Dosage of Antibody-Drug Conjugates

Abstract

The present disclosure relates to the field of pharmaceutical preparations, dosage regimens, and administration of antibody-drug conjugates (ADCs). More specifically, ADCs are composed of anti-trophoblast cell surface antigen 2 (TROP2) antibodies linked via a linker to anti-cancer drugs, such as topoisomerase I inhibitors.

Description

Technical Field

[0001] The present disclosure relates to the field of pharmaceutical preparations, dosages, and administrations of antibody-drug conjugates (ADCs). More specifically, the ADC is composed of an anti-trophoblast cell surface antigen 2 (TROP2) antibody linked to a topoisomerase I inhibitor such as a derivative of exatecan via a linker.

[0002] Related Applications This application claims priority to U.S. Provisional Application No. 62 / 853,970, filed on May 29, 2019, and U.S. Provisional Application No. 62 / 896,478, filed on September 5, 2019, under 35 U.S.C. § 119(e), the entire contents of which are incorporated herein by reference.

Background Art

[0003] The following description is provided merely to assist the reader in understanding the present disclosure and is not admitted to describe or constitute the prior art of the present disclosure.

[0004] Trophoblast cell surface antigen 2 (TROP2) is a transmembrane glycoprotein of 323 amino acids encoded by the Tacstd2 gene. It is an intracellular calcium signal transducer that is differentially expressed in many cancers (Ripani E et al., Int. J. Cancer, 76(5), 671-676 (1998) and El Sewedy T et al., Int. J. Cancer, 75(2), 324-330 (1998)). It signals cells for self-renewal, proliferation, invasion, and survival. TROP2 is further involved in immune tolerance common to human trophoblast cells and cancer cells (Faulk WP et al., Proc. Natl. Acad. Sci. 75(4), 1947-1951 (1978) and Lipinski M et al., Proc. Natl. Acad. Sci. 78(8), 5147-5150 (1981)). The DNA sequence and amino acid sequence of human TROP2 are available in public databases, for example, accession numbers NM_002353 and NP_002344 (NCBI).

[0005] TROP2 has been found to be overexpressed in various epithelial cell carcinomas compared to its low-level expression in normal epithelial cells. The expression of TROP2 has also been reported to be correlated with poor prognosis in, among others, colorectal cancer (Ohmachi T et al., Clin. Cancer Res., 12(10), 3057-3063 (2006)), gastric cancer (Muhlmann G et al., J. Clin. Pathol., 62(2), 152-158 (2009)), pancreatic cancer (Fong D et al., Br. J. Cancer, 99(8), 1290-1295 (2008)), oral cancer (Fong D et al., Mod. Pathol., 21(2), 186-191 (2008)), and glioma (Ning S et al., Neurol. Sci., 34(10), 1745-1750 (2013)). Using colorectal cancer cells as a model, it has been further reported that the expression of TROP2 is involved in anchorage-independent cell proliferation and tumor formation of tumor cells in immunodeficient mice (Wang J et al., Mol. Cancer Ther., 7(2), 280-285 (2008)).

[0006] Considering that TROP2 is associated with various cancers, multiple anti-TROP2 antibodies have been prepared and investigated. Among these antibodies, there are reports of unconjugated antibodies showing some antitumor activity in nude mouse xenograft models (International Patent Publication No. 2008 / 144891, International Patent Publication No. 2011 / 145744, International Patent Publication No. 2011 / 155579, and International Patent Publication No. 2013 / 077458) and reports of antibodies showing antitumor activity as antibody-drug conjugates (ADCs) (International Patent Publication No. 2003 / 074566, International Patent Publication No. 2011 / 068845, International Patent Publication No. 2013 / 068946, and U.S. Patent No. 7999083). However, the strength and scope of application of anti-TROP2 antibodies and ADCs are still insufficient, and the medical need to utilize TROP2 as a therapeutic target remains unmet.

[0007] The present disclosure provides TROP2-specific ADCs and their dosages for treating various cancers. Accordingly, the present disclosure meets the above-mentioned need in the art of safe and effective cancer treatment targeting TROP2.

Summary of the Invention

Problems to be Solved by the Invention

[0008] Antitumor antibodies targeting TROP2 have not been successful to date, and many antitumor small molecule compounds have safety problems due to unacceptable side effects and toxicities (even when using compounds with excellent antitumor effects). Therefore, there remains a need to achieve excellent therapeutic effects while at the same time enhancing safety. Accordingly, an object of the present disclosure is to provide an antitumor drug having excellent therapeutic efficacy and safety.

Means for Solving the Problems

[0009] When the anti-tumor compound exatecan is converted into an antibody-drug conjugate via a linker structure moiety by conjugation to an anti-TROP2 antibody capable of targeting, recognizing, binding to, or internalizing in tumor cells, it is possible to acquire cell-killing activity based on the antibody, and the anti-tumor compound can be more reliably delivered to tumor cells and specifically exhibit an anti-tumor effect. Therefore, it is possible to surely exhibit an anti-tumor effect, and it is possible to reduce the dosage of the anti-tumor compound as compared with the single administration of the compound, whereby the negative side effects on normal cells are reduced and the safety is increased.

[0010] Disclosed herein are novel TROP2-targeted ADCs comprising an exatecan derivative and an anti-TROP2 antibody, and methods of using the same.

[0011] In one aspect, the present disclosure provides an anti-TROP2 antibody-drug conjugate for use in the treatment or prevention of cancer, the antibody-drug conjugate comprising an anti-TROP2 antibody and an anti-tumor compound linked by a linker.

[0012] In another aspect, the present disclosure provides a method of treating or preventing cancer in a subject, the method comprising administering to the subject an anti-TROP2 antibody-drug conjugate comprising an anti-TROP2 antibody and an anti-tumor compound linked by a linker.

[0013] In another aspect, the present disclosure provides the use of an anti-TROP2 antibody-drug conjugate in the manufacture of a medicament for treating or preventing cancer, the antibody-drug conjugate comprising an anti-TROP2 antibody and an anti-tumor compound linked by a linker.

[0014] In some embodiments, the linker and the anti-tumor compound have the following formula: -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2-O-CH2-C(=O)-(NH-DX) (In the formula, -(Succinimid-3-yl-N)- is linked to the antibody at its 3-position and linked to the methylene group in the linker structure containing this structure on the nitrogen atom at the 1-position by the following formula:

[0015]

Chemical formula

[0016] It has a structure represented by, and (NH-DX) is the following formula:

[0017]

Chemical formula

[0018] represents a group represented by, wherein the nitrogen atom of the amino group at the 1-position is the linking position).

[0019] In some embodiments, the anti-TROP2 antibody comprises, within its heavy chain variable region, CDRH1 consisting of the amino acid sequence of SEQ ID NO: 23, CDRH2 consisting of the amino acid sequence of SEQ ID NO: 24, and CDRH3 consisting of the amino acid sequence of SEQ ID NO: 25, and, within its light chain variable region, CDRL1 consisting of the amino acid sequence of SEQ ID NO: 26, CDRL2 consisting of the amino acid sequence of SEQ ID NO: 27, and CDRL3 consisting of the amino acid sequence of SEQ ID NO: 28.

[0020] In some embodiments, the average number of binding per antibody of the anti-tumor compound is in the range of 2 to 8, or 3 to 8. In some embodiments, the average number of binding per antibody of the anti-tumor compound is in the range of 3.4 to 4.5. In some embodiments, the average number of binding per antibody of the anti-tumor compound is 4.

[0021] In some embodiments, the antibody comprises a heavy chain variable region comprising amino acids 1 to 121 of SEQ ID NO: 45 and a light chain variable region comprising amino acids 1 to 109 of SEQ ID NO: 46. In some embodiments, the antibody comprises a heavy chain comprising SEQ ID NO: 45 and a light chain comprising SEQ ID NO: 46. In some embodiments, the anti-TROP2 antibody lacks a lysine residue at the carboxyl terminus of the heavy chain.

[0022] In some embodiments, the antibody-drug conjugate is administered to a cancer subject at a dosage within the range of 2 mg / kg to 10 mg / kg. In some embodiments, the antibody-drug conjugate at a dosage of about 4 mg / kg is administered to a cancer subject. In some embodiments, the antibody-drug conjugate at a dosage of about 6 mg / kg is administered to a cancer subject. In some embodiments, the antibody-drug conjugate at a dosage of about 8 mg / kg is administered to a cancer subject.

[0023] In some embodiments, the antibody-drug conjugate is administered by intravenous administration.

[0024] In some embodiments, the antibody-drug conjugate is administered once every three weeks or once every four weeks.

[0025] In some embodiments, the cancer is selected from the group consisting of lung cancer, renal cancer, urothelial cancer, colorectal cancer, prostate cancer, glioblastoma multiforme, ovarian cancer, pancreatic cancer, breast cancer, melanoma, liver cancer, bladder cancer, gastric cancer, cervical cancer, head and neck cancer, and esophageal cancer. In some embodiments, the lung cancer is non-small cell lung cancer (NSCLC).

[0026] In some embodiments, the cancer is resistant or refractory. In some embodiments, the resistance or the refractoriness is the resistance or refractoriness acquired by the cancer for treatment with an anti-cancer drug. In some embodiments, the anti-cancer drug is an EGFR inhibitor, an ALK inhibitor, a platinum-based chemotherapeutic agent or a checkpoint inhibitor. In some embodiments, the anti-cancer drug is gefitinib, erlotinib, osimertinib, afatinib, alectinib, crizotinib, ceritinib, cisplatin, carboplatin, nivolumab, pembrolizumab, atezolizumab, avelumab, ipilimumab, durvalumab, tislelizumab, sintilimab or semaprimab.

[0027] In some embodiments, the cancer is a TROP2-expressing cancer. In some embodiments, the TROP2-expressing cancer is a TROP2-overexpressing cancer. In some embodiments, the TROP2-overexpressing cancer is a cancer that has been given a high score for the expression of TROP2 in immunohistochemistry. In some embodiments, the TROP2-overexpressing cancer is a cancer that has been given a high score for the expression of TROP2 in in situ hybridization.

[0028] In some embodiments, the cancer is inoperable cancer or recurrent cancer.

[0029] Also provided herein is a pharmaceutical composition comprising the antibody-drug conjugate or a salt thereof according to any one of the above-described modes or embodiments as an active ingredient and a pharmaceutically acceptable formulation ingredient.

[0030] The above general description and the following "Mode for Carrying Out the Invention" are exemplary and explanatory, and are intended to provide further explanation of the present disclosure as claimed. Other objects, advantages and novel features will be readily apparent to those skilled in the art from the following "Brief Description of the Drawings" and "Mode for Carrying Out the Invention" of the present disclosure.

Brief Description of the Drawings

[0031]

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[0032] Various embodiments of the novel TROP2-targeted ADC and methods of using the same will be described below with reference to the drawings. The embodiments described below are presented as typical examples of embodiments of the present invention and are not intended to limit the scope of the present invention.

[0033] The anti-TROP2 antibody-drug conjugate of the present invention is an antitumor drug in which an anti-TROP2 antibody is conjugated to an antitumor compound via a linker structure moiety, and will be described in detail below.

[0034] **DEFINITIONS** It should be understood that the methods are not limited to the specific embodiments described, and may themselves be different. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. The scope of the present technology will be limited only by the appended claims.

[0035] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, but representative and exemplary methods and materials are described herein.

[0036] When a range of values is provided, each value between the upper and lower limits of that range, to one tenth of the unit of the lower limit, unless the context clearly dictates otherwise, and any other stated value or values between the stated ranges are understood to be included within the present invention. The upper and lower limits of these smaller ranges may independently be included within the smaller ranges and are also included within the present invention, subject to any specifically excluded limit values within the stated range. When the stated range includes one or both of the limits, the range excluding one or both of those included limits is also included within the present invention.

[0037] As used in the specification and claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise.

[0038] As used herein, the term "comprising" is intended to mean that the compositions and methods include the recited elements but do not exclude others. "Consisting essentially of" when used to define compositions and methods, means excluding any other elements of any substantial importance to the composition or method. "Consisting of" means excluding more than trace elements of other components for the claimed compositions and substantial method steps. Embodiments defined by each of these transitional terms are within the scope of the present disclosure. Thus, methods and compositions can be intended to include (comprising) additional steps and components, alternatively include (consisting essentially of) unimportant steps and compositions, or alternatively, be intended to consist of only the recited method steps or compositions (consisting of).

[0039] As used herein, "about" means plus or minus 10% and the specified number. For example, "about 10" should be understood to mean both "10" and "9 - 11".

[0040] As used herein, "optional" or "optionally" means that the subsequently described event or situation may or may not occur, and that the description includes examples where the event or situation occurs and examples where the event or situation does not occur.

[0041] The terms "individual", "subject", and "patient" are used interchangeably herein and refer to any mammalian individual to be treated according to the methods or uses of the present disclosure, e.g., cows, dogs, cats, horses, monkeys, pigs, camels, bats, or humans. In a preferred embodiment, the subject is a human.

[0042] As used herein, the phrases "effective amount", "therapeutically effective amount", and "therapeutic level" mean the dosage or concentration in a subject in which an ADC is administered in a subject in need of such treatment, i.e., to provide a particular pharmacological effect for treating or preventing cancer (e.g., lung cancer, TROP2-expressing cancer, or resistant or refractory cancer). It is emphasized that the therapeutically effective amount or therapeutic level of an ADC may not necessarily be effective for treating the cancers described herein, even if such dosage is considered by one of ordinary skill in the art to be a therapeutically effective amount. For the sake of convenience only, exemplary dosages, drug delivery amounts, therapeutically effective amounts, and therapeutic levels are provided below. One of ordinary skill in the art can adjust such amounts according to standard practice methods as needed for treating a particular subject and / or condition. The therapeutically effective amount may vary based on the route of administration and dosage form, the age and weight of the subject, and / or the condition of the subject, including the type and severity of the cancer.

[0043] As used herein with respect to cancer, the terms "treatment" or "treating" refer to reducing, suppressing, or eliminating cancer, reducing, suppressing, or eliminating cancer cell proliferation, reducing, suppressing, or eliminating cancer metastasis, or causing a tumor or metastasis to regress or die. Treatment and treating may also, optionally, mean improving the subject's QOL (quality of life) or overall survival rate, even if cancer cell proliferation is not inhibited and / or cancer does not die.

[0044] As used herein with respect to cancer, the terms "prevent" or "preventing" refer to preventing or precluding the occurrence of metastasis (i.e., the growth of cancer at secondary sites where cancer is not present at the start of treatment), and, if the subject achieves remission or the cancer / tumor is completely destroyed or killed, preventing or precluding cancer recurrence.

[0045] As used herein, the term "pharmaceutical composition" refers to a complex of an active agent and an inert or active carrier that renders the composition suitable, inter alia, for diagnostic or therapeutic use in vivo or ex vivo.

[0046] As used herein, the term "pharmaceutically acceptable carrier" refers to any of the standard pharmaceutical carriers such as phosphate buffered saline, water, emulsions (e.g., oil-in-water emulsions or water-in-oil emulsions), and various types of wetting agents. The composition may also contain stabilizers and preservatives. For examples of carriers, stabilizers, and adjuvants, see, e.g., Martin, Remington’s Pharmaceutical Sciences, 15th Ed., Mack Publ. Co., Easton, PA

[1975] .

[0047] As used herein, the terms "parenteral administration" and "administered parenterally" mean a mode of administration other than enteral administration and topical administration, usually by injection, including, but not limited to, intravenous injection and infusion, intramuscular injection and infusion, intraarterial injection and infusion, intrathecal injection and infusion, intracapsular injection and infusion, retrobulbar injection and infusion, intracardiac injection and infusion, intradermal injection and infusion, intraperitoneal injection and infusion, transtracheal injection and infusion, subcutaneous injection and infusion, subepidermal injection and infusion, intraarticular injection and infusion, subcapsular injection and infusion, subdural injection and infusion, intrathecal injection and infusion, intramedullary injection and infusion, and intrasternal injection and infusion.

[0048] As used herein, the terms "systemic administration", "administered systemically", "peripheral administration" and "administered peripherally" mean the administration of a compound, drug or other material, other than direct administration into the central nervous system, such that the compound, drug or other material enters the system of the affected body and undergoes metabolism or other similar processes, for example, subcutaneous administration.

[0049] As used herein, the term "gene" includes not only DNA, but also its mRNA, its cDNA and its cRNA.

[0050] As used herein, the term "polynucleotide" is used in the same sense as nucleic acid and includes DNA, RNA, probes, oligonucleotides and primers.

[0051] As used herein, the terms "polypeptide" and "protein" are used interchangeably.

[0052] As used herein, the term "cell" includes cells in an animal's body and cultured cells.

[0053] As used herein, the term "TROP2" is used in the same sense as the TROP2 protein.

[0054] As used herein, the term "CDR" refers to the complementarity-determining region (CDR). Each of the heavy and light chains of an antibody molecule is known to have three complementarity-determining regions (CDRs). CDRs are also called hypervariable domains and are present within the variable regions of each of the heavy and light chains of an antibody. It is a site with significantly high variability in the primary structure, and there are three separate CDRs in the primary structure of each of the heavy polypeptide chain and the light polypeptide chain. As used herein, with respect to the CDRs of an antibody, the CDRs of the heavy chain are represented as CDRH1, CDRH2, and CDRH3 from the amino-terminal side of the amino acid sequence of the heavy chain, and the CDRs of the light chain are represented as CDRL1, CDRL2, and CDRL3 from the amino-terminal side of the amino acid sequence of the light chain. These sites are proximal to each other in the tertiary structure and determine the specificity for the antigen to which the antibody binds.

[0055] As used herein, the phrase "hybridization is carried out under stringent conditions" refers to a process in which hybridization is carried out at 68 °C in a commercially available hybridization solution, ExpressHyb Hybridization Solution (manufactured by Clontech, Inc.), or hybridization is carried out at 68 °C in the presence of 0.7 - 1.0 M NaCl using the filter having DNA immobilized thereon, followed by washing at 68 °C or under equivalent conditions using a 0.1 - 2× SSC solution (1× SSC solution is composed of 150 mM NaCl and 15 mM sodium citrate), and identification can be achieved under the conditions where hybridization is carried out.

[0056] As used herein, "several" refers to 1 - 10, 1 - 9, 1 - 8, 1 - 7, 1 - 6, 1 - 5, 1 - 4, 1 - 3, or 1 - 2.

[0057] As amino acid substitutions in this specification, conservative amino acid substitutions are preferred. Conservative amino acid substitutions refer to substitutions that occur among groups of amino acids related to the amino acid side chain. Preferred amino acid groups are as follows. Acidic groups (aspartic acid and glutamic acid), basic groups (lysine, arginine and histidine), non-polar groups (alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine and tryptophan) and uncharged polar families (glycine, asparagine, glutamine, cysteine, serine, threonine and tyrosine). More preferred amino acid groups are as follows. Aliphatic hydroxyl groups (serine and threonine), amide-containing groups (asparagine and glutamine), aliphatic groups (alanine, valine, leucine and isoleucine) and aromatic groups (phenylalanine, tryptophan and tyrosine). Such amino acid substitutions are preferably made within a range that does not impair the properties of the substance having the original amino acid sequence.

[0058] Throughout this specification, when a composition is described as having, including, or comprising specific components, or when a process and method are described as having, including, or comprising specific steps, it is contemplated that there are also compositions of the present disclosure consisting essentially of, or consisting of, the recited components, and processes and methods according to the present disclosure consisting essentially of, or consisting of, the recited process steps.

[0059] Generally, compositions specifying percentages are by weight unless otherwise specified. Further, when a variable is not defined, the previous definition of the variable prevails.

[0060] TROP2 TROP2 is a member of the TACSTD family expressed in human trophoblast cells and is a type I transmembrane protein involved in the immune tolerance common to human trophoblast cells and cancer cells.

[0061] For the purposes of the present disclosure, it is possible to directly purify and use TROP2 protein from TROP2-expressing cells of a human or non-human mammal (e.g., rat or mouse), or to prepare and use the cell membrane fraction of the above cells. Further, TROP2 can be obtained by its in vitro synthesis or its production in a host cell by genetic engineering. Specifically, in genetic engineering, after TROP2 cDNA is integrated into a vector capable of expressing TROP2 cDNA, the TROP2 protein can be synthesized in a solution containing enzymes, substrates, and energy substances necessary for transcription and translation, or by expressing TROP2 in another prokaryotic transformation host cell or eukaryotic transformation host cell. Alternatively, the above genetically engineered TROP2-expressing cells, or a cell line expressing TROP2, may be used as the TROP2 protein.

[0062] The DNA sequence and amino acid sequence of TROP2 are available in public databases and can be referenced, for example, by accession numbers NM_002353 and NP_002344 (NCBI).

[0063] Furthermore, TROP2 also includes a protein consisting of an amino acid sequence in which one or more amino acids are substituted, deleted, and / or added in any of the above amino acid sequences of TROP2 and having a biological activity equivalent to that of the said protein.

[0064] The human TROP2 protein includes a signal sequence consisting of 26 amino acid residues at the N-terminus, an extracellular domain consisting of 248 amino acid residues, a transmembrane domain consisting of 23 amino acid residues, and an intracellular domain consisting of 26 amino acid residues.

[0065] Anti-TROP2 antibody The anti-TROP2 antibody used in the anti-TROP2 antibody-drug conjugate of the present disclosure may be derived from any species, and preferred examples of said species include humans, rats, mice, and rabbits. When it is derived from a species other than the human species, it is preferably chimerized or humanized using well-known techniques. The antibody of the present invention may be a polyclonal antibody or a monoclonal antibody, and is preferably a monoclonal antibody.

[0066] The anti-TROP2 antibody can target tumor cells, recognize tumor cells, bind to tumor cells, or internalize into tumor cells, etc., and can be converted into an antibody-drug conjugate by conjugating a compound having antitumor activity via a linker.

[0067] The binding activity of the antibody to tumor cells can be confirmed using flow cytometry. Examples of methods for confirming the internalization of the antibody into tumor cells include: (1) an assay for visualizing the antibody incorporated into cells under a fluorescence microscope using the binding of a secondary antibody (fluorescently labeled) to the therapeutic antibody (Cell Death and Differentiation (2008) 15, 751-761); (2) an assay for measuring the fluorescence intensity incorporated into cells using the binding of a secondary antibody (fluorescently labeled) to the therapeutic antibody (Molecular Biology of the Cell, Vol. 15, 5268-5282, December 2004); or (3) the Mab-ZAP assay using the binding of an immunotoxin to the therapeutic antibody, in which a toxin is released upon incorporation into cells and inhibits cell growth (Bio Techniques 28: 162-165, January 2000). The catalytic region of diphtheria toxin and the recombinant complex protein of protein G may be used as the immunotoxin.

[0068] Since the drug conjugated in the antibody-drug conjugate exerts an antitumor effect, it is preferable but not essential for the antibody itself to have an antitumor effect. For the purpose of specifically and selectively exerting the cell-destroying activity of the antitumor compound against tumor cells, it is important and also preferable for the antibody to have the property of internalizing to move into tumor cells.

[0069] An anti-TROP2 antibody can be obtained using methods commonly practiced in the art, including immunizing an animal with an antigenic polypeptide and recovering and purifying the antibody produced in vivo. The origin of the antigen is not limited to humans, and the animal may be immunized with an antigen derived from non-human animals such as mice, rats, and the like. In this case, the cross-reactivity of the obtained antibody binding to the heterologous antigen with a human antigen can be tested to screen for antibodies applicable to human diseases.

[0070] Alternatively, according to methods known in the art (e.g., Kohler and Milstein, Nature (1975) 256, pp. 495 - 497, and Kennet, R. et al., Monoclonal Antibodies, pp. 365 - 367, Plenum Press, N.Y. (1980)), antibody-producing cells producing antibodies against an antigen are fused with myeloma cells, and then hybridomas capable of obtaining monoclonal antibodies are established.

[0071] An antigen can be obtained by genetically engineering a host cell to produce a gene encoding the antigen protein. Specifically, a vector enabling the expression of the antigen gene is prepared and transferred into the host cell so that the gene is expressed. The antigen thus expressed can be purified. The antibody can also be obtained using a method of immunizing an animal having the genetically engineered antigen-expressing cell or a cell line expressing the antigen.

