containing the ADC in plasma for a predetermined time period (e.g., 2, 4, 6, 8, 16, or 24 hours) and subsequently quantifying the amount of free drug fraction present in the plasma. Stability allows the ADC time to localize to target cancer cells and prevents premature drug release, which could reduce the therapeutic index of the ADC by indiscriminately damaging both normal and cancerous tissues. The linker moiety is stable outside the target cells and releases eribulin from the intracellular ADC, allowing eribulin to bind to its target (e.g., to microtubules). Therefore, the linker moiety according to embodiments of the invention: (i) maintains the specific binding properties of the antibody fraction; (ii) allows delivery of eribulin via stable binding to the antibody fraction, e.g., intracellular delivery; (iii) remains stable and intact until the ADC has been transported or delivered to the target site; and (iv) allows the therapeutic effects of eribulin after lysis, e.g., cytotoxic effects.

[0094] The linker can cleave under intracellular conditions, such that cleavage of the linker fully releases eribulin from the antibody moiety in the intracellular environment to activate eribulin and / or make the drug therapeutically effective. In some embodiments, eribulin is not cleaved from the antibody moiety until the ADC enters a cell expressing an antigen specific to the ADC antibody moiety, and eribulin is not cleaved from the antibody moiety after entering the cell. In some embodiments, the linker includes a cleavable portion positioned such that after cleavage, neither the linker nor any portion of the antibody moiety retains binding to eribulin.

[0095] The linker portion comprises the amino acid unit of valine-citrulline (Val-Cit). This amino acid unit can be cleaved by lysins (e.g., intracellular peptidases or proteases) present in the intracellular environment (e.g., in lysosomes, endosomes, or caveoleas of the cell membrane). Peptidases such as cathepsins (e.g., cathepsins B, C, F, H, K, L, O, S, V, X, or W) can cleave the valine-citrulline (Val-Cit) sequence or the alanine-alanine-asparagine (Ala-Ala-Asn) sequence (Dubowchik et al. (2002) Bioconjugate Chem. [Bioconjugate Chemistry] 13: 855-69). Enzymatic cleavage of the linker portion in target cells allows the ADC to separate into the antibody side and the eribulin side. Compared to ADCs containing alternative amino acid units (e.g., Gly-Gly), ADCs containing Val-Cit exhibit increased stability, reduced off-target cell killing, increased on-target cell killing, decreased aggregation levels, and / or increased drug loading.

[0096] Citrulline and eribulin are linked via a p-aminobenzyloxycarbonyl group (pAB). Specifically, the amino group of the p-aminobenzyloxycarbonyl group forms an amide bond with citrulline, and the carbonyl group is covalently bonded to the primary amine (C-35 amine) on eribulin. The p-aminobenzyloxycarbonyl group is self-degrading. Unbound by theory, it is thought that the self-degradation of pAB involves a spontaneous 1,6-elimination reaction (Jain et al. (2015) Pharm Res. [Pharmaceutical Research] 32:3526-40). When the linker portion in the ADC (e.g., Val-Cit) cleaves in the target cell due to the self-degradation of the self-degrading unit (pAB), unmodified eribulin can be released without retaining excess functional groups on the eribulin side.

[0097] Self-ablation chemistry is known in the art and can be readily selected for use in ADCs according to this disclosure. In various embodiments, the spacer subunit that combines the cleavable portion in the connector with the drug portion (e.g., eribulin) is self-ablative and undergoes self-ablation under intracellular conditions simultaneously with or shortly before / after the cleavage of the cleavable portion.

[0098] On the other hand, maleimide units (Mal), ethylene oxide units (PEG), and propionic acid units (PA) are arranged between valine and the antibody Ab. The α,β-unsaturated carbonyl group in the maleimide is reactive with the cysteine ​​residues (specifically, the thiol group (-SH)) in the antibody and functions to bind the linker moiety and eribulin to the antibody. The linker moiety contains the maleimide unit and is linked to the antibody via this maleimide unit. As a result, the antibody drug loading can be further improved (p: the number of eribulin moieties on each antibody moiety).

[0099] Ethylene oxide units (PEGs) are units composed of one or more ethylene oxide groups and are substantially hydrophilic. Eribulin can be used to reduce the extent to which eribulin may be pumped out of drug-resistant cancer cells via multidrug resistance (MDR) transporters or functionally similar transporters. In some embodiments, the linker portion is a shorter PEG linker, which provides improved stability and reduced aggregation compared to longer PEG linkers. In Formula (I), the number of ethylene oxide groups is represented by m, and m is an integer from 1 to 10. m is preferably 2. When m is an integer less than 2, the length of the linker portion is reduced, and lower aggregation levels and / or higher drug loading can be exhibited compared to ADCs containing longer linker portions (e.g., m = 8).

[0100] In some embodiments, ADCs containing cleavable peptide moieties exhibit lower aggregation levels, improved antibody:drug ratios, increased on-target killing of cancer cells, reduced off-target killing of non-cancer cells, and / or increased drug loading (p) compared to ADCs containing alternative cleavable moieties. In some embodiments, the addition of a cleavable moieties increases cytotoxicity and / or potency compared to non-cleavable linkers. In some embodiments, increased potency and / or cytotoxicity can be observed in cancers expressing moderate levels of antigens targeted by the ADC antibody moieties (e.g., moderate FRA expression). In some embodiments, the cleavable peptide moieties are enzymatically cleavable, and the linker is an enzymatically cleavable linker. In some embodiments, the enzyme is a cathepsin, and the linker is a cathepsin-cleavable linker. In some embodiments, an enzymatically cleavable linker (e.g., a cathepsin-cleavable linker) exhibits one or more of the aforementioned improved properties compared to alternative cleavage mechanisms.

[0101] Furthermore, the linker portion can affect the physicochemical properties of the ADC. Since many cytotoxic agents are inherently hydrophobic, linking them to antibodies with additional hydrophobic portions can cause aggregation. ADC aggregates are insoluble and typically limit the achievable drug loading on the antibody, which can adversely affect the ADC's potency. Typically, protein aggregates of biologics are also associated with increased immunogenicity. The linker according to embodiments of the present invention yields an ADC with low aggregation levels and the desired drug loading levels.

[0102] In various embodiments, the adapter is designed to promote cell internalization, post-internalization lysis, and bypass killing (killing of neighboring cells) by diffusion to neighboring cells with adapter-eribulin and / or eribulin alone. In some embodiments, the adapter is designed to minimize lysis in the extracellular environment, thereby reducing toxicity to off-target tissues (e.g., non-cancerous tissues) while maintaining ADC binding to target tissues and bypass killing of cancerous tissues surrounding target cancer tissues that do not express the antigen targeted by the ADC antibody portion. The adapter portion according to embodiments of the invention is particularly effective in providing these functional features, for example, when conjugating an anti-FRA antibody portion such as MORAb-003 with a pharmaceutical portion such as eribulin. In some embodiments, at least some of these functional features may also be observed in the absence of the anti-FRA antibody portion and / or the absence of anti-MORAb-003. The adapter portion according to embodiments of the invention can effectively provide some or all of these functional features, for example, when conjugating an anti-her2 antibody portion such as trastuzumab with a pharmaceutical portion such as eribulin.

