Method for producing eribulin-based antibody-drug conjugates

Abstract

The present invention provides a method for producing an antibody-drug conjugate represented by formula (I) [wherein: Ab represents an antibody or an antigen-binding fragment thereof; D represents eribulin; m is an integer of 1-10; and p is an integer of 1-8], said method comprising step 1 for reacting eribulin or a salt thereof with a compound represented by formula (A) [wherein: m is an integer of 1-10; and X represents a phenoxy group or a nitrophenoxy group] to give a compound represented by formula (B) [wherein m is an integer of 1-10], and step 2 for reacting the compound represented by formula (B) with the Ab to give the antibody-drug conjugate represented by formula (I).　

Description

[Technical Field]

[0001] This invention relates to a method for producing an eribulin-based antibody-drug conjugate (ADC). [Background technology]

[0002] Cancer is one of the leading causes of disease incidence and death worldwide, with approximately 14 million new cases and 8.2 million cancer-related deaths in 2012. The most common causes of cancer death are lung cancer (1.59 million 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% in the next 20 years, reaching approximately 22 million new cases per year (Non-Patent Literature 1).

[0003] Microtubules are dynamic, fibrous cytoskeletal proteins involved in a variety of cellular functions, including intracellular migration and transport, cell signaling, and maintenance of cell shape. Microtubules also play a crucial role in mitotic cell division by forming the mitotic spindle, which is necessary for separating chromosomes into two daughter cells. The biological function of microtubules in all cells is largely regulated by their polymerization dynamics, which result from the reversible non-covalent addition of α and β tubulin dimers at both ends of the microtubule. This dynamic behavior and the resulting length-long regulation of microtubules are critical to proper spindle function. Even slight changes in microtubule dynamics can trigger spindle checkpoints, halting cell cycle progression in mitosis and subsequently leading to cell death (Non-Patent Literature 2). Due to their rapid cell division, cancer cells are generally more sensitive than normal cells to compounds that bind to tubulin and disrupt its normal function. For this reason, tubulin inhibitors and other microtubule-targeting drugs represent a promising class of drugs for treating cancer (Non-Patent Literature 3).

[0004] Folate receptor alpha (FRA) is a glycophosphatidylinositol (GPI)-binding membrane protein that binds to folate. While the role of FRA in the biology of normal and cancerous tissues is not fully understood, it is highly overexpressed in a high percentage of epithelial ovarian cancers (Non-Patent Literature 4) and in a certain percentage of non-small cell lung cancers (Non-Patent Literature 5). FRA expression is also restricted in normal tissues. These characteristics make FRA an attractive target for cancer immunotherapy.

[0005] 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, thereby serving as an oncogenic driver in several cancers, including approximately 20% of invasive breast cancers (Non-Patent Literature 7-8). Because HER2 amplification mediates a transformative phenotype, HER2 is another promising target for cancer treatment. [Prior art documents] [Patent Documents]

[0006] [Patent Document 1] International Publication No. 2017 / 151979 [Non-patent literature]

[0007] [Non-Patent Document 1] World Cancer Report 2014. [Non-Patent Document 2] Mukhtar et al. (2014) Mol. Cancer Ther. 13:275-84. [Non-Patent Document 3] Dumontet and Jordan (2010) Nat. Rev. Drug Discov. 9:790-803.

Non-Patent Document 4

Non-Patent Document 5

Non-Patent Document 6

Non-Patent Document 7

Non-Patent Document 8

Non-Patent Document 9

Summary of the Invention

Problems to be Solved by the Invention

[0008] An object of the present invention is to provide a method for producing an antibody-drug conjugate with a higher yield, and a synthetic intermediate useful therefor.

Means for Solving the Problems

[0009] The present invention provides the following [1] to [8]. [1] A method for producing an antibody-drug conjugate represented by formula (I),

Chemical Formula

[0010] The present invention provides a method for producing antibody-drug conjugates in higher yield. It also provides synthetic intermediates useful for producing antibody-drug conjugates in higher yield. [Modes for carrying out the invention]

[0011] Embodiments of the present invention will be described in detail below.

[0012] One embodiment of the present invention relates to a method for producing an antibody-drug conjugate (ADC) represented by formula (I).

[0013] First, let's explain the ADC represented by equation (I).

[0014] ADCs can bind to tumor cells (e.g., FRA-expressing tumor cells), internalize them, and kill them. Furthermore, the antibody portion (Ab) used in ADCs is preferably an antibody or its antigen-binding fragment, which targets tumor cells. The antibody or its antigen-binding fragment may be, for example, (a) comprising three heavy chain CDRs containing the amino acid sequences of heavy chain complementarity determining region (heavy chain CDR) 1 represented by SEQ ID NO: 2, heavy chain CDR2 represented by SEQ ID NO: 3, and heavy chain CDR3 represented by SEQ ID NO: 4, as defined by the Kabat numbering system (Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991))), and three light chain CDRs containing the amino acid sequences of light chain complementarity determining region (light chain CDR) 1 represented by SEQ ID NO: 7, light chain CDR2 represented by SEQ ID NO: 8, and light chain CDR3 represented by SEQ ID NO: 9, or, (b) comprising three heavy chain CDRs including the amino acid sequences of heavy chain CDR1 represented by SEQ ID NO: 13, heavy chain CDR2 represented by SEQ ID NO: 14, and heavy chain CDR3 represented by SEQ ID NO: 15, as defined by the IMGT numbering system (International ImMunoGeneTics Information System (IMGT®)), and three light chain CDRs including the amino acid sequences of light chain CDR1 represented by SEQ ID NO: 16, light chain CDR2 represented by SEQ ID NO: 17, and light chain CDR3 represented by SEQ ID NO: 18.

[0015] In this specification, the terms “antibody-drug conjugate,” “antibody conjugate,” “conjugate,” “immunoconjugate,” and “ADC” are used interchangeably and refer to a compound or derivative thereof that is conjugated to an antibody (e.g., an anti-FRA antibody), as defined by Formula I [wherein 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 eribulins per antibody moiety].

[0016] The term "antibody," in its broadest sense, is used to refer to an immunoglobulin molecule that recognizes and specifically binds to a target, such as a protein, polypeptide, carbohydrate, polynucleotide, lipid, or a combination thereof, via at least one antigen recognition site within the variable region of the immunoglobulin molecule. The heavy chain of an antibody is the heavy chain variable region (VH ) and heavy chain constant region (C H The antibody light chain is composed of the light chain variable region (V). L ) and light chain constant region (C L ) is composed of. For the purposes of this application, the mature heavy chain and light chain variable regions each include three complementarity-determining regions (CDR1, CDR2, and CDR3) within four framework regions (FR1, FR2, FR3, and FR4) arranged from N-terminus to C-terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4, respectively. “Antibodies” may be naturally occurring or artificial, such as monoclonal antibodies produced by conventional hybridoma technology. The term “antibody” includes full-length monoclonal antibodies, full-length polyclonal antibodies, and single-chain antibodies. Antigen-binding fragments of antibodies include, for example, Fab, Fab', F(ab')2, and Fv. An antibody may be any one of the five main groups of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, or their subclasses (e.g., isotypes IgG1, IgG2, IgG3, and IgG4). This term further includes human antibodies, chimeric antibodies, humanized antibodies, and any modified immunoglobulin molecules containing antigen recognition sites, insofar as they demonstrate the desired biological activity.

[0017] 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 naturally occurring, possible variations that may be present in small amounts. Monoclonal antibodies are highly specific and target a single antigen epitope. In contrast, conventional (polyclonal) antibody preparations typically contain a number of antibodies that target (or are specific to) various epitopes. The modifying phrase “monoclonal” indicates a characteristic of the antibody that it is obtained from a substantially homogeneous population of antibodies, and should not be interpreted as requiring the production of the antibody by any particular method. For example, the monoclonal antibodies used herein may be produced by the hybridoma method first described by Kohler et al. (1975) Nature 256: 495, or by the recombinant DNA method (see, for example, U.S. Patent No. 4,816,567). Monoclonal antibodies can also be isolated from phage antibody libraries using techniques described, for example, Clackson et al. (1991) Nature 352: 624-8 and Markset al. (1991) J. Mol. Biol. 222: 581-97.

[0018] 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 a corresponding sequence in an antibody derived from a particular species or belonging to a particular group or subclass of antibodies, while the remainder of the chain(s) is identical or homologous to a corresponding sequence in an antibody derived from another species or belonging to another group or subclass of antibodies, or even to a corresponding sequence in a fragment of such an antibody, insofar as they specifically bind to a target antigen and / or exhibit the desired biological activity.

[0019] The term "homologous" refers to a molecule that exhibits homology to another molecule, for example, by having the same or similar sequences of chemical residues at corresponding positions.

[0020] As used herein, the term "human antibody" refers to an antibody produced by a human or an antibody having the amino acid sequence of a human-produced antibody.

[0021] As used herein, the term "chimeric antibody" refers to an antibody whose immunoglobulin molecule's amino acid sequence originates from two or more species. In some cases, both the heavy and light chain variable regions correspond to the variable regions of an antibody from one species, possessing desired specificity, affinity, and activity, while the constant region is homologous to an antibody from another species (e.g., human), minimizing the immune response in the latter species.

[0022] As used herein, the term “humanized antibody” refers to a form of antibody that contains sequences from non-human (e.g., mouse) antibodies, and even from human antibodies. Such antibodies are chimeric antibodies containing minimal sequences derived from non-human immunoglobulins. Generally, a humanized antibody contains substantially all of at least one, typically two, variable regions, where all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin, and all or substantially all of the framework region (FR) is a human immunoglobulin sequence. A humanized antibody optionally also contains at least a portion of the immunoglobulin constant region (Fc), typically the constant region (Fc) of a human immunoglobulin. Humanized antibodies may be further modified by residue substitutions in the Fv framework region and / or within the replaced non-human residues to refine and optimize antibody specificity, affinity, and / or activity.