[0072] An anti-TROP2 antibody can be obtained by techniques known in the art.

[0073] The anti-TROP2 antibodies that can be used in the present invention are not particularly limited. For example, preferably, those specified by the amino acid sequences shown in the sequence listing of this application can be used. The anti-TROP2 antibodies used in the present invention preferably have the characteristics as described below. (1) The following characteristics: (a) Specifically binding to TROP2, and (b) Having the activity of internalizing into TROP2-expressing cells by binding to TROP2 An antibody having the above. (2) The antibody according to (1), wherein TROP2 is human TROP2. (3) The antibody according to (1) or (2), wherein the antibody has the heavy chain complementarity determining regions (CDRs) H1, CDRH2 and CDRH3 of SEQ ID NO: 45, and / or the light chain CDRL1, CDRL2 and CDRL3 of SEQ ID NO: 46. Alternatively or additionally, the antibody has, as the heavy chain complementarity determining region, CDRH1 containing the amino acid sequence represented by SEQ ID NO: 23, CDRH2 containing the amino acid sequence represented by SEQ ID NO: 24, CDRH3 containing the amino acid sequence represented by SEQ ID NO: 25, and, as the light chain complementarity determining region, CDRL1 containing the amino acid sequence represented by SEQ ID NO: 26, CDRL2 containing the amino acid sequence represented by SEQ ID NO: 27, and CDRL3 containing the amino acid sequence represented by SEQ ID NO: 28. The antibody according to (1) or (2). (4) The antibody according to any one of (1) to (3), wherein the constant region thereof is a human-derived constant region. (5) The antibody according to any one of (1) to (4), wherein the antibody is a humanized antibody. (6) The antibody has a heavy chain variable region comprising an amino acid sequence selected from the group consisting of (a) the amino acid sequence set forth at amino acid positions 1 to 121 in SEQ ID NO: 45, (b) an amino acid sequence having at least 95% homology with (a), and (c) an amino acid sequence derived from either of the sequences (a) or (b) by deletion, substitution or addition of at least one amino acid, and a light chain variable region comprising an amino acid sequence selected from the group consisting of (d) the amino acid sequence set forth at amino acid positions 1 to 109 in SEQ ID NO: 46, (e) an amino acid sequence having at least 95% homology with (d), and (f) an amino acid sequence derived from either of the sequences (d) or (e) by deletion, substitution or addition of at least one amino acid, the antibody according to (5). Alternatively or additionally, the antibody has a heavy chain variable region comprising an amino acid sequence selected from the group consisting of (a) the amino acid sequence set forth at amino acid positions 20 to 140 in SEQ ID NO: 12, (b) the amino acid sequence set forth at amino acid positions 20 to 140 in SEQ ID NO: 14, (c) the amino acid sequence set forth at amino acid positions 20 to 140 in SEQ ID NO: 16, (d) an amino acid sequence having at least 95% homology with any of the sequences (a) to (c), and (e) an amino acid sequence derived from any of the sequences (a) to (c) by deletion, substitution or addition of at least one amino acid, and a light chain variable region comprising an amino acid sequence selected from the group consisting of (f) the amino acid sequence set forth at amino acid positions 21 to 129 in SEQ ID NO: 18, (g) the amino acid sequence set forth at amino acid positions 21 to 129 in SEQ ID NO: 20, (h) the amino acid sequence set forth at amino acid positions 21 to 129 in SEQ ID NO: 22, (i) an amino acid sequence having at least 95% homology with any of the sequences (f) to (h), and (j) an amino acid sequence derived from any of the sequences (f) to (h) by deletion, substitution or addition of at least one amino acid, the antibody according to (5). (7) The antibody according to (6), having a heavy chain variable region comprising the amino acid sequence set forth in amino acid positions 1 to 121 in SEQ ID NO: 45 and a light chain variable region comprising the amino acid sequence set forth in amino acid positions 1 to 109 in SEQ ID NO: 46. Alternatively or additionally, the antibody has a heavy chain variable region comprising the amino acid sequence set forth in amino acid positions 20 to 140 in SEQ ID NO: 12 and a light chain variable region comprising the amino acid sequence set forth in amino acid positions 21 to 129 in SEQ ID NO: 18, a heavy chain variable region comprising the amino acid sequence set forth in amino acid positions 20 to 140 in SEQ ID NO: 12 and a light chain variable region comprising the amino acid sequence set forth in amino acid positions 21 to 129 in SEQ ID NO: 20, a heavy chain variable region comprising the amino acid sequence set forth in amino acid positions 20 to 140 in SEQ ID NO: 12 and a light chain variable region comprising the amino acid sequence set forth in amino acid positions 21 to 129 in SEQ ID NO: 22, a heavy chain variable region comprising the amino acid sequence set forth in amino acid positions 20 to 140 in SEQ ID NO: 14 and a light chain variable region comprising the amino acid sequence set forth in amino acid positions 21 to 129 in SEQ ID NO: 18, a heavy chain variable region comprising the amino acid sequence set forth in amino acid positions 20 to 140 in SEQ ID NO: 14 and a light chain variable region comprising the amino acid sequence set forth in amino acid positions 21 to 129 in SEQ ID NO: 20, a heavy chain variable region comprising the amino acid sequence set forth in amino acid positions 20 to 140 in SEQ ID NO: 14 and a light chain variable region comprising the amino acid sequence set forth in amino acid positions 21 to 129 in SEQ ID NO: 22, a heavy chain variable region comprising the amino acid sequence set forth in amino acid positions 20 to 140 in SEQ ID NO: 16 and a light chain variable region comprising the amino acid sequence set forth in amino acid positions 21 to 129 in SEQ ID NO: 18, a heavy chain variable region comprising the amino acid sequence set forth in amino acid positions 20 to 140 in SEQ ID NO: 16 and a light chain variable region comprising the amino acid sequence set forth in amino acid positions 21 to 129 in SEQ ID NO: 20, and a heavy chain variable region comprising the amino acid sequence set forth in amino acid positions 20 to 140 in SEQ ID NO: 16 and a light chain variable region comprising the amino acid sequence set forth in amino acid positions 21 to 129 in SEQ ID NO: 22, and having a heavy chain variable region and a light chain variable region selected from the group consisting of these. The antibody according to (6). (8) The antibody according to (7), having a heavy chain variable region and a light chain variable region selected from the group consisting of a heavy chain variable region containing the amino acid sequence set forth at amino acid positions 20 to 140 in SEQ ID NO: 12 and a light chain variable region containing the amino acid sequence set forth at amino acid positions 21 to 129 in SEQ ID NO: 18; a heavy chain variable region containing the amino acid sequence set forth at amino acid positions 20 to 140 in SEQ ID NO: 14 and a light chain variable region containing the amino acid sequence set forth at amino acid positions 21 to 129 in SEQ ID NO: 18; a heavy chain variable region containing the amino acid sequence set forth at amino acid positions 20 to 140 in SEQ ID NO: 14 and a light chain variable region containing the amino acid sequence set forth at amino acid positions 21 to 129 in SEQ ID NO: 20; and a heavy chain variable region containing the amino acid sequence set forth at amino acid positions 20 to 140 in SEQ ID NO: 16 and a light chain variable region containing the amino acid sequence set forth at amino acid positions 21 to 129 in SEQ ID NO: 22. (9) The antibody according to (6) or (7), comprising a heavy chain comprising the amino acid sequence set forth at amino acid positions 1 to 451 in SEQ ID NO: 45 and a light chain comprising the amino acid sequence set forth at amino acid positions 1 to 214 in SEQ ID NO: 46. Alternatively or additionally, the antibody comprises a heavy chain comprising the amino acid sequence set forth at amino acid positions 20 to 470 in SEQ ID NO: 12 and a light chain comprising the amino acid sequence set forth at amino acid positions 21 to 234 in SEQ ID NO: 18, a heavy chain comprising the amino acid sequence set forth at amino acid positions 20 to 470 in SEQ ID NO: 12 and a light chain comprising the amino acid sequence set forth at amino acid positions 21 to 234 in SEQ ID NO: 20, a heavy chain comprising the amino acid sequence set forth at amino acid positions 20 to 470 in SEQ ID NO: 12 and a light chain comprising the amino acid sequence set forth at amino acid positions 21 to 234 in SEQ ID NO: 22, a heavy chain comprising the amino acid sequence set forth at amino acid positions 20 to 470 in SEQ ID NO: 14 and a light chain comprising the amino acid sequence set forth at amino acid positions 21 to 234 in SEQ ID NO: 18, a heavy chain comprising the amino acid sequence set forth at amino acid positions 20 to 470 in SEQ ID NO: 14 and a light chain comprising the amino acid sequence set forth at amino acid positions 21 to 234 in SEQ ID NO: 20, a heavy chain comprising the amino acid sequence set forth at amino acid positions 20 to 470 in SEQ ID NO: 14 and a light chain comprising the amino acid sequence set forth at amino acid positions 21 to 234 in SEQ ID NO: 22, a heavy chain comprising the amino acid sequence set forth at amino acid positions 20 to 470 in SEQ ID NO: 16 and a light chain comprising the amino acid sequence set forth at amino acid positions 21 to 234 in SEQ ID NO: 18, a heavy chain comprising the amino acid sequence set forth at amino acid positions 20 to 470 in SEQ ID NO: 16 and a light chain comprising the amino acid sequence set forth at amino acid positions 21 to 234 in SEQ ID NO: 20, and a heavy chain comprising the amino acid sequence set forth at amino acid positions 20 to 470 in SEQ ID NO: 16 and a light chain comprising the amino acid sequence set forth at amino acid positions 21 to 234 in SEQ ID NO: 22, wherein the heavy chain and light chain are selected from the group consisting of: the antibody according to (6) or (7). (10) The antibody according to (6) or (7), wherein the antibody comprises a heavy chain comprising the amino acid sequence represented by SEQ ID NO: 45 and a light chain comprising the amino acid sequence represented by SEQ ID NO: 46. Alternatively or additionally, the antibody comprises a heavy chain comprising the amino acid sequence represented by SEQ ID NO: 12 and a light chain comprising the amino acid sequence represented by SEQ ID NO: 18, a heavy chain comprising the amino acid sequence represented by SEQ ID NO: 12 and a light chain comprising the amino acid sequence represented by SEQ ID NO: 20, a heavy chain comprising the amino acid sequence represented by SEQ ID NO: 12 and a light chain comprising the amino acid sequence represented by SEQ ID NO: 22, a heavy chain comprising the amino acid sequence represented by SEQ ID NO: 14 and a light chain comprising the amino acid sequence represented by SEQ ID NO: 18, a heavy chain comprising the amino acid sequence represented by SEQ ID NO: 14 and a light chain comprising the amino acid sequence represented by SEQ ID NO: 20, a heavy chain comprising the amino acid sequence represented by SEQ ID NO: 14 and a light chain comprising the amino acid sequence represented by SEQ ID NO: 22, a heavy chain comprising the amino acid sequence represented by SEQ ID NO: 16 and a light chain comprising the amino acid sequence represented by SEQ ID NO: 18, a heavy chain comprising the amino acid sequence represented by SEQ ID NO: 16 and a light chain comprising the amino acid sequence represented by SEQ ID NO: 20, and a heavy chain comprising the amino acid sequence represented by SEQ ID NO: 16 and a light chain comprising the amino acid sequence represented by SEQ ID NO: 22; the antibody according to (6) or (7) comprising a heavy chain and a light chain selected from the group consisting of these. (11) The antibody according to (8), wherein the antibody comprises a heavy chain comprising the amino acid sequence set forth at amino acid positions 20 to 470 in SEQ ID NO: 12 and a light chain comprising the amino acid sequence set forth at amino acid positions 21 to 234 in SEQ ID NO: 18, a heavy chain comprising the amino acid sequence set forth at amino acid positions 20 to 470 in SEQ ID NO: 14 and a light chain comprising the amino acid sequence set forth at amino acid positions 21 to 234 in SEQ ID NO: 18, a heavy chain comprising the amino acid sequence set forth at amino acid positions 20 to 470 in SEQ ID NO: 14 and a light chain comprising the amino acid sequence set forth at amino acid positions 21 to 234 in SEQ ID NO: 20, and a heavy chain comprising the amino acid sequence set forth at amino acid positions 20 to 470 in SEQ ID NO: 16 and a light chain comprising the amino acid sequence set forth at amino acid positions 21 to 234 in SEQ ID NO: 22; the antibody comprising a heavy chain and a light chain selected from the group consisting of these. (12) The antibody according to any one of (1) to (11), wherein the antibody lacks a lysine residue at the carboxyl terminus of the heavy chain. (13) An antibody obtained by a method for producing an antibody according to any one of (1) to (12), the method comprising culturing a host cell transformed with an expression vector containing a polynucleotide encoding the antibody, and recovering the antibody of interest from the culture obtained in the above step.

[0074] For the purposes of the present disclosure, the entire sequences of SEQ ID NOs: 45 and 46 are shown in Table 1 (and FIG. 2) below.

[0075]

Table 1

[0076] Production of anti-TROP2 antibody The antibody against TROP2 of the present invention can usually be obtained by using a method practiced in the art, which includes immunizing an animal with TROP2 or any polypeptide selected from the amino acid sequence of TROP2, and recovering and purifying the antibody produced in vivo. The biological species of TROP2 used as an antigen is not limited to humans, and animals can be immunized with TROP2 derived from non-human animals such as mice or rats. In this case, by examining the cross-reactivity between the obtained heterologous TROP2 and antibody binding to human TROP2, an antibody applicable to human diseases can be selected.

[0077] Furthermore, a monoclonal antibody can be obtained from a hybridoma established by fusing one or more antibody-producing cells that produce an antibody against TROP2 with myeloma cells according to known methods (e.g., Kohler and Milstein, Nature, (1975) 256, pp. 495 - 497; Kennet, R., ed., Monoclonal Antibodies, pp. 365 - 367, Plenum Press, N.Y. (1980)).

[0078] By expressing the TROP2 gene in a host cell using genetic engineering, TROP2 can be obtained for use as an antigen. Specifically, it is possible to create a vector capable of expressing the TROP2 gene, transfect the obtained vector into a host cell to express the gene, and then purify the expressed TROP2.

[0079] Alternatively, the above genetically engineered TROP2-expressing cells or cell lines expressing TROP2 may be used as the TROP2 protein. Hereinafter, the method for obtaining an antibody against TROP2 will be specifically described.

[0080] (1) Preparation of Antigen

[0081] Examples of antigens used for producing an anti-TROP2 antibody include TROP2, or a polypeptide consisting of a partial amino acid sequence containing at least 6 consecutive amino acids of TROP2, or a derivative obtained by adding a predetermined amino acid sequence or carrier thereto.

[0082] TROP2 can be directly purified from human tumor tissue or human tumor cells and used. Furthermore, TROP2 can be obtained by synthesizing it in vitro or by producing it in a host cell by genetic engineering.

[0083] Specifically, regarding genetic engineering, after the TROP2 cDNA is integrated into a vector capable of expressing the TROP2 cDNA, the antigen can be obtained by synthesizing it in a solution containing enzymes, substrates, and energy substances necessary for transcription and translation, or by expressing TROP2 in another prokaryotic transformed host cell or eukaryotic transformed host cell.

[0084] Furthermore, the antigen can also be obtained as a secreted protein by expressing a fusion protein obtained by linking the extracellular domain of a membrane protein, TROP2, to the constant region of an antibody in an appropriate host-vector system.

[0085] For example, TROP2 cDNA can be obtained by the so-called PCR method in which a polymerase chain reaction (hereinafter referred to as "PCR"; see Saiki, R. K. et al., Science, (1988) 239, pp. 487-489) is performed using a cDNA library expressing TROP2 cDNA as a template and primers that specifically amplify TROP2 cDNA.

[0086] Examples of the in vitro synthesis of the polypeptide include, but are not limited to, the Rapid Translation System (RTS) manufactured by Roche Diagnostics, Inc.

[0087] Examples of prokaryotic host cells include Escherichia coli and Bacillus subtilis. To transform a host cell with a target gene, the host cell is transformed with a plasmid vector containing a replicon, i.e., an origin of replication derived from a species compatible with the host, and regulatory sequences. Furthermore, the vector preferably has a sequence capable of conferring phenotypic selectivity to the transformed cell.

[0088] Examples of eukaryotic host cells include vertebrate cells, insect cells, and yeast cells. As vertebrate cells, for example, monkey COS cells (Gluzman, Y., Cell, (1981) 23, 175-182, ATCC CRL-1650; ATCC: American Type Culture Collection), mouse fibroblast NIH3T3 (ATCC No. CRL-1658), dihydrofolate reductase-deficient strain of Chinese hamster ovary cells (CHO cells; ATCC: CCL-61) (Urlaub, G. and Chasin, L.A., Proc. Natl. Acad. Sci. USA (1980) 77, 4126-4220) and the like are often used, but the cells are not limited thereto.

[0089] The transformant thus obtained can be cultured according to methods commonly practiced in the art, and by culturing the transformant, the target polypeptide is produced intracellularly or extracellularly.

[0090] Suitable media for the culture can be selected by those skilled in the art from various commonly used culture media depending on the host cell used. When Escherichia coli is used, for example, an LB medium supplemented with antibiotics such as ampicillin or IPMG can be used as needed.

[0091] The recombinant protein produced intracellularly or extracellularly by the transformant by such culture can be separated and purified by any of various known separation methods that utilize the physical or chemical properties of the protein.

[0092] Specific examples of the method include treatment with common protein precipitants, ultrafiltration, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, and various types of liquid chromatography such as affinity chromatography, dialysis, and combinations thereof.

[0093] Furthermore, by attaching a label of six histidine residues to the recombinant protein to be expressed, the protein can be efficiently purified using a nickel affinity column. Alternatively, by attaching an IgG Fc region to the recombinant protein to be expressed, the protein can be efficiently purified using a protein A column.

[0094] By combining the above methods, a large amount of target polypeptide can be easily produced in high yield and high purity.

[0095] The above transformant itself can also be used as an antigen. Alternatively, a cell line expressing TROP2 may be used as an antigen. Examples of such cell lines include human lung cancer lines NCI-H322, PC14, NCIH-H2122 and LCAM1, human prostate cancer line PC3, human pancreatic cancer lines BxPC-3, Capan-1 and PK-1, human ovarian cancer line SKOV3, and human colorectal cancer line COLO205. However, the cell lines according to the present invention are not limited to these cell lines as long as they express TROP2.

[0096] (2) Production of anti-TROP2 monoclonal antibody

[0097] Examples of antibodies that specifically bind to TROP2 include monoclonal antibodies that specifically bind to TROP2, and methods for obtaining such antibodies are as described below.

[0098] The production of monoclonal antibodies involves the following (a) An operation step of purifying the biopolymer used as an antigen or preparing antigen-expressing cells, (b) An operation step of preparing antibody-producing cells by immunizing an animal by injecting the antigen, collecting blood, assaying its antibody titer, and determining the time point for removing the spleen, (c) An operation step of preparing myeloma cells (hereinafter referred to as "myeloma"), (d) The step of fusing the antibody-producing cells with the myeloma; (e) The step of screening a group of hybridomas that produce the desired antibody; (f) The step of dividing (cloning) the hybridomas into single cell clones; (g) Optionally, the step of culturing the hybridomas to produce a large amount of monoclonal antibody or breeding the animals transplanted with the hybridomas; (h) The step of examining the monoclonal antibody thus produced for biological activity and binding specificity, or assaying its properties using it as a labeling reagent, and the like are generally required.

[0099] Hereinafter, the method for producing monoclonal antibody after the above steps will be described in detail. However, the method is not limited thereto. For example, antibody-producing cells other than spleen cells and myeloma can be used.

[0100] (a) Purification of antigen

[0101] As the antigen, TROP2 prepared by the method as described above, or its partial peptide can be used.

[0102] Furthermore, a membrane fraction prepared from recombinant cells expressing TROP2, the recombinant cells expressing TROP2 themselves, or partial peptides of the protein of the present invention chemically synthesized by methods known to those skilled in the art can also be used as the antigen.

[0103] Furthermore, a cell line expressing TROP2 can also be used as the antigen.

[0104] (b) Preparation of antibody-producing cells

[0105] The antigen obtained in step (a) is mixed with an adjuvant such as Freund's complete adjuvant or Freund's incomplete adjuvant, or an adjuvant such as aluminum potassium sulfate, and the resulting mixture is used as an immunogen to immunize experimental animals. In an alternative method, the experimental animals are immunized with antigen-expressing cells as the immunogen. As the experimental animals, any animals used in known hybridoma production methods can be used without any problem. Specifically, for example, mice, rats, goats, sheep, cows, horses, or the same species can be used. However, from the viewpoint of the ease of availability of myeloma cells to be fused with the extracted antibody-producing cells, mice or rats are preferably used as the animals to be immunized.

[0106] Furthermore, the strains of mice or rats to be used are not particularly limited. In the case of mice, for example, various strains such as A, AKR, BALB / c, BDP, BA, CE, C3H, 57BL, C57BL, C57L, DBA, FL, HTH, HT1, LP, NZB, NZW, RF, R III, SJL, SWR, WB, and 129, and the same species can be used. In the case of rats, for example, Wistar, Low, Lewis, Sprague, Dolly, ACI, BN, Fischer, and the same species can be used.

[0107] These mice and rats can be obtained from experimental animal breeders / distributors, for example, CLEA Japan, Inc. and Charles River Laboratories Japan, Inc.

[0108] In consideration of the compatibility with the myeloma cells described below, the BALB / c strain is particularly preferred for mice and the Wistar strain and the Low strain are particularly preferred for rats as the animals to be immunized.

[0109] Furthermore, in consideration of the antigenic homology between humans and mice, it is also preferable to use mice with a reduced biological function of removing autoantibodies, that is, mice with autoimmune diseases.

[0110] The age of such a mouse or rat at the time of immunization is preferably 5 to 12 weeks old, more preferably 6 to 8 weeks old.

[0111] To immunize an animal with TROP2 or a recombinant thereof, for example, methods known in the art such as, for example, Weir, D.M., Handbook of Experimental Immunology, Volumes I, II, III, Blackwell Scientific Publications, Oxford (1987); Kabat, E.A. and Mayer, M.M., Experimental Immunochemistry, Charles C Thomas Publisher Springfield, Illinois (1964) or the like can be used.

[0112] Among these immunization methods, preferred specific methods in the present invention are, for example, as follows.

[0113] That is, first, a membrane protein fraction serving as an antigen or cells expressing the antigen are administered to the animal intradermally or intraperitoneally. However, in order to increase the immunization efficiency, it is preferable to use a combination of both administration routes. When intradermal administration is performed in the first half and intraperitoneal administration is performed only in the second half or at the last administration, the immunization efficiency may particularly increase.

[0114] The antigen administration schedule varies depending on the type of animal to be immunized, individual differences or the like. However, generally, an administration schedule in which the frequency of antigen administration is 3 to 6 times and the administration interval is 2 to 6 weeks is preferable, and an administration schedule in which the frequency of antigen administration is 3 to 4 times and the administration interval is 2 to 4 weeks is more preferable.

[0115] Furthermore, the antigen dosage varies depending on the type of animal, individual differences or the like, but the dosage is generally set at 0.05 to 5 mg, preferably about 0.1 to 0.5 mg.

[0116] Booster immunization is carried out 1 to 6 weeks, preferably 1 to 4 weeks, more preferably 1 to 3 weeks after administration of the antigen as described above. When the immunogen is a cell, 1×10 6 ~1×10 7 cells are used.

[0117] The dosage of the antigen when performing booster immunization varies depending on the type or size of the animal or the same kind, but for example, in the case of a mouse, the dosage is generally set to 0.05 to 5 mg, preferably 0.1 to 0.5 mg, more preferably about 0.1 to 0.2 mg. When the immunogen is a cell, 1×10 6 ~1×10 7 cells are used.

[0118] Spleen cells or lymphocytes containing antibody-producing cells are aseptically removed from the immunized animal 1 to 10 days, preferably 2 to 5 days, more preferably 2 to 3 days after booster immunization. At this time, when measuring the antibody titer and using an animal with a sufficiently increased antibody titer as a source of antibody-producing cells, subsequent procedures can be carried out more efficiently.