[0103] ADCs have been found to exhibit specific combinations of desired properties, particularly when paired with anti-FRA antibodies such as MORAb-003 or its antigen-binding fragment. These properties include, but are not limited to, effective drug loading levels (p ≥ about 4), low aggregation levels, stability under storage conditions or in vivo circulation (e.g., serum stability), maintaining affinity for target-expressing cells comparable to unconjugated antibodies, strong cytotoxicity against target-expressing cells, low off-target cell killing levels, high bystander effect levels, and / or effective in vivo anticancer activity, all of which are compared to ADCs using other linker-eribulin and / or antibody moieties.

[0104] 3. Eriblin As used herein, the term "eribrin" refers to a synthetic analogue of spongin B, which is derived from the marine sponge *Spongyoma okadaense* (…). Halichondria okadais The isolated macrocyclic compound is eribulin. Eribulin is a microtubule dynamics inhibitor, believed to bind to tubulin and induce cell cycle arrest at the G2 / M phase by inhibiting the mitotic spindle assembly. The term "eribulin mesylate" refers to the mesylate of eribulin, marketed under the trade name Halaven (trademark). In the ADC according to embodiments of the present invention, eribulin binds to the ADC linker via the primary amino group of eribulin.

[0105] 4. Antibody-drug conjugates (ADCs) In the ADC according to embodiments of the present invention, eribulin binds to the antibody portion via the aforementioned linker portion. The antibody portion may be, for example, an anti-FRA antibody, an anti-mesothelin antibody, or an anti-HER2 antibody such as trastuzumab.

[0106] An ADC according to embodiments of the present invention can selectively deliver an effective dose of a cytotoxic agent or a cell growth inhibitor to cancer cells or to tumor tissue. ADCs have been found to have strong cytotoxic activity and / or cell growth inhibitory activity against cells expressing a corresponding target antigen (e.g., FRA or her2). In some embodiments, the cytotoxic activity and / or cell growth inhibitory activity of the ADC depends on the expression level of the target antigen in the cells. In some embodiments, the ADC according to this disclosure can kill cancer cells expressing intermediate levels of the target antigen particularly effectively compared to cancer cells expressing low levels of the same antigen.

[0107] The term "cancer" refers to a physiological condition in mammals characterized by uncontrolled cell growth in a population of cells. Examples of cancer include, but are not limited to, carcinoma, lymphoma, germ cell tumor, sarcoma, and leukemia. More specific examples of such cancers include squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, squamous cell carcinoma of the lung, peritoneal carcinoma, hepatocellular carcinoma, gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatocellular carcinoma, breast cancer (e.g., triple-negative breast cancer), osteosarcoma, melanoma, colon cancer, colorectal cancer, endometrial (e.g., serous) or uterine cancer, salivary gland cancer, kidney cancer, liver cancer, prostate cancer, vulvar cancer, thyroid cancer, hepatic carcinoma, and various types of head and neck cancer. Triple-negative breast cancer is breast cancer that is negative for the expression of genes for estrogen receptor (ER), progesterone receptor (PR), or Her2 / neu.

[0108] The term "tumor" refers to any benign or malignant mass of tissue caused by excessive cell growth or proliferation, including precancerous lesions.

[0109] The terms “cancer cell” and “tumor cell” refer to a single cell or a population of cells derived from a tumor, including non-tumorigenic cells and cancer stem cells. When referring only to tumor cells that lack the capacity for renewal and differentiation, the term “tumor cell” as used herein is modified by the term “non-tumorigenic” to distinguish those tumor cells from cancer stem cells.

[0110] Exemplary cancers with high FRA expression include, but are not limited to, ovarian cancer (e.g., serous ovarian cancer, clear cell ovarian cancer), lung carcinoid, triple-negative breast cancer, endometrial cancer, and non-small cell lung cancer (e.g., adenocarcinoma). Exemplary cancers with moderate FRA expression include, but are not limited to, gastric cancer and colorectal cancer. Exemplary cancers with low FRA expression include, but are not limited to, melanoma and lymphoma. Exemplary cancers with high her2 expression include, but are not limited to, breast cancer, gastric cancer, esophageal cancer, ovarian cancer, and endometrial cancer. Exemplary cancers with moderate her2 expression include, but are not limited to, lung cancer and bladder cancer.

[0111] As used herein, the term “inhibit or inhibition of” means a reduction in a measurable amount and may include, but does not require, complete prevention or inhibition.

[0112] As disclosed herein, an “effective amount” of an ADC is an amount sufficient to achieve the stated purpose (e.g., to produce a therapeutic effect upon administration, such as reducing tumor growth rate or tumor volume, reducing cancer symptoms, or certain other indicators of therapeutic efficacy). An effective amount can be determined in a conventional manner relevant to the stated purpose. The term “therapeutic effective amount” refers to the amount of ADC that effectively treats a subject’s disease or disorder. In the case of cancer, a therapeutically effective amount of ADC may reduce the number of cancer cells, reduce tumor size, inhibit (e.g., slow or stop) tumor metastasis, inhibit (e.g., slow or stop) tumor growth, and / or alleviate one or more symptoms. A “preventive effective amount” refers to an effective amount that achieves the desired preventive effect within the necessary dose and time period. Typically, because the preventive dose is administered to the subject before or at an early stage of the disease, the preventive effective amount will be less than the therapeutic effective amount.

[0113] As used herein, the terms "treatment" or "therapeutic" and grammatically related terms refer to any improvement in any outcome of a disease, such as prolonged survival, lower morbidity, and / or reduced side effects caused by alternative treatment modalities. As is readily understood in the art, complete eradication is preferred but not required for therapeutic actions. As used herein, "treatment" means administering the ADC to a subject, such as a patient. Treatment can be curative, healing, relief, mitigation, alteration, remedy, improvement, amelioration, modification, or influence of a disorder (e.g., cancer), its symptoms, or a predisposition to the disorder.

[0114] In some embodiments, labeled ADCs are used. Suitable “labels” include radionuclides, enzymes, acceptors, cofactors, inhibitors, fluorescent portions, chemiluminescent portions, magnetic particles, etc.