[0023] As used herein, the term "antigen-binding fragment" of an antibody refers to a fragment of one or more antibodies that retains the ability to specifically bind to an antigen (e.g., FRA). The antigen-binding fragment preferably also retains the ability to internalize into antigen-expressing cells. In some embodiments, the antigen-binding fragment also retains immune effector activity. Fragments of full-length antibodies have been found to perform the antigen-binding function of the full-length antibody. Examples of binding fragments included within the scope of the term "antigen-binding fragment" of an antibody include: (i) a monovalent fragment consisting of the V L and V H , C L , and C H1 regions; (ii) an F(ab’)2 fragment, a bivalent fragment comprising two Fab fragments linked by disulfide bridges in the hinge region; (iii) an Fd fragment consisting of the V H and C H1 regions; (iv) an Fv fragment consisting of the V L and V H regions of one arm of an antibody; (v) a dAb fragment (see, e.g., Ward et al. (1989) Nature 341: 544-6 and Winter et al. WO90 / 05144) comprising one variable region, e.g., the V H region), and (vi) isolated complementarity-determining regions (CDRs). Further, the two regions of an Fv fragment, V L and V H are encoded by separate genes, but using recombinant methods, they are made such that they are V L and V HThese can be linked by synthetic linkers, which allow them to be constructed as a single protein chain (known as single-chain Fv (scFv)) in which regions pair up to form a monovalent molecule. See, for example, Bird et al. (1988) Science 242: 423-6 and Hustonet al. (1988) Proc. Natl. Acad. Sci. USA 85: 5879-83. Such single-chain antibodies are also intended to be included within the scope of the term “antigen-binding fragment” of an antibody, and are known in the art as exemplary types of binding fragments that can be internalized into cells upon 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 certain embodiments, the scFv molecule may be incorporated into the fusion protein. Other forms of single-chain antibodies, such as diabodies, are also included. Diabodies are V H and V L The region is expressed on a single polypeptide chain, but uses a linker that is too short to allow pairing between two regions on the same chain, thereby forcing the region to pair with a complementary region on another chain to create two antigen-binding sites, resulting in a bivalent diabody (see, e.g., Holliger et al. (1993) Proc. Natl. Acad. Sci. USA 90: 6444-8 and Poljak et al. (1994) Structure 2: 1121-3). Antigen-binding fragments are obtained using conventional techniques known to those skilled in the art, and are screened for usefulness (e.g., binding affinity, internalization) in the same manner as intact antibodies. Antigen-binding fragments can also be prepared by cleavage of intact proteins, e.g., by protease or chemical cleavage.

[0024] In this specification, antibodies or their antigen-binding fragments include internalized antibodies or internalized-binding fragments, where "internalized," as used herein in relation to antibodies or their antigen-binding fragments, means that, upon binding to a cell, the antibody or its antigen-binding fragment can be taken up (i.e., internalized) through the cell's lipid bilayer membrane into an internal compartment, preferably into a degradation compartment such as an intracellular lysosome. For example, an internalized anti-FRA antibody can be taken up into the cell after binding to FRA on the cell membrane.

[0025] The terms “folate receptor alpha” or “FRA” as used herein refer to any native form of human FRA. This term includes full-length FRA (e.g., NCBI Reference Sequence: NP_000793; SEQ ID NO: 19), as well as any form of human FRA resulting from cell processing. The term also includes, but is not limited to, naturally occurring variants of FRA, including splicing variants, allele variants, and isoforms. FRA may be isolated from humans or produced recombinantly or synthetically.

[0026] The terms “anti-FRA antibody” or “antibody that specifically binds to FRA” refer to any form of antibody or antigen-binding fragment that specifically binds to FRA, and include monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, and antibody fragments that have biological function insofar as 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 “specific” and “specifically bind” refer to the selective binding of an antibody to a target antigen epitope. An antibody can be tested for binding specificity by comparing its binding to a suitable antigen with its binding to an unrelated antigen or antigen mixture under given conditions. An antibody is understood to “specifically bind” if it binds to a suitable antigen with at least 2, 5, or 7, preferably 10, higher affinity than an unrelated antigen or antigen mixture. In one embodiment, a specific antibody binds only to the FRA antigen and does not bind to (or shows only slight binding to) other antigens.

[0027] The terms “human epidermal growth factor receptor 2,” “her2,” or “her2 / neu,” as used herein, refer to any native form of human her2. This term includes full-length her2 (e.g., NCBI Reference Sequence: NP_004439.2; SEQ ID NO: 21), as well as any form of human her2 arising from cell processing. The term also includes, but is not limited to, naturally occurring variants of her2, including splicing mutants, allele mutants, and isoforms. Her2 can be isolated from humans or produced recombinantly or synthetically.

[0028] The terms “anti-HER2 antibody” or “antibody that specifically binds to HER2” refer to any form of antibody or antigen-binding fragment that specifically binds to HER2, and include monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, and antibody fragments that have biological function insofar as 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 an antigen-binding fragment. Trastuzumab is an exemplary anti-human HER2 antibody.

[0029] The term "epitope" refers to a portion of an antigen that an antibody can recognize and specifically bind to. When the antigen is a polypeptide, the epitope can be formed from a sequence of amino acids or from discontinuous amino acids that are parallel due to the three-dimensional folding of the polypeptide. Any epitope mapping technique known in the art can be used to identify the epitopes to which antibodies bind. Such epitope mapping techniques include, for example, X-ray crystallography for epitope identification by direct visualization of the antigen-antibody complex, monitoring the binding of antibodies to antigen fragments or variants, and monitoring the solvent exposure of various portions of the antibody and antigen. Exemplary methods used to map epitopes include, but are not limited to, array-based oligo-peptide scanning, limited proteolysis, site-directed mutagenesis, high-throughput mutagenesis mapping, hydrogen-deuterium exchange, and mass spectrometry (see, e.g., Gershoni et al. (2007) 21: 145-56 and Hager-Braun and Tomer (2005) Expert Rev. Proteomics 2: 745-56).

[0030] Competitive binding and epitope binning can also be used to determine antibodies that share the same or overlapping epitopes. Competitive binding can be evaluated using cross-blocking assays, such as the assay 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 a test antibody or its antigen-binding fragment is evaluated as competitive if it reduces the binding of a reference antibody or its antigen-binding fragment (e.g., a binding protein containing a CDR and / or variable region selected from those identified in Tables 2, 4, and 6) to a target antigen, e.g., FRA or HER2, by at least about 50% (e.g., 50%, 60%, 70%, 80%, 90%, 95%, 99%, 99.5%, or more, or any percentage in between) and / or vice versa in a cross-blocking assay. In some embodiments, competitive binding may occur due to identical or similar (e.g., partially overlapping) epitopes, or due to steric hindrance when each antibody or its antigen-binding fragment binds to a nearby 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 that have similar epitopes; for example, those whose binding competes can be "binned" as a group of binding proteins with overlapping or nearby epitopes, and those that do not compete can be distinguished as a separate group of binding proteins that do not have overlapping or nearby epitopes.

[0031] "k on " or "k aThe term "rate constant" refers to the rate of association between an antibody and an antigen to form an antibody / antigen complex. This rate can be determined using a standard assay, such as a Biacore or ELISA assay.

[0032] "k off " or "k d The term "off-rate constant" refers to the off-rate constant for the dissociation of an antibody from an antibody / antigen complex. This rate can be determined using a standard assay, such as a Biacore or ELISA assay.

[0033] "K D The term "K" refers to the equilibrium dissociation constant of a specific antibody-antigen interaction. D is, k a / k d This rate is calculated by [formula]. This rate can be determined using a standard assay, such as a Biacore or ELISA assay.

[0034] The term "p" or "antibody:drug ratio" refers to the number of structural units consisting of the linker moiety and eribulin per antibody moiety (Ab) (i.e., drug load). In some embodiments, p is an integer between 1 and 10, 1 and 9, 1 and 8, 1 and 7, 1 and 6, 1 and 5, 1 and 4, 1 and 3, or 1 and 2, preferably an integer between 3 and 4. In compositions comprising multiple ADCs represented by formula I, "p" refers to the average number of structural units consisting of the linker moiety and eribulin per antibody moiety (Ab) (also called the average drug load). When p represents the average drug load, p may be 3 and 4, 3.2 and 3.8, 3.5 and 4.5, 3.6 and 4.4, or 4.

[0035] In some embodiments, p is an integer between 1 and 6, 2 and 5, or 3 and 4. A larger number for p results in a greater number of eribulins per antibody moiety, allowing a single antibody to deliver more eribulins to target cells and further enhancing its pharmacological effect.

[0036] ADCs remain intact when outside the cell, but when internalized into a cell (e.g., a cancer cell), the linker portion of the ADC is cleaved, releasing eribulin into the cell. The linker portion is usually stable outside the cell. In other words, ADCs recognize cells (e.g., cancer cells) that express antigens specific to their antibody portion (Ab) and invade the interior of those cells. Once inside the cell, the linker portion connecting eribulin and the antibody portion (Ab) is cleaved, releasing eribulin and exerting its pharmacological effect.

[0037] 1.Antibody part (Ab) The antibody portion (Ab) in the ADC is an antibody or antigen-binding fragment, particularly an anti-folate receptor alpha (FRA) antibody or its antigen-binding fragment, which can bind to FRA-expressing tumor cells.

[0038] In some embodiments, the antibody or its antigen-binding fragment may bind to folate receptor alpha (FRA) and target FRA-expressing tumor cells. In some embodiments, the antibody or its antigen-binding fragment may include (a) three heavy chain CDRs defined by the Kabat numbering system (heavy chain CDR1 represented by SEQ ID NO: 2, heavy chain CDR2 represented by SEQ ID NO: 3, and heavy chain CDR3 represented by SEQ ID NO: 4) and three light chain CDRs (light chain CDR1 represented by SEQ ID NO: 7, light chain CDR2 represented by SEQ ID NO: 8, and light chain CDR3 represented by SEQ ID NO: 9), or (b) three heavy chain CDRs defined by the IMGT numbering system (heavy chain CDR1 represented by SEQ ID NO: 13, heavy chain CDR2 represented by SEQ ID NO: 14, and heavy chain CDR3 represented by SEQ ID NO: 15) and three light chain CDRs (light chain CDR1 represented by SEQ ID NO: 16, light chain CDR2 represented by SEQ ID NO: 17, and light chain CDR3 represented by SEQ ID NO: 18). In some embodiments, the antibody or its antigen-binding fragment may include a human framework sequence. In some embodiments, the antibody or its antigen-binding fragment includes a heavy chain variable region represented by SEQ ID NO: 23 and a light chain variable region represented by SEQ ID NO: 24. In some embodiments, the antibody or its antigen-binding fragment includes a human IgG1 heavy chain constant region and an Ig kappa light chain constant region. In some embodiments, the antibody or its antigen-binding fragment competes for and / or binds to the same epitope as an antibody containing the heavy chain variable region represented by SEQ ID NO: 23 and the light chain variable region represented by SEQ ID NO: 24. In some embodiments, the antibody or its antigen-binding fragment binds to an epitope containing alanine-histidine-lysine-aspartic acid (Ala-His-Lys-Asp, SEQ ID NO: 345) (Non-Patent Literature 9). In some embodiments, the antibody or antigen-binding fragment binds to an epitope containing NTSQEAHKDVSYL (Asn-Thr-Ser-Gln-Glu-Ala-His-Lys-Asp-Val-Ser-Tyr-Leu, SEQ ID NO: 346).

[0039] In other embodiments, the antibody or its antigen-binding fragment may bind to human epidermal growth factor receptor 2 (HER2) and target HER2-expressing tumor cells. In some embodiments, the antibody or its antigen-binding fragment comprises (a) three heavy chain CDRs (heavy chain CDR1 represented by SEQ ID NO: 71, heavy chain CDR2 represented by SEQ ID NO: 72, and heavy chain CDR3 represented by SEQ ID NO: 73) and three light chain CDRs (light chain CDR1 represented by SEQ ID NO: 74, light chain CDR2 represented by SEQ ID NO: 75, and light chain CDR3 represented by SEQ ID NO: 76) as defined by the Kabat numbering system, or (b) three heavy chain CDRs (heavy chain CDR1 represented by SEQ ID NO: 191, heavy chain CDR2 represented by SEQ ID NO: 192, and heavy chain CDR3 represented by SEQ ID NO: 193) and three light chain CDRs (light chain CDR1 represented by SEQ ID NO: 194, light chain CDR2 represented by SEQ ID NO: 195, and light chain CDR3 represented by SEQ ID NO: 196). In some embodiments, the antibody or its antigen-binding fragment includes a human framework sequence. In some embodiments, the antibody or its antigen-binding fragment includes a heavy chain variable region represented by SEQ ID NO: 27 and a light chain variable region represented by SEQ ID NO: 28. In some embodiments, the antibody or its antigen-binding fragment includes a human IgG1 heavy chain constant region and an Ig kappa light chain constant region. In some embodiments, the antibody or its antigen-binding fragment competes for and / or binds to the same epitope as an antibody containing the heavy chain variable region represented by SEQ ID NO: 27 and the light chain variable region represented by SEQ ID NO: 28.