[0119] Examples of methods for measuring the antibody titer used here include the RIA method and the ELISA method, but the methods are not limited thereto. For example, when using the ELISA method, the measurement of the antibody titer in the present invention can be carried out according to the procedures as described below.

[0120] First, a purified or partially purified antigen is adsorbed on the surface of a solid phase such as a 96-well plate for ELISA, and the surface of the solid phase without the adsorbed antigen is covered with a protein irrelevant to the antigen such as bovine serum albumin (BSA). After washing the surface, the surface is contacted with a serially diluted sample (for example, mouse serum) as the primary antibody to bind the antibody in the sample to the antigen.

[0121] Furthermore, as the secondary antibody, an antibody labeled with an enzyme against the mouse antibody is added to and bound to the mouse antibody. After washing, a substrate for the enzyme is added, and the change in absorbance caused by the color development induced by the decomposition of the substrate or a similar one is measured, and the antibody titer is calculated based on the measurement.

[0122] Separation of antibody-producing cells from the spleen cells or lymphocytes of immunized animals can be carried out according to known methods (for example, Kohler et al., Nature (1975), 256, page 495; Kohler et al., Eur. J. Immunol. (1977), 6, page 511; Milstein et al., Nature (1977), 266, page 550; Walsh, Nature (1977), 266, page 495). For example, in the case of spleen cells, a general method can be used in which the spleen is homogenized to obtain cells by filtration through a stainless-steel mesh, and the cells are separated by suspending them in Eagle's minimum essential medium (MEM).

[0123] (c) Preparation of myeloma cells (hereinafter referred to as "myeloma")

[0124] The myeloma cells used for cell fusion are not particularly limited, but appropriate cells can be selected from known cell lines. However, considering convenience, when selecting hybridomas from fused cells, it is preferable to use an HGPRT (hypoxanthine-guanine phosphoribosyltransferase) -deficient strain for which a selection method has been established.

[0125] More specifically, examples of HGPRT-deficient strains include X63-Ag8 (X63), NS1-ANS / 1 (NS1), P3X63-Ag8.U1 (P3U1), X63-Ag8.653 (X63.653), SP2 / 0-Ag14 (SP2 / 0), MPC11-45.6TG1.7 (45.6TG), FO, S149 / 5XXO, and BU.1, which are derived from mice; 210.RSY3.Ag.1.2.3 (Y3), which is derived from rats; and U266AR (SKO-007), GM1500·GTG-A12 (GM1500), UC729-6, LICR-LOW-HMy2 (HMy2), and 8226AR / NIP4-1 (NP41), which are derived from humans. These HGPRT-deficient strains are available, for example, from ATCC or the like.

[0126] These cell lines are subcultured in an appropriate medium such as a medium obtained by adding 8-azaguanine to RPMI1640 medium supplemented with glutamine, 2-mercaptoethanol, gentamicin, and fetal bovine serum (hereinafter referred to as "FBS"), Iscove's modified Dulbecco's medium (IMDM), or Dulbecco's modified Eagle's medium (DMEM). In this case, 3 to 4 days before cell fusion, the cells are subcultured in a normal medium (for example, ASF104 medium (manufactured by Ajinomoto Co., Ltd.) containing 10% FCS) to ensure 2 × 10 7 or more cells on the day of cell fusion.

[0127] (d) Cell fusion

[0128] The fusion between antibody-producing cells and myeloma cells can be appropriately carried out under conditions such that the cell viability does not decrease excessively, according to known methods (Weir, D.M., Handbook of Experimental Immunology, Volumes I, II, III, Blackwell Scientific Publications, Oxford (1987); Kabat, E.A. and Mayer, M.M., Experimental Immunochemistry, Charles C Thomas Publisher, Springfield, Illinois (1964), etc.).

[0129] As such methods, for example, a chemical method of mixing antibody-producing cells and myeloma cells in a solution containing a polymer such as polyethylene glycol at a high concentration, a physical method using electric stimulation, or a method using the same species can be used. Among these methods, specific examples of the chemical method are as described below.

[0130] That is, when polyethylene glycol is used in a solution containing a polymer at a high concentration, the antibody-producing cells and myeloma cells are mixed in a solution of polyethylene glycol having a molecular weight of 1500 to 6000, more preferably 2000 to 4000, at a temperature of 30 to 40 °C, preferably 35 to 38 °C, for 1 to 10 minutes, preferably 5 to 8 minutes.

[0131] (e) Selection of a group of hybridomas

[0132] The method for selecting hybridomas obtained by the above cell fusion is not particularly limited. Usually, the HAT (hypoxanthine, aminopterin, thymidine) selection method (Kohler et al., Nature (1975), 256, page 495; Milstein et al., Nature (1977), 266, page 550) is used.

[0133] This method is effective when obtaining hybridomas using myeloma cells of HGPRT-deficient strains that cannot survive in the presence of aminopterin. That is, by culturing non-fused cells and hybridomas in HAT medium, only hybridomas resistant to aminopterin can be selectively survived and proliferated.

[0134] (f) Division into single cell clones (cloning)

[0135] As a method for cloning hybridomas, known methods such as the methylcellulose method, soft agarose method, or limiting dilution method can be used (see, for example, Barbara, B.M. and Stanley, M.S.: Selected Methods in Cellular Immunology, W.H.Freeman and Company, San Francisco (1980)). Among these methods, in particular, three-dimensional culture methods such as the methylcellulose method are preferred. For example, a group of hybridomas produced by cell fusion is suspended and cultured in a methylcellulose medium such as ClonaCell-HY Selection Medium D (manufactured by StemCell Technologies, Inc., #03804). Then, monoclonal hybridomas can be obtained by collecting the formed hybridoma colonies. Each of the collected hybridoma colonies is cultured, and hybridomas confirmed to have a stable antibody titer in the obtained hybridoma culture supernatant are selected as TROP2 monoclonal antibody-producing hybridoma strains.

[0136] Examples of hybridoma strains established in this way include the TROP2 hybridoma TINA1. In this specification, the antibody produced by the TROP2 hybridoma TINA1 is referred to as the "TINA1 antibody" or simply "TINA1".

[0137] The heavy chain variable region of the TINA1 antibody has the amino acid sequence represented by SEQ ID NO: 2 in the sequence listing. Further, the light chain variable region of the TINA1 antibody has the amino acid sequence represented by SEQ ID NO: 4 in the sequence listing.

[0138] (g) Preparation of monoclonal antibodies by culturing hybridomas

[0139] By culturing the hybridomas thus selected, monoclonal antibodies can be efficiently obtained. However, prior to culturing, it is preferable to screen for hybridomas that produce the target monoclonal antibody.

[0140] In such screening, known methods can be used.

[0141] Measurement of the antibody titer in the present invention can be carried out, for example, by the ELISA method described in item (b) above.

[0142] The hybridomas obtained by the above method can be stored in a frozen state in liquid nitrogen or in a freezer at -80°C or lower.

[0143] After completion of cloning, the medium is changed from HT medium to normal medium, and the hybridomas are cultured.

[0144] Large-scale culture is carried out by rotary culture using a large culture flask or by stirring culture. From the supernatant obtained by large-scale culture, a monoclonal antibody that specifically binds to the protein of the present invention can be obtained by purification using methods known to those skilled in the art, such as gel filtration.

[0145] Furthermore, by injecting the hybridomas into the abdominal cavity of a mouse of the same strain as the hybridoma (for example, the above-mentioned BALB / c) or a Nu / Nu mouse to proliferate the hybridomas, ascites containing a large amount of the monoclonal antibody of the present invention can be obtained.

[0146] When administering hybridomas intraperitoneally, if a mineral oil such as 2,6,10,14 - tetramethylpentadecane (Pristane) is administered 3 to 7 days before, a larger amount of ascites can be obtained.

[0147] For example, an immunosuppressant is injected in advance into the peritoneal cavity of a mouse of the same strain as the hybridoma to inactivate T cells. Twenty days later, 10 6 ~10 7 individual hybridoma clone cells are suspended in a serum - free medium (0.5 mL), and the suspension is administered into the peritoneal cavity of the mouse. Generally, when the abdomen is distended and filled with ascites, the ascites is collected from the mouse. By this method, monoclonal antibodies can be obtained at a concentration about 100 - fold or much higher than the concentration in the culture solution.

[0148] The monoclonal antibody obtained by the above - mentioned method can be purified, for example, by the method described in Weir, D.M.: Handbook of Experimental Immunology, Volumes I, II, III, Blackwell Scientific Publications, Oxford (1978).

[0149] The monoclonal antibody obtained in this way has high antigen specificity for TROP2.

[0150] (h) Assay of monoclonal antibody

[0151] The isotype and subclass of the monoclonal antibody obtained in this way can be determined as follows.

[0152] First, examples of identification methods include the Ouchterlony method, ELISA method, and RIA method.

[0153] The Ouchterlony method is simple, but when the concentration of the monoclonal antibody is low, a concentration - enrichment operation is required.

[0154] On the one hand, when the ELISA method or the RIA method is used, the culture supernatant is directly reacted with the antigen-adsorbed solid phase, and antibodies corresponding to various types of immunoglobulin isotypes and subclasses are used as the secondary antibody, whereby the isotype and subclass of the monoclonal antibody can be identified.

[0155] Furthermore, as a more convenient method, commercially available identification kits (for example, Mouse Typer Kit manufactured by Bio-Rad Laboratories, Inc.) or the like can also be used.

[0156] Furthermore, the quantification of the protein can be carried out by the Folin Lowry method and the calculation method based on the absorbance at 280 nm (1.4 (OD280) = 1 mg / mL of immunoglobulin).

[0157] Furthermore, even when monoclonal antibodies are obtained separately and independently by performing the steps (a) to (h) of (2) again, it is possible to obtain an antibody having cytotoxic activity equivalent to that of the TINA1 antibody, or an antibody comprising a heavy chain containing SEQ ID NO: 45 and a light chain containing SEQ ID NO: 46. As an example of such an antibody, an antibody that binds to the same epitope as the TINA1 antibody, or an antibody comprising a heavy chain containing SEQ ID NO: 45 and a light chain containing SEQ ID NO: 46. When the newly produced monoclonal antibody binds to a partial peptide or a partial tertiary structure to which the TINA1 antibody or an antibody comprising a heavy chain containing SEQ ID NO: 45 and a light chain containing SEQ ID NO: 46 binds, it can be determined that the monoclonal antibody binds to the same epitope. Furthermore, by confirming that the monoclonal antibody competes with the TINA1 antibody or an antibody comprising a heavy chain containing SEQ ID NO: 45 and a light chain containing SEQ ID NO: 46 for binding to TROP2 (i.e., the monoclonal antibody inhibits the binding between the TINA1 antibody or an antibody comprising a heavy chain containing SEQ ID NO: 45 and a light chain containing SEQ ID NO: 46 and TROP2), even when the specific epitope sequence or structure has not been determined, it can be determined that the monoclonal antibody binds to the same epitope as the anti-TROP2 antibody. When it is confirmed that the monoclonal antibody binds to the same epitope as the anti-TROP2 antibody, the monoclonal antibody is strongly expected to have antigen-binding affinity and biological activity equivalent to those of the antibody comprising the TINA1 antibody or a heavy chain containing SEQ ID NO: 45 and a light chain containing SEQ ID NO: 46.

[0158] (3) Other antibodies

[0159] The antibodies of the present invention include not only the above-mentioned monoclonal antibodies against TROP2, but also recombinant antibodies obtained by artificial modification for the purpose of reducing heterologous antigenicity to humans, such as chimeric antibodies, humanized antibodies, and human antibodies. These antibodies can be produced using known methods.

[0160] Examples of chimeric antibodies include antibodies in which the antibody variable region and the antibody constant region are derived from different species, for example, chimeric antibodies in which a mouse-derived antibody variable region or a rat-derived antibody variable region is linked to a human-derived antibody constant region (see Proc. Natl. Acad. Sci. USA, 81, 6851-6855, (1984)).

[0161] Examples of humanized antibodies include antibodies obtained by integrating only the complementarity-determining regions (CDRs) into a human-derived antibody (see Nature (1986) 321, pages 522-525), and antibodies obtained by grafting a part of the framework amino acid residues and the CDR sequences onto a human antibody by the CDR grafting method (International Publication No. 90 / 07861).

[0162] However, the humanized antibody derived from the TINA1 antibody is not limited to a specific humanized antibody as long as the humanized antibody has the CDR sequences of all six types of the TINA1 antibody. The heavy chain variable region of the TINA1 antibody has CDRH1 (TAGMQ) consisting of the amino acid sequence represented by SEQ ID NO: 23 in the Sequence Listing, CDRH2 (WINTHSGVPKYAEDFKG) consisting of the amino acid sequence represented by SEQ ID NO: 24 in the Sequence Listing, and CDRH3 (SGFGSSYWYFDV) consisting of the amino acid sequence represented by SEQ ID NO: 25 in the Sequence Listing. Further, the light chain variable region of the TINA1 antibody has CDRL1 (KASQDVSTAVA) consisting of the amino acid sequence represented by SEQ ID NO: 26 in the Sequence Listing, CDRL2 (SASYRYT) consisting of the amino acid sequence represented by SEQ ID NO: 27 in the Sequence Listing, and CDRL3 (QQHYITPLT) consisting of the amino acid sequence represented by SEQ ID NO: 28 in the Sequence Listing.

[0163] As an example of a humanized antibody of the mouse antibody TINA1, a heavy chain comprising a heavy chain variable region consisting of any one of (1) an amino acid sequence consisting of amino acid residues 20 to 140 of SEQ ID NO: 12, 14 or 16 in the sequence listing or amino acid residues 1 to 121 of SEQ ID NO: 45, (2) an amino acid sequence having at least 95% homology with the above amino acid sequence (1), and (3) an amino acid sequence in which one or more amino acids in the above amino acid sequence (1) are deleted, substituted, or added, and a light chain comprising a light chain variable region consisting of any one of (4) an amino acid sequence consisting of amino acid residues 21 to 129 of SEQ ID NO: 18, 20 or 22 in the sequence listing or amino acid residues 1 to 109 of SEQ ID NO: 46, (5) an amino acid sequence having at least 95% homology with the above amino acid sequence (4), and (6) an amino acid sequence in which one or more amino acids in the above amino acid sequence (4) are deleted, substituted, or added can be exemplified.

[0164] As antibodies having the preferred combination of the above heavy chain and light chain, an antibody comprising a heavy chain containing a variable region comprising amino acids 1 to 121 of SEQ ID NO: 45 and a light chain containing a variable region comprising amino acids 1 to 109 of SEQ ID NO: 46; an antibody comprising a heavy chain containing a variable region consisting of the amino acid sequence from amino acid position 20 to 140 of SEQ ID NO: 12 and a light chain containing a variable region consisting of the amino acid sequence from amino acid position 21 to 129 of SEQ ID NO: 18; an antibody comprising a heavy chain containing a variable region consisting of the amino acid sequence from amino acid position 20 to 140 of SEQ ID NO: 12 and a light chain containing a variable region consisting of the amino acid sequence from amino acid position 21 to 129 of SEQ ID NO: 20; an antibody comprising a heavy chain containing a variable region consisting of the amino acid sequence from amino acid position 20 to 140 of SEQ ID NO: 12 and a light chain containing a variable region consisting of the amino acid sequence from amino acid position 21 to 129 of SEQ ID NO: 22; an antibody comprising a heavy chain containing a variable region consisting of the amino acid sequence from amino acid position 20 to 140 of SEQ ID NO: 14 and a light chain containing a variable region consisting of the amino acid sequence from amino acid position 21 to 129 of SEQ ID NO: 18; an antibody comprising a heavy chain containing a variable region consisting of the amino acid sequence from amino acid position 20 to 140 of SEQ ID NO: 14 and a light chain containing a variable region consisting of the amino acid sequence from amino acid position 21 to 129 of SEQ ID NO: 20; an antibody comprising a heavy chain containing a variable region consisting of the amino acid sequence from amino acid position 20 to 140 of SEQ ID NO: 14 and a light chain containing a variable region consisting of the amino acid sequence from amino acid position 21 to 129 of SEQ ID NO: 22; an antibody comprising a heavy chain containing a variable region consisting of the amino acid sequence from amino acid position 20 to 140 of SEQ ID NO: 16 and a light chain containing a variable region consisting of the amino acid sequence from amino acid position 21 to 129 of SEQ ID NO: 18; an antibody comprising a heavy chain containing a variable region consisting of the amino acid sequence from amino acid position 20 to 140 of SEQ ID NO: 16 and a light chain containing a variable region consisting of the amino acid sequence from amino acid position 21 to 129 of SEQ ID NO: 20; and an antibody comprising a heavy chain containing a variable region consisting of the amino acid sequence from amino acid position 20 to 140 of SEQ ID NO: 16 and a light chain containing a variable region consisting of the amino acid sequence from amino acid position 21 to 129 of SEQ ID NO: 22 can be exemplified.

[0165] Furthermore, as antibodies having a more preferred combination of the above heavy chain and light chain, an antibody comprising a heavy chain containing SEQ ID NO: 45 and a light chain containing SEQ ID NO: 46, an antibody comprising a heavy chain consisting of the amino acid sequence consisting of amino acid positions 20 to 470 of SEQ ID NO: 12 and a light chain consisting of the amino acid sequence consisting of amino acid positions 21 to 234 of SEQ ID NO: 18, an antibody comprising a heavy chain consisting of the amino acid sequence consisting of amino acid positions 20 to 470 of SEQ ID NO: 12 and a light chain consisting of the amino acid sequence consisting of amino acid positions 21 to 234 of SEQ ID NO: 20, an antibody comprising a heavy chain consisting of the amino acid sequence consisting of amino acid positions 20 to 470 of SEQ ID NO: 12 and a light chain consisting of the amino acid sequence consisting of amino acid positions 21 to 234 of SEQ ID NO: 22, an antibody comprising a heavy chain consisting of the amino acid sequence consisting of amino acid positions 20 to 470 of SEQ ID NO: 14 and a light chain consisting of the amino acid sequence consisting of amino acid positions 21 to 234 of SEQ ID NO: 18, an antibody comprising a heavy chain consisting of the amino acid sequence consisting of amino acid positions 20 to 470 of SEQ ID NO: 14 and a light chain consisting of the amino acid sequence consisting of amino acid positions 21 to 234 of SEQ ID NO: 20, an antibody comprising a heavy chain consisting of the amino acid sequence consisting of amino acid positions 20 to 470 of SEQ ID NO: 14 and a light chain consisting of the amino acid sequence consisting of amino acid positions 21 to 234 of SEQ ID NO: 22, an antibody comprising a heavy chain consisting of the amino acid sequence consisting of amino acid positions 20 to 470 of SEQ ID NO: 16 and a light chain consisting of the amino acid sequence consisting of amino acid positions 21 to 234 of SEQ ID NO: 18, an antibody comprising a heavy chain consisting of the amino acid sequence consisting of amino acid positions 20 to 470 of SEQ ID NO: 16 and a light chain consisting of the amino acid sequence consisting of amino acid positions 21 to 234 of SEQ ID NO: 20, and an antibody comprising a heavy chain consisting of the amino acid sequence consisting of amino acid positions 20 to 470 of SEQ ID NO: 16 and a light chain consisting of the amino acid sequence consisting of amino acid positions 21 to 234 of SEQ ID NO: 22 can be exemplified.

[0166] As antibodies having a more excellent and preferable combination of the above heavy chain and light chain, there can be exemplified an antibody comprising a heavy chain containing a variable region comprising amino acids 1 to 121 of SEQ ID NO: 45 and a light chain containing a variable region comprising amino acids 1 to 109 of SEQ ID NO: 46; an antibody comprising a heavy chain containing a variable region consisting of the amino acid sequence consisting of amino acid residues 20 to 140 of SEQ ID NO: 12 and a light chain containing a variable region consisting of the amino acid sequence consisting of amino acid residues 21 to 129 of SEQ ID NO: 18; an antibody comprising a heavy chain containing a variable region consisting of the amino acid sequence consisting of amino acid residues 20 to 140 of SEQ ID NO: 14 and a light chain containing a variable region consisting of the amino acid sequence consisting of amino acid residues 21 to 129 of SEQ ID NO: 18; an antibody comprising a heavy chain containing a variable region consisting of the amino acid sequence consisting of amino acid residues 20 to 140 of SEQ ID NO: 14 and a light chain containing a variable region consisting of the amino acid sequence consisting of amino acid residues 21 to 129 of SEQ ID NO: 20; and an antibody comprising a heavy chain containing a variable region consisting of the amino acid sequence consisting of amino acid residues 20 to 140 of SEQ ID NO: 16 and a light chain containing a variable region consisting of the amino acid sequence consisting of amino acid residues 21 to 129 of SEQ ID NO: 22.

[0167] Furthermore, as antibodies having another more preferable combination of the above heavy chain and light chain, an antibody consisting of a heavy chain containing SEQ ID NO: 45 and a light chain containing SEQ ID NO: 46, an antibody consisting of a heavy chain consisting of the amino acid sequence of SEQ ID NO: 12 and a light chain consisting of the amino acid sequence of SEQ ID NO: 18, an antibody consisting of a heavy chain consisting of the amino acid sequence of SEQ ID NO: 12 and a light chain consisting of the amino acid sequence of SEQ ID NO: 20, an antibody consisting of a heavy chain consisting of the amino acid sequence of SEQ ID NO: 12 and a light chain consisting of the amino acid sequence of SEQ ID NO: 22, an antibody consisting of a heavy chain consisting of the amino acid sequence of SEQ ID NO: 14 and a light chain consisting of the amino acid sequence of SEQ ID NO: 18, an antibody consisting of a heavy chain consisting of the amino acid sequence of SEQ ID NO: 14 and a light chain consisting of the amino acid sequence of SEQ ID NO: 20, an antibody consisting of a heavy chain consisting of the amino acid sequence of SEQ ID NO: 14 and a light chain consisting of the amino acid sequence of SEQ ID NO: 22, an antibody consisting of a heavy chain consisting of the amino acid sequence of SEQ ID NO: 16 and a light chain consisting of the amino acid sequence of SEQ ID NO: 18, an antibody consisting of a heavy chain consisting of the amino acid sequence of SEQ ID NO: 16 and a light chain consisting of the amino acid sequence of SEQ ID NO: 20, an antibody consisting of a heavy chain consisting of the amino acid sequence of SEQ ID NO: 16 and a light chain consisting of the amino acid sequence of SEQ ID NO: 22 can be exemplified.

[0168] As antibodies having an even more excellent and preferable combination of the above heavy chain and light chain, an antibody containing a heavy chain containing SEQ ID NO: 45 and a light chain containing SEQ ID NO: 46, an antibody consisting of a heavy chain consisting of the amino acid sequence from amino acid positions 20 to 470 of SEQ ID NO: 12 and a light chain consisting of the amino acid sequence from amino acid positions 21 to 234 of SEQ ID NO: 18, an antibody consisting of a heavy chain consisting of the amino acid sequence from amino acid positions 20 to 470 of SEQ ID NO: 14 and a light chain consisting of the amino acid sequence from amino acid positions 21 to 234 of SEQ ID NO: 18, an antibody consisting of a heavy chain consisting of the amino acid sequence from amino acid positions 20 to 470 of SEQ ID NO: 14 and a light chain consisting of the amino acid sequence from amino acid positions 21 to 234 of SEQ ID NO: 20, and an antibody consisting of a heavy chain consisting of the amino acid sequence from amino acid positions 20 to 470 of SEQ ID NO: 16 and a light chain consisting of the amino acid sequence from amino acid positions 21 to 234 of SEQ ID NO: 22 can be exemplified.