[0115] As used herein, “protein” means at least two covalently linked amino acids. The term encompasses polypeptides, oligopeptides, and peptides. In some embodiments, two or more covalently linked amino acids are linked via peptide bonds. Proteins can be composed of naturally occurring amino acids and peptide bonds, for example, when proteins are recombinantly produced using an expression system and host cells. Alternatively, proteins may comprise synthetic amino acids (e.g., homophenylalanine, citrulline, ornithine, and oroleucine) or peptide-mimicking structures (i.e., “peptide or protein analogs,” such as peptide-like structures). Peptide-like structures are a class of exemplary peptide mimics whose side chains are attached to the nitrogen atom of the peptide backbone rather than to the α-carbon (as is the case with amino acids), and which have different hydrogen bonding and conformational characteristics compared to peptides (see, for example, Simon et al. (1992) Proc Natl Acad Sci. USA [Proceedings of the National Academy of Sciences] 89:9367). Therefore, peptide-like structures may be resistant to proteolysis or other physiological or storage conditions and can efficiently penetrate cell membranes. Such synthetic amino acids can be incorporated, especially when antibodies are synthesized in vitro using conventional methods well known in the art. Furthermore, any combination of peptide-mimicking, synthetic, and naturally occurring residues / structures can be used. "Amino acid" also includes imino acid residues such as proline and hydroxyproline. The amino acid "R group" or "side chain" can be in (L)- or (S)-configuration. In specific embodiments, the amino acid is in (L)- or (S)-configuration.

[0116] "Recombinant protein" is a protein produced using recombinant technology, using any technology and method known in the art, i.e., a protein produced by expressing recombinant nucleic acids. Methods and techniques for producing recombinant proteins are well known in the art.

[0117] The “isolated” protein is not accompanied by at least one material with which it normally associates in its native state, for example, comprising at least about 5% by weight or at least about 50% by weight of the total protein in a given sample. It should be understood that, depending on the circumstances, the isolated protein may comprise from 5% by weight to 99.9% by weight of the total protein content. For example, the protein can be produced at a significantly higher concentration using an inducible promoter or a highly expressed promoter, thereby enabling the production of the protein at increased concentration levels. This definition includes the production of antibodies in a wide variety of organisms and / or host cells known in the art.

[0118] For amino acid sequences, sequence identity and / or similarity can be determined using standard techniques known in the art, including but not limited to the local sequence identity algorithm of Smith and Waterman (1981) Adv. Appl. Math. 2:482, the sequence identity alignment algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443, the similarity method retrieval of Pearson and Lipman (1988) Proc. Nat. Acad. Sci. USA 85:2444, computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package of the Genetics Computer Group at 575 Science Drive, Madison, Wisconsin), and by Devereux et al. (1984). The best-fit sequence procedure described in Nucl. Acid Res. 12:387-95 is preferred, using the default settings or by validation. The percentage of agreement can be calculated with reference to “Current Methods in Sequence Comparison and Analysis”, Macromolecule Sequencing and Synthesis, Selected Methods and Applications, pp. 127-149 (1988), Alan R. Liss, Inc., or preferably calculated using the Fast DB database based on the following parameters.

[0119] Mismatch penalty: 1 Open shot penalty: 1 Vacancy penalty: 0.33 Engagement Penalty: 30 An example of an applicable algorithm is PILEUP. PILEUP uses progressive pairwise alignment to generate multiple sequence alignments from a set of related sequences. It can also draw a tree diagram showing the clustering relationships used to generate the alignments. PILEUP uses a simplified form of the progressive alignment method described by Feng and Doolittle (1987) J. Mol. Evol. [Journal of Molecular Evolution] 35:351-60; this method is similar to that described by Higgins and Sharp (1989) CABIOS 5:151-3. Applicable PILEUP parameters include a preset gap weight of 3.00, a preset gap length weight of 0.10, and weighted end gaps.

[0120] Another example of a suitable algorithm is the BLAST algorithm described below: Altschul et al. (1990) J. Mol. Biol. [Journal of Molecular Biology] 215:403-10; Altschul et al. (1997) Nucleic Acids Res. [Nucleic Acid Research] 25:3389-402; and Karin et al. (1993) Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences of the United States of America] 90:5873-87. A particularly suitable BLAST program is the WU-BLAST-2 program obtained from Altschul et al. (1996) Methods in Enzymology [Enzymological Methods] 266:460-80. WU-BLAST-2 uses several search parameters, most of which are set to default values. The adjustable parameters are set with the following values: overlap interval = I, overlap score = 0.125, and word threshold (T) = II. The HSP S and HSP S2 parameters are dynamic values ​​and are determined by the program itself based on the composition of the specific sequence and the composition of the specific database against which the target sequence is compared; however, these values ​​can be adjusted to improve sensitivity.

[0121] An additional applicable algorithm is the gapped BLAST reported by Altschul et al. (1993) Nucl. Acids Res. 25:3389-402. The gapped BLAST uses BLOSUM-62 instead of a scoring system; the threshold parameter T is set to 9; a two-hit method is used to trigger the gapless extension, adding a gap length k at a cost of 10 + k; Xu is set to 16, and Xg is set to 40 during the database retrieval phase and 67 during the algorithm output phase. Gap alignment is triggered by a scoring system corresponding to approximately 22.

[0122] Typically, the proteins and their variants disclosed herein (including variants of FRA, variants of her2, variants of the tubulin sequence, and variants of antibody variable domains (including single variants CDR)) share at least 80% amino acid homology, similarity, or identity with the sequences described herein, and more typically preferably have at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, almost 100%, or 100% increased homology or identity.

[0123] Similarly, the "percentage of nucleic acid sequence identity (%)" for nucleic acid sequences of antibodies and other proteins identified herein is defined as the percentage of nucleotide residues in the candidate sequence that are identical to nucleotide residues in the coding sequence of the antigen-binding protein. Specifically, the method utilizes the BLASTN module of WU-BLAST-2 with default parameters set, where the overlap interval and overlap fraction are set to 1 and 0.125, respectively.

[0124] Although the sites or regions where amino acid sequence changes are introduced are predetermined, the mutation itself does not need to be predetermined. For example, to optimize the efficacy of mutations at a given site, the best combination of random mutagenesis at target codons or regions and screening for the desired activity of expressed antigen-binding protein CDR variants can be achieved. Techniques for substitution mutations at predetermined sites in DNA with known sequences are well-known, such as MI3 primer mutagenesis and PCR mutagenesis.

[0125] Next, a method for preparing the ADC shown in formula (I) according to an embodiment of the present invention will be described.

[0126] The present invention provides a method for preparing the antibody-drug conjugate (ADC) shown in formula (I), and includes steps 1 and 2.

[0127] In this formula, Ab represents an antibody or its antigen-binding fragment. D stands for Eriblin. m is an integer from 1 to 10, and p is an integer from 1 to 8. Step 1: The step of obtaining the compound shown in formula (B) by reacting eribrin or its salt with the compound shown in formula (A).