[0040] In other embodiments, the antibody or its antigen-binding fragment may bind to mesothelin (MSLN) and target MSLN-expressing tumor cells. In some embodiments, the antibody or its antigen-binding fragment comprises (a) three heavy chain CDRs (heavy chain CDR1 represented by SEQ ID NO: 65, heavy chain CDR2 represented by SEQ ID NO: 66, and heavy chain CDR3 represented by SEQ ID NO: 67) and three light chain CDRs (light chain CDR1 represented by SEQ ID NO: 68, light chain CDR2 represented by SEQ ID NO: 69, and light chain CDR3 represented by SEQ ID NO: 70) as defined by the Kabat numbering system, or (b) three heavy chain CDRs (heavy chain CDR1 represented by SEQ ID NO: 185, heavy chain CDR2 represented by SEQ ID NO: 186, and heavy chain CDR3 represented by SEQ ID NO: 187) and three light chain CDRs (light chain CDR1 represented by SEQ ID NO: 188, light chain CDR2 represented by SEQ ID NO: 189, and light chain CDR3 represented by SEQ ID NO: 190) as defined by the IMGT numbering system. In some embodiments, the antibody or antigen-binding fragment includes a heavy chain variable region represented by SEQ ID NO: 25 and a light chain variable region represented by SEQ ID NO: 26. In some embodiments, the antibody or antigen-binding fragment includes a human IgG1 heavy chain constant region and an Ig kappa light chain constant region. In some embodiments, the antibody or antigen-binding competes for and / or binds to the same epitope as the antibody containing the heavy chain variable region represented by SEQ ID NO: 25 and the light chain variable region represented by SEQ ID NO: 26.

[0041] In this embodiment, the preferred antibody portion (Ab) is an anti-folate receptor alpha antibody or its antigen-binding fragment, comprising (a) three heavy chain CDRs containing amino acid sequences represented by 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 containing amino acid sequences represented by 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 containing amino acid sequences represented by 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 containing amino acid sequences represented by SEQ ID NO: 16 (light chain CDR1), SEQ ID NO: 17 (light chain CDR2), and SEQ ID NO: 18 (light chain CDR3). Other preferred antibody moieties (Ab) are (c) three heavy chain CDRs comprising the amino acid sequences represented by 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 represented by 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 represented by 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 the amino acid sequences represented by SEQ ID NO: 194 (light chain CDR1), SEQ ID NO: 195 (light chain CDR2), and SEQ ID NO: 196 (light chain CDR3), an anti-human epidermal growth factor receptor 2 (HER2) antibody or its antigen-binding fragment. More preferably, it is an anti-folate receptor alpha antibody or an antigen-binding fragment thereof.

[0042] The antibody portion (Ab) contains, within its range, any antibody or antigen-binding fragment that specifically binds to the target antigen on cancer cells. The antibody or antigen-binding fragment, as measured by, for example, BIAcore® analysis, has a dissociation constant (K) of ≤1 mM, ≤100 nM, or ≤10 nM. D) can bind to the target antigen in any amount between ) and any amount in between. In certain embodiments, K D The concentration is 1 pM to 500 pM. In some embodiments, K D These ranges are 500 pM to 1 μM, 1 μM to 100 nM, or 100 mM to 10 nM.

[0043] In some embodiments, the antibody portion is a quadruple-chain antibody (also called immunoglobulin) containing two heavy chains and two light chains. In some embodiments, the antibody portion is a double-chain half-body (one light chain and one heavy chain) or antigen-binding fragment of immunoglobulin.

[0044] The amino acid and nucleic acid sequences of the exemplary antibodies described herein are shown in Tables 1-9.

[0045] [Table 1] Abbreviations: xi stands for chimeric antibody, and zu stands for humanized antibody.

[0046] Amino acid sequence of the mAb variable region [Table 2]

[0047] Nucleic acid sequence encoding the mAb variable region [Table 3]

[0048] Amino acid sequence of mAb Kabat CDR [Table 4] TIFF0007856432000011.tif137149

[0049] Nucleic acid sequences encoding mAb Kabat CDRs [Table 5] TIFF0007856432000013.tif137149

[0050] Amino acid sequence of mAb IMGT CDR [Table 6] TIFF0007856432000015.tif137149

[0051] Nucleic acid sequence encoding mAb IMGT CDR [Table 7] TIFF0007856432000017.tif137149

[0052] Amino acid sequence of the full-length mAb Ig chain [Table 8]

[0053] Nucleic acid sequence encoding the full-length mAb Ig chain [Table 9] The enumerated arrays do not include the leader array.

[0054] The ADC may comprise any set of heavy and light chain variable regions listed in the table above (e.g., MORAb-003 heavy and light chain variable regions, or trastuzumab heavy and light chain variable regions), or a set of six CDR sequences from the heavy and light chain set. In some embodiments, the ADC further comprises human heavy and light chain constant regions or fragments thereof. For example, the ADC may comprise a human IgG heavy chain constant region (such as IgG1) and a human kappa or lambda light chain constant region. The antibody moiety comprises a human immunoglobulin G subtype 1 (IgG1) heavy chain constant region together with a human Ig kappa light chain constant region.

[0055] The cancer antigen targeted by ADCs may be folate receptor alpha (FRA).

[0056] The anti-FRA antibody or its antigen-binding fragment may include three heavy chain CDRs (heavy chain CDR1 represented by SEQ ID NO: 2, heavy chain CDR2 represented by SEQ ID NO: 3, and heavy chain CDR3 represented by SEQ ID NO: 4) and three light chain CDRs (light chain CDR1 represented by SEQ ID NO: 7, light chain CDR2 represented by SEQ ID NO: 8, and light chain CDR3 represented by SEQ ID NO: 9), as defined by the Kabat numbering system.

[0057] Furthermore, the anti-FRA antibody or its antigen-binding fragment may also include three heavy chain CDRs (heavy chain CDR1 represented by SEQ ID NO: 13, heavy chain CDR2 represented by SEQ ID NO: 14, and heavy chain CDR3 represented by SEQ ID NO: 15) and three light chain CDRs (light chain CDR1 represented by SEQ ID NO: 16, light chain CDR2 represented by SEQ ID NO: 17, and light chain CDR3 represented by SEQ ID NO: 18), as defined by the IMGT numbering system.

[0058] In various embodiments, the anti-FRA antibody or its antigen-binding fragment includes a heavy chain variable region containing the amino acid sequence represented by SEQ ID NO: 23 and a light chain variable region containing the amino acid sequence represented by SEQ ID NO: 24, or includes a heavy chain variable region containing the amino acid sequence represented by SEQ ID NO: 1 and a light chain variable region containing the amino acid sequence represented by SEQ ID NO: 6. In some embodiments, the anti-FRA antibody or its antigen-binding fragment includes the heavy chain variable region amino acid sequence represented by SEQ ID NO: 23 and the light chain variable region amino acid sequence represented by SEQ ID NO: 24, or a sequence that is at least 95% identical to the above sequences. In some embodiments, the anti-FRA antibody or its antigen-binding fragment has a heavy chain variable region amino acid sequence that is at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 23 and a light chain variable region amino acid sequence that is at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 24.

[0059] Anti-FRA antibodies may contain the human IgG1 heavy chain constant region together with the human Ig kappa light chain constant region.

[0060] The anti-FRA antibody comprises a heavy chain amino acid sequence represented by SEQ ID NO: 1 or a sequence that is at least 95% identical to SEQ ID NO: 1, and a light chain amino acid sequence represented by SEQ ID NO: 6 or a sequence that is at least 95% identical to SEQ ID NO: 6. In certain embodiments, the antibody comprises a heavy chain amino acid sequence represented by SEQ ID NO: 1 and a light chain amino acid sequence represented by SEQ ID NO: 6, or a sequence that is at least 95% identical to the above sequences. In some embodiments, the anti-FRA antibody has a heavy chain amino acid sequence that is at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 1 and / or a light chain amino acid sequence that is at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 6. In some embodiments, the anti-FRA antibody comprises a heavy chain encoded by a nucleotide sequence represented by SEQ ID NO: 11 (containing a nucleotide encoding the leader sequence) or SEQ ID NO: 325 (not containing a nucleotide encoding the leader sequence), and a light chain encoded by a nucleotide represented by SEQ ID NO: 12 (containing a nucleotide encoding the leader sequence) or SEQ ID NO: 326 (not containing a nucleotide encoding the leader sequence). In some embodiments, the heavy chain amino acid sequence is C-terminal lysine deleted. In various embodiments, the anti-FRA antibody has the amino acid sequence of an antibody produced by a cell line deposited on April 24, 2006, under accession number PTA-7552, in accordance with the provisions of the Budapest Convention, or a similar sequence with a deletion of the heavy chain C-terminal lysine. 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.

[0061] The cancer antigen targeted by ADCs may be human epidermal growth factor receptor 2 (HER2).

[0062] The anti-HER2 antibody or its antigen-binding fragment may include three heavy chain CDRs (heavy chain CDR1 represented by SEQ ID NO: 71, heavy chain CDR2 represented by SEQ ID NO: 72, and heavy chain CDR3 represented by SEQ ID NO: 73) and three light chain CDRs (light chain CDR1 represented by SEQ ID NO: 74, light chain CDR2 represented by SEQ ID NO: 75, and light chain CDR3 represented by SEQ ID NO: 76), as defined by the Kabat numbering system.

[0063] The anti-HER2 antibody or its antigen-binding fragment may include three heavy chain CDRs (heavy chain CDR1 represented by SEQ ID NO: 191, heavy chain CDR2 represented by SEQ ID NO: 192, and heavy chain CDR3 represented by SEQ ID NO: 193) and three light chain CDRs (light chain CDR1 represented by SEQ ID NO: 194, light chain CDR2 represented by SEQ ID NO: 195, and light chain CDR3 represented by SEQ ID NO: 196), as defined by the IMGT numbering system.

[0064] In various embodiments, the anti-HER2 antibody or its antigen-binding fragment includes a heavy chain variable region containing the amino acid sequence represented by SEQ ID NO: 27 and a light chain variable region containing the amino acid sequence represented by SEQ ID NO: 28, or includes a heavy chain region containing the amino acid sequence represented by SEQ ID NO: 347 and a light chain region containing the amino acid sequence represented by SEQ ID NO: 308. In some embodiments, the anti-HER2 antibody or its antigen-binding fragment includes the heavy chain variable region amino acid sequence represented by SEQ ID NO: 27 and the light chain variable region amino acid sequence represented by SEQ ID NO: 28, or a sequence that is at least 95% identical to the above sequences. In some embodiments, the anti-HER2 antibody or its antigen-binding fragment has a heavy chain variable region amino acid sequence that is at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 27 and / or a light chain variable region amino acid sequence that is at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 28.