[0169] Furthermore, as antibodies having even more excellent and preferable combinations of the above heavy and light chains, antibodies comprising a heavy chain consisting of the amino acid sequence consisting of amino acid positions 20 to 469 of SEQ ID NO: 12 and a light chain consisting of the amino acid sequence consisting of amino acid positions 21 to 234 of SEQ ID NO: 18, antibodies comprising a heavy chain consisting of the amino acid sequence consisting of amino acid positions 20 to 469 of SEQ ID NO: 14 and a light chain consisting of the amino acid sequence consisting of amino acid positions 21 to 234 of SEQ ID NO: 18, antibodies comprising a heavy chain consisting of the amino acid sequence consisting of amino acid positions 20 to 469 of SEQ ID NO: 14 and a light chain consisting of the amino acid sequence consisting of amino acid positions 21 to 234 of SEQ ID NO: 20, and antibodies comprising a heavy chain consisting of the amino acid sequence consisting of amino acid positions 20 to 469 of SEQ ID NO: 16 and a light chain consisting of the amino acid sequence consisting of amino acid positions 21 to 234 of SEQ ID NO: 22 can be exemplified.

[0170] By combining a sequence having high homology with the above heavy chain amino acid sequence with a sequence having high homology with the above light chain amino acid sequence, it is possible to select an antibody having biological activity equivalent to each of the above antibodies. Such homology is generally 80% or more homology, preferably 90% or more homology, more preferably 95% or more homology, and most preferably 99% or more homology. Furthermore, by combining amino acid sequences in which one to several amino acid residues are substituted, deleted, or added in the heavy chain amino acid sequence or the light chain amino acid sequence, it is also possible to select an antibody having biological activity equivalent to each of the above antibodies.

[0171] The homology between two amino acid sequences can be determined using the default parameters of the Blast algorithm version 2.2.2 (Altschul, Stephen F., Thomas L. Madden, Alejandro A. Schaeffer, Jinghui Zhang, Zheng Zhang, Webb Miller, and David J. Lipman (1997), "Gapped BLAST and PSI-BLAST: a new generation of protein database search programs", Nucleic Acids Res. 25: 3389-3402). The Blast algorithm can be used through the Internet by accessing the site ncbi.nlm.nih.gov / blast.

[0172] In the heavy chain amino acid sequence represented by SEQ ID NO: 12, 14 or 16 in the Sequence Listing, the amino acid sequence consisting of amino acid residues 1 to 19 is a signal sequence, the amino acid sequence consisting of amino acid residues 20 to 140 is a variable region, and the amino acid sequence consisting of amino acid residues 141 to 470 is a constant region.

[0173] Furthermore, in the light chain amino acid sequence represented by SEQ ID NO: 18, 20 or 22 in the Sequence Listing, the amino acid sequence consisting of amino acid residues 1 to 20 is a signal sequence, the amino acid sequence consisting of amino acid residues 21 to 129 is a variable region, and the amino acid sequence consisting of amino acid residues 130 to 234 is a constant region.

[0174] Furthermore, the antibodies of the present invention include human antibodies that bind to TROP2. An anti-TROP2 human antibody refers to a human antibody having only the sequence of an antibody derived from the human chromosome. An anti-TROP2 human antibody can be obtained by a method using a human antibody-producing mouse having a human chromosome fragment containing a human antibody heavy chain gene and a light chain gene (see Tomizuka, K. et al., Nature Genetics (1997) 16, pp. 133-143; Kuroiwa, Y. et al., Nucl. Acids Res. (1998) 26, pp. 3447-3448; Yoshida, H. et al., Animal Cell Technology: Basic and Applied Aspects, Vol. 10, pp. 69-73 (edited by Kitagawa, Y., Matuda, T. and Iijima, S.), Kluwer Academic Publishers, 1999; Tomizuka, K. et al., Proc. Natl. Acad. Sci. USA (2000) 97, pp. 722-727, etc.).

[0175] Such a human antibody-producing mouse can be specifically produced as follows. A genetically modified animal in which the endogenous immunoglobulin heavy chain locus and the endogenous immunoglobulin light chain locus are disrupted and, instead, a human immunoglobulin heavy chain locus and a human immunoglobulin light chain locus are introduced via a yeast artificial chromosome (YAC) vector or the like is produced by producing knockout animals and transgenic animals and mating these animals.

[0176] Furthermore, according to recombinant DNA technology, eukaryotic cells are transformed by using cDNA encoding each of such heavy and light chains of a human antibody and preferably a vector containing such cDNA, and the transformed cells producing a recombinant human monoclonal antibody are cultured, whereby an antibody can also be obtained from the culture supernatant.

[0177] Here, as the host, for example, eukaryotic cells, preferably mammalian cells such as CHO cells, lymphocytes or myeloma cells can be used.

[0178] Furthermore, methods for obtaining phage display-derived human antibodies selected from human antibody libraries (see Wormstone, I.M. et al., Investigative Ophthalmology & Visual Science. (2002) 43(7), pages 2301-2308; Carmen, S. et al., Briefings in Functional Genomics and Proteomics (2002), 1(2), pages 189-203; Siriwardena, D. et al., Ophthalmology (2002) 109(3), pages 427-431, etc.) are also known.

[0179] For example, the phage display method (Nature Biotechnology (2005), 23, (9), 1105-1116) can be used, in which the variable region of a human antibody is expressed on the surface of a phage as a single-chain antibody (scFv), and phages that bind to an antigen are selected.

[0180] By analyzing the gene of the phage selected based on binding to the antigen, a DNA sequence encoding the variable region of the human antibody that binds to the antigen can be determined.

[0181] When determining the DNA sequence of an scFv that binds to an antigen, a human antibody can be obtained by preparing an expression vector containing the said sequence, introducing the vector into an appropriate host and expressing it (WO 92 / 01047, WO 92 / 20791, WO 93 / 06213, WO 93 / 11236, WO 93 / 19172, WO 95 / 01438, WO 95 / 15388; Annu. Rev. Immunol. (1994) 12, 433-455; Nature Biotechnology (2005) 23(9), 1105-1116).

[0182] When a newly generated human antibody binds to a partial peptide or a partial tertiary structure to which the TINA1 antibody binds, it can be determined that the human antibody binds to the same epitope as the TINA1 antibody. Further, even when a specific epitope sequence or epitope structure has not been determined, by confirming that the human antibody competes with the TINA1 antibody for binding to TROP2 (i.e., the human antibody inhibits the binding between the TINA1 antibody and TROP2), it can be determined that the human antibody binds to the same epitope as the TINA1 antibody. When it is confirmed that the human antibody binds to the same epitope as the TINA1 antibody, the human antibody is strongly expected to have biological activity equivalent to that of the TINA1 antibody.

[0183] The chimeric antibody, humanized antibody, or human antibody obtained by the above method can be evaluated for its binding characteristics to an antigen by a known method or a similar method, and a preferred antibody can be selected.

[0184] As an example of another index for use in comparing antibody characteristics, the stability of an antibody can be exemplified. Differential scanning calorimetry (DSC) is a means capable of rapidly and accurately measuring the midpoint temperature of thermal denaturation (Tm), which is a good index of the relative conformational stability of a protein. By measuring the Tm value using DSC and comparing the values, differences in thermal stability can be compared. The storage stability of an antibody is known to show some correlation with the thermal stability of the antibody (Lori Burton et al., Pharmaceutical Development and Technology (2007) 12, pages 265 - 273), and a preferred antibody can be selected by using the thermal stability as an index. Examples of other indices for selecting an antibody include the following characteristics: high yield in a suitable host cell and low aggregability in an aqueous solution. For example, the antibody showing the highest yield does not necessarily show the highest thermal stability. Therefore, it is necessary to select the most suitable antibody for administration to humans by comprehensively evaluating based on the above indices.

[0185] In the present invention, modified variants of the antibody are also included. The modified variants refer to variants obtained by chemically modifying or biologically modifying the antibody of the present invention. Examples of chemically modified variants include variants obtained by subjecting the antibody to chemical modification by linking a chemical moiety to the amino acid backbone, and variants obtained by subjecting the N-linked carbohydrate chain or O-linked carbohydrate chain to chemical modification. Examples of biologically modified variants include variants obtained by post-translational modification (e.g., N-linked glycosylation or O-linked glycosylation, N-terminal processing or C-terminal processing, amide cleavage, aspartic acid isomerization, or methionine oxidation), and variants obtained by adding a methionine residue to the N-terminus by expressing in a prokaryotic host cell.

[0186] Furthermore, antibodies labeled so as to enable detection or isolation of the antibody or antigen of the present invention, such as enzyme-labeled antibodies, fluorescent-labeled antibodies, and affinity-labeled antibodies, are also included in the meaning of modified variants. Such modified variants of the antibody of the present invention are useful for improving the stability and blood storage of the antibody, reducing its antigenicity, detecting or isolating the antibody or antigen, and the like.

[0187] Furthermore, it is possible to enhance the antibody-dependent cell cytotoxic activity by regulating the modification (such as glycosylation, defucosylation, etc.) of the glycan linked to the antibody of the present invention. International Publication No. WO1999 / 54342, International Publication No. WO2000 / 61739, International Publication No. WO2002 / 31140, etc. are known as methods for regulating the modification of the glycan of the antibody. However, the methods are not limited thereto. In the antibody of the present invention, antibodies in which the modification of the glycan is regulated are also included.

[0188] When producing an antibody by first isolating the antibody gene and then introducing the gene into a suitable host, a combination of a suitable host and a suitable expression vector can be used. Specific examples of antibody genes include combinations of a gene encoding the heavy chain sequence of an antibody described herein and a gene encoding its light chain sequence. When transforming host cells, it is possible to insert the heavy chain sequence gene and the light chain sequence gene into the same expression vector, or to insert them separately into different expression vectors.

[0189] When using eukaryotic cells as hosts, animal cells, plant cells, and eukaryotic microorganisms can be used. Examples of animal cells include mammalian cells, such as monkey COS cells (Gluzman, Y., Cell, (1981) 23, 175-182, ATCC CRL-1650), mouse fibroblast NIH3T3 (ATCC number CRL-1658), and a dihydrofolate reductase-deficient strain of Chinese hamster ovary cells (CHO cells; ATCC: CCL-61) (Urlaub, G. and Chasin, L.A., Proc. Natl. Acad. Sci. USA (1980) 77, 4126-4220).

[0190] When using prokaryotic cells, for example, Escherichia coli and Bacillus subtilis can be exemplified.

[0191] By introducing the desired antibody gene into these cells by transformation and culturing the thus-transformed cells in vitro, an antibody can be obtained. In the above culture method, the yield may vary depending on the antibody sequence. Therefore, it is possible to select an antibody that can be easily produced as a pharmaceutical by using the yield as an index for antibodies having equivalent binding activity. Therefore, the antibody of the present invention also includes an antibody obtained by a method for producing an antibody, which comprises a step of culturing the transformed host cells and a step of recovering the desired antibody from the culture product obtained in the culturing step.

[0192] It is known to delete the lysine residue at the carboxyl terminus of the heavy chain of an antibody produced in cultured mammalian cells (Journal of Chromatography A, 705: 129-134 (1995)), and to delete the two amino acid residues (glycine and lysine) at the carboxyl terminus of the heavy chain of an antibody produced in cultured mammalian cells and amidate the newly located proline residue at the carboxyl terminus (Analytical Biochemistry, 360: 75-83 (2007)). However, such deletions and modifications of the heavy chain sequence do not affect the antigen-binding affinity and effector functions (such as complement activation, antibody-dependent cell cytotoxicity, etc.) of the antibody. Therefore, the antibodies of the present invention include antibodies that have been subjected to such modifications and functional fragments of said antibodies, including deletion mutants in which one or two amino acids are deleted at the carboxyl terminus of the heavy chain, mutants obtained by amidation of said deletion mutants (e.g., heavy chains in which the carboxyl-terminal proline residue is amidated), and the like. The type of deletion mutant having a deletion at the carboxyl terminus of the heavy chain of the antibody according to the present invention is not limited to the above mutants as long as the antigen-binding affinity and effector functions are conserved. The two heavy chains constituting the antibody according to the present invention may be of one type selected from the group consisting of full-length heavy chains and the above deletion mutants, or may be of two types in a combination selected therefrom. The ratio of the amount of each deletion mutant may be affected by the type of cultured mammalian cells producing the antibody according to the present invention and the culture conditions, but a case can be exemplified where one amino acid residue at the carboxyl terminus is deleted in both of the two heavy chains that are included as the main components in the antibody according to the present invention.

[0193] Examples of the isotype of the antibody of the present invention include, for example, IgG (IgG1, IgG2, IgG3, IgG4), and preferably IgG1 or IgG2 can be exemplified.

[0194] As the biological activities of an antibody, generally, antigen-binding activity, the activity of internalizing within cells expressing an antigen by binding to the antigen, the activity of neutralizing the activity of an antigen, the activity of enhancing the activity of an antigen, antibody-dependent cell cytotoxicity (ADCC) activity, complement-dependent cytotoxicity (CDC) activity, and antibody-dependent cell-mediated phagocytosis (ADCP) activity can be exemplified. The function of the antibody of the present invention is the binding activity to TROP2, preferably the activity of internalizing within TROP2-expressing cells by binding to TROP2. Further, in addition to the cell internalization activity, the antibody of the present invention may have ADCC activity, CDC activity, and / or ADCP activity.

[0195] The obtained antibody can be purified until it becomes homogeneous. Separation and purification of the antibody may be carried out using conventional protein separation and purification methods. For example, column chromatography, filter filtration, ultrafiltration, salting out, dialysis, preparative polyacrylamide gel electrophoresis, isoelectric focusing electrophoresis, and the like can be appropriately selected and combined to separate and purify the antibody (Strategies for Protein Purification and Characterization: A Laboratory Course Manual, edited by Daniel R. Marshak et al., Cold Spring Harbor Laboratory Press (1996); Antibodies: A Laboratory Manual. Ed Harlow and David Lane, Cold Spring Harbor Laboratory (1988)), but the methods are not limited thereto.

[0196] Examples of such chromatography include affinity chromatography, ion exchange chromatography, hydrophobic chromatography, gel filtration chromatography, reverse phase chromatography, and adsorption chromatography.

[0197] Such chromatography can be carried out using liquid chromatography such as HPLC or FPLC.

[0198] Examples of columns used in affinity chromatography include protein A columns and protein G columns. For example, examples of columns using a protein A column include Hyper D, POROS, Sepharose FF (Pharmacia), and the like.

[0199] Furthermore, by using the carrier having an antigen immobilized thereon as the carrier, the antibody can also be purified by utilizing the binding property of the antibody to the antigen.

[0200] Anticancer compound In this section, an antitumor compound conjugated to an anti-TROP2 antibody as part of the disclosed antibody-drug conjugate of the present invention will be described.

[0201] The antitumor compound used in the present invention is not particularly limited as long as it has an antitumor effect and a partial structure that enables linkage to a substituent or a linker structure. When a partial or entire linker is cleaved within a tumor cell, the antitumor compound moiety is released to exhibit the antitumor effect of the antitumor compound. When the linker is cleaved at the drug-linking position, the antitumor compound is released within its unmodified structure to exhibit its intrinsic antitumor effect.

[0202] As the antitumor compound used in the present invention, exatecan, which is one of the camptothecin derivatives ((1S,9S)-1-amino-9-ethyl-5-fluoro-2,3-dihydro-9-hydroxy-4-methyl-1H,12H-benzo[de]pyrano[3’,4’:6,7]indolizino[1,2-b]quinoline-10,13(9H,15H)-dione) shown in the following formula, can preferably be used. Exatecan is shown below in Formula 1.

[0203]

Chemical formula

[0204] Exatecan has excellent antitumor effects but has not been commercialized as an antitumor drug. The compound can be easily obtained by known methods, and preferably, the amino group at the 1-position can be used as the linking position to the linker structure. Furthermore, it is also possible to still attach a part of the linker and release exatecan in tumor cells, and it remains an excellent anticancer compound showing excellent antitumor effects even in such a structure.

[0205] Since exatecan has a camptothecin structure, it is known that the equilibrium shifts to a structure having a closed lactone ring (closed ring) in an aqueous acidic medium (for example, about pH 3), but shifts to a structure having an open lactone ring (open ring) in an aqueous basic medium (for example, about pH 10). When an exatecan residue corresponding to the closed ring structure is introduced into the drug conjugate, it is also expected that the open ring structure has the same antitumor effect, and any of these situations is within the scope of the present invention.

[0206] Other examples of antitumor compounds include doxorubicin, daunorubicin, mitomycin C, bleomycin, cytarabine, vincristine, vinblastine, methotrexate, platinum-based antitumor agents (cisplatin or its derivatives), taxol or its derivatives, and camptothecin or its derivatives (antitumor agents described in JP-A-6-87746).

[0207] Regarding antibody-drug conjugates, the number of conjugated drug molecules per antibody molecule is a major factor affecting efficacy and safety. The production of antibody-drug conjugates is carried out by clarifying the reaction conditions including the amounts of raw materials and reagents for the reaction so as to have a certain number of conjugated drug molecules. Antibody-drug conjugates are generally obtained as a mixture containing different numbers of conjugated drug molecules, different from the chemical reactions of low-molecular compounds. The number of drugs conjugated to the antibody molecule is represented by or specified as an average value, i.e., the average number of conjugated drug molecules. Unless specifically described as a principle, the number of conjugated drug molecules means an average value, except when it represents an antibody-drug conjugate having a specific number of conjugated drug molecules contained in an antibody-drug conjugate mixture having different numbers of conjugated drug molecules. The number of exatecan molecules conjugated to the antibody molecule is controllable, and about 1 to 10 exatecans can be linked as the average number of conjugated drug molecules per antibody. In some embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 exatecans can be linked. Preferably, it is 2 to 8, more preferably 3 to 8, and even more preferably 3.5 to 4.5, or 4. On the other hand, those skilled in the art can design a reaction for conjugating a required number of drug molecules to an antibody molecule based on the description of the examples of this application and obtain an antibody-drug conjugate having a controlled number of exatecan molecules.

[0208] Linker structure Regarding the anti-TROP2 antibody-drug conjugate of the present invention, a linker structure for conjugating an antitumor compound to an anti-TROP2 antibody is described. The linker has a structure of the following formula. -L 1 -L 2 -L P -NH-(CH2)n 1 -L a -(CH2)n 2 -C(=O)-

[0209] The antibody is linked to the end of L 1 (the end opposite to the linking part to L 2 ), and the antitumor compound is -La -(CH2)n 2 is linked to the carbonyl group of the -C(=O)- moiety.

[0210] n 1 represents an integer from 0 to 6, preferably an integer from 1 to 5, more preferably an integer from 1 to 3.

[0211] L 1

[0212] L 1 is represented by the structure of -(Succinimid-3-yl-N)-(CH2)n 3 -C(=O)-.

[0213] In the above, n 3 is an integer from 2 to 8, and "-(Succinimid-3-yl-N)-" has a structure represented by the following formula.

[0214]

Chemical formula

[0215] The 3-position of the above partial structure is the linking position to the anti-TROP2 antibody. The binding to the anti-TROP2 antibody at the 3-position is characterized by binding through thioether formation. The nitrogen atom at the 1-position of the structural moiety is linked to the carbon atom of the methylene present in the linker containing the said structure. Specifically, -(Succinimid-3-yl-N)-(CH2)n 3 -C(=O)-L 2 - is a structure represented by the following formula (in this specification, "antibody-S-" is derived from an antibody).

[0216]

Chemical formula

[0217] In the said formula, n 3 is an integer from 2 to 8, preferably an integer from 2 to 5.

[0218] L 1 Specific examples of -(Succinimid-3-yl-N)-CH2CH2-C(=O)-, -(Succinimid-3-yl-N)-CH2CH2CH2-C(=O)-, -(Succinimid-3-yl-N)-CH2CH2CH2CH2-C(=O)-, -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)- can be cited.

[0219] L 2

[0220] L 2 is a linker represented by the following structure. -NH-(CH2CH2-O)n 4 -CH2CH2-C(=O)-,

[0221] L 2 may not exist, and in such a case, L 2 is a single bond. In the above, n 4 is an integer from 1 to 6, preferably from 2 to 4. L 2 is linked to L 1 by its terminal amino group, and is linked to L P by its carbonyl group at the other terminal.

[0222] L 2 Specific examples of -NH-CH2CH2-O-CH2CH2-C(=O)-, -NH-CH2CH2-O-CH2CH2-O-CH2CH2-C(=O)-, -NH-CH2CH2-O-CH2CH2-O-CH2CH2-O-CH2CH2-C(=O)-, -NH-CH2CH2-O-CH2CH2-O-CH2CH2-O-CH2CH2-O-CH2CH2-C(=O)-, -NH-CH2CH2-O-CH2CH2-O-CH2CH2-O-CH2CH2-O-CH2CH2-O-CH2CH2-C(=O)-, -NH-CH2CH2-O-CH2CH2-O-CH2CH2-O-CH2CH2-O-CH2CH2-O-CH2CH2-O-CH2CH2-C(=O)- can be exemplified.

[0223] L P

[0224] L P is a peptide residue consisting of 2 to 7 amino acids. Specifically, it consists of an oligopeptide residue in which 2 to 7 amino acids are linked by peptide bonds. L P is linked to L 2 at its N-terminus and, at its C-terminus, to the amino group of the -NH-(CH2)n 1 -L a -(CH2)n 2 -C(=O)- moiety of the linker.

[0225] In the linker, the amino acids constituting L P are not particularly limited, and examples thereof include L-amino acids or D-amino acids, preferably L-amino acids. And, in addition to α-amino acids, it can be an amino acid having a structure such as β-alanine, ε-aminocaproic acid or γ-aminobutyric acid, and furthermore, it can be an unnatural amino acid such as an N-methylated amino acid.

[0226] L P The amino acid sequence of is not particularly limited, and examples of the constituent amino acids include phenylalanine (Phe; F), tyrosine (Tyr; Y), leucine (Leu; L), glycine (Gly; G), alanine (Ala; A), valine (Val; V), lysine (Lys; K), citrulline (Cit), serine (Ser; S), glutamic acid (Glu; E) and aspartic acid (Asp; D).

[0227] Among them, preferred examples include phenylalanine, glycine, valine, lysine, citrulline, serine, glutamic acid, and aspartic acid. Depending on the type of amino acid, the drug release pattern can be controlled. The number of amino acids can be between 2 and 7.

[0228] L P Specific examples of -GGF- -DGGF- -(D-)D-GGF- -EGGF- -GGFG- -SGGF- -KGGF- -DGGFG- -GGFGG- -DDGGFG- -KDGGFG- -GGFGGGF can be mentioned.

[0229] In the above, "(D-)D" represents D-aspartic acid.

[0230] L for the antibody-drug conjugate of the present invention P Particularly preferred examples of

[0231] L a -(CH2)n 2 -C(=O)-

[0232] L a -(CH2)n 2 In L in -C(=O)- a is a structure of -O- or a single bond. n 2 is an integer of 0 to 5, more preferably 0 to 3, and even more preferably 0 or 1.

[0233] L a -(CH2)n 2 Examples of -C(=O)- include the following structures, -O-CH2-C(=O)-, -O-CH2CH2-C(=O)-, -O-CH2CH2CH2-C(=O)-, -O-CH2CH2CH2CH2-C(=O)-, -O-CH2CH2CH2CH2CH2-C(=O)-, -CH2-C(=O)-, -CH2CH2-C(=O)-, -CH2CH2CH2-C(=O)-, -CH2CH2CH2CH2-C(=O)-, -CH2CH2CH2CH2CH2-C(=O)- Those having the following can be mentioned.

[0234] Among them, -O-CH2-C(=O)-, -O-CH2CH2-C(=O)-, or L a is a single bond and n 2 is preferably 0.

[0235] -NH-(CH2)n in the linker 1 -L a -(CH2)n 2 -C(=O)-, specific examples of the structure represented by -NH-CH2-C(=O)-, -NH-CH2CH2-C(=O)-, -NH-CH2-O-CH2-C(=O)-, -NH-CH2CH2-O-C(=O)-, -NH-CH2CH2-O-CH2-C(=O)-, -NH-CH2CH2CH2-C(=O)-, -NH-CH2CH2CH2CH2-C(=O)-, -NH-CH2CH2CH2CH2CH2-C(=O)- can be mentioned, -NH-CH2CH2CH2-C(=O)-, -NH-CH2-O-CH2-C(=O)-, or -NH-CH2CH2-O-C(=O)- is preferred.