[0128] [In this formula, m is an integer from 1 to 10, and X is phenoxy or nitrophenoxy.] [In this formula, m is an integer from 1 to 10.] Step 2: The step of obtaining the antibody-drug conjugate shown in formula (I) by reacting the compound shown in formula (B) with Ab.

[0129] Step 1 is the step of obtaining the compound shown in formula (B) by reacting eribulin or its salt with the compound shown in formula (A).

[0130] Eribulin may be in its free form or as a salt of eribulin. Examples of salts of eribulin include eribulin methanesulfonate. In the case of free eribulin, the amount of eribulin or its salt used may be from 1 g to 1000 g, or from 10 g to 300 g.

[0131] The compound shown in formula (A) corresponds to the linker portion of an ADC, with its terminal maleimide structure reactive to the antibody and its other terminal X reactive to eribulin. X is phenoxy or nitrophenoxy. Nitrophenoxy can be any of ortho-nitrophenoxy, m-nitrophenoxy, and p-nitrophenoxy. When X is phenoxy or nitrophenoxy, X is reactive to the primary amino group at the eribulin terminal. The compound shown in formula (A) can be prepared using the method described below.

[0132] The amount of the compound represented by formula (A) used relative to 1 mol of eribulin can be from 0.5 mol to 2.0 mol, 0.6 mol to 3.0 mol, 1.0 mol to 2.0 mol, or 1.3 mol to 1.8 mol.

[0133] In step 1, a base may be used. The base is not limited, as long as it does not inhibit the reaction of eribulin with the compound shown in formula (A), and examples of bases include tertiary amines (such as triethylamine or N,N-diisopropylethylamine) and nitrogen-containing aromatic compounds (such as pyridine or 2,6-dimethylpyridine). The amount of base used relative to 1 mol of eribulin may be from 0.5 mol to 3.0 mol, 1.0 mol to 2.0 mol, or 1.1 mol to 1.5 mol.

[0134] Step 1 can be carried out in the absence of a solvent or in a solvent, and is preferably carried out in a solvent. The solvent is not limited, as long as it does not inhibit the reaction of eribulin with the compound shown in formula (A), and examples of solvents include N,N-dimethylformamide (DMF), dimethyl sulfoxide (DMSO), N-methylpyrrolidone (NMP), and pyridine. The amount of solvent used relative to 1 g of eribulin can be from 5 mL to 20 mL or from 6 mL to 15 mL.

[0135] The reaction temperature in step 1 is not limited, as long as it is the temperature at which the reaction of eribulin with the compound shown in formula (A) takes place. The reaction temperature can be normal temperature or 20°C to 25°C.

[0136] To promote the reaction in step 1, a promoter such as 4-dimethylaminopyridine can be used. The amount of promoter used relative to 1 mol eribulin can be from 0.01 g to 0.8 g or from 0.1 g to 0.3 g.

[0137] An example of a purification method will be described below. However, the purification method is not limited to this example, and purification can be performed using methods known in chemistry. When the method of step 1 is complete, the reaction mixture can be purified on a reversed-phase column. A reversed-phase column is, for example, a column in which silica gel with octadecyl (C18) groups is used as the packing material. The reaction mixture is placed on top of the reversed-phase column and extracted with a polar solvent such as acetonitrile or water. Optionally, preferably, acetic acid or the like is used to make the pH of the extraction solvent acidic. Dichloromethane is added to the obtained extract, and the compound represented by formula (B) is conveyed to a dichloromethane layer (organic layer). During extraction, optionally, the pH can be adjusted with an aqueous sodium bicarbonate solution. The obtained organic layer can be neutralized with the remaining acid, such as an aqueous sodium bicarbonate solution. The obtained organic layer is concentrated under reduced pressure to dissolve the residue in dichloromethane, methanol, or the like, and the solution is added dropwise to pentane. As a result, the compound represented by formula (B) precipitates. The obtained solid is washed with pentane and dried under reduced pressure. As a result, the desired compound is obtained.

[0138] Step 2 involves reacting the compound of formula (B) with Ab to obtain the antibody-drug conjugate of formula (I). The cysteine ​​residues in the antibody moiety (Ab) undergo Michael addition with the maleimide structure of the compound of formula (B), thereby forming a covalent bond therebetween. Multiple compounds of formula (B) can bind to one antibody moiety Ab. The number of bonds formed corresponds to p in formula (A).

[0139] For the antibody portion Ab, the amount of the compound represented by formula (B) used may be from 1.0 mol to 8.0 mol or from 3.0 mol to 5.0 mol.

[0140] Typically, step 2 is performed in a buffer solution. The buffer solution is not limited, as long as it does not alter the antibody Ab to be used and does not inhibit binding to the compound shown in formula (B). The buffer solution may be a phosphate buffer solution, a borate buffer solution, a tris(hydroxymethyl)aminomethane buffer solution, an ethylenediaminetetraacetic acid buffer solution, or any combination thereof. The pH of the buffer solution is preferably from 6.0 to 8.0, and more preferably from 6.5 to 7.5.

[0141] In step 2, a polar organic solvent may be added to the buffer solution. The polar organic solvent is not limited, as long as it is miscible with the buffer solution and improves the solubility of the compound shown in formula (B). For example, a solution of the compound shown in formula (B) dissolved in a polar organic solvent may be added to a buffer solution in which antibody Ab has been dissolved.

[0142] Examples of polar organic solvents include N,N-dimethylformamide, N,N-dimethylacetamide, and dimethyl sulfoxide. The amount of organic solvent used relative to 1 mg of the compound shown in formula (B) can be 60 mL to 300 mL, 110 mL to 240 mL, 130 mL to 210 mL, or 140 mL to 180 mL.

[0143] The reaction temperature in step 2 is not limited, as long as it is the temperature at which the compound shown in formula (B) reacts with the antibody Ab. The reaction temperature can be the normal temperature or 20°C to 25°C.

[0144] More specifically, the reaction temperature is as follows. First, the antibody Ab is filtered through a filter (permeable membrane) and dissolved by adding a buffer solution. Partial reduction is performed by adding TCEP-HCl (tris(2-carboxyethyl)phosphonic acid hydrochloride) to the resulting solution, cleaving the disulfide bonds in the antibody molecule to form thiol groups. Next, a compound of formula (B) or a solution of the compound of formula (B) dissolved in a polar organic solvent is added to allow the compound of formula (B) to bind to the antibody. When the reaction in step 2 is complete, for example, N-acetylcysteine ​​may be added. N-acetylcysteine ​​is reacted with the remaining compound of formula (B) and also with the thiol groups formed through partial reduction.

[0145] An example of the purification method will be described below. However, the purification method is not limited to this example, and purification can be performed using methods known in chemistry. After the reaction is complete, the buffer solution of the reaction solution is replaced with another buffer solution using a tangential flow filtration (TFF) method. Next, an aqueous solution of citric acid is added, filtered through a filter, and purified with a buffer solution.