[0065] Anti-HER2 antibodies may contain the human IgG1 heavy chain constant region and the human Ig kappa light chain constant region.

[0066] In various embodiments, the anti-HER2 antibody comprises a heavy chain amino acid sequence represented by SEQ ID NO: 307 or a sequence that is at least 95% identical to SEQ ID NO: 307, and a light chain amino acid sequence represented by SEQ ID NO: 308 or a sequence that is at least 95% identical to SEQ ID NO: 308. In certain embodiments, the antibody comprises a heavy chain amino acid sequence represented by SEQ ID NO: 307 and a light chain amino acid sequence represented by SEQ ID NO: 308, or a sequence that is at least 95% identical to the above sequences. In some embodiments, the anti-HER2 antibody has a heavy chain amino acid sequence that is at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 307, and a light chain amino acid sequence that is at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 308. In various embodiments, the anti-HER2 antibody is trastuzumab or its antigen-binding fragment.

[0067] 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 in which one, two, three, four, five, or six amino acids are added, deleted, or substituted to heavy chain CDR1 (SEQ ID NO: 2 according to Kabat definition, or SEQ ID NO: 13 according to IMGT definition), heavy chain CDR2 (SEQ ID NO: 3 according to Kabat definition, or SEQ ID NO: 14 according to IMGT definition), heavy chain CDR3 (SEQ ID NO: 4 according to Kabat definition, or SEQ ID NO: 15 according to IMGT definition), light chain CDR1 (SEQ ID NO: 7 according to Kabat definition, or SEQ ID NO: 16 according to IMGT definition), light chain CDR2 (SEQ ID NO: 8 according to Kabat definition, or SEQ ID NO: 17 according to IMGT definition), and light chain CDR3 (SEQ ID NO: 9 according to Kabat definition, or SEQ ID NO: 18 according to IMGT definition).

[0068] 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 an amino acid sequence in which one, two, three, four, five, or six amino acids are added, deleted, or substituted to heavy chain CDR1 (SEQ ID NO: 71 according to Kabat definition, or SEQ ID NO: 191 according to IMGT definition), heavy chain CDR2 (SEQ ID NO: 72 according to Kabat definition, or SEQ ID NO: 192 according to IMGT definition), heavy chain CDR3 (SEQ ID NO: 73 according to Kabat definition, or SEQ ID NO: 193 according to IMGT definition), light chain CDR1 (SEQ ID NO: 74 according to Kabat definition, or SEQ ID NO: 194 according to IMGT definition), light chain CDR2 (SEQ ID NO: 75 according to Kabat definition, or SEQ ID NO: 195 according to IMGT definition), and light chain CDR3 (SEQ ID NO: 76 according to Kabat definition, or SEQ ID NO: 196 according to IMGT definition).

[0069] In various embodiments, amino acid substitutions are the substitution of a single residue. Insertions are typically about 1 to 20 amino acid residues, but considerably larger insertions may be permitted as long as biological function (e.g., binding to FRA or HER2) is maintained. Deletions are typically in the range of about 1 to 20 amino acid residues, but in some cases, deletions may be larger. Substitutions, deletions, insertions, or any combination thereof may be used to arrive at the final derivative or variant. Generally, these changes are made with only a few amino acids to minimize alteration of the molecule, particularly the specificity of immunogenic and antigen-binding proteins. However, larger changes may be permitted in certain circumstances. Conservative substitutions are generally made according to the following chart, as shown in Table 10.

[0070] [Table 10]

[0071] Substantially altering function or immunoidentity is achieved by selecting substitutions that are less conserved than those shown in Table 10. For example, substitutions that have a more significant effect on the structure of the polypeptide backbone in the altered region, e.g., α-helix or β-sheet structure, the molecular charge or hydrophobicity at the target site, or the bulk of the side chains may be used. In general, the substitutions predicted to bring about the greatest change in polypeptide properties are (a) to (d) below. (a) A hydrophilic amino acid residue (e.g., Ser or Thr) is replaced by a hydrophobic amino acid residue (e.g., Leu, Ile, Phe, Val or Ala). (b) Cys or Pro is replaced by any other residue. (c) An amino acid residue with an electronegative side chain (e.g., Lys, Arg, or His) is replaced by an electronegative amino acid residue (e.g., Gln or Asn). (d) A residue with a bulky side chain (e.g., Phe) is replaced by an amino acid without a side chain (e.g., Gly).

[0072] In various embodiments, when a variant antibody sequence is used in an ADC, the variant typically exhibits the same qualitative biological activity and induces the same immune response, but if necessary, the variant may be selected to modify the characteristics of the antigen-binding protein. Alternatively, the variant may be designed to alter the biological activity of the antigen-binding protein. For example, the glycosylation site may be modified or removed, as discussed herein.

[0073] In the ADC according to this embodiment, various antibodies may be used to target cancer cells. Suitable antigens that the antibodies target and that are expressed on tumor cells rather than on healthy cells, or expressed on tumor cells at higher levels than on healthy cells, are known in the art. These antibodies can be used together with the linker and eribulin disclosed herein.

[0074] The antibody portion of the ADC may be an FRA-targeted antibody portion such as MORAb-003. In some embodiments, the linker and eribulin of this disclosure may be unexpectedly effective with multiple different tumor-targeting antibodies. When the antibody portion of the ADC is an FRA-targeted antibody portion such as MORAb-003, the ADC may result in a specific improvement in drug-to-antibody ratio, tumor targeting, bystander killing, treatment efficacy, and a reduction in off-target killing. The improvement in treatment efficacy can be measured in vitro or in vivo and may include a reduction in tumor growth rate and / or tumor volume.

[0075] When the antibody portion of the ADC is a HER2-targeted antibody such as trastuzumab, some or all of these desirable functional properties are observed. Furthermore, the antibody portion of the ADC may be a HER2-targeted antibody such as trastuzumab. Additionally, when the antibody portion of the ADC is an MSLN-targeted antibody such as MORAb-009, some or all of these desirable functional properties are observed.

[0076] In some embodiments, free cysteine ​​residues are introduced into the amino acid sequence of the antibody portion. For example, an antibody in which one or more amino acids in the amino acid sequence of the parent antibody are replaced with cysteine ​​(cysteine-modified antibody) can be prepared. For example, cysteine ​​can be introduced into a fragment of the parent Fab antibody as described above to form a cysteine-modified Fab antibody (also known as "ThioFab"). Similarly, cysteine ​​can be introduced into a parent monoclonal antibody to form a cysteine-modified monoclonal antibody (also known as "ThioMab"). A single-site mutation results in a single modified cysteine ​​residue in ThioFab, while a single-site mutation results in two modified cysteine ​​residues in ThioMab due to the dimeric nature of the IgG antibody. The DNA encoding the amino acid sequence variant of the parent polypeptide can be prepared by various methods known in the art (e.g., the method described in WO2006 / 034488). These methods include, but are not limited to, site-directed (or oligonucleotide-mediated) mutagenesis, PCR mutagenesis, and cassette mutagenesis of the initially prepared DNA encoding the polypeptide. Variants of recombinant antibodies can also be constructed by restriction fragment modification or by overlap extension PCR with synthetic oligonucleotides. ADCs of formula I include, but are not limited to, antibodies having one, two, three, or four modified cysteine ​​residues (Lyon et al. (2012) Methods Enzymol. 502: 123-38). In some embodiments, if 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 to eribulin without modification.

[0077] 2. Linker section The linker portion in this embodiment has the following chemical structure and is composed of five units, from the side adjacent to the antibody portion: a maleimide unit (Mal), an oxyethylene unit (PEG), a propionic acid unit (PA), an amino acid unit (Val-Cit), and a self-destructing unit (pAB: p-aminobenzyloxycarbonyl). [ka]

[0078] The linker portion is stable extracellularly to ensure sufficient therapeutic efficacy. In some embodiments, the linker portion is stable extracellularly so that the ADC remains intact when present under extracellular conditions (e.g., before transport or delivery to cells). As used herein, the term “intact” means that the antibody portion remains bound to eribulin. As used herein, the term “stable” in the context of ADC means that, when the ADC is present under extracellular conditions, less than 20%, about 15%, about 10%, about 5%, about 3%, or about 1% of the linkers in the ADC sample (or any percentage in between) are cleaved (or the entire ADC is not otherwise intact).

[0079] Whether the linker portion is stable extracellularly can be determined, for example, by introducing the ADC into plasma for a predetermined period (e.g., 2, 4, 6, 8, 16, or 24 hours) and then quantifying the amount of free drug portion present in the plasma. Stability allows the ADC to localize to target tumor cells and prevents premature release of the drug, which would reduce the therapeutic index of the ADC by indiscriminately damaging both normal and tumor tissue. The linker portion is stable outside the target cell but releases eribulin from the ADC inside the cell so that eribulin can bind to its target (e.g., microtubules). Therefore, the linker portion according to this embodiment (i) maintains the specific binding properties of the antibody portion, (ii) enables delivery of eribulin, e.g., intracellular delivery, via stable binding to the antibody portion, (iii) remains stable and intact until the ADC is transported or delivered to its target site, and (iv) enables the therapeutic effect of eribulin after cleavage, e.g., cytotoxic effect.

[0080] The linker is cleavable under intracellular conditions such that cleavage of the linker sufficiently releases eribulin from the antibody moiety in the intracellular environment, thereby activating eribulin and / or making eribulin therapeutically effective. In some embodiments, eribulin is not cleaved from the antibody moiety until the ADC enters a cell expressing an antigen specific to its antibody moiety, and once it enters the cell, eribulin is cleaved from the antibody moiety. In some embodiments, the linker includes a cleavable moiety that, once cleaved, is positioned so that no portion of the linker or antibody moiety remains bound to eribulin.

[0081] The linker portion contains a valine-citrulline (Val-Cit) amino acid unit. The valine-citrulline (Val-Cit) amino acid unit can be cleaved by cleavage agents (e.g., intracellular peptidases, proteases) present in the intracellular environment (e.g., within lysosomes, endosomes, or caveolae). Peptidases such as cathepsins (e.g., cathepsins B, C, F, H, K, L, O, S, V, X, and W) can cleave valine-citrulline (Val-Cit) sequences and alanine-alanine-asparagine (Ala-Ala-Asn) sequences (Dubowchik et al. (2002) Bioconjugate Chem. 13: 855-69). In the target cell, the linker portion of the ADC is cleaved by enzymes, separating it into the antibody side and the eribulin side. ADCs containing Val-Cit may enable increased stability, reduced off-target cell death, increased target cell death, lower aggregation levels, and / or higher drug load compared to ADCs containing alternative amino acid units (e.g., Gly-Gly).