[0236] In the linker, -NH-(CH2)n 1 -L a -(CH2)n 2 The chain length of -C(=O)- is preferably a chain length of 4 to 7 atoms, more preferably a chain length of 5 or 6 atoms.

[0237] Regarding the anti-TROP2 antibody-drug conjugate of the present invention, when the anti-TROP2 antibody-drug conjugate is transferred into tumor cells, the linker portion is cleaved, and NH2-(CH2)n 1 -L a -(CH2)n 2 It is considered that a drug derivative having a structure represented by -C(=O)-(NH-DX) is released to exhibit an antitumor effect. Examples of antitumor derivatives that exhibit an antitumor effect by release from the antibody-drug conjugate of the present invention include -NH-(CH2)n of the linker 1 -L a -(CH2)n 2 Antitumor derivatives having a structural portion having a terminal amino group in the structure represented by -C(=O)- are exemplified, and particularly preferred examples include the following. NH2-CH2CH2-C(=O)-(NH-DX), NH2-CH2CH2CH2-C(=O)-(NH-DX), NH2-CH2-O-CH2-C(=O)-(NH-DX), NH2-CHCH2-O-CH2-C(=O)-(NH-DX)

[0238] On the other hand, in the case of NH2-CH2-O-CH2-C(=O)-(NH-DX), if the aminals structure in the molecule is unstable, it will undergo self-decomposition again to give the following HO-CH2-C(=O)-(NH-DX) It was confirmed that was released. Preferably, these compounds can also be used as production intermediates of the antibody-drug conjugate of the present invention.

[0239] Regarding the antibody-drug conjugate of the present invention in which exatecan is used as a drug, a drug-linker structural portion [-L having the following structure1 -L 2 -L P -NH-(CH2)n 1 -L a -(CH2)n 2 It is preferable that -C(=O)-(NH-DX) is linked to the antibody. The average number of complexations of the drug-linker structural moiety per antibody can be 1 to 10, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. Preferably, it is 2 to 8, more preferably 3 to 8, and still more preferably 3.5 to 4.5, or 4. -(Succinimid-3-yl-N)-CH2CH2-C(=O)-GGFG-NH-CH2CH2-C(=O)-(NH-DX), -(Succinimid-3-yl-N)-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-(NH-DX), -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2-C(=O)-(NH-DX), -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-(NH-DX), -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2CH2CH2-C(=O)-(NH-DX), -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2-O-CH2-C(=O)-(NH-DX), -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2-O-CH2-C(=O)-(NH-DX), -(Succinimid-3-yl-N)-CH2CH2-C(=O)-NH-CH2CH2-O-CH2CH2-O-CH2CH2-C(=O)-GGFG-NH-CH2CH2-C(=O)-(NH-DX), -(N-Succinimid-3-yl)-CH2CH2-C(=O)-NH-CH2CH2-O-CH2CH2-O-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-(NH-DX), -(N-Succinimid-3-yl)-CH2CH2-C(=O)-NH-CH2CH2-O-CH2CH2-O-CH2CH2-O-CH2CH2-O-CH2CH2-C(=O)-GGFG-NH-CH2CH2-C(=O)-(NH-DX), -(N-Succinimid-3-yl)-CH2CH2-C(=O)-NH-CH2CH2-O-CH2CH2-O-CH2CH2-O-CH2CH2-O-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-(NH-DX)

[0240] Among them, the more preferred ones are as follows. -(N-Succinimid-3-yl)-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-(NH-DX), -(N-Succinimid-3-yl)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-(NH-DX), -(N-Succinimid-3-yl)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2-O-CH2-C(=O)-(NH-DX), -(N-Succinimid-3-yl)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2-O-CH2-C(=O)-(NH-DX), -(N-Succinimid-3-yl)-CH2CH2-C(=O)-NH-CH2CH2-O-CH2CH2-O-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-(NH-DX), -(Succinimid-3-yl-N)-CH2CH2-C(=O)-NH-CH2CH2-O-CH2CH2-O-CH2CH2-O-CH2CH2-O-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-(NH-DX)

[0241] Particularly preferred are the following. -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2-O-CH2-C(=O)-(NH-DX), -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2-O-CH2-C(=O)-(NH-DX), -(Succinimid-3-yl-N)-CH2CH2-C(=O)-NH-CH2CH2-O-CH2CH2-O-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-(NH-DX)

[0242] Regarding the linker structure for conjugating the anti-TROP2 antibody and the drug in the antibody-drug conjugate of the present invention, a preferred linker can be constructed by linking the preferred structures shown for each part of the linker described above. Regarding the linker structure, preferably, those having the following structure can be used. On the other hand, the left end of the said structure is the linking position with the antibody, and the right end is the linking position with the drug. -(Succinimid-3-yl-N)-CH2CH2-C(=O)-GGFG-NH-CH2CH2-C(=O)-, -(Succinimid-3-yl-N)-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-, -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2-C(=O)-, -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-, -(N-Succinimid-3-yl)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2CH2CH2-C(=O)-、 -(N-Succinimid-3-yl)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2-O-CH2-C(=O)-、 -(N-Succinimid-3-yl)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2-O-CH2-C(=O)-、 -(N-Succinimid-3-yl)-CH2CH2-C(=O)-NH-CH2CH2-O-CH2CH2-O-CH2CH2-C(=O)-GGFG-NH-CH2CH2-C(=O)-、 -(N-Succinimid-3-yl)-CH2CH2-C(=O)-NH-CH2CH2-O-CH2CH2-O-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-、 -(N-Succinimid-3-yl)-CH2CH2-C(=O)-NH-CH2CH2-O-CH2CH2-O-CH2CH2-O-CH2CH2-O-CH2CH2-C(=O)-GGFG-NH-CH2CH2-C(=O)-、 -(N-Succinimid-3-yl)-CH2CH2-C(=O)-NH-CH2CH2-O-CH2CH2-O-CH2CH2-O-CH2CH2-O-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-

[0243] Among them, the more preferred ones are as follows. -(N-Succinimid-3-yl)-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-、 -(N-Succinimid-3-yl)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-、 -(N-Succinimid-3-yl)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2-O-CH2-C(=O)-、 -(N-Succinimid-3-yl)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2-O-CH2-C(=O)- -(N-Succinimid-3-yl)-CH2CH2-C(=O)-NH-CH2CH2-O-CH2CH2-O-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)- -(N-Succinimid-3-yl)-CH2CH2-C(=O)-NH-CH2CH2-O-CH2CH2-O-CH2CH2-O-CH2CH2-O-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-

[0244] Particularly preferred ones include the following. -(N-Succinimid-3-yl)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2-O-CH2-C(=O)- -(N-Succinimid-3-yl)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2-O-CH2-C(=O)- -(N-Succinimid-3-yl)-CH2CH2-C(=O)-NH-CH2CH2O-CH2CH2O-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-

[0245] Regarding the anti-TROP2 antibody-drug conjugate used in the present invention, when it is transferred into the interior of tumor cells, the linker moiety is cleaved, and a drug derivative having a structure represented by the formula: NH2-CH2-O-CH2-C(=O)-(NH-DX) can be released. When the aminol structure in the molecule of the drug derivative is unstable, it was confirmed that it undergoes self-degradation again to release a compound represented by the formula: HO-CH2-C(=O)-(NH-DX).

[0246] The said compound has the following formula:

[0247]

Chemical formula

[0248] It can be represented by (hereinafter also referred to as "Compound 1" in the present invention).

[0249] Compound 1 is considered to be the main pharmaceutical active substance of the antitumor activity possessed by the antibody-drug conjugate used in the present invention, and it has been confirmed to have a topoisomerase I inhibitory effect (Ogitani Y. et al., Clinical Cancer Research, October 15, 2016; 22(20):5097~5108, Epub March 29, 2016).

[0250] Production method Next, a representative method for producing the antibody-drug conjugate of the present invention or its production intermediate will be described. On the other hand, the above compounds are described herein below together with the compound numbers shown in each chemical reaction formula. Specifically, they are referred to as "the compound of formula (1)", "compound (1)" or the like. Compounds with other numbers are described in the same way.

[0251] Production method A

[0252] The antibody-drug conjugate represented by formula (1) linked to the drug-linker structure via a thioether can be produced, for example, by the following method.

[0253]

Chemical formula

[0254] In the above formula, AB represents an antibody having a sulfhydryl group, and L 1’ represents L whose linker terminal is a maleimidyl group 1 and represents a linker structure (formula shown below).

[0255]

Chemical formula

[0256] In the above formula, the nitrogen atom is the linking position. Specifically, L 1 of -(Succinimid-3-yl-N)-(CH2)n 3 -C(=O)- in which the -(Succinimid-3-yl-N)- moiety is a maleimidyl group. Further, -(NH-DX) is the following formula:

[0257]

Chemical formula

[0258] represents a structure represented by, which is a group derived by removing one hydrogen atom of the amino group at the 1-position of exatecan.

[0259] Furthermore, the compound of formula (1) in the above chemical reaction formula is interpreted as having a structure in which one structural moiety corresponding from the drug to the linker end is linked to one antibody. However, this is only a description shown for convenience, and in reality, multiple structural moieties are often linked to one antibody molecule. The same applies to the description of the production method described below.

[0260] Antibody-drug conjugate (1) can be produced by reacting a compound (2) obtainable by the method described below with an antibody (3a) having a sulfhydryl group.

[0261] Antibodies having sulfhydryl groups (3a) can be obtained by methods well known in the art (Hermanson, G.T., Bioconjugate Techniques, pages 56 - 136, 456 - 493, Academic Press (1996)). Examples include: reacting a trout reagent with the amino groups of the antibody; reacting N-succinimidyl S-acetylthioalkanoate with the amino groups of the antibody and then reacting with hydroxylamine; reacting with N-succinimidyl 3-(pyridyldithio)propionate and then reacting the antibody with a reducing agent; reacting the antibody with a reducing agent such as dithiothreitol, 2-mercaptoethanol, and tris(2-carboxyethyl)phosphine hydrochloride (TCEP) to reduce the disulfide bonds in the antibody to form sulfhydryl groups, but are not limited thereto.

[0262] Specifically, for each disulfide in the antibody, 0.3 to 3 molar equivalents of TCEP can be used as a reducing agent and reacted with the antibody in a buffer solution containing a chelating agent to obtain an antibody having partially or fully reduced disulfides in the antibody. Examples of chelating agents include ethylenediaminetetraacetic acid (EDTA) and diethylenetriaminepentaacetic acid (DTPA). It can be used at a concentration of 1 mM to 20 mM. Examples of buffer solutions that can be used include solutions of sodium phosphate, sodium borate, or sodium acetate. Specifically, an antibody having partially or fully reduced sulfhydryl groups (3a) can be obtained by reacting the antibody with TCEP at 4°C to 37°C for 1 to 4 hours.

[0263] On the other hand, by performing a reaction for adding a sulfhydryl group to the drug-linker moiety, the drug-linker moiety can be conjugated with a thioether bond.

[0264] Using 2 to 20 molar equivalents of compound (2) per antibody having a sulfhydryl group (3a), an antibody-drug conjugate (1) in which 2 to 8 drug molecules are conjugated per antibody can be produced. Specifically, it is sufficient to add a solution containing compound (2) dissolved therein to a buffer solution containing the antibody having a sulfhydryl group (3a) for the reaction. In this specification, examples of buffer solutions that can be used include sodium acetate solution, sodium phosphate, and sodium borate. The pH for the reaction is 5 to 9, and more preferably, the reaction is carried out at approximately pH 7. Examples of solvents for dissolving compound (2) include organic solvents such as dimethyl sulfoxide (DMSO), dimethylformamide (DMF), dimethylacetamide (DMA), and N-methyl-2-pyridone (NMP).

[0265] It is sufficient to add an organic solvent solution containing compound (2) dissolved therein to a buffer solution containing the antibody having a sulfhydryl group (3a) for the reaction at 1 to 20% v / v. The reaction temperature is 0 to 37 °C, more preferably 10 to 25 °C, and the reaction time is 0.5 to 2 hours. The reaction can be terminated by inactivating the reactivity of unreacted compound (2) with a thiol-containing reagent. Examples of thiol-containing reagents include cysteine and N-acetyl-L-cysteine (NAC). More specifically, the reaction can be terminated by adding 1 to 2 molar equivalents of NAC to the compound (2) used and incubating at room temperature for 10 to 30 minutes.

[0266] For the generated antibody-drug conjugate (1), after concentration, buffer exchange, purification, measurement of the antibody concentration and the average number of conjugated drug molecules per antibody molecule by the general methods described below, and identification of the antibody-drug conjugate (1) can be performed.

[0267] General method A: Concentration of an aqueous solution of an antibody or an antibody-drug conjugate

[0268] The solution of the antibody or antibody-drug conjugate was added to an Amicon Ultra (50,000 MWCO, Millipore Corporation) vessel, and the solution of the antibody or antibody-drug conjugate was concentrated by centrifugation (centrifugation at 2000G to 3800G for 5 to 20 minutes) using a centrifuge (Allegra X-15R, Beckman Coulter, Inc.).

[0269] General method B: Measurement of antibody concentration

[0270] The antibody concentration was measured using a UV detector (Nanodrop1000, Thermo Fisher Scientific Inc.) according to the method specified by the manufacturer. At that time, different 280 nm absorption coefficients (1.3 mL mg -1 cm -1 ~1.8 mL mg -1 cm -1 ) were used for each antibody.

[0271] General method C-1: Buffer exchange for antibodies

[0272] A NAP-25 column (Cat. No. 17-0852-02, GE Healthcare Japan Corporation) using Sephadex G-25 carrier was equilibrated with a phosphate buffer (10 mM, pH 6.0 containing sodium chloride (137 mM) and ethylenediaminetetraacetic acid (EDTA, 5 mM); it is referred to as PBS6.0 / EDTA in this specification) according to the method specified by the manufacturer. An aqueous solution of the antibody was applied to a single NAP-25 column in an amount of 2.5 mL, and then a fraction (3.5 mL) eluted with 3.5 mL of PBS6.0 / EDTA was collected. The obtained fraction was concentrated by general method A. After measuring the antibody concentration using general method B, the antibody concentration was adjusted to 10 mg / mL using PBS6.0 / EDTA.

[0273] General method C-2: Buffer exchange for antibodies

[0274] A NAP-25 column (Cat. No. 17-0852-02, GE Healthcare Japan Corporation) using Sephadex G-25 carrier was equilibrated with a phosphate buffer (50 mM, pH 6.5; which is referred to as PBS6.5 / EDTA herein) containing sodium chloride (50 mM) and EDTA (2 mM) according to the method specified by the manufacturer. An aqueous solution of the antibody was applied to a single NAP-25 column in an amount of 2.5 mL, and then a fraction (3.5 mL) eluted with 3.5 mL of PBS6.5 / EDTA was collected. The obtained fraction was concentrated by general method A. After measuring the antibody concentration using general method B, the antibody concentration was adjusted to 20 mg / mL using PBS6.5 / EDTA.

[0275] General method D: Purification of antibody-drug conjugate

[0276] The NAP-25 column was equilibrated with any buffer selected from commercially available phosphate buffer (PBS7.4, Cat. No. 10010-023, Invitrogen), sodium phosphate buffer (10 mM, pH 6.0; which is referred to as PBS6.0 herein) containing sodium chloride (137 mM), and acetate buffer (10 mM, pH 5.5; which is referred to as ABS herein) containing sorbitol (5%). An aqueous solution of the antibody-drug conjugate reaction was applied to the NAP-25 column in an amount of about 1.5 mL, and then an antibody fraction was collected by eluting with the amount of buffer specified by the manufacturer. The collected fraction was applied to the NAP-25 column again, and the gel filtration purification process for elution with buffer was repeated a total of 2 to 3 times to obtain an antibody-drug conjugate excluding uncomplexed drug linker and low molecular weight compounds (tris(2-carboxyethyl)phosphine hydrochloride (TCEP), N-acetyl-L-cysteine (NAC) and dimethyl sulfoxide).

[0277] General method E: Measurement of antibody concentration in antibody-drug conjugate and average number of conjugated drug molecules per antibody molecule (1).

[0278] The concentration of the conjugated drug in the antibody-drug conjugate was calculated by measuring the UV absorbance of an aqueous solution of the antibody-drug conjugate at two wavelengths of 280 nm and 370 nm, and then the calculation shown below can be performed.

[0279] Since the total absorbance at any wavelength is equal to the sum of the absorbances of all the light-absorbing chemical species present in the system (additivity of absorbance), when the molar absorption coefficients of the antibody and the drug do not change before and after the conjugation between the antibody and the drug, the antibody concentration and the drug concentration in the antibody-drug conjugate are represented by the following equations. A 280 =A D,280 +A A,280 =ε D,280 C D +ε A,280 C A Equation (I) A 370 =A D,370 +A A,370 =ε D,370 C D +ε A,370 C A Equation (II)

[0280] In the above, A 280 represents the absorbance of the aqueous solution of the antibody-drug conjugate at 280 nm, A 370 represents the absorbance of the aqueous solution of the antibody-drug conjugate at 370 nm, A A,280 represents the absorbance of the antibody at 280 nm, A A,370 represents the absorbance of the antibody at 370 nm, A D,280 represents the absorbance of the conjugate precursor at 280 nm, A D,370 represents the absorbance of the conjugate precursor at 370 nm, ε A,280 represents the molar absorption coefficient of the antibody at 280 nm, ε A,370 represents the molar absorption coefficient of the antibody at 370 nm, ε D,280 represents the molar absorption coefficient of the conjugate precursor at 280 nm, ε D,370 represents the molar absorption coefficient of the conjugate precursor at 370 nm, C A represents the antibody concentration in the antibody-drug conjugate, C Drepresents the drug concentration in the antibody-drug conjugate.

[0281] ε in the above A,280 ε A,370 ε D,280 and ε D,370 For, use pre-prepared values (estimated values based on calculated or measured values obtained by UV measurement of the compound). For example, ε can be estimated from the amino acid sequence of the antibody using known calculation methods (Protein Science, 1995, Vol. 4, 2411 - 2423). ε is generally zero. By measuring the absorbance of a solution in which the precursor complex used dissolves at a specific molar concentration, ε and ε can be obtained based on Lambert-Beer's law (absorbance = molar concentration × molar absorption coefficient × cell optical path length). By measuring A and A of the aqueous solution of the antibody-drug conjugate and solving the simultaneous equations (I) and (II) using the above values, C and C can be obtained. Further, by dividing C by C, the average number of conjugated drugs per antibody can be obtained. A,280 A,370 D,280 and ε D,370 280 370 A and C D D A

[0282] General method F: Measurement of the average number of conjugated drug molecules per antibody molecule in an antibody-drug conjugate - (2).

[0283] In addition to the above general method E, the average number of conjugated drug molecules per antibody molecule in an antibody-drug conjugate can also be determined by high performance liquid chromatography (HPLC) analysis using the method described below.

[0284] [F-1. Preparation of sample for HPLC analysis (reduction of antibody-drug conjugate)]

[0285] Mix an antibody-drug conjugate solution (about 1 mg / mL, 60 μL) with an aqueous dithiothreitol (DTT) solution (100 mM, 15 μL). Incubate the mixture at 37 °C for 30 minutes to cleave the disulfide bonds between the L-chain and H-chain of the antibody-drug conjugate. Use the resulting sample for HPLC analysis.

[0286] [F-2. HPLC Analysis]

[0287] Perform HPLC analysis under the following measurement conditions.

[0288] HPLC System: Agilent 1290 HPLC System (Agilent Technologies, Inc.)

[0289] Detector: UV absorption spectrometer (measurement wavelength: 280 nm)

[0290] Column: PLRP-S (2.1 × 50 mm, 8 μm, 1000 angstroms; Agilent Technologies, Inc., P / N PL1912-1802)

[0291] Column Temperature: 80 °C

[0292] Mobile Phase A: 0.04% trifluoroacetic acid (TFA) aqueous solution

[0293] Mobile Phase B: Acetonitrile solution containing 0.04% TFA

[0294] Gradient Program: 29% - 36% (0 min - 12.5 min), 36% - 42% (12.5 - 15 min), 42% - 29% (15 min - 15.1 min), 29% - 29% (15.1 min - 25 min)

[0295] Sample Injection Volume: 15 μL

[0296] [F-3. Data Analysis]

[0297] [F-3-1] Compared with the L-chain (L0) and H-chain (H0) of the non-conjugated antibody, the drug-conjugated L-chain (L-chain linked to one drug molecule: L1) and H-chain (H-chain linked to one drug molecule: H1, H-chain linked to two drug molecules: H2, H-chain linked to three drug molecules: H3) show higher hydrophobicity in proportion to the number of conjugated drug molecules, and thus have longer retention times. Therefore, these chains elute in the order of L0 and L1, or H0, H1, H2, and H3. By comparing the retention times with L0 and H0, the detection peaks can be assigned to any of L0, L1, H0, H1, H2, and H3.

[0298] [F-3-2] Since the drug linker has UV absorption, the peak area values are corrected according to the following formula according to the number of conjugated drug linker molecules, using the molar absorption coefficients of the L-chain, H-chain, and drug linker.

[0299] [Number]

[0300] [Number]

[0301] Here, regarding the molar absorption coefficient (280 nm) of the L-chain or H-chain of each antibody, values estimated from the amino acid sequence of the L-chain or H-chain of each antibody by a known calculation method (Protein Science, 1995, Vol. 4, 2411-2423) can be used. In the case of hTINA, according to its amino acid sequence, molar extinction coefficients of 34690 and 95000 were used as the estimated values for the L-chain and H-chain, respectively. Regarding the molar absorption coefficient (280 nm) of the drug linker, the measured molar absorption coefficient (280 nm) of the compound in which the maleimide group was converted to succinimide thioether by the reaction of each drug linker with mercaptoethanol or N-acetylcysteine was used.

[0302] [F-3-3] Calculate the peak area ratio (%) of each chain for the total corrected value of the peak area according to the following formula.

[0303]

Equation

[0304] [F-3-4] In the antibody-drug conjugate, the average number of conjugated drug molecules per antibody molecule is calculated according to the following formula.

[0305] Average number of conjugated drug molecules = (L0 peak area ratio × 0 + L0 peak area ratio × 1 + H0 peak area ratio × 0 + H1 peak area ratio × 1 + H2 peak area ratio × 2 + H3 peak area ratio × 3) / 100 × 2

[0306] The compound represented by formula (2) in production method 1 is the compound represented by the following formula.

[0307] (Maleimid-N-yl)-(CH2)n 3 -C(=O)-L 2 -L P -NH-(CH2)n 1 -L a -(CH2)n 2 -C(=O)-(NH-DX)

[0308] In the formula, n 3 represents an integer from 2 to 8, L 2 represents -NH-(CH2CH2-O)n 4 -CH2CH2-C(=O)- or a single bond. In the formula, n 4 represents an integer from 1 to 6, L P represents a peptide residue consisting of 2 to 7 amino acids selected from phenylalanine, glycine, valine, lysine, citrulline, serine, glutamic acid, and aspartic acid, n 1 represents an integer from 0 to 6, n2 represents an integer from 0 to 5, L a represents -O- or a single bond, (Maleimid-N-yl)- is a maleimidyl group (2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl group) represented by the following formula,

[0309] [Chemical formula] (wherein the nitrogen atom is the linking position)

[0310] -(NH-DX) is a group represented by the following formula.

[0311] [Chemical formula] (wherein the nitrogen atom of the amino group at the 1-position is the linking position)

[0312] L 2 when L is a single bond or -NH-(CH2CH2-O)n 4 -CH2CH2-C(=O)-, n 4 is preferably an integer from 2 to 4 for the resulting intermediate compound.

[0313] L P Regarding the peptide residue of L, a compound having a peptide residue containing an amino acid selected from phenylalanine, glycine, valine, lysine, citrulline, serine, glutamic acid, and aspartic acid is preferably the resulting intermediate. Among those peptide residues, L P is preferably a compound having a peptide residue consisting of 4 amino acids as the resulting intermediate. More specifically, L P is preferably a compound having a tetrapeptide residue of -GGFG- as the resulting intermediate.