[0146] Here, a method for preparing the compound shown in formula (A) will be described. The compound shown in formula (A) can be prepared, for example, from the compound shown in formula (1) and the compound shown in formula (2) by the two steps described below.

[0147] Step 3 is the step of activating the carboxyl group of the compound shown in formula (1) and condensing the compound shown in formula (1) with the compound shown in formula (2). The compound shown in formula (3) is obtained by condensing the compound shown in formula (1) and the compound shown in formula (2).

[0148] The compound shown in formula (1) is available from Tokyo Chemical Industry Co., Ltd. under the trade name "Mal-PEG2-acid" (catalog code: M3203). Furthermore, the compound shown in formula (1) can be prepared using methods known in organic chemistry, employing tert-butyl acrylate and its analogues with polyethylene glycol of appropriate length as starting materials.

[0149] The amount of the compound represented by formula (1) used may be 1.0 mol to 1.5 mol or 1.1 mol to 2.0 mol relative to 1 mol of the compound represented by formula (2).

[0150] The compound shown in formula (2) is available from Tokyo Chemical Industry Co., Ltd., under the trade name "Fmoc-Val-Cit-PAB-OH" (catalog code: F1223). Furthermore, the compound shown in formula (2) can be prepared using methods known in organic chemistry, employing N-Fmoc-citrulline, p-aminobenzyl alcohol, and N-Fmoc-valine. "Fmoc" is an amino protecting group, which is 9-fluorenylmethoxycarbonyl.

[0151] Preferably, step 3 is carried out in the presence of a condensing agent. The condensing agent is not limited, as long as it can accelerate the condensation reaction of the carboxylic acid with the primary amine, and examples of condensing agents include dichlorohexylcarbodiimide (DCC), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, and 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM).

[0152] The amount of condensing agent used relative to 1 mol of the compound shown in formula (1) can be from 1.0 mol to 2.0 mol or from 1.3 mol to 1.5 mol.

[0153] In step 3, a base may be used. The base is not limited, as long as it does not inhibit the reaction between the compound shown in formula (1) and the compound shown in formula (2), and examples of bases include tertiary amines (such as triethylamine or N,N-diisopropylethylamine) and nitrogen-containing aromatic compounds (such as pyridine or 2,6-dimethylpyridine). The amount of base used relative to 1 mol of the compound shown in formula (1) may be from 0.5 mol to 2.0 mol, 1.0 mol to 1.5 mol, or 1.1 mol to 1.3 mol.

[0154] Step 3 can be carried out in the absence of a solvent or in a solvent, and preferably in a solvent. The solvent is not limited, as long as it does not inhibit the reaction of the compound shown in formula (1) with the compound shown in formula (2), and examples of solvents include N,N-dimethylformamide (DMF), tetrahydrofuran (THF), methanol (MeOH), and mixtures thereof. The amount of solvent used relative to 1 g of the compound shown in formula (1) can be from 10 mL to 40 mL or from 20 mL to 30 mL.

[0155] The reaction temperature in step 3 is not limited, as long as it is the temperature at which the reaction between the compound shown in formula (1) and the compound shown in formula (2) takes place. The reaction temperature can be the normal temperature or between 15°C and 35°C.

[0156] Step 4 is the step of acylation of the hydroxyl groups in the compound of formula (3) by using an acylation agent to obtain the compound of formula (A).

[0157] The amount of the compound represented by formula (3) used can be appropriately selected according to the reaction. For example, the amount of the compound represented by formula (3) used can be from 10 g to 500 g or from 100 g to 300 g.

[0158] Examples of acylating agents include diphenyl carbonate, di(4-nitrophenyl) carbonate, di(2-nitrophenyl) carbonate, di(3-nitrophenyl) carbonate, phenyl chloroformate, 4-nitrophenyl chloroformate, 2-nitrophenyl chloroformate, and 3-nitrophenyl chloroformate. A portion of the acylating agent corresponds to X in formula (A), and the type of acylating agent can be selected based on the desired choice of the X group.

[0159] The amount of acylation agent used relative to 1 mol of the compound shown in formula (3) can be from 1.0 mol to 5.0 mol or from 2.0 mol to 4.0 mol.

[0160] Step 4 can be carried out in the absence of a solvent or in a solvent, and preferably in a solvent. The solvent is not limited, as long as it does not inhibit the acylation reaction of the compound shown in formula (3). Examples of solvents include N,N-dimethylformamide (DMF), N,N-dimethylacetamide, toluene, dimethyl sulfoxide (DMSO), and N,N-dimethylacetamide (DMA). The amount of solvent used relative to 1 g of the compound shown in formula (3) can be from 5 mL to 20 mL or from 6 mL to 15 mL.

[0161] The reaction temperature in step 4 is not limited, as long as it is the temperature at which the acylation reaction of the compound shown in formula (3) takes place. The reaction temperature can be the normal temperature or 20°C to 25°C.

[0162] Example The invention will be described in more detail below using production examples and comparative production examples.

[0163] The abbreviations used in the examples are the same as those used in the field, for example, as shown below.

[0164] DIPEA: N,N-Diisopropylethylamine DMA: N,N-dimethylacetamide DMF: N,N-dimethylformamide DMT-MM: 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride EDTA: Ethylenediaminetetraacetic acid THF: Tetrahydrofuran <Production Example 1> (11S,14S)-14-[3-(carbamoylamino)propyl]-1-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)-N-[4-(hydroxymethyl)phenyl]-9,12-dioxo-11-(propyl-2-yl)-3,6-dioxa-10,13-diazapentadecane-15-amide A mixture of (2S)-2-{[(2S)-2-amino-3-methylbutyryl]amino}-5-(carbamoylamino)-N-[4-(hydroxymethyl)phenyl]pentanamide (184 g), 3-{2-[2-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)ethoxy]ethoxy}propionic acid (150 g), DMT-MM (174 g), methanol (2.76 L), and THF (2.76 L) was stirred at 15°C to 35°C for 24 hours under a nitrogen atmosphere. The reaction mixture was concentrated under reduced pressure, and acetonitrile was added. The precipitate was filtered and further washed with acetonitrile. The obtained solid was mixed with acetonitrile and filtered to obtain a solid. The obtained solid was dried under reduced pressure to obtain the title compound (267 g, 89% yield).