[0082] Citrulline and eribulin are linked via p-aminobenzyloxycarbonyl (pAB), specifically, the amino group of p-aminobenzyloxycarbonyl is amide-bonded to citrulline, and the carbonyl group is covalently bonded to a primary amine (C-35 amine) on eribulin. p-aminobenzyloxycarbonyl is self-immolative. While not bound by theory, the self-destruction of pAB is thought to be related to a spontaneous 1,6-removal reaction (Jain et al. (2015) Pharm. Res. 32: 3526-40). When the linker portion of the ADC (e.g., Val-Cit) is cleaved in the target cell, the self-destructive unit (pAB) self-destructs, releasing unmodified eribulin without leaving any extra functional groups on the eribulin side.

[0083] Self-destruction chemistry is known in the art and can be readily selected for the ADCs of this disclosure. In various embodiments, a spacer unit that binds a cleavable portion within the linker to a drug portion (e.g., eribulin) is self-destructive and self-destructs simultaneously with, or immediately before or after, the cleavable portion under intracellular conditions.

[0084] On the other hand, a maleimide unit (Mal), an oxyethylene unit (PEG), and a propionic acid unit (PA) are positioned between valine and the antibody Ab. The α,β-unsaturated carbonyl group of maleimide is reactive with cysteine ​​residues in the antibody (especially sulfhydryl groups (-SH)) and plays a role in binding the linker moiety and eribulin to the antibody. The linker moiety has a maleimide unit, and by linking to the antibody via this, the drug load of the antibody (p: number of eribulins per antibody moiety) can be increased.

[0085] An oxyethylene unit (PEG) is a unit consisting of one or more oxyethylene groups and is substantially hydrophilic. It can reduce the extent to which eribulin is effluxed from resistant cancer cells by multidrug-resistant (MDR) or functionally similar transporters. In some embodiments, the linker portion is a short PEG linker, which results in improved stability and reduced aggregation compared to a long PEG linker. In formula (I), the number of oxyethylene groups is denoted by m, where m is an integer from 1 to 10. m is preferably 2. A smaller integer m results in a shorter linker portion, but may allow for lower aggregation levels and / or higher drug loading compared to ADCs having a longer linker portion (e.g., m is 8).

[0086] In some embodiments, ADCs containing a cleavable peptide moiety demonstrate lower aggregation levels, improved antibody-to-drug ratio, increased on-target cancer cell death, decreased off-target non-cancer cell death, and / or higher drug load (p) compared to ADCs containing an alternative cleavable moiety. In some embodiments, the addition of a cleavable moiety increases cytotoxicity and / or potency compared to a non-cleavable linker. In some embodiments, the increased potency and / or cytotoxicity may be observed in cancers expressing the antigen targeted by the antibody moiety of the ADC at moderate levels (e.g., moderate FRA expression). In some embodiments, the cleavable peptide moiety is 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 certain embodiments, an enzymatically cleavable linker (e.g., a cathepsin-cleavable linker) exhibits one or more of the improved properties described above compared to an alternative cleavage mechanism.

[0087] Furthermore, the linker portion can affect the physicochemical properties of the ADC. Since many cytotoxic drugs are inherently hydrophobic, binding them to antibodies with additional hydrophobic portions can lead to aggregation. ADC aggregates are insoluble and, in many cases, limit the achievable drug load on the antibody, thus negatively impacting the efficacy of the ADC. Protein aggregation of biologics generally leads to increased immunogenicity. The linker according to this embodiment results in an ADC with a low aggregation level and a desirable level of drug load.

[0088] In various embodiments, the linker is designed to facilitate intracellular integration, subsequent cleavage, and bystander death (death of adjacent cells) by diffusion of the linker-eribulin and / or eribulin alone to adjacent cells. In some embodiments, the linker is designed to minimize cleavage in the extracellular environment, thereby reducing toxicity to off-target tissues (e.g., non-cancerous tissues), while maintaining bystander death of cancerous tissue surrounding the target cancer tissue that does not express the antigen targeted by the antibody portion of the ADC but does express the antigen. The linker portion according to this embodiment is particularly effective when, for example, an anti-FRA antibody portion such as MORAb-003 and a drug portion such as eribulin are linked to obtain these functional features. In some embodiments, at least some of these functional features can also be observed without the anti-FRA antibody portion and / or without MORAb-003. The linker portion according to this embodiment is effective when, for example, connecting an anti-HER2 antibody portion such as trastuzumab and a drug portion such as eribulin, in order to obtain some or all of these functional features.

[0089] ADCs have been found to demonstrate a specific combination of desirable 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 levels of drug loading (p ≥ approximately 4), low levels of aggregation, stability under storage conditions or in systemic circulation (e.g., serum stability), maintenance of affinity to target-expressing cells comparable to non-conjugated antibodies, potent cytotoxicity against target-expressing cells, low levels of off-target cell death, high levels of bystander death, and / or effective in vivo anticancer activity, compared to all other ADCs using other linker-eribulin and / or antibody moieties.

[0090] 3. Eriblin In this specification, eribulin refers to a synthetic analog of halichondrin B, a macrocyclic compound isolated from the sponge Halichondria okadais. Eribulin is a microtubule dynamics inhibitor and is thought to induce cell cycle arrest in the G2 / M phase by binding to tubulin and inhibiting the construction of the mitotic spindle. The term "eribulin mesylate" refers to the mesylate salt of eribulin, which is marketed under the trade name Halaven®. In the ADC according to this embodiment, eribulin is bound to the linker of the ADC via its primary amino group. [ka]

[0091] 4. Antibody-drug conjugates (ADCs) In this embodiment, the ADC has eribulin bound to the antibody moiety via the linker moiety described above. The antibody moiety may be, for example, an anti-FRA antibody, an anti-mesothelin antibody, or an anti-HER2 antibody such as trastuzumab.

[0092] The ADCs according to this embodiment can selectively deliver effective doses of cytotoxic or cell proliferation inhibitory agents to cancer cells or tumor tissue. The ADCs have been found to have potent cytotoxic and / or cell proliferation inhibitory activity against cells expressing each target antigen (e.g., FRA or HER2). In some embodiments, the cytotoxic and / or cell proliferation inhibitory activity of the ADCs depends on the level of target antigen expression in the cells. In some embodiments, the ADCs of this disclosure are particularly effective in killing cancer cells expressing the target antigen at moderate levels compared to cancer cells expressing the same antigen at low levels.

[0093] 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, carcinomas, lymphomas, blastomas, sarcomas, and leukemias. More specific examples of such cancers include squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, peritoneal cancer, 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, liver cancer, and various types of head and neck cancers. Triple-negative breast cancer refers to breast cancer that is negative for the expression of genes for estrogen receptor (ER), progesterone receptor (PR), or Her2 / neu.

[0094] The term "tumor" refers to any mass of tissue resulting from excessive cell growth or proliferation, whether benign or malignant, including precancerous lesions.

[0095] The terms “cancer cells” and “tumor cells” refer to individual cells or entire populations of cells derived from a tumor, including both non-tumor cells and cancer stem cells. As used herein, the term “tumor cells” is modified by the term “non-tumor” if it refers only to tumor cells that lack the ability to regenerate and differentiate in order to distinguish them from cancer stem cells.

[0096] Exemplary high-FRA-expressing cancers 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 moderate-FRA-expressing cancers include, but are not limited to, gastric cancer and colorectal cancer. Exemplary low-FRA-expressing cancers include, but are not limited to, melanoma and lymphoma. Exemplary high-HER2-expressing cancers include, but are not limited to, breast cancer, gastric cancer, esophageal cancer, ovarian cancer, and endometrial cancer. Exemplary moderate-HER2-expressing cancers include, but are not limited to, lung cancer and bladder cancer.

[0097] The terms "inhibit" or "inhibit," as used herein, mean to reduce by a measurable amount and may include, but do not require, complete prevention or inhibition.

[0098] In this specification, “effective dose” of ADC is a sufficient amount to produce a therapeutic effect after administration, such as a reduction in tumor growth rate or tumor volume, a reduction in cancer symptoms, or some other sign of treatment effectiveness, in order to achieve the purposes specifically stated. The effective dose can be determined by routine methods in relation to the purposes stated. The term “therapeutic effective dose” refers to the amount of ADC that is effective in treating a disease or disorder in a subject. In the case of cancer, a therapeutic effective dose of ADC can reduce the number of cancer cells, reduce tumor size, inhibit tumor metastasis (e.g., slow or stop), inhibit tumor growth (e.g., slow or stop), and / or alleviate one or more symptoms. “Preventive effective dose” refers to the amount that is effective in achieving the desired preventive outcome over the required period of time, in the required dosage. Typically, since preventive doses are used in a subject before the disease or in the early stages of the disease, the preventive effective dose will be less than the therapeutic effective dose.

[0099] As used herein, “to treat” or “therapeutic” and grammatically related terms refer to any improvement in any prognosis of a disease, such as sustained survival, lower morbidity, and / or mitigation of side effects that are byproducts of an alternative therapeutic modality. Complete eradication of the disease is preferred but not a requirement of a treatment action, as is readily apparent in the art. “Treatment” or “to treat” as used herein refers to a subject, e.g., administration of the ADC described to a patient. Treatment may be for the purpose of treating, curing, alleviating, reducing, modifying, correcting, improving, mitigating, or influencing a disorder, a symptom of a disorder, or a disorder, e.g., a predisposition to cancer.

[0100] In some embodiments, labeled ADCs are used. Suitable "labels" include radionuclides, enzymes, substrates, cofactors, inhibitors, fluorescent moieties, chemiluminescent moieties, magnetic particles, and the like.

[0101] As used herein, “protein” means at least two covalently bonded amino acids. This term includes polypeptides, oligopeptides, and peptides. In some embodiments, two or more covalently bonded amino acids are linked by peptide bonds. For example, if a protein is recombinantly produced using an expression system and host cells, the protein may consist of naturally occurring amino acids and peptide bonds. Alternatively, a protein may include synthetic amino acids (e.g., homophenylalanine, citrulline, ornithine, and norleucine) or peptide-mimicking structures, i.e., “peptides or protein analogs,” such as peptoids. Peptoids are an exemplary group of peptide mimetic substances whose side chains are attached to the nitrogen atom of the peptide backbone rather than the α-carbon (as in amino acids) and which have different hydrogen bonding and conformational isomerism characteristics compared to peptides (see, for example, Simon et al. (1992) Proc. Natl. Acad. Sci. USA 89:9367). Therefore, peptoids can be effective in that they may be resistant to proteolysis or other physiological or storage conditions and can permeate cell membranes. In particular, such synthetic amino acids can be incorporated when antibodies are synthesized in vitro by conventional methods well known in the art. In addition, any combination of peptide mimes, synthetic and naturally occurring residues / structures can be used. "Amino acids" also include imino acid residues, such as proline and hydroxyproline. The amino acid "R group" or "side chain" may be in either an (L)- or (S)- configuration. In one specific embodiment, the amino acid is in either an (L)- or (S)- configuration.

[0102] A "recombinant protein" is a protein produced using recombinant techniques, i.e., by the expression of recombinant nucleic acids, using any techniques and methods known in the art. Methods and techniques for producing recombinant proteins are well known in the art.