[0314] Furthermore, -NH-(CH2)n 1 -L a -(CH2)n2 Regarding -, compounds having -NH-CH2CH2-, -NH-CH2CH2CH2-, -NH-CH2CH2CH2CH2-, -NH-CH2CH2CH2CH2CH2-, -NH-CH2-O-CH2-, or -NH-CH2CH2-O-CH2- are preferred as production intermediates. Compounds having -NH-CH2CH2CH2-, -NH-CH2-O-CH2-, or -NH-CH2CH2-O-CH2- are more preferred.

[0315] Furthermore, in the compound represented by the formula (2), n 3 is an integer of 2 to 5, and L 2 is a single bond, and -NH-(CH2)n 1 -L a -(CH2)n 2 - being -NH-CH2CH2-, -NH-CH2CH2CH2-, -NH-CH2CH2CH2CH2-, -NH-CH2CH2CH2CH2CH2-, -NH-CH2-O-CH2-, or -NH-CH2CH2-O-CH2- is preferred as a production intermediate. -NH-(CH2)n 1 -L a -(CH2)n 2 - being -NH-CH2CH2-, -NH-CH2CH2CH2-, -NH-CH2-O-CH2-, or -NH-CH2CH2-O-CH2- is more preferred. n 3 being an integer of 2 or 5 is even more preferred.

[0316] Furthermore, in the compound represented by the formula (2), n 3 is an integer of 2 to 5, and L 2 is -NH-(CH2CH2-O)n 4 -CH2CH2-C(=O)-, and n 4 is an integer of 2 to 4, and -NH-(CH2)n 1 -L a -(CH2)n 2Compounds in which - is -NH-CH2CH2-, -NH-CH2CH2CH2-, -NH-CH2CH2CH2CH2-, -NH-CH2CH2CH2CH2CH2-, -NH-CH2-O-CH2-, or -NH-CH2CH2-O-CH2- are preferred as production intermediates. n 4 Compounds in which n is an integer of 2 or 4 are more preferred. -NH-(CH2)n 1 -L a -(CH2)n 2 Compounds in which - is -NH-CH2CH2CH2-, -NH-CH2-O-CH2-, or -NH-CH2CH2-O-CH2- are even more preferred.

[0317] Examples of such preferred intermediates useful in the production of the compounds of the present invention include the following. (Maleimid-N-yl)-CH2CH2-C(=O)-GGFG-NH-CH2CH2-C(=O)-(NH-DX), (Maleimid-N-yl)-CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2-C(=O)-(NH-DX), (Maleimid-N-yl)-CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2-C(=O)-(NH-DX), (Maleimid-N-yl)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2-C(=O)-(NH-DX), (Maleimid-N-yl)-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-(NH-DX), (Maleimid-N-yl)-CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-(NH-DX), (Maleimid-N-yl)-CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-(NH-DX), (Maleimid-N-yl)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-(NH-DX), (Maleimid-N-yl)-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2CH2-C(=O)-(NH-DX), (Maleimid-N-yl)-CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2CH2-C(=O)-(NH-DX), (Maleimid-N-yl)-CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2CH2-C(=O)-(NH-DX), (Maleimid-N-yl)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2CH2-C(=O)-(NH-DX), (Maleimid-N-yl)-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2CH2CH2-C(=O)-(NH-DX), (Maleimid-N-yl)-CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2CH2CH2-C(=O)-(NH-DX), (Maleimid-N-yl)-CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2CH2CH2-C(=O)-(NH-DX), (Maleimid-N-yl)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2CH2CH2-C(=O)-(NH-DX), (Maleimid-N-yl)-CH2CH2-C(=O)-GGFG-NH-CH2-O-CH2-C(=O)-(NH-DX), (Maleimid-N-yl)-CH2CH2CH2-C(=O)-GGFG-NH-CH2-O-CH2-C(=O)-(NH-DX), (Maleimid-N-yl)-CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2-O-CH2-C(=O)-(NH-DX), (Maleimid-N-yl)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2-O-CH2-C(=O)-(NH-DX), (Maleimid-N-yl)-CH2CH2-C(=O)-GGFG-NH-CH2CH2-O-CH2-C(=O)-(NH-DX), (Maleimid-N-yl)-CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2-O-CH2-C(=O)-(NH-DX), (Maleimid-N-yl)-CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2-O-CH2-C(=O)-(NH-DX), (Maleimid-N-yl)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2-O-CH2-C(=O)-(NH-DX), (Maleimid-N-yl)-CH2CH2-C(=O)-NH-CH2CH2-O-CH2CH2-O-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-(NH-DX), (Maleimid-N-yl)-CH2CH2-C(=O)-NH-CH2CH2-O-CH2CH2-O-CH2CH2-O-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-(NH-DX), (Maleimid-N-yl)-CH2CH2-C(=O)-NH-CH2CH2-O-CH2CH2-O-CH2CH2-O-CH2CH2-O-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-(NH-DX)

[0318] By reacting a drug-linker compound selected from the above group of intermediate compounds with an anti-TROP2 antibody or a reactive derivative thereof to form a thioether bond at a disulfide bond site present in the anti-TROP2 antibody, the anti-TROP2 antibody-drug conjugate of the present invention can be produced. In this case, a reactive derivative of the anti-TROP2 antibody is preferably used. In particular, a reactive derivative obtained by reducing the anti-TROP2 antibody is preferred.

[0319] The following are more preferred compounds as intermediate products. (Maleimid-N-yl)-CH2CH2-C(=O)-GGFG-NH-CH2CH2-C(=O)-(NH-DX), (Maleimid-N-yl)-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-(NH-DX), (Maleimid-N-yl)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2-C(=O)-(NH-DX), (Maleimid-N-yl)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-(NH-DX), (Maleimid-N-yl)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2CH2CH2-C(=O)-(NH-DX), (Maleimid-N-yl)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2-O-CH2-C(=O)-(NH-DX), (Maleimid-N-yl)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2-O-CH2-C(=O)-(NH-DX), (Maleimid-N-yl)-CH2CH2-C(=O)-NH-CH2CH2-O-CH2CH2-O-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-(NH-DX), (Maleimid-N-yl)-CH2CH2-C(=O)-NH-CH2CH2-O-CH2CH2-O-CH2CH2-C(=O)-GGFG-NH-CH2CH2-C(=O)-(NH-DX), (Maleimid-N-yl)-CH2CH2-C(=O)-NH-CH2CH2-O-CH2CH2-O-CH2CH2-O-CH2CH2-O-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-(NH-DX)

[0320] Among the above groups of intermediate compounds, the following formula: (Maleimid-N-yl)-CH2CH2-C(=O)-NH-CH2CH2-O-CH2CH2-O-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-(NH-DX), (Maleimid-N-yl)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2-O-CH2-C(=O)-(NH-DX), or Compounds represented by (Maleimid-N-yl)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2-O-CH2-C(=O)-(NH-DX) are more preferred compounds.

[0321] To ensure the amount of the complex, a plurality of complexes obtained under the same production conditions can be mixed to have an equal number (e.g., about ±1) of drugs to prepare a new lot. In this case, the average number of drugs is between the average numbers of drugs in the complexes before the mixing.

[0322] Production Method 2

[0323] The compound represented by formula (2) and its pharmacologically acceptable salts as intermediates used in the previous production method can be produced, for example, by the following method.

[0324]

Chemical formula

[0325] In the above formula, L 1’ represents a maleimidyl group, and P 1 , P 2 and P 3 each represent a protecting group.

[0326] Compound (6) can be produced by derivatizing carboxylic acid (5) into an active ester, mixed acid anhydride, acid halide or the like, and reacting it with NH2-DX (4) or a pharmaceutically acceptable salt thereof in the presence of a base. NH2-DX (4) represents exatecan (chemical name: (1S,9S)-1-amino-9-ethyl-5-fluoro-2,3-dihydro-9-hydroxy-4-methyl-1H,12H-benzo[de]pyrano[3’,4’:6,7]indolizino[1,2-b]quinoline-10,13(9H,15H)-dione).

[0327] The reaction reagents and reaction conditions generally used for peptide synthesis can be used for the said reaction. There are various active esters. For example, it can be produced by reacting a phenol such as p-nitrophenol, N-hydroxybenzotriazole, N-hydroxysuccinimide or the like with carboxylic acid (5) using a condensing agent such as N,N’-dicyclohexylcarbodiimide or 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride. Further, the said active ester can also be produced by the reaction of carboxylic acid (5) with pentafluorophenyl trifluoroacetate or the like, the reaction of carboxylic acid (5) with 1-benzotriazolyloxytris(pyrrolidino)phosphonium hexafluorophosphate, the reaction of carboxylic acid (5) with diethyl cyanophosphonate (salt-in method), the reaction of carboxylic acid (5) with triphenylphosphine and 2,2’-dipyridyl disulfide (Mukaiyama's method), the reaction of carboxylic acid (5) with a triazine derivative such as 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMTMM) or the like. Further, the said reaction can also be carried out by, for example, the acid halide method in which carboxylic acid (5) is treated with an acid halide such as thionyl chloride and oxalyl chloride in the presence of a base.

[0328] The active ester, mixed acid anhydride or acid halide of the carboxylic acid (5) obtained as described above is reacted with the compound (4) in an inert solvent in the presence of a suitable base at a reaction temperature of -78 °C to 150 °C, whereby the compound (6) can be produced. On the other hand, "inert solvent" refers to a solvent that does not inhibit the target reaction in which the solvent is used.

[0329] Specific examples of the base used for each of the above steps include carbonates, alkoxides, hydroxides or hydrides of alkali metals or alkaline earth metals including sodium carbonate, potassium carbonate, sodium ethoxide, potassium butoxide, sodium hydroxide, potassium hydroxide, sodium hydride and potassium hydride, organometallic bases represented by alkyllithiums including n-butyllithium and dialkylaminolithiums including lithium diisopropylamide, organometallic bases of bissilylamines including lithium bis(trimethylsilyl)amide, and tertiary amines or nitrogen-containing heterocyclic compounds, for example, organic bases including pyridine, 2,6-lutidine, collidine, 4-dimethylaminopyridine, triethylamine, N-methylmorpholine, diisopropylethylamine and 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU).

[0330] Examples of the inert solvent used for the reaction of the present invention include halogenated hydrocarbon solvents such as dichloromethane, chloroform and carbon tetrachloride, ether solvents such as tetrahydrofuran, 1,2-dimethoxyethane and dioxane, aromatic hydrocarbon solvents such as benzene and toluene, and amide solvents such as N,N-dimethylformamide, N,N-dimethylacetamide and N-methylpyrrolidin-2-one. In addition, in some cases, sulfoxide solvents such as dimethyl sulfoxide and sulfolane, ketone solvents such as acetone and methyl ethyl ketone, and alcohol solvents such as methanol and ethanol may be used. Furthermore, these solvents may be mixed for use.

[0331] Protecting group P for the terminal amino group of the compound (6)1 Regarding 1 , protecting groups for amino groups generally used for peptide synthesis, such as tert-butyloxycarbonyl group, 9-fluorenylmethyloxycarbonyl group, and benzyloxycarbonyl group, can be used. Examples of other protecting groups for amino groups include alkanoyl groups such as acetyl group, alkoxycarbonyl groups such as methoxycarbonyl group and ethoxycarbonyl group, arylmethoxycarbonyl groups such as paramethoxybenzyloxycarbonyl group and para (or ortho) nitroybenzyloxy carbonyl group, arylmethyl groups such as benzyl group and triphenylmethyl group, aroyl groups such as benzoyl group, and arylsulfonyl groups such as 2,4-dinitrobenzenesulfonyl group and orthonitrobenzenesulfonyl group. The protecting group P 1 can be selected, for example, according to the properties of the compound having the amino group to be protected.

[0332] By deprotecting the protecting group P for the terminal amino group of the obtained compound (6), compound (7) can be produced. For this deprotection, reagents and conditions can be selected according to the protecting group. 1 By derivatizing the peptide carboxylic acid (8) having an N-terminus protected with P 2 into an active ester, mixed acid anhydride or the like, and reacting it with the obtained compound (7), compound (9) can be produced. The reaction conditions, reagents, bases, and inert solvents used to form the peptide bond between the peptide carboxylic acid (8) and compound (7) can be appropriately selected and used from those described for the synthesis of compound (6). The protecting group P from those described for the protecting groups of compound (6)

[0333] P 2 2 ​It can be appropriately selected and used, and for example, the selection can be made based on the properties of the compound having the amino group to be protected. As generally used in peptide synthesis, compound (9) can also be produced by continuously repeating the reactions and deprotections of the amino acids or peptides constituting the peptide carboxylic acid (8) for elongation.

[0334] The protecting group P for the amino group of the obtained compound (9) 2 Compound (10) can be produced by deprotecting it. For this deprotection, reagents and conditions can be selected according to the protecting group.

[0335] Compound (2) can be produced by derivatizing carboxylic acid (11) into an active ester, mixed acid anhydride, acid halide or the like and reacting it with the obtained compound (10). The reaction conditions, reagents, bases and inert solvents used for forming the peptide bond between carboxylic acid (11) and compound (10) can be appropriately selected and used from those described for the synthesis of compound (6).

[0336] For example, compound (9) can also be produced by the following method.

[0337] P 2 Derivatize the peptide carboxylic acid (8) having an N-terminus protected with P into an active ester, mixed acid anhydride or the like, and react it with an amine compound (12) having a carboxy group protected with P 3 in the presence of a base to produce compound (13). The reaction conditions, reagents, bases and inert solvents used for forming the peptide bond between peptide carboxylic acid (8) and compound (12) can be appropriately selected and used from those described for the synthesis of compound (6).

[0338] The protecting group P for the amino group of compound (13) 2 may be protected with a generally used protecting group.

[0339] Specifically, examples of protecting groups for a hydroxyl group include alkoxymethyl groups such as a methoxymethyl group, arylmethyl groups such as a benzyl group, a 4-methoxybenzyl group, and a triphenylmethyl group, alkanoyl groups such as an acetyl group, aroyl groups such as a benzoyl group, and silyl groups such as a tert-butyldiphenylsilyl group. The carboxy group can be protected as an ester having an alkyl group such as a methyl group, an ethyl group, and a tert-butyl group, an allyl group, or an arylmethyl group such as a benzyl group. Examples of protecting groups for an amino group include alkyloxycarbonyl groups such as a tert-butyloxycarbonyl group, a methoxycarbonyl group, and an ethoxycarbonyl group, an allyloxycarbonyl group, or an arylmethyloxycarbonyl group such as a 9-fluorenylmethyloxycarbonyl group, a benzyloxycarbonyl group, a paramethoxybenzyloxycarbonyl group, and a para (or ortho) nitrobenzyloxycarbonyl group, alkanoyl groups such as an acetyl group, arylmethyl groups such as a benzyl group and a triphenylmethyl group, aroyl groups such as a benzoyl group, and arylsulfonyl groups such as a 2,4-dinitrobenzenesulfonyl group or an orthonitrobenzenesulfonyl group.

[0340] Protecting group P for a carboxy group 3 Regarding the protecting group P for a carboxy group, a protecting group generally used as a protecting group for a carboxy group in organic synthetic chemistry, particularly peptide synthesis, can be used. Specific examples include esters having an alkyl group such as a methyl group, an ethyl group, or a tert-butyl group, allyl esters, and benzyl esters, and the protecting group can be appropriately selected from the above protecting groups. In such a case, it is preferable that the protecting group for an amino group and the protecting group for a carboxy group can be removed by different methods or different conditions. For example, as a representative example, P 2 is a tert-butyloxycarbonyl group, and P 3Examples of the combination where it is a benzyl group can be mentioned. The protecting group can be selected from those described above, for example, according to the properties of the compound having an amino group and a carboxy group to be protected. For the removal of the protecting group, the reagent and conditions can be selected according to the protecting group.

[0341] Protecting group P for the carboxy group of the obtained compound (13) 3 By deprotecting, compound (14) can be produced. For this deprotection, the reagent and conditions are selected according to the protecting group.

[0342] Compound (9) can be produced by derivatizing the obtained compound (14) into an active ester, mixed acid anhydride, acid halide or the like and reacting it with compound (4) in the presence of a base. For said reaction, it is also possible to use the reaction reagents and reaction conditions generally used for peptide synthesis, and it is possible to appropriately select the reaction conditions, reagents, base and inert solvent used for said reaction from those described for the synthesis of compound (6).

[0343] For example, compound (2) can also be produced by the following method.

[0344] Protecting group P for the amino group of compound (13) 2 By deprotecting, compound (15) can be produced. For this deprotection, the reagent and conditions can be selected according to the protecting group.

[0345] The carboxylic acid derivative (11) is derivatized into an active ester, mixed acid anhydride, acid halide or the like and reacted with the obtained compound (15) in the presence of a base, whereby compound (16) can be produced. The reaction conditions, reagents, base and inert solvent used for forming the amide bond between the peptide carboxylic acid (11) and compound (15) can be appropriately selected from those described for the synthesis of compound (6).

[0346] Compound (17) can be produced by deprotecting the protecting group for the carboxy group of the obtained compound (16). This deprotection can be carried out in the same manner as the deprotection at the carboxy group for producing compound (14).

[0347] Compound (2) can be produced by derivatizing compound (17) into an active ester, mixed acid anhydride, acid halide or the like and reacting it with compound (4) in the presence of a base. For said reaction, it is also possible to use the reaction reagents and reaction conditions generally used for peptide synthesis, and the reaction conditions, reagents, base and inert solvent used for said reaction can be appropriately selected from those described for the synthesis of compound (6).

[0348] Production Method 3

[0349] The compound represented by formula (2) of the intermediate can also be produced by the following method.

[0350]

Chemical formula

[0351] In the above formula, L 1’ is converted at the end to a maleimidyl group and corresponds to L 4 having a structure in which P 1 represents a protecting group.

[0352] Compound (19) can be produced by derivatizing compound (11) into an active ester, mixed acid anhydride or the like and reacting it with a peptide carboxylic acid (18) having a C-terminus protected with P 4 in the presence of a base. The reaction conditions, reagents, base and inert solvent used for forming the peptide bond between peptide carboxylic acid (18) and compound (11) can be appropriately selected from those described for the synthesis of compound (6). The protecting group P 4can be appropriately selected from the above protecting groups.

[0353] Compound (20) can be produced by deprotecting the protecting group for the carboxy group of the obtained compound (19). This deprotection can be carried out in the same manner as the deprotection of the carboxy group for producing compound (14).

[0354] Compound (2) can be produced by derivatizing the obtained compound (20) into an active ester, mixed acid anhydride or the like and reacting it with compound (7). For this reaction, it is also possible to use reaction reagents and reaction conditions generally used for peptide synthesis, and the reaction conditions, reagents, bases and inert solvents used for this reaction can be appropriately selected from those described for the synthesis of compound (6).

[0355] Production method 4

[0356] Hereinafter, in this specification, n in the production intermediate (10) described in production method 2 1 = 1, L a = O, a method for producing compound (10b) will be described in detail. For example, according to the following method, a compound represented by formula (10b), a salt or solvate thereof can be produced.

[0357]

Chemical formula

[0358] In the above formula, L P is as defined above, L represents an acyl group such as an alkanoyl group such as an acetyl group or an aroyl group such as a benzoyl group, a hydrogen atom or the like, X and Y each represent an oligopeptide consisting of 1 to 3 amino acids, P 5 and P 7 each represent a protecting group for an amino group, and P 6 represents a protecting group for a carboxy group.

[0359] Use the method described in JP-A-2002-60351 or the literature (J. Org. Chem., Vol. 51, p. 3196, 1986), or apply it, and if necessary, remove the protecting group or modify the functional group to produce the compound represented by formula (21). Alternatively, it can also be obtained by treating an amino acid with a protected terminal amino group, or by treating an acid amide of an oligopeptide with a protected amino group having an aldehyde or a ketone.

[0360] Compound (23) can be produced by reacting compound (21) with a compound (22) having a hydroxyl group in an inert solvent in the presence of an acid or a base at a temperature ranging from a cooling temperature condition to room temperature.

[0361] Examples of the acid that can be used here include inorganic acids such as hydrofluoric acid, hydrogen chloride, sulfuric acid, nitric acid, phosphoric acid, and boric acid, organic acids such as acetic acid, citric acid, p-toluenesulfonic acid, and methanesulfonic acid, and Lewis acids such as tetrafluoroborate, zinc chloride, tin chloride, aluminum chloride, and iron chloride. Among them, sulfonic acid, particularly p-toluenesulfonic acid, is preferred. Regarding the base, any one of the above-mentioned bases can be appropriately selected and used. Preferred examples thereof include alkali metal alkoxides such as potassium tert-butoxide, alkali metal hydroxides such as sodium hydroxide and potassium hydroxide, alkali metal hydrides such as sodium hydride and potassium hydride, organometallic bases represented by dialkylaminolithium such as lithium diisopropylamide, and organometallic bases of bissilylamine such as lithium bis(trimethylsilyl)amide. Examples of the solvent used for the reaction include ether solvents such as tetrahydrofuran and 1,4-dioxane, and aromatic hydrocarbon solvents such as benzene and toluene. These solvents can be prepared as a mixture with water. Further, P 5The protecting group for the amino group as exemplified by 5 is not particularly limited as long as it is a group generally used for the protection of amino groups. Representative examples include the protecting groups for amino groups described in Production Method 2. However, in this reaction, the protecting group for the amino group as exemplified by P

[0362] may be cleaved. In such a case, it may be necessary to reintroduce the protecting group, so it is necessary to carry out a reaction with an appropriate reagent for protecting the amino group. 6 Compound (24) can be produced by removing the protecting group P 6 Representative examples of the protecting groups for carboxy groups as exemplified by 5 are described in Production Method 2, and appropriate ones can be selected therefrom. In Compound (23), the protecting group P 6 for the amino group and the protecting group P 5 for the carboxy group are desirably protecting groups that can be removed by different methods or under different conditions. For example, representative examples include a combination where P 6 is a 9-fluorenylmethyloxycarbonyl group and P

[0363] is a benzyl group. For example, the protecting group can be selected according to the properties of the compound having the amino group and carboxy group to be protected. For the removal of the protecting group, the reagents and conditions are selected according to the protecting group. 5 Compound (26) can be produced by derivatizing carboxylic acid (24) into an active ester, mixed acid anhydride, acid halide or the like and reacting it with Compound (4) or a pharmaceutically acceptable salt thereof to produce Compound (25), and then removing the protecting group P 6 of the obtained Compound (25). For the reaction between Compound (4) and carboxylic acid (24) and for the reaction for removing the protecting group P

[0364] React the compound (26) with an amino acid having a protected terminal amino group or an oligopeptide (27) having a protected amino group to produce the compound (9b), and the protecting group P of the obtained compound (9b) 7 can be removed to produce the compound (10b). The protecting group represented by P 7 for the amino group is not particularly limited when generally used for the protection of the amino group. Representative examples thereof include the protecting groups for the amino group described in Production Method 2. For removing the protecting group, the reagent and conditions are selected according to the protecting group. For the reaction between the compound (26) and the compound (27), the reaction reagents and reaction conditions generally used for peptide synthesis can be used. The compound (10b) produced by the above method can be derivatized into the compound (1) of the present invention according to the above method.

[0365] The anti-TROP2 antibody-drug conjugate of the present invention may remain in the air or, for example, when recrystallized for purification, absorb moisture, have adsorbed water, or change to a hydrate, and such compounds, and water containing salts are also included in the present invention.

[0366] Compounds labeled with various radioisotopes or non-radioisotopes are also included in the present invention. One or more atoms constituting the antibody-drug conjugate of the present invention may contain atomic isotopes in non-natural ratios. Examples of atomic isotopes include deuterium ( 2 H), tritium ( 3 H), iodine-125 ( 125 I) and carbon-14 ( 14 C). Further, the compounds of the present invention include tritium ( 3 H), iodine-125 ( 125 I), carbon-14 ( 14 C), copper-64 ( 64 Cu), zirconium-89 ( 89 Zr), iodine-124 ( 124 I), fluorine-18 ( 18 F), indium-111 ( 111I), Carbon 11( 11 C) and Iodine 131( 131 I) etc. may be radiolabeled with radioisotopes. Compounds labeled with radioisotopes are useful as therapeutic or prophylactic agents, research reagents such as assay reagents, and diagnostic agents such as in vivo diagnostic imaging agents. Regardless of radioactivity, any isotope isomers of the antibody-drug conjugates of the present invention are within the scope of the present invention.