[0165] <Production Example 2> (4-{[(11S,14S)-14-[3-(carbamoylamino)propyl]-1-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)-9,12,15-trioxo-11-(propyl-2-yl)-3,6-dioxa-10,13-diazapentadecan-15-yl]amino}phenyl)methyl=4-nitrophenyl=carbonate A mixture of the compound obtained in Production Example 1 (250 g), di(4-nitrophenyl) carbonate (615 g), DIPEA (157 g), and DMF (5.0 L) was stirred at 15°C to 35°C for 3 hours under a nitrogen atmosphere. The reaction mixture was concentrated under reduced pressure and purified on a silica gel column (dichloromethane / acetone), and the fraction containing the title compound was concentrated under reduced pressure. As a result, the title compound (162 g, 51% yield) was obtained as a solid.

[0166] <Production Example 3> (4-{[(11S,14S)-14-[3-(carbamoylamino)propyl]-1-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)-9,12,15-trioxo-11-(propyl-2-yl)-3,6-dioxa-10,13-diazapentadecan-15-yl]amino}phenyl)methyl={(2S)-2-hydroxy-3-[(2R,3R,3aS,7R,8aS,9S,10aR,11S,12R,13aR,13b]amino}phenyl)methyl={(2S)-2-hydroxy-3-[(2R,3R,3aS,7R,8aS,9S,10aR,11S,12R,13aR,13b]amino}phenyl)methyl={(2S)-2-hydroxy-3-[(2R,3R,3aS,7R,8aS,9S,10aR,11S,12R,13aR,13b]amino}phenyl} ...phenyl}phenyl}phenyl}phenyl}phenyl}phenyl}phenyl}phenyl}phenyl}phenyl}phenyl}phenyl}phenyl}phenyl}phenyl}phenyl}phenyl}phenyl}phenyl}phenyl}phenyl}phenyl}phenyl}phenyl}phenyl}phenyl}phenyl}phenyl}phenyl}phenyl}phenyl}phenyl}phenyl}phenyl}phenyl}phenyl}phenyl}phenyl}phenyl}phenyl}phenyl}phenyl}phenyl}phenyl} S,15S,18S,21S,24S,26R,28R,29aS)-3-methoxy-26-methyl-20,27-dimethylenehexahydro-11,15:18,21:24,28-tricyclooxy-7,9-bridged ethylene-12,15-bridged methylene-9H,15H-furano[3,2-i]furano[2',3':5,6]pyrano[4,3-b][1,4]dioxane-2-(4H)-keto-2-yl]propyl}carbamate A mixture of iribulin methanesulfonate (93 g), DIPEA (26 mL), the compound obtained in Production Example 2 (116 g), and DMF (930 mL) was stirred at 20°C to 25°C for 20 hours under a nitrogen atmosphere. The reaction mixture was purified on a reversed-phase column (trade name: Kromasil C18) (acetonitrile / water / acetic acid), and the fraction containing the title compound was extracted with dichloromethane. The organic layer was washed sequentially with aqueous sodium bicarbonate solution and water, and then concentrated under reduced pressure. The concentrated residue was dissolved in a mixed solvent of dichloromethane, methanol, and acetic acid, and the solution was added dropwise to pentane. The precipitate was filtered, and the resulting solid was washed with pentane and dried under reduced pressure. As a result, the title compound was obtained (109.6 g, 71% yield).

[0167] 1¹H-NMR (400 MHz, CD₃OD): δ (ppm) 7.59 (d, J = 8.4 Hz, 2H), 7.31 (d, J = 8.4 Hz, 2H), 6.81 (s, 2H), 5.13 (s, 1H), 5.06 (d, J = 12.4 Hz, 1H), 5.02 (s, 1H), 5.01 (d, J = 12.4 Hz, 1H), 4.87 (s, 1H), 4.82 (s, 1H), 4.71 (t, J = 4.0 Hz, 1H), 4.61 (t, J = 4.4 Hz, 1H), 4.50 (dd, J = 5.2, 9.2 Hz, 1H), 4.47 (d, J = 10.8 Hz, 1H), 4.32-4.27 (m, 2H), 4.19 (dd, J = 6.8, 11.6 Hz, 1H), 4.13-4.07 (m, 2H), 3.98 (t, J = 10.4 Hz, 1H), 3.88-3.82 (m, 3H), 3.76-3.64 (m, 6H), 3.62-3.51 (m, 6H), 3.38 (s, 3H), 3.22-3.08 (m, 4H), 2.93 (dd, J = 2.4, 9.6 Hz, 1H), 2.92-2.84 (m, 1H), 2.76-2.63 (m, 2H), 2.52 (t, J = 6.0 Hz, 2H), 2.44-2.29 (m, 5H), 2.21-1.97 (m, 8H), 1.93-1.83 (m, 3H), 1.80-1.66 (m, 5H), 1.66-1.28 (m, 10H), 1.11 (d, J = 6.4 Hz, 3H), 1.07-1.01 (m, 1H), 0.99 (d, J = 6.8 Hz, 3H), 0.97 (d, J = 6.4 Hz, 3H).

[0168] LCMS (M+H): m / z 1374.9 <Comparative Preparation Example 1> <Comparative Preparation Example 1-1> 4-{[(2S)-5-(carbamoylamino)-2-{[(2S)-2-{[(9H-fluoren-9-ylmethoxy)carbonyl]amino}3-methylbutyryl]amino}pentanoyl]amino}benzyl={(2S)-2-hydroxy-3-[(2R,3R,3aS,7R,8aS,9S,10aR,11S,12R,13aR,13bS,15S,18S,21S,24S,26R,28 R,29aS)-3-methoxy-26-methyl-20,27-dimethylenehexahydro-11,15:18,21:24,28-tricyclooxy-7,9-bridged ethylene-12,15-bridged methylene-9H,15H-furano[3,2-i]furano[2',3':5,6]pyrano[4,3-b][1,4]dioxane-2-penta-5(4H)-keto-2-yl]propyl}carbamate A mixture of iribulin methanesulfonate (93 g), DIPEA (25 mL), 4-{[(2S)-5-(carbamoylamino)-2-{[(2S)-2-{[(9H-fluoren-9-ylmethoxy)carbonyl]amino}3-methylbutyryl]amino}pentanoyl]amino}benzyl=4-nitrophenyl carbonate (103.6 g) and DMF (930 mL) was stirred at 20°C to 25°C for 16 hours under a nitrogen atmosphere.

[0169] <Comparison of Production Examples 1-2> 4-{[(2S)-2-{[(2S)-2-amino-3-methylbutyryl]amino}-5-(carbamoylamino)pentanoyl]amino}benzyl={(2S)-2-hydroxy-3-[(2R,3R,3aS,7R,8aS,9S,10aR,11S,12R,13aR,13bS,15S,18S,21S,24S,26R,28R,29aS)-3 -Methoxy-26-methyl-20,27-dimethylenehexahydro-11,15:18,21:24,28-triepoxy-7,9-bridged ethylene-12,15-bridged methylene-9H,15H-furano[3,2-i]furano[2',3':5,6]pyrano[4,3-b][1,4]dioxane-2-penta-5(4H)-keto-2-yl]propyl}carbamate Diethylamine (234 mL) was added to the reaction mixture and stirred at 20°C to 25°C for 0.5 hours. Ethyl acetate and hydrochloric acid were added to the reaction mixture, and the aqueous layer was washed with heptane. Methyltetrahydrofuran and an aqueous solution of potassium carbonate and sodium chloride were added to the aqueous layer, and the organic layer was washed with an aqueous solution of sodium chloride. The organic layer was dried over magnesium sulfate and concentrated under reduced pressure.