[0103] An “isolated” protein is, for example, without any accompanying substances that normally accompany it in its native state, constituting at least about 5% by weight, or at least about 50% by weight, of the total protein in a given sample. It is understood that an isolated protein may, depending on the circumstances, constitute 5 to 99.9% by weight of the total protein content. For example, a protein may be produced at fairly high concentrations by the use of an inducible promoter or a high-expression promoter, so that the protein is produced at high concentration levels. This definition includes the production of antibodies in a wide range of organisms and / or host cells that are known in the art.

[0104] Sequence identity and / or similarity in amino acid sequences are not limited to these, but include the local sequence identity algorithm described in Smith and Waterman (1981) Adv. Appl. Math. 2: 482, the sequence identity alignment algorithm described in Needleman and Wunsch (1970) J. Mol. Biol. 48: 443, the similarity search method described in Pearson and Lipman (1988) Proc. Nat. Acad. Sci. USA 85: 2444, and computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA at the Wisconsin Genetics Software Package (GeneticsComputer Group, 575 Science Drive, Madison, Wis.), and BestFIT, FASTA, and TFASTA described in Develeux et al. (1984) Nucl. Acid Res. 12: 387-95). The identity percentage can be determined using standard techniques known in the art, including Fit sequence programs, preferably using default settings or by inspection. The identity percentage may be calculated by referring to “CurrentMethods in Sequence Comparison and Analysis”, MacromoleculeSequencing and Synthesis, Selected Methods and Applications, pp.127-149 (1988), Alan R. Liss, Inc., preferably by FastDB based on the following parameters. Mismatch penalty: 1 Gap penalty: 1 Gap size penalty: 0.33 Connection penalty: 30

[0105] An example of a useful algorithm is PILEUP. PILEUP uses progressive pairwise alignment to construct multiple sequence alignments from a group of related sequences. It can also plot a phylogenetic tree showing the clustering relationships used to construct the alignments. PILEUP uses a simplified version of the progressive alignment method from Feng & Doolittle (1987) J. Mol. Evol. 35: 351-60; this method is similar to the method described by Higgins and Sharp (1989) CABIOS 5:151-3. Useful PILEUP parameters include a default gap weight = 3.00, a default gap length weight = 0.10, and a weight-end gap.

[0106] Another example of a useful algorithm is the BLAST algorithm described in Altschul et al. (1990) J. Mol. Biol. 215: 403-10, Altschul et al. (1997) Nucleic Acids Res. 25: 3389-402, and Karin et al. (1993) Proc. Natl. Acad. Sci. USA 90: 5873-87. A particularly useful BLAST program is the WU-BLAST-2 program derived from Altschul et al. (1996) Methods in Enzymology 266: 460-80. WU-BLAST-2 uses several search parameters, many of which are set to their default values. The adjustable parameters are set as overlap span = l, overlap fraction = 0.125, and word threshold (T) = II. The HSP S and HSP S2 parameters are dynamic values ​​established by a program that itself depends on the composition of the specific sequence being searched for and the composition of the specific database; however, their values ​​may be adjusted to increase sensitivity.

[0107] An additional useful algorithm is Gap BLAST, as reported by Altschul et al. (1993) Nucl. Acids Res. 25: 3389-402. Gap BLAST uses a BLOSUM-62 substitution score; a threshold T parameter set to 9; a two-hit method to initiate gapless extension, loading a cost of 10+k onto the gap length k; Xu set to 16; and Xg set to 40 during the algorithm's database search phase and 67 during the output phase. Gap alignment is initiated by a score corresponding to approximately 22 bits.

[0108] Generally, the amino acid homology, similarity, or identity between the proteins and their variants disclosed herein, including variants of FRA, her2, tubulin sequence, and antibody variable region (including individual variant CDRs), is at least 80% with respect to the sequences shown herein, and more typically, it is preferable that the homology or identity increases to at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and nearly 100% or 100%.

[0109] Similarly, the "percentage of nucleic acid sequence identity (%)" for the nucleic acid sequences of antibodies and other proteins identified herein is defined as the percentage of nucleotide residues in a candidate sequence that are identical to the nucleotide residues of the coding sequence in the antigen-binding protein. The specific method utilizes the BLASTN module of WU-BLAST-2, set to default parameters with overlap span and overlap fraction set to 1 and 0.125, respectively.

[0110] Even if the site or region for introducing an amino acid sequence change is predetermined, the mutation itself does not need to be predetermined. For example, to optimize the performance of a mutation at a given site, random mutagenesis may be performed at the target codon or region, and the expressed antigen-binding protein CDR variants may be screened for the optimal combination of desired activity. Techniques for generating substitutional mutations at predetermined sites in DNA with known sequences, such as MI3 primer mutagenesis and PCR mutagenesis, are well known.

[0111] Next, a method for manufacturing an ADC represented by formula (I), which is one embodiment of the present invention, will be described.

[0112] This embodiment is a method for producing an antibody-drug conjugate (ADC) represented by formula (I), and includes the following steps 1 and 2. [ka] [In the formula, Ab is an antibody or its antigen-binding fragment, D is Eribrin, m is an integer between 1 and 10. p is an integer between 1 and 8. Step 1: A step to obtain a compound represented by formula (B) by reacting eribulin or a salt thereof with a compound represented by formula (A). [ka] [In the formula, m is an integer between 1 and 10, and X is a phenoxy group or a nitrophenoxy group.] [ka] [In the formula, m is an integer between 1 and 10.] Step 2: A step to obtain an antibody-drug conjugate represented by formula (I) by reacting the compound represented by formula (B) with Ab.

[0113] Step 1 is a step in which a compound represented by formula (B) is obtained by reacting eribulin or a salt thereof with a compound represented by formula (A).

[0114] Eribulin may be used in its free form or as a salt of eribulin. An example of an eribulin salt is eribulin mesylate. The amount of eribulin or its salt used may be 1 to 1000 g or 10 to 300 g when converted to the free form of eribulin.

[0115] The compound represented by formula (A) is the linker portion of the ADC, exhibiting reactivity with the antibody at its terminal maleimide structure and reactivity with eribulin at its opposite terminal X. X is a phenoxy group or a nitrophenoxy group. The nitrophenoxy group may be an o-nitrophenoxy group, an m-nitrophenoxy group, or a p-nitrophenoxy group. When X is a phenoxy group or a nitrophenoxy group, it can react with the primary amino group at the terminal of eribulin. The compound represented by formula (A) can be prepared by the method described later.

[0116] The amount of compound represented by formula (A) used may be 0.5 to 2.0 moles, 0.6 to 3.0 moles, 1.0 to 2.0 moles, or 1.3 to 1.8 moles per mole of eribulin.

[0117] Step 1 may use a base. The base can be any base that does not inhibit the reaction between eribulin and the compound represented by formula (A), and examples include tertiary amines such as triethylamine and N,N-diisopropylethylamine, and nitrogen-containing aromatic compounds such as pyridine and 2,6-lutidine. The amount of base used may be 0.5 to 3.0 moles, 1.0 to 2.0 moles, or 1.1 to 1.5 moles per mole of eribulin.

[0118] Step 1 can be carried out without a solvent or in a solvent, and is preferably carried out in a solvent. The solvent can be any solvent that does not inhibit the reaction between eribulin and the compound represented by formula (A), such as N,N-dimethylformamide (DMF), dimethyl sulfoxide (DMSO), N-methylpyrrolidone (NMP), pyridine, etc. The amount of solvent used may be 5 to 20 mL or 6 to 15 mL per 1 g of eribulin.

[0119] The reaction temperature in step 1 should be any temperature at which the reaction between eribulin and the compound represented by formula (A) proceeds. The reaction temperature may be room temperature or between 20 and 25°C.

[0120] To accelerate the reaction in step 1, an accelerator such as 4-dimethylaminopyridine may be used. The amount of accelerator used may be 0.01 to 0.8 g or 0.1 to 0.3 g per mole of eribulin.

[0121] An example of a purification method is shown below, but the method is not limited to this, and any method well known in the field of chemistry may be used for purification. When the reaction in step 1 is completed, the reaction mixture can be purified by placing it on a reversed-phase column. A reversed-phase column is, for example, a column using silica gel having an octadecyl group (C18) as a packing material. After placing the reaction mixture on top of the reversed-phase column, it is extracted with a polar solvent such as acetonitrile or water. If necessary, it is preferable to add acetic acid or the like to make the pH of the extraction solvent acidic. Methylene chloride is added to the obtained extract to transfer the compound represented by formula (B) to the methylene chloride layer (organic layer). During extraction, the pH may be adjusted with an aqueous sodium bicarbonate solution or the like if necessary. The obtained organic layer may be neutralized to remove any remaining acid with an aqueous sodium bicarbonate solution or the like. The obtained organic layer is concentrated under reduced pressure, the residue is dissolved in methylene chloride, methanol, etc., and this is added dropwise to pentane, causing the compound represented by formula (B) to precipitate. The obtained solid is washed with pentane and dried under reduced pressure to obtain the target compound.

[0122] Step 2 is a step to obtain an antibody-drug conjugate represented by formula (I) by a reaction between the compound represented by formula (B) and Ab described above. The cysteine ​​residue in the antibody moiety (Ab) undergoes Michael addition to the maleimide structure of the compound represented by formula (B), thereby forming a covalent bond between the two. Multiple compounds represented by formula (B) can be bound to one antibody moiety (Ab). The number of bonds formed corresponds to p in formula (A).

[0123] The amount of compound represented by formula (B) used may be 1.0 to 8.0 moles or 3.0 to 5.0 moles relative to the antibody portion Ab.

[0124] Step 2 is typically carried out in a buffer. The buffer should not be one that does not denature the antibody Ab used and does not inhibit its binding to the compound represented by formula (B). The buffer may be a phosphate buffer, a borate buffer, a tris(hydroxymethyl)aminomethane buffer, an ethylenediaminetetraacetic acid buffer, or a combination thereof. The pH of the buffer is preferably 6.0 to 8.0, and more preferably 6.5 to 7.5.

[0125] In step 2, a polar organic solvent may be added to the buffer. Any polar organic solvent that is miscible with the buffer can be used to further improve the solubility of the compound represented by formula (B). For example, a solution in which the compound represented by formula (B) is dissolved in a polar organic solvent may be added to the buffer in which the antibody Ab is dissolved.

[0126] Examples of polar organic solvents include N,N-dimethylformamide, N,N-dimethylacetamide, and dimethyl sulfoxide. The amount of organic solvent used may be 60 to 300 mL, 110 to 240 mL, 130 to 210 mL, or 140 to 180 mL per 1 mg of the compound represented by formula (B).

[0127] The reaction temperature in step 2 should be any temperature at which the reaction between the compound represented by formula (B) and antibody Ab proceeds. The reaction temperature may be room temperature or 20-25°C.