[0367] Antibody-drug conjugate (ADC) The present disclosure provides a TROP2-targeted antibody-drug conjugate (ADC) comprising an anti-TROP2 antibody and an anticancer compound such as a topoisomerase I inhibitor (DXd). See Figure 1. In some embodiments, the TROP2-targeted ADC may comprise Formula 13 as shown below.

[0368] [Chemical formula]

[0369] In some embodiments, the heavy chain of the ADC is QVQLVQSGAEVKKPGASVKVSCKASGYTFTTAGMQWVRQAPGQGLEWMGWINTHSGVPKYAEDFKGRVTISADTSTSTAYLQLSSLKSEDTAVYYCARSGFGSSYWYFDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 45) may include.

[0370] In some embodiments, the light chain of the ADC is DIQMTQSPSSLSASVGDRVTITCKASQDVSTAVAWYQQKPGKAPKLLIYSASYRYTGVPSRFSGSGSGTDFTLTISSLQPEDFAVYYCQQHYITPLTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 46) may include.

[0371] The anti-TROP2 antibody-drug conjugate of the present invention exhibits cytotoxic activity against cancer cells and thus can be used as a drug, particularly as a therapeutic and / or prophylactic agent for cancer.

[0372] That is, the anti-TROP2 antibody-drug conjugate of the present invention can be selectively used as a drug for chemotherapy, which is a major method for treating cancer. As a result, it can delay the growth of cancer cells, inhibit their proliferation, and further kill cancer cells. By this, the symptoms caused by cancer in the affected body may disappear, or it may be possible to achieve an improvement in the QOL of the affected body, and the therapeutic effect is achieved by prolonging the life of the affected body. Even when the anti-TROP2 antibody-drug conjugate of the present invention does not achieve the death of cancer cells, it may be possible to achieve a longer survival period of the affected body and a higher QOL of the affected body by inhibiting or controlling the proliferation of cancer cells.

[0373] In such drug therapy, it can be used as a drug alone and as a drug in combination with additional therapy in adjuvant therapy, and can be used in combination with surgical operation, radiotherapy, hormone therapy or the like. Furthermore, it can also be used as a drug for drug therapy in neoadjuvant therapy.

[0374] In addition to the therapeutic uses as described above, the effect of suppressing the growth of micrometastatic cancer cells and further killing them by binding to these cancer cells can also be expected by the binding characteristics of the antibody to the antigen. In particular, when the expression of TROP2 is confirmed in primary cancer cells, the administration of the anti-TROP2 antibody-drug conjugate of the present invention can be expected to have an inhibitory or preventive effect on cancer metastasis. For example, it can be expected to have an effect of inhibiting and killing cancer cells in body fluids during the process of metastasis, or an effect of inhibiting and killing microcancer cells immediately after implantation in any tissue, for example. Furthermore, in particular, after surgical resection of cancer, an inhibitory or preventive effect on cancer metastasis can be expected. Therefore, an effect of inhibiting cancer metastasis can be expected.

[0375] The anti-TROP2 antibody-drug conjugate of the present invention can be expected to exert a therapeutic effect by administration as a systemic treatment to the affected body and, additionally, by local administration to the cancer tissue.

[0376] Examples of cancer types to which the anti-TROP2 antibody-drug conjugate of the present invention is applied include lung cancer, kidney cancer, urothelial cancer, colorectal cancer, prostate cancer, glioblastoma multiforme, ovarian cancer, pancreatic cancer, breast cancer, melanoma, liver cancer, bladder cancer, gastric cancer, cervical cancer, head and neck cancer, or esophageal cancer. However, as long as the cancer cells to be treated express a protein that can be recognized by the antibody in the antibody-drug conjugate within the cancer cells, they are not limited thereto.

[0377] The anti-TROP2 antibody-drug conjugate of the present invention can preferably be administered to mammals, more preferably to humans.

[0378] Pharmaceutical compositions and modes of administration The substances used in the pharmaceutical composition containing the anti-TROP2 antibody-drug conjugate of the present invention can be appropriately selected from formulation additives generally used in the art or the like in view of the dosage or administration concentration and can be applied.

[0379] The anti-TROP2 antibody-drug conjugate of the present invention can be administered as a pharmaceutical composition containing at least one pharmaceutically appropriate component. For example, the pharmaceutical composition typically contains at least one pharmaceutical carrier (e.g., a sterilized liquid). In some embodiments, the liquid includes, for example, water and oils (petroleum, and oils derived from animals, plants, or synthetic sources). The oil may be, for example, peanut oil, soybean oil, mineral oil, or sesame oil. When the pharmaceutical composition is administered intravenously, water is a more typical carrier. As a liquid carrier for injection solutions in particular, saline solutions, aqueous dextrose solutions, and aqueous glycerol solutions can also be used. Appropriate pharmaceutical vehicles are known in the art. Optionally, the above compositions may contain trace amounts of humectants, emulsifiers, or pH buffering agents. Examples of suitable pharmaceutical carriers are disclosed in "Remington’s Pharmaceutical Sciences" by E.W. Martin. The formulation corresponds to the mode of administration.

[0380] Pharmacologically acceptable carriers for various dosage forms are known in the art. For example, excipients, lubricants, binders, and disintegrants for solid preparations are known, and solvents, solubilizers, suspending agents, isotonic agents, buffers, and soothing agents for liquid preparations are known. In some embodiments, the pharmaceutical composition includes one or more additional components such as one or more preservatives, antioxidants, stabilizers, and the like.

[0381] Additionally, the disclosed pharmaceutical compositions can be formulated as solutions, microemulsions, liposomes, or other ordered structures suitable for high drug concentrations. The carrier can be, for example, a solvent or dispersion medium including water, ethanol, polyols (such as glycerol, propylene glycol, and liquid polyethylene glycol and the like), and suitable mixtures thereof. For example, the use of coatings such as lecithin, the maintenance of the required particle size in the case of dispersions, and the use of surfactants can maintain appropriate fluidity. In some embodiments, it is preferable to include in the composition an isotonic agent such as sugar, a polyhydric alcohol such as mannitol, sorbitol, or sodium chloride. By including in the composition an agent for delaying absorption such as a monostearate salt and gelatin, sustained absorption of the injectable composition can be achieved.

[0382] If necessary, a sterile injectable solution can be prepared by incorporating the required amount of the active compound into a suitable solvent having one or a combination of the components listed above and then performing sterile microfiltration. Generally, a dispersion is prepared by incorporating the active compound into a sterile vehicle containing a basic dispersion medium and the other necessary components from those listed above. In the case of a sterile powder for the preparation of a sterile injectable solution, the preferred method of preparation is vacuum drying and freeze-drying (lyophilization) to obtain a powder of the active ingredient and any additional desired components from its previously sterile-filtered solution.

[0383] Various delivery systems are known and can be used to administer the anti-TROP2 antibody-drug conjugate of the present invention. Examples of administration routes include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, and subcutaneous routes. Said administration can be effected, for example, by injection or bolus injection. According to a specific preferred embodiment, the administration of the antibody-drug conjugate is effected by injection. Parenteral administration is the preferred route of administration.

[0384] According to a representative embodiment, the pharmaceutical composition is defined as a pharmaceutical composition suitable for intravenous administration to humans according to conventional techniques. The composition for intravenous administration is typically a solution in a sterile and isotonic aqueous buffer. Optionally, the drug may contain a solubilizing agent and a local anesthetic (e.g., lidocaine) to relieve pain at the injection site. Generally, the above components are provided individually as either a lyophilized powder or an anhydrous concentrate contained within a container obtained by sealing within an ampoule or sachet having an amount of the active agent, or as a mixture of unit dosage forms. When the drug is in the form of administration by injection, it may be administered from an injection vial containing sterile pharmaceutical grade water or saline. When the drug is administered by injection, an ampoule of sterile water or sterile saline for injection may be provided so that the above components are mixed with each other prior to administration.

[0385] The pharmaceutical composition of the present invention may be a pharmaceutical composition containing only the anti-TROP2 antibody-drug conjugate of the present application, or a pharmaceutical composition containing the anti-TROP2 antibody-drug conjugate and at least one cancer treatment agent other than the conjugate. The anti-TROP2 antibody-drug conjugate of the present invention can be administered to an individual together with, simultaneously with, or sequentially with other cancer treatment agents, and accordingly, the anti-cancer effect can be enhanced. Another anti-cancer agent used for such a purpose may be administered simultaneously with, separately from, or following the antibody-drug conjugate, and may be administered while varying the administration interval for each. Examples of cancer treatment agents include Abraxane, paclitaxel, cisplatin, gemcitabine, irinotecan (CPT-11), paclitaxel, pemetrexed, sorafenib, vinorelbine, the drugs described in WO 2003 / 038043, LH-RH analogs (leuprolide, goserelin or the like), estramustine phosphate, estrogen antagonists (tamoxifen, raloxifene or the like) and aromatase inhibitors (anastrozole, letrozole, exemestane or the like), but it is not limited as long as it is a drug having anti-tumor activity.

[0386] The pharmaceutical composition can be formulated into a lyophilized preparation or a liquid preparation as a preparation having a desired composition and a required purity. When it is formulated as a lyophilized preparation, it may be a preparation containing appropriate formulation additives used in the art. For the liquid preparation as well, it can be formulated as a liquid preparation containing various formulation additives used in the art.

[0387] The composition and concentration of the pharmaceutical composition may vary depending on the administration method. However, the anti-TROP2 antibody-drug conjugate contained in the pharmaceutical composition of the present invention exhibits a pharmaceutical effect event even at a low dose when the antibody-drug conjugate has a higher affinity for the antigen, that is, a higher affinity (i.e., a lower Kd value) with respect to the dissociation constant for the antigen (i.e., the Kd value). Therefore, in order to determine the dose of the antibody-drug conjugate, the dose can be determined in view of the situation regarding the affinity between the antibody-drug conjugate and the antigen. When administering the antibody-drug conjugate of the present invention to a human, for example, about 0.001 to 100 mg / kg can be administered once, or it can be administered several times at intervals of 1 to 180 days each time.

[0388] TROP2-expressing cancer TROP2 is highly expressed in epithelial cancers, and its expression is associated with a low survival rate. Examples of TROP2-expressing cancers include, but are not limited to, lung cancer, kidney cancer, urothelial cancer, colorectal cancer, prostate cancer, glioblastoma multiforme, ovarian cancer, pancreatic cancer, breast cancer, melanoma, liver cancer, bladder cancer, gastric cancer, cervical cancer, head and neck cancer, and esophageal cancer. Any of these cancers may be treated with the disclosed ADCs and their dosages. However, it should be understood that cancer cells may be treated according to the disclosed methods even if they do not fall into the above-listed categories of cancer as long as they express TROP2.

[0389] Non-small cell lung cancer (NSCLC) is a type of lung cancer that is particularly suitable for treatment using the disclosed ADCs and dosages. For example, in Examples 5 to 7, a phase 1 clinical trial of administering the disclosed ADC to subjects with NSCLC is described in detail.

[0390] The disclosed TROP2-targeting ADCs can be used to treat any of the above TROP2-expressing cancers.

[0391] Treatment methods and uses The present disclosure provides a method for treating cancer comprising administering an anti-TROP2 antibody-drug conjugate as disclosed herein. Also provided herein is any of the disclosed anti-TROP ADCs for use in the treatment of cancer.

[0392] In some embodiments, the cancer is a TROP2-expressing cancer. Examples of TROP2-expressing cancers include, but are not limited to, lung cancer (e.g., non-small cell lung cancer or NSCLC), kidney cancer, urothelial cancer, colorectal cancer, prostate cancer, glioblastoma multiforme, ovarian cancer, pancreatic cancer, breast cancer, melanoma, liver cancer, bladder cancer, gastric cancer, cervical cancer, head and neck cancer, and esophageal cancer.

[0393] For the purposes of the present disclosure, the term "TROP2-overexpressing cancer" is not particularly limited as long as it is recognized as a TROP2-overexpressing cancer by those skilled in the art. Preferred examples of TROP2-overexpressing cancers include cancers with a high score for TROP2 expression in immunohistochemistry (IHC) or in situ hybridization (ISH). Examples of in situ hybridization methods of the present invention include fluorescence in situ hybridization (FISH) and dual-color in situ hybridization (DISH).

[0394] The method for scoring the degree of TROP2 expression by immunohistochemistry or the method for determining positive or negative for TROP2 expression by in situ hybridization is not particularly limited as long as it is recognized by those skilled in the art.

[0395] The ADCs and the treatment methods and uses of the present invention can preferably be used for the treatment of inoperable cancer or recurrent cancer.

[0396] In some embodiments, the ADC and the treatment methods and uses of the present invention can also be used as a pharmaceutical composition for the treatment of cancer, comprising an antibody-drug conjugate used in the present invention, a salt thereof, or a hydrate thereof as an active ingredient, and a pharmaceutically acceptable formulation ingredient.

[0397] In some embodiments, the ADC and the treatment methods and uses of the present invention exhibit excellent antitumor activity against cancers that show resistance to existing anticancer drugs (i.e., resistant cancers), particularly cancers that have acquired resistance to existing anticancer drugs (i.e., secondary resistant cancers). Therefore, when the ADC for the treatment of the present invention is applied to a group of cancer patients with resistance to existing anticancer drugs (patients with a history of treatment with existing anticancer drugs) among cancer patients, it exerts a significant antitumor effect. In particular, the cancer to be treated may be resistant or refractory to treatment with an EGFR inhibitor (i.e., gefitinib, erlotinib, osimertinib, afatinib), an ALK inhibitor (i.e., alectinib, crizotinib, ceritinib), platinum-based chemotherapy (i.e., cisplatin, carboplatin), and / or a checkpoint inhibitor (i.e., nivolumab, pembrolizumab, atezolizumab, avelumab, ipilimumab, durvalumab, tislelizumab, sintilimab, semaprilumab).

[0398] The ADC for the treatment of the present invention can show a high therapeutic effect, for example, against cancers that have acquired resistance to these existing anticancer drugs, by being administered to cancer patients instead of or in combination with these existing anticancer drugs.

[0399] Therefore, in some embodiments of the disclosed methods and uses, the cancer to be treated may be a resistant form of lung cancer (e.g., non-small cell lung cancer or NSCLC), renal cancer, urothelial cancer, colorectal cancer, prostate cancer, glioblastoma multiforme, ovarian cancer, pancreatic cancer, breast cancer, melanoma, liver cancer, bladder cancer, gastric cancer, cervical cancer, head and neck cancer, and esophageal cancer.

[0400] The ADC, and the method or use for the treatment of the present invention, are capable of delaying the growth of cancer cells, inhibiting their proliferation, and further killing cancer cells. Due to these effects, the cancer patient may be free from the symptoms caused by cancer or achieve an improvement in the quality of life (QOL) of the cancer patient, and the therapeutic effect is achieved by prolonging the lifespan of the cancer patient. Even when the anti-TROP2 antibody-drug conjugate of the present invention does not achieve the death of cancer cells, it may achieve a longer survival period and provide a higher QOL for the cancer patient by inhibiting or controlling the proliferation of cancer cells.

[0401] In some embodiments of the disclosed methods and uses, the ADC can be used as a drug alone or as a drug in combination with additional treatments in adjuvant therapy, and can be combined with surgical procedures, radiotherapy, hormonal therapy or the like. Further, it can also be used as a drug for drug therapy in neoadjuvant therapy. In some embodiments, the ADC may be combined with anti-cancer agents such as, for example, Abraxane, paclitaxel, cisplatin, carboplatin, gemcitabine, irinotecan (CPT-11), pemetrexed, sorafenib, vinorelbine, the drugs described in WO 2003 / 038043, LH-RH analogs (leuprolide, goserelin or the like), estramustine phosphate, estrogen antagonists (tamoxifen, raloxifene or the like), aromatase inhibitors (anastrozole, letrozole, exemestane or the like), EGFR inhibitor treatments (gefitinib, erlotinib, osimertinib, afatinib), ALK inhibitor treatments (alectinib, crizotinib, ceritinib) and / or checkpoint inhibitor treatments (nivolumab, pembrolizumab, atezolizumab, avelumab, ipilimumab, durvalumab, tislelizumab, sintilimab, semiprimumab), but is not limited thereto.

[0402] In addition to the above-described treatment method and use, it is also possible to expect a preventive effect of suppressing the growth of small metastatic cancer cells and further killing them. In particular, when the expression of TROP2 is confirmed in primary cancer cells, by administering the anti-TROP2 antibody-drug conjugate of the present invention, an inhibitory or preventive effect on cancer metastasis can be expected. For example, an effect of inhibiting and killing cancer cells in body fluids during the process of metastasis, or an effect of inhibiting and killing small cancer cells immediately after implantation in any tissue can be expected. Therefore, in particular, after surgical removal of cancer, an inhibitory or preventive effect on cancer metastasis can be expected.

[0403] In some embodiments of the methods and uses, the ADC may be administered to a subject with cancer (e.g., TROP2-expressing cancer) at about 0.1 to about 15 mg / kg, about 0.5 to about 12 mg / kg, about 1.0 to about 10 mg / kg, or about 4 to about 8 mg / kg. That is, in some embodiments, the dosage of the ADC administered to the subject may be about 0.1 mg / kg or more, about 0.2 mg / kg or more, about 0.3 mg / kg or more, about 0.4 mg / kg or more, about 0.5 mg / kg or more, about 0.6 mg / kg or more, about 0.7 mg / kg or more, about 0.8 mg / kg or more, about 0.9 mg / kg or more, about 1.0 mg / kg or more, about 1.25 mg / kg or more, about 1.5 mg / kg or more, about 1.75 mg / kg or more, about 2.0 mg / kg or more, about 2.25 mg / kg or more, about 2.5 mg / kg or more, about 2.75 mg / kg or more, about 3.0 mg / kg or more, about 3.25 mg / kg or more, about 3.5 mg / kg or more, about 3.75 mg / kg or more, about 4.0 mg / kg or more, about 4.25 mg / kg or more, about 4.5 mg / kg or more, about 4.75 mg / kg or more, about 5.0 mg / kg or more, about 5.25 mg / kg or more, about 5.5 mg / kg or more, about 5.75 mg / kg or more, about 6.0 mg / kg or more, about 6.25 mg / kg or more, about 6.5 mg / kg or more, about 6.75 mg / kg or more, about 7.0 mg / kg or more, about 7.25 mg / kg or more, about 7.5 mg / kg or more, about 7.75 mg / kg or more, about 8.0 mg / kg or more, about 8.25 mg / kg or more, about 8.5 mg / kg or more, about 8.75 mg / kg or more, about 9.0 mg / kg or more, about 9.25 mg / kg or more, about 9.5 mg / kg or more, about 9.75 mg / kg or more, about 10.0 mg / kg or more, about 10.25 mg / kg or more, about 10.5 mg / kg or more, about 10.75 mg / kg or more, about 11.0 mg / kg or more, about 11.25 mg / kg or more, about 11.5 mg / kg or more, about 11.75 mg / kg or more, or about 12 mg / kg or more.In some embodiments, the dosage of the ADC administered to the subject may be 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.25, 1.5, 1.75, 2.0, 2.25, 2.5, 2.75, 3.0, 3.25, 3.5, 3.75, 4.0, 4.25, 4.5, 4.75, 5.0, 5.25, 5.5, 5.75, 6.0, 6.25, 6.5, 6.75, 7.0, 7.25, 7.5, 7.75, 8.0, 8.25, 8.5, 8.75, 9.0, 9.25, 9.5, 9.75, 10.0, 10.25, 10.5, 10.75, 11.0, 11.25, 11.5, 11.75, or 12 mg / kg or more. In some embodiments, the dosage may be about 2 mg / kg to about 10 mg / kg, about 2 mg / kg to about 8 mg / kg, about 4 mg / kg to about 10 mg / kg, about 4 mg / kg to about 8 mg / kg, about 6 mg / kg to about 10 mg / kg, or about 6 mg / kg to about 8 mg / kg. In a preferred embodiment, the dosage may be 2 mg / kg, 3 mg / kg, 4 mg / kg, 5 mg / kg, 6 mg / kg, 7 mg / kg, 8 mg / kg, 9 mg / kg, or 10 mg / kg, more preferably 4 mg / kg, 6 mg / kg, or 8 mg / kg.

[0404] In some embodiments of the method and the use, the anti-TROP2 ADC or its pharmaceutical composition is administered to a cancer subject via parenteral administration. Preferred parenteral routes of administration include, but are not limited to, injections such as intravenous injection, intramuscular injection, and subcutaneous injection. The anti-TROP2 antibody-drug conjugate used in the present invention is expected to exert a therapeutic effect by systemic application to the affected body and, additionally, by local application to cancer tissue.

[0405] The timing of administration or the dosage regimen may be once every week (q1w), once every two weeks (q2w), once every three weeks (q3w), once every four weeks (q4w), once every five weeks (q5w), once every six weeks (q6w), once every seven weeks (q7w), once every eight weeks (q8w), once every nine weeks (q9w) or once every ten weeks (q10w), but is preferably once every three weeks or once every four weeks.

[0406] The dosage regimen may be adjusted to provide an optimal desired response (e.g., a therapeutic response such as tumor regression or tumor remission). For example, in some embodiments, the dosage regimen may be 2 mg / kg once every three weeks (q3w), 4 mg / kg once every three weeks (q3w), 6 mg / kg once every three weeks (q3w), 8 mg / kg once every three weeks (q3w), 2 mg / kg once every four weeks (q4w), 4 mg / kg once every four weeks (q4w), 6 mg / kg once every four weeks (q4w), or 8 mg / kg once every four weeks (q4w). And in some embodiments, a single bolus may be administered, while in some embodiments, several divided doses may be administered over time, or the dosage may be proportionally decreased or increased as indicated by the situation.

[0407] Furthermore, the subjects of the method and the use are generally cancer sufferers, but the age of the sufferers is not limited. The disclosed method and use are useful for treating cancers, malignant diseases or cancer cell proliferation with various recurrence outcomes and prognostic outcomes across all age groups and age cohorts. Thus, in some embodiments, the subject may be a pediatric subject, while in other embodiments, the subject may be an adult subject.

[0408] To illustrate the present invention, the following examples are shown. However, it should be understood that the present invention is not limited to the specific conditions or details described in these examples. [Examples]

Examples

[0409] Generation of Antibody-Drug Conjugates According to the generation methods disclosed in International Publication No. WO 2015 / 098099 and International Publication No. WO 2017 / 002776, using an anti-TROP2 antibody (for example, an antibody comprising a heavy chain consisting of the amino acid sequence at amino acid positions 1 to 451 in SEQ ID NO: 45 and a light chain consisting of the amino acid sequence at amino acid positions 1 to 214 in SEQ ID NO: 46), The anti-TROP2 antibody is linked via a thioether bond to the following formula:

[0410]

Chemical Formula

[0411] (In the formula, n represents the average drug-antibody ratio (DAR) per single antibody molecule. The value of n for the antibody-drug conjugate (1) is within the range of 3.5 to 4.5, and the value of n for the antibody-drug conjugate (2) is within the range of 6.5 to 8.0), antibody-drug conjugates (1) and (2) (hereinafter referred to as "antibody-drug conjugate (1)" and "antibody-drug conjugate (2)") were generated, which are bound to a drug linker represented by the formula.)

[0412] The schematic structures and sequences of antibody-drug conjugate (1) and antibody-drug conjugate (2) can be found in FIGS. 1 and 2.

Examples

[0413] Test on the Antitumor Effect of Antibody-Drug Conjugates For the purpose of this experiment, the experiment was conducted on 5- to 6-week-old female BALB / c nude mice (Charles River Laboratories Japan). A human pancreatic adenocarcinoma cell line (CFPAC-1 cells) purchased from ATCC was suspended in saline, and 4×10 6Individual cells were transplanted into the right side of each body of the female nude mice. Fourteen days after transplantation, the mice were grouped (Day0). In the single-dose group (once every three weeks), antibody-drug conjugates (1) and (2) were administered at a dose of 0.3 mg / kg or 1 mg / kg on Day0. In the frequent-dose group (once a week for three weeks), antibody-drug conjugates (1) and (2) were administered at a dose of 0.3 mg / kg on Day0, Day8, and Day14. A vehicle-administered group was determined as the control group. Tumor growth inhibition (TGI) on Day22 was obtained by calculation. No particularly significant findings such as weight loss were confirmed in any of the administered groups.