[0170] 1H-NMR (400 MHz, CD3OD): δ (ppm) 7.56 (d, J = 8.4 Hz, 2H), 7.32 (d, J= 8.4 Hz, 2H), 5.14 (s, 1H), 5.06 (d, J = 12.4 Hz, 1H), 5.03 (s, 1H), 5.01(d, J = 12.4 Hz,1H), 4.87 (s, 1H), 4.83 (s, 1H), 4.71 (t, J = 4.4 Hz, 1H),4.62 (t, J = 4.4 Hz, 1H), 4.57 (dd, J = 4.8, 8.8 Hz, 1H), 4.47 (d, J = 10.8Hz, 1H), 4.32-4.27 (m, 2H), 4.18 (dd, J = 4.8, 6.4 Hz, 1H), 4.13-4.07 (m,2H), 3.98 (t, J = 10.4 Hz, 1H), 3.88-3.82 (m, 3H), 3.76-3.70 (m, 4H), 3.60(d, J = 6.0 Hz, 1H), 3.38 (s, 3H), 3.26-3.10 (m, 3H), 2.93 (dd, J =2.0, 11.2Hz, 1H), 2.91-2.84 (m, 1H), 2.75-2.64 (m, 2H), 2.44-2.29 (m, 5H), 2.21-1.97(m, 8H), 1.93-1.83 (m, 3H), 1.79-1.72 (m, 5H), 1.68-1.29 (m, 8H), 1.11 (d, J= 6.8 Hz, 3H), 1.07-1.01 (m, 1H), 1.06 (d, J = 7.2 Hz, 3H), 1.02 (d, J = 7.2Hz, 3H) LCMS(M+H): m / z1135.7 <Comparative Production Examples 1-3> (4-{[(11S,14S)-14-[3-(carbamoylamino)propyl]-1-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)-9,12,15-trioxo-11-(propyl-2-yl)-3,6-dioxa-10,13-diazapentadecan-15-yl]amino}phenyl)methyl={(2S)-2-hydroxy-3-[(2R,3R,3aS,7R,8aS,9S,10aR,11S,12R,13aR,13b]amino}phenyl)methyl={(2S)-2-hydroxy-3-[(2R,3R,3aS,7R,8aS,9S,10aR,11S,12R,13aR,13b]amino}phenyl)methyl={(2S)-2-hydroxy-3-[(2R,3R,3aS,7R,8aS,9S,10aR,11S,12R,13aR,13b]amino}phenyl} ...phenyl}phenyl}phenyl}phenyl}phenyl}phenyl}phenyl}phenyl}phenyl}phenyl}phenyl}phenyl}phenyl}phenyl}phenyl}phenyl}phenyl}phenyl}phenyl}phenyl}phenyl}phenyl}phenyl}phenyl}phenyl}phenyl}phenyl}phenyl}phenyl}phenyl}phenyl}phenyl}phenyl}phenyl}phenyl}phenyl}phenyl}phenyl}phenyl}phenyl}phenyl}phenyl}phenyl}phenyl} S,15S,18S,21S,24S,26R,28R,29aS)-3-methoxy-26-methyl-20,27-dimethylenehexahydro-11,15:18,21:24,28-tricyclooxy-7,9-bridged ethylene-12,15-bridged methylene-9H,15H-furano[3,2-i]furano[2',3':5,6]pyrano[4,3-b][1,4]dioxane-2-(4H)-keto-2-yl]propyl}carbamate 41-[2-(2-{3-[(2,5-dioxopyrrolidine-1-yl)oxy]-3-oxopropoxy}ethoxy)ethyl]-1H-pyrrole-2,5-dione (47.9 g), DIPEA (20 mL), and DMF (913 mL) were added to the concentrated residue obtained in comparative production examples 1-2, and the mixture was stirred at 20°C to 25°C for 3 hours.

[0171] The reaction mixture was purified on a reversed-phase column (trade name: SILMERCK 10013) (acetonitrile / water / acetic acid), and the fraction containing the title compound was extracted with dichloromethane. The organic layer was washed with an aqueous solution of sodium bicarbonate and water, and then concentrated under reduced pressure. The concentrated residue was dissolved in dichloromethane, methanol, and acetic acid, and the solution was added dropwise to pentane. As a result, the precipitate was filtered off. The obtained solid was washed with pentane and dried under reduced pressure. As a result, the title compound was obtained (88.1 g, 57% yield in three steps).

[0172] <Production Example 4> 1493 mL of a buffer solution for dilution (10 mmol / L trisodium phosphate, 100 mmol / L sodium chloride, 3 w / v sucrose, pH 6.5) and 1098 mL of a buffer solution for pH adjustment (0.25 mol / L tris(hydroxymethyl)aminomethane hydrochloride, 20 mmol / L EDTA, pH 7.7) were added to 7854 mL of a buffer solution containing MORAb-003 (26.6 mg / mL MORAb-003, 10 mmol / L trisodium phosphate, 100 mmol / L sodium chloride, 3 w / v sucrose, pH 6.5). A 10 mmol / L (311 g) aqueous solution of tris(2-carboxyethyl)phosphine was added at 20 °C and the mixture was stirred for 2.5 hours.

[0173] Add a DMA solution (8.37 mmol / L, 809 mL) of the compound obtained in Production Example 3 and stir for 0.5 h. Add an aqueous solution of N-acetylcysteine ​​(30 mmol / L, 452 g) to stop the reaction and stir for 0.5 h. Purify the antibody-drug conjugate (ADC) by dialysis filtration under the following conditions.

[0174] (Purification conditions) Filter (permeable membrane): Pellicon 3 (1.14 m) 2 30 kDa × 1, 0.57 m 2 30 kDa × 1) TMP (Transmembrane Pressure Difference): 15 psi Additional buffer solution: 25 mmol / L citric acid aqueous solution (pH 6.3) Temperature: 17.0℃ to 23.0℃ Osmosis: 163.2 kg An aqueous solution of ADC (protein concentration: 10.6 mg / mL, total weight: 20.9 kg, ADC content: 213.430 g, 99% yield) was obtained by adding 25 mmol / L citric acid aqueous solution (3013 g, pH 6.3) and 5668 g of buffer solution (25 mmol / L citric acid, 1 mol / L sucrose, 0.24 w / v%, polysorbate 80, pH 6.3). The ADC aqueous solution was then sterile filtered (Millipore Durapore PVDF).