[0128] More specifically, the process is as follows: First, the antibody Ab is filtered through a filter (permeable membrane) and then dissolved in buffer. TCEP-HCl (tri(2-carboxyethyl)phosphine hydrochloride) is added to the resulting solution and partially reduced to cleave the intramolecular disulfide bond in the antibody, generating a sulfhydryl group. Next, the compound represented by formula (B) or a solution of the compound in a polar organic solvent is added to bind the compound represented by formula (B) to the antibody. To terminate the reaction in step 2, for example, N-acetylcysteine ​​can be added. The N-acetylcysteine ​​reacts with the remaining compound represented by formula (B) and also with the sulfhydryl group generated by the partial reduction.

[0129] An example of a purification method is shown below, but any well-known method in the field of chemistry may be used. After the reaction is complete, the buffer solution in the reaction mixture is replaced with another buffer solution by tangential flow filtration (TFF). Then, an aqueous citric acid solution is added, filtered, and washed with buffer solution.

[0130] Here, we will explain how to produce the compound represented by formula (A). The compound represented by formula (A) can be produced in two steps from the compound represented by formula (1) and the compound represented by formula (2), for example, as shown below. [ka]

[0131] Step 3 is a step in which the carboxyl group of the compound represented by formula (1) is activated and condensed with the compound represented by formula (2). The condensation of the compound represented by formula (1) and the compound represented by formula (2) yields the compound represented by formula (3).

[0132] The compound represented by formula (1) is available from Tokyo Chemical Industry Co., Ltd. under the trade name "Mal-PEG2-acid" (catalog code: M3203). Alternatively, the compound represented by formula (1) may be produced using polyethylene glycol of the corresponding length as a starting material and tert-butyl acrylate, etc., by methods well known in the field of organic chemistry.

[0133] The amount of compound represented by formula (1) used may be 1.0 to 1.5 moles or 1.1 to 2.0 moles per mole of compound represented by formula (2).

[0134] The compound represented by formula (2) is available from Tokyo Chemical Industry Co., Ltd. under the trade name "Fmoc-Val-Cit-PAB-OH" (catalog code: F1223). Alternatively, the compound represented by formula (2) may be produced using N-Fmoc-citrulline, p-aminobenzyl alcohol, and N-Fmoc-valine by methods well known in the field of organic chemistry. "Fmoc" is a protecting group for amino groups, specifically a 9-fluorenylmethyloxycarbonyl group.

[0135] Step 3 is preferably carried out in the presence of a condensing agent. The condensing agent can be any reagent that can promote the condensation reaction between a carboxylic acid and a primary amine, such as dicyclohexylcarbodiimide (DCC), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, 4-(4,6-dimethoxy-1,3,5-triazine-2-yl)-4-methylmorpholinium chloride (DMT-MM), etc.

[0136] The amount of condensing agent used may be 1.0 to 2.0 moles or 1.3 to 1.5 moles per mole of the compound represented by formula (1).

[0137] Step 3 may use a base. The base can be any base that does not inhibit the reaction between the compound represented by formula (1) and the compound represented by formula (2), and examples include tertiary amines such as triethylamine and N,N-diisopropylethylamine, and nitrogen-containing aromatic compounds such as pyridine and 2,6-lutidine. The amount of base used may be 0.5 to 2.0 moles, 1.0 to 1.5 moles, or 1.1 to 1.3 moles per mole of the compound represented by formula (1).

[0138] Step 3 can be carried out without a solvent or in a solvent, and is preferably carried out in a solvent. The solvent can be any solvent that does not inhibit the reaction between the compound represented by formula (1) and the compound represented by formula (2), such as N,N-dimethylformamide (DMF), tetrahydrofuran (THF), methanol (MeOH), or a mixture thereof. The amount of solvent used may be 10 to 40 mL or 20 to 30 mL per 1 g of the compound represented by formula (1).

[0139] The reaction temperature in step 3 should be the temperature at which the reaction between the compound represented by formula (1) and the compound represented by formula (2) proceeds. The reaction temperature may be room temperature or between 15 and 35°C.

[0140] Step 4 is a step in which the hydroxyl group of the compound represented by formula (3) is acylated with an acylating agent to obtain the compound represented by formula (A).

[0141] The amount of compound represented by formula (3) used can be appropriately selected depending on the reaction. For example, it may be 10 to 500 g, or 100 to 300 g.

[0142] Examples of acyling agents include diphenyl carbonate, bis(4-nitrophenyl) carbonate, bis(2-nitrophenyl) carbonate, bis(3-nitrophenyl) carbonate, phenyl chloroformate, 4-nitrophenyl chloroformate, 2-nitrophenyl chloroformate, and 3-nitrophenyl chloroformate. Some of the acyling agents correspond to X in formula (A), and the type of acyling agent can be selected according to the desired X group selection.

[0143] The amount of acylating agent used may be 1.0 to 5.0 moles or 2.0 to 4.0 moles per mole of the compound represented by formula (3).

[0144] Step 4 can be carried out without a solvent or in a solvent, and is preferably carried out in a solvent. The solvent can be any solvent that does not inhibit the acylation reaction of the compound represented by formula (3), such as N,N-dimethylformamide (DMF), N,N-dimethylacetamide, toluene, dimethyl sulfoxide (DMSO), and N,N-dimethylacetamide (DMA). The amount of solvent used may be 5 to 20 mL or 6 to 15 mL per 1 g of the compound represented by formula (3).

[0145] The reaction temperature in step 4 should be any temperature at which the acylation reaction of the compound represented by formula (3) proceeds. The reaction temperature may be room temperature or 20-25°C. [Examples]

[0146] The present invention will be described in more detail below with reference to manufacturing examples and comparative manufacturing examples. The abbreviations used in the examples are used in the sense commonly used in this industry, for example, as follows: DIPEA: N,N-diisopropylethylamine DMA: N,N-dimethylacetamide DMF: N,N-dimethylformamide DMT-MM: 4-(4,6-dimethoxy-1,3,5-triazine-2-yl)-4-methylmorpholinium chloride EDTA: Ethylenediaminetetraacetic acid THF: Tetrahydrofuran

[0147] <Manufacturing Example 1> (11S,14S)-14-[3-(carbamoylamino)propyl]-1-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)-N-[4-(hydroxymethyl)phenyl]-9,12-dioxo-11-(propan-2-yl)-3,6-dioxa-10,13-diazapentadecane-15-amide

[0148] A mixture of (2S)-2-{[(2S)-2-amino-3-methylbutanoyl]amino}-5-(carbamoylamino)-N-[4-(hydroxymethyl)phenyl]pentanamide (184 g), 3-{2-[2-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)ethoxy]ethoxy}propanoic acid (150 g), DMT-MM (174 g), methanol (2.76 L), and THF (2.76 L) was stirred under a nitrogen atmosphere at 15-35°C for 24 hours. The reaction mixture was concentrated under reduced pressure, and then acetonitrile was added. The precipitated material was filtered, and the precipitate was further washed with acetonitrile. The resulting 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, yield 89%).

[0149] <Manufacturing Example 2> (4-{[(11S,14S)-14-[3-(carbamoylamino)propyl]-1-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)-9,12,15-trioxo-11-(propan-2-yl)-3,6-dioxa-10,13-diazapentadecane-15-yl]amino}phenyl)methyl=4-nitrophenyl=carbonate

[0150] A mixture of the compound obtained in Preparation Example 1 (250 g), bis(4-nitrophenyl)carbonate (615 g), DIPEA (157 g), and DMF (5.0 L) was stirred under a nitrogen atmosphere at 15-35°C for 3 hours. The reaction mixture was concentrated under reduced pressure and then purified by silica gel column (methylene chloride / acetone). The fraction containing the title compound was concentrated under reduced pressure to obtain the title compound (162 g, yield 51%) as a solid.

[0151] <Manufacturing Example 3> (4-{[(11S,14S)-14-[3-(carbamoylamino)propyl]-1-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)-9,12,15-trioxo-11-(propan-2-yl)-3,6-dioxa-10,13-diazapentadecane-15-yl]amino}phenyl)methyl={(2S)-2-hydroxy-3-[(2R,3R,3aS,7R,8aS,9S,10aR,11S,12R,13 aR,13bS,15S,18S,21S,24S,26R,28R,29aS)-3-Methoxy-26-methyl-20,27-DimethylideneHexacosahydro-11,15:18,21:24,28-Trepoxy-7,9-Ethano-12,15-Methane-9H,15H-Flo[3,2-i]Flo[2',3':5,6]Pyrano[4,3-b][1,4]Dioxacyclopentacosin-5(4H)-on-2-yl]propyl}Carbamate [ka]

[0152] A mixture of eribulin mesylate (93 g), DIPEA (26 mL), the compound obtained in Preparation Example 2 (116 g), and DMF (930 mL) was stirred under a nitrogen atmosphere at 20-25°C for 20 hours. The reaction mixture was purified using a reversed-phase column (trade name: Kromasil C18) (acetonitrile / water / acetic acid), and the fraction containing the title compound was extracted with methylene chloride. The organic layer was sequentially washed with aqueous sodium bicarbonate solution and water, and then concentrated under reduced pressure. The concentrated residue was dissolved in a mixed solvent of methylene chloride, methanol, and acetic acid, and this was added dropwise to pentane. The precipitated solid was filtered, washed with pentane, and dried under reduced pressure to obtain the title compound (109.6 g, yield 71%). 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). LCMS (M + H): m / z 1374.9.

[0153] <Comparative Production Example 1>

Chemical Structure

[0154] <Comparative Production Example 1-1> 4-{[(2S)-5-(carbamoylamino)-2-{[(2S)-2-{[(9H-fluoren-9-ylmethoxy)carbonyl]amino}-3-methylbutanoyl]amino}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-DimethylideneHexacosahydro-11,15:18,21:24,28-Trepoxy-7,9-Ethano-12,15-Methane-9H,15H-Flo[3,2-i]Flo[2',3':5,6]Pyrano[4,3-b][1,4]Dioxacyclopentacosin-5(4H)-on-2-yl]propyl}Carbamate

[0155] A mixture of eribulin mesylate (93 g), DIPEA (25 mL), 4-{[(2S)-5-(carbamoylamino)-2-{[(2S)-2-{[(9H-fluoren-9-ylmethoxy)carbonyl]amino}-3-methylbutanoyl]amino}pentanoyl]amino}benzyl=4-nitrophenylcarbonate (103.6 g), and DMF (930 mL) was stirred under a nitrogen atmosphere at 20-25°C for 16 hours.

[0156] <Comparative Manufacturing Example 1-2> 4-{[(2S)-2-{[(2S)-2-amino-3-methylbutanoyl]amino}-5-(carbamoylamino)pentanoyl]aminobenzyl={(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-dimethylidenehexacosahydro-11,15:18,21:24,28-triepoxy-7,9-ethano-12,15-methano-9H,15H-fl[3,2-i]fl[2',3':5,6]pyrano[4,3-b][1,4]dioxacyclopentacosin-5(4H)-on-2-yl]propyl}carbamate

[0157] Diethylamine (234 mL) was added to the reaction mixture and stirred at 20-25°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 potassium carbonate / sodium chloride aqueous solution were added to the aqueous layer, and the organic layer was washed with sodium chloride aqueous solution. The organic layer was dried over magnesium sulfate and then concentrated under reduced pressure. 1 H-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 = 10.8 Hz, 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, 11.2 Hz, 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 =7.2 Hz, 3H). LCMS(M+H): m / z 1135.7.