[0175] All sequences set forth in the sequence list of the parent application of this application are incorporated herein by reference, and include at least the following sequences: SEQ ID NO: 17 <210> 17 <211> 3 <212> PRT <213> Artificial sequence <220> <223> MORAb-003 LC CDR2; IMGT <400> 17 Gly Thr Ser 1 SEQ ID NO: 189 <210> 189 <211> 3 <212> PRT <213> Artificial sequence <220> <223> MORAb-009 LC CDR2 (Table 6) <400> 189 Asp Thr Ser 1 SEQ ID NO: 195 <210> 195 <211> 3 <212> PRT <213> Artificial sequence <220> <223> Trastuzumab LC CDR2 (Table 6) <400> 195 Ser Ala Ser 1 SEQ ID NO: 201 <210> 201 <211> 3 <212> PRT <213> Artificial sequence <220> <223> 33011-xi LC CDR2 (Table 6) <400> 201 Leu Ala Ser 1 SEQ ID NO: 207 <210> 207 <211> 3 <212> PRT <213> Artificial sequence <220> <223> 33011-zu LC CDR2 (Table 6) <400> 207 Leu Ala Ser 1 SEQ ID NO: 213 <210> 213 <211> 3 <212> PRT <213> Artificial sequence <220> <223> 111B10-xi LC CDR2 (Table 6) <400> 213 Ser Ala Ser 1 SEQ ID NO: 219 <210> 219 <211> 3 <212> PRT <213> Artificial sequence <220> <223> 111B10-zu LC CDR2 (Table 6) <400> 219 Ser Ala Ser 1 SEQ ID NO: 225 <210> 225 <211> 3 <212> PRT <213> Artificial sequence <220> <223> 201C15-xi LC CDR2 (Table 6) <400> 225 Asp Thr Ser 1 SEQ ID NO: 231 <210> 231 <211> 3 <212> PRT <213> Artificial sequence <220> <223> 201C15-zu LC CDR2 (Table 6) <400> 231 Asp Thr Ser 1 SEQ ID NO: 237 <210> 237 <211> 3 <212> PRT <213> Artificial sequence <220> <223> 346C6-xi LC CDR2 (Table 6) <400> 237 Leu Ala Ser 1 SEQ ID NO: 243 <210> 243 <211> 3 <212> PRT <213> Artificial sequence <220> <223> 346C6-zu LC CDR2 (Table 6) <400> 243 Leu Ala Ser 1 SEQ ID NO: 249 <210> 249 <211> 9 <212> DNA <213> Artificial sequence <220> <223> MORAb-003 LC CDR2 (Table 7) <400> 249 ggcacatcc 9 SEQ ID NO: 255 <210> 255 <211> 9 <212> DNA <213> Artificial sequence <220> <223> MORAb-009 LC CDR2 (Table 7) <400> 255 gacacatcc 9 SEQ ID NO: 261 <210> 261 <211> 9 <212> DNA <213> Artificial sequence <220> <223> 33011-xi LC CDR2 (Table 7) <400> 261 ctggcatct 9 SEQ ID NO: 267 <210> 267 <211> 9 <212> DNA <213> Artificial sequence <220> <223> 33011-zu LC CDR2 (Table 7) <400> 267 ttggcctcc 9 SEQ ID NO: 273 <210> 273 <211> 9 <212> DNA <213> Artificial sequence <220> <223> 111B10-xi LC CDR2 (Table 7) <400> 273 tctgcatcc 9 SEQ ID NO: 279 <210> 279 <211> 9 <212> DNA <213> Artificial sequence <220> <223> 111B10-zu LC CDR2 (Table 7) <400> 279 tctgcctcc 9 SEQ ID NO: 285 <210> 285 <211> 9 <212> DNA <213> Artificial sequence <220> <223> 201C15-xi LC CDR2 (Table 7) <400> 285 gatacatcc 9 SEQ ID NO: 291 <210> 291 <211> 9 <212> DNA <213> Artificial sequence <220> <223> 201C15-zu LC CDR2 (Table 7) <400> 291 gacaccagc 9 SEQ ID NO: 297 <210> 297 <211> 9 <212> DNA <213> Artificial sequence <220> <223> 346C6-xi LC CDR2 (Table 7) <400> 297 ctggcatcc 9 SEQ ID NO: 303 <210> 303 <211> 9 <212> DNA <213> Artificial sequence <220> <223> 346C6-zu LC CDR2 (Table 7) <400> 303 ctggcctcc 9

Claims

1. A method for preparing the antibody-drug conjugate shown in formula (I), In this formula, Ab is an antibody or its antigen-binding fragment, D is eribulin, m is an integer from 1 to 10, and p is an integer from 1 to 8. The method includes: Step 1: The compound shown in formula (B) is obtained by reacting eribulin or its salt with the compound shown in formula (A). In this formula, m is an integer from 1 to 10, and X is phenoxy or nitrophenoxy. In this formula, m is an integer from 1 to 10; and Step 2: Obtain the antibody-drug conjugate shown in formula (I) by reacting the compound shown in formula (B) with Ab.

2. The method according to claim 1, Wherein Ab comprises: (i) a heavy chain domain comprising the amino acid sequence shown in SEQ ID NO: 1 and a light chain domain comprising the amino acid sequence shown in SEQ ID NO: 6, (ii) a heavy chain variable domain comprising the amino acid sequence shown in SEQ ID NO: 23 and a light chain variable domain comprising the amino acid sequence shown in SEQ ID NO: 24, (iii) a heavy chain variable domain comprising the amino acid sequence shown in SEQ ID NO: 27 and a light chain variable domain comprising the amino acid sequence shown in SEQ ID NO: 28, or (iv) a heavy chain domain comprising the amino acid sequence shown in SEQ ID NO: 347 and a light chain domain comprising the amino acid sequence shown in SEQ ID NO:

308.

3. The method according to claim 1 or 2, wherein p is 3 or 4.

4. The method according to any one of claims 1 to 3, wherein eribulin or a salt thereof is eribulin methanesulfonate.

5. The method according to any one of claims 1 to 4, wherein step 1 is carried out in the presence of an alkali.

6. A method for preparing the compound shown in formula (B), In this formula, m is an integer from 1 to 10. The method includes: The steps for obtaining the compound shown in formula (B) by reacting eribulin or its salt with the compound shown in formula (A) are as follows: In this formula, m is an integer from 1 to 10, and X is phenoxy or nitrophenoxy.

7. The method of claim 6, wherein the step is carried out in the presence of a base.

8. A compound represented by formula (A), In this formula, m is an integer from 1 to 10, and X is phenoxy or nitrophenoxy.

Images