[0158] <Comparative Manufacturing Example 1-3> (4-{[(11S,14S)-14-[3-(carbamoylamino)propyl]-1-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)-9,12,15-trioxo-11-(propan-2-yl)-3,6-dioxa-10,13-diazapentadecane-15-yl]amino}phenyl)methyl={(2S)-2-hydroxy-3-[(2R,3R,3aS,7R,8aS,9S,10aR,11S,12R,13 aR,13bS,15S,18S,21S,24S,26R,28R,29aS)-3-Methoxy-26-methyl-20,27-DimethylideneHexacosahydro-11,15:18,21:24,28-Trepoxy-7,9-Ethano-12,15-Methane-9H,15H-Flo[3,2-i]Flo[2',3':5,6]Pyrano[4,3-b][1,4]Dioxacyclopentacosin-5(4H)-on-2-yl]propyl}Carbamate

[0159] To the concentrated residue obtained in Comparative Production Example 1-2, 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, and the mixture was stirred at 20-25°C for 3 hours. The reaction mixture was purified using a reversed-phase column (product name: SILMERCK 10013) (acetonitrile / water / acetic acid), and the fraction containing the title compound was extracted with methylene chloride. The organic layer was washed with aqueous sodium bicarbonate solution and water, and then concentrated under reduced pressure. The concentrated residue was dissolved in methylene chloride, methanol, and acetic acid, and the precipitate was filtered after being added dropwise to pentane. The resulting solid was washed with pentane and dried under reduced pressure to obtain the title compound (88.1 g, 3-step yield 57%).

[0160] <Manufacturing Example 4> 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) was mixed with 1493 mL of a dilution buffer solution (10 mmol / L trisodium phosphate, 100 mmol / L sodium chloride, 3 w / v% sucrose, pH 6.5) and 1098 mL of a pH adjustment buffer solution (0.25 mol / L tris(hydroxymethyl)aminomethane hydrochloride, 20 mmol / L EDTA, pH 7.7). At 20°C, an aqueous solution of tris(2-carboxylethyl)phosphine (10 mmol / L, 311 g) was added and the mixture was stirred for 2.5 hours. A DMA solution (8.37 mmol / L, 809 mL) of the compound obtained in Production Example 3 was added and the mixture was stirred for 0.5 hours. An aqueous solution of N-acetylcysteine ​​(30 mmol / L, 452 g) was added to stop the reaction, and the mixture was stirred for 0.5 hours. The antibody-drug conjugate (ADC) was purified by dialysfiltration under the following conditions. (Purification conditions) Filter: Pellicon 3(1.14m 2 30kDa x1, 0.57m 2 30kDa x1) TMP (Transmembrane Pressure Difference): 15 psi Additional buffer: 25 mmol / L citric acid aqueous solution (pH 6.3) Temperature: 17.0~23.0℃ Permeate: 163.2kg

[0161] By adding 3013 g of a 25 mmol / L citric acid aqueous solution (pH 6.3) and 5668 g of a conditioning buffer solution (25 mmol / L citric acid, 1 mol / L sucrose, 0.24 w / v% polysorbate 80, pH 6.3), an aqueous solution of ADC (protein concentration: 10.6 mg / mL, total weight: 20.9 kg, ADC content: 213.430 g, yield: 99%) was obtained. The aqueous solution of ADC was then sterile filtered (Millipore Durapore PVDF).

Claims

1. A method for producing a compound represented by formula (B), 【Chemistry 1】 [In equation (B), m is an integer between 1 and 10.] (i) The compound represented by formula (1) 【Chemistry 2】 [In equation (1), m is an integer between 1 and 10.] By reacting with the compound represented by formula (2), 【Transformation 3】 To obtain the compound represented by formula (3), 【Chemistry 4】 [In equation (3), m is an integer between 1 and 10.] (ii) Producing a compound represented by formula (A) by acyling a hydroxyl group in a compound represented by formula (3) using an acyling agent, wherein the acyling agent contains a phenoxy group or a nitrophenoxy group, (iii) Compound represented by formula (A) 【Transformation 5】 [In formula (A), m is an integer between 1 and 10, and X is a phenoxy group or a nitrophenoxy group.] A method comprising reacting eribulin or a salt thereof with a compound represented by formula (B).

2. A method for producing a compound represented by formula (B), 【Transformation 6】 [In equation (B), m is an integer between 1 and 10.] (a) an acylating agent containing a phenoxy group or a nitrophenoxy group, (b) a compound represented by formula (3). 【Transformation 7】 [In equation (3), m is an integer between 1 and 10.] , and (c) preparing a mixture containing eribulin or a salt thereof, Using the aforementioned acylating agent, the hydroxyl group in the compound represented by formula (3) is acylated to obtain the compound represented by formula (A). 【Transformation 8】 [In formula (A), m is an integer between 1 and 10, and X is a phenoxy group or a nitrophenoxy group.] To obtain, A method comprising reacting a compound represented by formula (A) with eribulin or a salt thereof to produce a compound represented by formula (B).

3. The method according to claim 1 or 2, wherein eribulin or a salt thereof is a methanesulfonate of eribulin.

4. The method according to any one of claims 1 to 3, wherein the reaction of the compound represented by formula (A) with eribulin or a salt thereof is carried out in the presence of a base.

5. The method according to claim 4, wherein the base is a tertiary amine or a nitrogen-containing aromatic compound.

6. The method according to claim 5, wherein the tertiary amine is triethylamine or N,N-diisopropylethylamine.

7. The method according to claim 5 or 6, wherein the nitrogen-containing aromatic compound is pyridine or 2,6-lutidine.

8. The method according to any one of claims 1 to 7, wherein the acylating agent is diphenyl carbonate, bis(4-nitrophenyl) carbonate, bis(2-nitrophenyl) carbonate, bis(3-nitrophenyl) carbonate, phenyl chloroformate, 4-nitrophenyl chloroformate, 2-nitrophenyl chloroformate, or 3-nitrophenyl chloroformate.

9. A method for producing an antibody-drug conjugate represented by formula (I), 【Chemistry 9】 [In the 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.] (i) The compound represented by formula (1) 【Chemistry 10】 [In equation (1), m is an integer between 1 and 10.] It is reacted with the compound represented by formula (2), 【Chemistry 11】 To obtain the compound represented by formula (3), 【Chemistry 12】 [In equation (3), m is an integer between 1 and 10.] (ii) Using an acylating agent to acylate a hydroxyl group in a compound represented by formula (3), wherein the acylating agent contains a phenoxy group or a nitrophenoxy group, thereby acylating a compound represented by formula (A). 【Chemistry 13】 [In formula (A), m is an integer between 1 and 10, and X is a phenoxy group or a nitrophenoxy group.] To manufacture, (iii) Reacting eribulin or a salt thereof with a compound represented by formula (A) to obtain a compound represented by formula (B), 【Chemistry 14】 [In equation (B), m is an integer between 1 and 10.] (iv) A method comprising reacting a compound represented by formula (B) with an antibody or an antigen-binding fragment thereof to obtain an antibody-drug conjugate represented by formula (I).

10. (A) The Ab comprises an antibody or antigen-binding fragment that binds to folate receptor alpha (FRA), and the antibody or antigen-binding fragment is (i) Three heavy chain CDR amino acid sequences represented by 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, and three light chain CDR amino acid sequences represented by SEQ ID NO: 7 (light chain CDR1), SEQ ID NO: 8 (light chain CDR2), and SEQ ID NO: 9 (light chain CDR3), (ii) Three heavy chain CDR amino acid sequences represented by 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, and three light chain CDR amino acid sequences represented by SEQ ID NO: 16 (light chain CDR1), SEQ ID NO: 17 (light chain CDR2), and SEQ ID NO: 18 (light chain CDR3), (iii) A heavy chain region containing the amino acid sequence represented by SEQ ID NO: 1, and a light chain region containing the amino acid sequence represented by SEQ ID NO: 6, or (iv) A heavy chain variable region containing the amino acid sequence represented by SEQ ID NO: 23, and a light chain variable region containing the amino acid sequence represented by SEQ ID NO: 24, Does it include, (B) The above Ab comprises an antibody or antigen-binding fragment that binds to human epidermal growth factor receptor 2 (HER2), and the above antibody or antigen-binding fragment is (v) Three heavy chain CDR amino acid sequences represented by 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, and three light chain CDR amino acid sequences represented by SEQ ID NO: 74 (light chain CDR1), SEQ ID NO: 75 (light chain CDR2), and SEQ ID NO: 76 (light chain CDR3), (vi) Three heavy chain CDR amino acid sequences represented by 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, and three light chain CDR amino acid sequences represented by SEQ ID NO: 194 (light chain CDR1), SEQ ID NO: 195 (light chain CDR2), and SEQ ID NO: 196 (light chain CDR3), (vii) A heavy chain variable region containing the amino acid sequence represented by SEQ ID NO: 27, and a light chain variable region containing the amino acid sequence represented by SEQ ID NO: 28, or (viiii) A heavy chain region containing the amino acid sequence represented by SEQ ID NO: 307 or 347, and a light chain region containing the amino acid sequence represented by SEQ ID NO: 308, including, or (C) The Ab comprises an antibody or antigen-binding fragment that binds to mesothelin, and the antibody or antigen-binding fragment is (ix) Three heavy chain CDR amino acid sequences represented by SEQ ID NO: 65 (heavy chain CDR1), SEQ ID NO: 66 (heavy chain CDR2), and SEQ ID NO: 67 (heavy chain CDR3), as defined by the Kabat numbering system, and three light chain CDR amino acid sequences represented by SEQ ID NO: 68 (light chain CDR1), SEQ ID NO: 69 (light chain CDR2), and SEQ ID NO: 70 (light chain CDR3), (x) Three heavy chain CDR amino acid sequences represented by SEQ ID NO: 185 (heavy chain CDR1), SEQ ID NO: 186 (heavy chain CDR2), and SEQ ID NO: 187 (heavy chain CDR3), as defined by the IMGT numbering system, and three light chain CDR amino acid sequences represented by SEQ ID NO: 188 (light chain CDR1), SEQ ID NO: 189 (light chain CDR2), and SEQ ID NO: 190 (light chain CDR3), (xi) Heavy chain variable region containing the amino acid sequence represented by SEQ ID NO: 25, and light chain variable region containing the amino acid sequence represented by SEQ ID NO: 26 (xi) Heavy chain region containing the amino acid sequence represented by SEQ ID NO: 305, and light chain region containing the amino acid sequence represented by SEQ ID NO: 306 The method according to claim 9, including the method described in claim 9.

11. The method according to claim 9 or 10, wherein p is 3 or 4.

12. The method according to any one of claims 9 to 11, wherein eribulin or a salt thereof is a methanesulfonate of eribulin.

13. The method according to any one of claims 9 to 12, wherein the acylating agent is diphenyl carbonate, bis(4-nitrophenyl) carbonate, bis(2-nitrophenyl) carbonate, bis(3-nitrophenyl) carbonate, phenyl chloroformate, 4-nitrophenyl chloroformate, 2-nitrophenyl chloroformate, or 3-nitrophenyl chloroformate.

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