[0414] Measurement / calculation formula: The major axis and minor axis of the tumor were measured twice a week using an electronic digital caliper (CD-15CX, Mitutoyo Corporation), and the tumor volume (mm 3 ) was calculated. The calculation formula is as shown below. Tumor volume (mm 3 ) = 1 / 2 × major axis (mm) × [minor axis (mm)] 2

[0415] Tumor growth inhibition (TGI) was calculated according to the following calculation formula. Tumor growth inhibition (%) = 100 × (1 - T / C) (where T represents the average tumor volume of the test substance-administered mouse group, and C represents the average tumor volume of the control mouse group).

[0416] Antibody-drug conjugates (1) and (2) were each diluted with acetate buffered saline (pH 5.5) (manufactured by Nacalai Tesque, Inc., hereinafter referred to as "ABS buffer"). The diluted solution (10 mL / kg) was administered via the tail vein.

[0417] The antitumor effects of antibody-drug conjugates (1) and (2) are shown in Figure 3. In antibody-drug conjugate (1), the TGI of the single-dose group at a dose of 0.3 mg / kg was 15%, and that of the single-dose group at a dose of 1 mg / kg was 86%, while the TGI of the multiple-dose group at a dose of (0.3 mg / kg) was 34%. In antibody-drug conjugate (2), the TGI of the single-dose group at a dose of 0.3 mg / kg was 43%, and that of the single-dose group at a dose of 1 mg / kg was 94%, while the TGI of the multiple-dose group at a dose of (0.3 mg / kg) was 80%.

[0418] From the above results, in both cases of antibody-drug conjugates (1) and (2), when comparing the single-dose group at a dose of 1 mg / kg with the multiple-dose group at a dose of 0.3 mg / kg, almost the same total dose was provided in both cases, but the TGI of the single-dose group was higher than that of the multiple-dose group. From this, it was shown that the single-dose method of administering the total dose only once every three weeks has better efficacy than the multiple-dose method of repeating the administration of the above dose three times a week. In the comparison between antibody-drug conjugates (1) and (2), the TGI of the single-dose group of antibody-drug conjugate (1) at a dose of 1 mg / kg was higher than the TGI of the single-dose group of antibody-drug conjugate (2) at a dose of 0.3 mg / kg and lower than the TGI of the single-dose group of antibody-drug conjugate (2) at a dose of 1 mg / kg. From this, it was shown that the difference in the therapeutic doses between antibody-drug conjugates (1) and (2) is within a three-fold range.

Example

[0419] Safety evaluation of antibody-drug conjugate The antibody-drug conjugates (1) and (2) generated according to Example 1 were separately administered to cross-reactive species (cynomolgus monkeys). More specifically, for the antibody-drug conjugate (1), it was administered a total of 3 times at intervals of once every 3 weeks, while for the antibody-drug conjugate (2), it was administered a total of 2 times at intervals of once a week. In the case of the antibody-drug conjugate (1), the observation was continued until the day after the final administration. In the case of the antibody-drug conjugate (2), the observation was continued until the week after the final administration. As a result, the maximum tolerated dose (HNSTD) at which severe toxicity of the antibody-drug conjugate (2) did not occur was less than 10 mg / kg, while the HNSTD of the antibody-drug conjugate (1) was 30 mg / kg. Therefore, it was shown that the antibody-drug conjugate (1) has better safety than the antibody-drug conjugate (2).

Example

[0420] Estimation of effective dose / administration dose of antibody-drug conjugate (1) in humans Hereinafter, "antibody-drug conjugate (1)" may also be referred to as "DS-1062a".

[0421] An antibody-drug conjugate (1) (i.e., DS-1062a) was intravenously administered once to cynomolgus monkeys at doses of 0.2 mg / kg, 0.6 mg / kg, 2 mg / kg, or 6 mg / kg. Subsequently, pharmacokinetic parameters were calculated using a target-mediated pharmacokinetic model based on the plasma concentration of the antibody-drug conjugate (1). Furthermore, the change in the plasma concentration of the antibody-drug conjugate over time during repeated administration to humans was estimated. The antibody-drug conjugate (1) was administered by repeating a dosing cycle consisting of 0.27 mg / kg, 0.54 mg / kg, 0.81 mg / kg, 1.6 mg / kg, 3.2 mg / kg, and 6.4 mg / kg once every three weeks (q3w×3). The results are shown in Figure 4. The change in the plasma concentration of the antibody-drug conjugate (1) over time estimated in humans was compared with the plasma concentration on Day 21 in the CFPAC-1 tumor-bearing mouse model. As a result, the plasma concentrations estimated at the time of administration to humans once every three weeks (q3w) exceeded the minimum concentration (1 mg / kg dosing group, 0.312 μg / mL) showing a tumor regression effect in mice in half and most of the dosing intervals, and the doses were 0.27 mg / kg and 0.81 mg / kg, respectively. From this, it was estimated that the effective dose / administration dose of the antibody-drug conjugate (1) in humans is once every three weeks and 0.27 mg / kg or more.

Example

[0422] Initial Phase 1 Clinical Trial

[0423] Preface DS-1062a is a trophoblast cell surface antigen 2 (TROP2)-targeted antibody-drug conjugate having a novel topoisomerase I inhibitor (exatecan derivative; DXd). DS-1062a binds to TROP2 on the cell surface, inhibits topoisomerase I, internalizes DXd into the cytoplasm, and releases it after enzymatic treatment that causes apoptosis of target cells.

[0424] TROP2 is highly expressed in epithelial cancers including lung cancer and is associated with low survival rates. In preclinical studies, DS-1062a showed promising antitumor activity in xenograft mouse models.

[0425] Purpose

[0426] The purpose of this study was to evaluate the safety and tolerability of DS-1062a and to determine the maximum tolerated dose (MTD) and the recommended dose escalation (RDE) in the dose expansion part (see clinicaltrials.gov identifier NCT03401385).

[0427] Study Design and Methods

[0428] The Phase 1 study was a multicenter, open-label, repeated-dose, first-in-human study of DS-1062a that enrolled subjects in the United States and Japan. As shown in Figure 5, the study included a dose-escalation cohort and a dose-expansion cohort. The dose-escalation cohort included a single intravenous infusion of DS-1062a and a 21-day dose-limiting toxicity (DLT) observation period (Cycle 1). The dose-expansion cohort included administration of the dose of DS-1062a at the RDE to subjects with NSCLC.

[0429] The primary objective of the dose-escalation cohort was to identify the MTD for the RDE and to evaluate the safety and tolerability of that dose.

[0430] The primary objective for the dose-expansion cohort was to confirm the safety and tolerability of DS-1062a at the RDE.

[0431] The secondary objectives included measuring the pharmacokinetic (PK) properties of DS-1062a, total TROP2 antibody, drug components, and the antitumor activity of DS1062a. The exploratory objective included evaluating biomarkers correlated with response to DS-1062a.

[0432] The inclusion criteria included patients aged 20 years or older (in Japan) or 18 years or older (in the United States) with pathologically confirmed metastatic NSCLC with no standard treatment options, an Eastern Cooperative Oncology Group Performance Status of 0 or 1, measurable disease based on RECIST version 1.1, a life expectancy of more than 3 months, and available tumor tissue for measurement of recent TROP2 levels by immunohistochemistry.

[0433] The exclusion criteria included patients with multiple primary malignancies (excluding appropriately resected non-melanoma skin cancer, in situ disease treated curatively, or other solid tumors treated curatively without evidence of disease for more than 3 years) or patients with clinically significant / suspected lung disease.

[0434] Patient evaluations included pre-specified echocardiogram or MUGA scan, 12-lead electrocardiogram, AE, PK, human anti-human antibody, biomarker, and tumor evaluation at the time of hospital visit. The demographic and baseline characteristics of the patients included in this initial Phase 1 trial are shown in Figure 6.

[0435] The dose escalation of DS-1062a to determine the MTD was derived by a modified continual reassessment method using a Bayesian logistic regression model following the principle of escalation with overdose control. The objective response rate (ORR) was summarized by 95% confidence intervals (CI) using the Clopper-Pearson method, and the progression-free survival (PFS) / overall survival (OS) was summarized using the Kaplan-Meier method. Safety assessment items, PK parameters of DS-1062a, anti-TROP2 antibody, DXd, and plasma anti-drug antibody were summarized using descriptive statistics.

[0436] Results

[0437] Thirty-nine affected subjects were included in seven DS-1062a treatment groups, as well as in the demographic and baseline characteristics of the affected subjects at the cut-off. The affected subjects (N = 39) were exposed to a median (range) of 3.0 (1 - 10) treatment cycles with DS-1062a over a median (range) period of 8.86 (3.0 - 31.1) weeks.

[0438] Two affected subjects required interruption of DS-1062a administration (one in the 4 mg / kg group and one in the 8 mg / kg group), and one affected subject in the 6.0 mg / kg group required dose reduction. Overall, 23 (54.8%) affected subjects discontinued treatment with DS-1062a. The main reason for discontinuation was RECIST-defined PD in 13 affected subjects ([0.5 mg / kg and 2.0 mg / kg], n = 4 each; [1.0 mg / kg], n = 3; [0.27 mg / kg and 4.0 mg / kg], n = 1 each). Two affected subjects discontinued due to clinical progression (one each in the 0.27 mg / kg and 6.0 mg / kg groups), two affected subjects discontinued (one each in the 0.5 mg / kg and 4.0 mg / kg groups), and one affected subject discontinued based on the physician's decision (1.0 mg / kg group). Five affected subjects (n = 3 in the 1.0 mg / kg group and n = 2 in the 0.27 mg / kg group) discontinued for "other" reasons.

[0439] Overall, 87.2% (34 / 39) of the affected subjects reported one or more TEAEs, and all but one of the reported TEAEs were considered grade 3 or lower (Figure 7). The most frequently occurring TEAE was fatigue, reported in 13 (33.3%) of the affected subjects. All grade 3 TEAEs were reported in only one affected subject each, except for grade 3 fatigue reported in two affected subjects (one each in the 0.5 mg / kg and 2.0 mg / kg treatment groups).

[0440] Drug-related TEAEs were reported in 23 / 39 (59.0%) of the affected subjects, and 21 / 23 (91.3%) of these affected subjects with these TEAEs had a severity of grade 1 or 2. The most frequently occurring TEAEs in descending order of frequency (in 3 or more affected subjects) were nausea (n = 10), infusion-related reactions (n = 8), fatigue (n = 7), alopecia (n = 6), vomiting (n = 5), anemia and rash (n = 4 each), and anorexia and stomatitis (n = 3 each). The infusion-related reactions were all grade 1 or 2 events and were manageable / reversible.

[0441] Serious TEAEs were reported in 10 / 39 (25.6%) of the affected subjects, with most (n = 8) being grade 3, and 1 each being grade 2 and grade 5 (grade 5 sepsis; 6.0 mg / kg dose group). There were no serious TEAEs that occurred in 2 or more affected subjects. Only 1 case of serious TEAE was considered drug-related (fever, grade 2; 4.0 mg / kg dose group).

[0442] One case of dose-limiting toxicity (DLT) (morbilliform papular rash, grade 3; resolved) occurred in a subject in the 6.0 mg / kg dose group, but the MTD was not reached.

[0443] As shown in Figure 8, among 35 tumor-evaluable subjects, 7 cases of PR (based on RECIST, including single-point PR but the efficacy has not yet been confirmed) were observed. After the data cut, 3 additional cases of PR (all in the 8.0 mg dose group) were observed for a total of 10 cases of PR.

[0444] Computerized tomography (Figures 9A, 9C, and 9D) and positron emission tomography (Figure 9B) were performed on three affected subjects. In two affected subjects in the 4.0 mg / kg dose group, the tumor size decreased by up to 36.6% (Figure 9A) and 38.4% (Figure 9B) at 4.5 months after the start of treatment with DS-1062a. Another affected subject in the 2 mg / kg dose group showed a maximum 65.5% decrease in tumor size at 3 months after the start of treatment with DS-1062a (Figure 9C), and a significant decrease in the number of multiple lung metastases (non-target lesions) at 3 months and 7 months after the start of treatment (Figure 9D).

[0445] Figure 10 shows the best percent change in the sum of the longest dimensions from baseline in target lesions. The best percent change (68% tumor reduction) was observed in an affected subject in the 2.0 mg / kg dose group.

[0446] Regarding pharmacokinetics, as shown in Figure 11, the systemic exposure to DS-1062a increased in an almost dose-proportional manner. The plasma levels of DS-1062a and total anti-TROP2 antibody were similar, suggesting that DS-1062a was stable in circulation. The exposure to DXd was lower than that of DS-1062a.

[0447] Summary

[0448] At the data cut-off time point, the tolerability of DS-1062a was good. One case of DLT of grade 3 skin rash, which was transient and reversible, was observed in the 6.0 mg / kg dose group. Ten cases of PR and 16 cases of stable disease were observed with DS-1062a. Two of the affected subjects with PR had received prior EGFR inhibitor treatment or ALK inhibitor treatment (i.e., alectinib, crizotinib, ceritinib, osimertinib). The overall response rate of the said trial is provided in Figure 12.

Example

[0449] Phase I Clinical Trial at the New Cut-off Date and Time After the initial data cut, additional affected subjects were incorporated into the Phase 1 trial, and the total number of subjects was 59 (N = 59). All affected subjects had non-resected progressive NSCLC tumors that had recurred and were refractory to standard of care (SOC). Of the affected subjects, 57.7% were male, 88.5% had stage IV disease, 73.1% had adenocarcinoma histology, 80.8% had an Eastern Cooperative Oncology Group Performance Status (ECOG PS) of 1, and 86.5% had previously been refractory to conventional immune checkpoint inhibitor treatment. The same dose escalation and dose expansion trial design was used.

[0450] Figure 13 shows the number of affected subjects in the Phase 1 trial at a new cut-off date and time point when treatment-emergent adverse events (TEAEs) occurred under investigational treatment regardless of causality. Briefly, the dose-limiting toxicity (DLT) reached 10 mg / kg, and the maximum tolerated dose (MTD) was established at 8 mg / kg, which was also the recommended dose expansion (RDE) in the dose expansion part of the future dose expansion part. The median exposure period for the affected subjects was 10.6 (range 3.0 - 43.1) weeks. Serious TEAEs occurred in 14 (26.9%) affected subjects, and 3 (5.8%) affected subjects died, but the deaths were not related to the investigational drug. TEAEs related to dose reduction, interruption, or discontinuation occurred in 5 (9.6%), 5 (9.6%), and 2 (3.8%) affected subjects, respectively. One affected subject (1.9%) with disease progression treated at a dose of 6.0 mg / kg developed a pulmonary adverse event of special interest, respiratory failure (grade 5) that was determined not to be interstitial lung disease (ILD). Reports of 4 cases of potential ILD that had not yet been determined were noted, including cases after the data cut-off (1 case of grade 2 pneumonitis [6.0 mg / kg], 1 case of grade 2 organizing pneumonia [8 mg / kg], 1 case of grade 2 pneumonitis [8 mg / kg], and 1 case of grade 5 [respiratory failure in an affected subject with disease progression; 8.0 mg / kg]).

[0451] In the dose-escalation group of the trial, 12 partial responses (at least 10 were confirmed) were observed across all doses. At 8 mg / kg, 5 / 7 of the affected subjects showed a partial response (PR), and 2 / 7 showed stable disease (SD). Six out of seven in this group continued treatment. Figure 14 shows the best percentage change in the sum of the longest dimension measurements from baseline in the target lesions of the subjects, and Figure 15 shows a clear dose-effect on the frequency of response when those affected subjects in the higher-dose groups showed a more consistent and significant decrease in tumor size. Figure 16 shows the antitumor activity observed in the various treatment groups (subjects previously treated with EGFR-targeted therapy, ALK-targeted therapy, and HER2-targeted therapy are shown).

[0452] Pretreatment tumor biopsies were evaluated by immunohistochemistry to determine TROP2 expression, and the subject responses are shown in Figure 17. As shown in Figures 12, 17, 21, and 26, some subjects had received prior EGFR inhibitor therapy or ALK inhibitor therapy, or immuno-oncology treatment. Six out of eight subjects who achieved a partial response (PR) had an H-score greater than the median, while 8 / 15 with stable disease (SD) and 4 / 12 with progressive disease had an H-score greater than the median. This is consistent with preclinical data (see Figure 18) showing that the antibody-drug conjugate (1), i.e., DS-1062a, had antitumor activity in a lung cancer xenograft mouse model with stronger antitumor activity in TROP2-positive tumors (NCI-H2170 and HCC827) compared to TROP2-negative tumors (Calu-6).

[0453] Changes in variant allele frequency (VAF) were also determined by evaluating cell-free DNA (cfDNA). VAF was confirmed at Cycle 3, Day 1 (C3D1) and end of treatment (EOT). These results shown in Figure 19 demonstrated that DS-1062a decreased cfDNA in subjects who achieved SD and PR.

[0454] In summary, DS-1062a had good tolerability at doses up to 8 mg / kg established as the MTD and RDE. At 10 mg / kg, 2 subjects developed grade 3 mucositis and tolerability was not demonstrated. Both 8 mg / kg and 6 mg / kg had good tolerability, but 8 mg / kg showed a better preliminary efficacy signal with a higher overall response rate (ORR) at 8 mg / kg compared to 6 mg / kg. Indeed, Figure 20 shows that the ORR was best in the 8 mg / kg treatment group.

[0455] A dose-dependent effect on antitumor activity was observed over the range of 2.0 - 8.0 mg / kg. Twelve partial responses were observed during dose escalation in heavily pretreated, non-selected NSCLC patients who had relapsed or progressed from standard of care (SOC) including immune checkpoint inhibitors. A summary of the efficacy results is provided in Figure 21.

Example

[0456] Preliminary Efficacy of Antibody-Drug Conjugates As of November 16, 2019, 88 out of 95 subjects treated with DS-1062a were evaluable for response.

[0457] The overall response rate (ORR; unconfirmed) evaluated by the principal investigator of the clinical trial was 27.8% (95% CI: 9.7, 53.5) in the 6 mg / kg treatment group (5 / 18 subjects responded, all PR) and 38.2% (95% CI: 22.2, 56.4) in the 8 mg / kg treatment group (13 / 34, all PR) (Table 2 and Figure 22). The disease control rate (DCR = CR + PR + SD) was 72.2% at 6 mg / kg and 79.4% at 8 mg / kg.

[0458] As of the data cut-off date, all 5 subjects with PR in the 6 mg / kg treatment group were on treatment without disease progression or death.

[0459] In the 8 mg / kg dosing group, among the 13 subjects with PR, 6 were in the process of treatment without disease progression or death, 2 developed progressive disease, 1 died, and 4 discontinued DS-1062a for reasons other than disease progression or death.

[0460]

Table 2

[0461] Subjects evaluable for efficacy were those who had both baseline tumor evaluations and post-baseline tumor evaluations, or those who discontinued the investigational treatment.

[0462] Pharmacokinetics

[0463] Using non-compartmental analysis in 61 subjects who received DS-1062a (0.27 mg / kg to 10 mg / kg), preliminary single-dose PK and preliminary repeated-dose PK were evaluated.

[0464] Figure 23 shows the plasma concentration of DS-1062a, total antibody, and free drug (referred to as payload in the figure) in repeated dosing of 8 mg / kg of DS-1062a. The mean values of mean AUClast, Cmax, and elimination half-life (t1 / 2) were 914 μg·d / mL, 196 μg / mL, and 5.45 days, respectively.

[0465] The plasma levels of DS-1062a and total anti-TROP2 antibody were similar, and the exposure of free drug was lower than that of DS-1062a, suggesting that DS-1062a was stable in circulation.

[0466] Conclusion

[0467] This DS-1062a was shown to be tolerable and safe at doses up to 8 mg / kg in the Phase I trial.

[0468] Among the 88 evaluable subjects, DS-1062a was effective at doses of 2 mg / kg or higher, achieving an ORR of 38.2% (13 / 34 subjects) and a DCR of 79.4% (27 / 34 subjects) in the 8 mg / kg group.

[0469] The results are superior to docetaxel, which is used as standard treatment after immune checkpoint inhibitors and platinum-based chemotherapy in NSCLC (Table 3).

[0470] Furthermore, 90.9% (20 / 22 subjects) of the PR subjects had been previously treated with immune checkpoint inhibitors (e.g., nivolumab, pembrolizumab, atezolizumab, avelumab, ipilimumab, durvalumab), and all subjects had been previously treated with platinum-based chemotherapeutic agents (e.g., cisplatin, carboplatin). Therefore, DS-1062a has shown the potential to replace docetaxel in subjects including those with NSCLC who are refractory or intolerant to these standard treatments.

[0471] Furthermore, sacituzumab govitecan, a competing antibody-drug conjugate targeting TROP2 developed in the United States, has an ORR of 19% in NSCLC in a phase 2 trial in subjects who received standard treatment, suggesting that DS-1062a may be more effective than this competing drug.

[0472] Therefore, the therapeutic agents and therapeutic pharmaceutical compositions containing DS-1062a used in the present invention, and the therapeutic method characterized by administering DS-1062a of the present invention, have been shown to be excellent for the treatment of subjects with unresectable advanced non-small cell lung cancer who are refractory or recurrent to standard treatment or for whom standard treatment is not applicable.

[0473] The evaluation of the safety and preliminary efficacy of 4 mg, 6 mg, and 8 mg is being continued in a phase I trial.

[0474] Furthermore, multiple Phase II trials are planned to start in 2020.

[0475]

Table 3

[0476] All patents and publications described herein are indicative of the level of those skilled in the art to which the present disclosure pertains. All patents and publications are incorporated herein by reference to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference.

[0477] Furthermore, those skilled in the art will readily recognize that the present disclosure is well-suited to carry out the object and obtain the described objects and advantages and those inherent therein. Those skilled in the art will envision modifications and other uses therein. These modifications are encompassed within the spirit of the present disclosure and are defined by the scope of the claims that set forth non-limiting embodiments of the present disclosure.

Claims

**Claim 1** A pharmaceutical composition comprising an anti-TROP2 antibody-drug conjugate for use in the treatment of cancer, wherein the antibody-drug conjugate comprises an anti-TROP2 antibody and an anti-tumor compound linked by a linker, the linker and the anti-tumor compound having the following formula: -(Succinimid-3-yl-N)-CH ２ CH ２ CH ２ CH ２ CH ２ -C(= O)-GGFG-NH-CH ２ -O-CH ２ -C(=O)-(NH-DX) (wherein -(Succinimid-3-yl-N)- is linked to the anti-TROP2 antibody by a thioether bond at its 3-position and is linked to a methylene group in a linker structure containing this structure on the nitrogen atom at the 1-position by the following formula: 【Chemical 1】 having the structure represented by, (NH-DX) represents a group represented by the following formula: 【Chemical 2】 wherein the nitrogen atom of the amino group at the 1-position is the linking position), the average number of conjugations of the anti-tumor compound per antibody being 3.5 to 4.5, the anti-TROP2 antibody being an anti-TROP2 antibody comprising a heavy chain containing SEQ ID NO: 45 and a light chain containing SEQ ID NO: 46 or an anti-TROP2 antibody lacking a lysine residue at the carboxyl terminus of the heavy chain, the antibody-drug conjugate being intravenously administered to a cancer subject once every 3 weeks or once every 4 weeks at a dose of 4 mg / kg or 6 mg / kg, the cancer being non-small cell lung cancer (NSCLC), a pharmaceutical composition. **Claim 2** The pharmaceutical composition according to claim 1, wherein the non-small cell lung cancer is adenocarcinoma. **Claim 3** The pharmaceutical composition according to claim 1, wherein the non-small cell lung cancer is large cell carcinoma. **Claim 4** The pharmaceutical composition according to any one of claims 1 to 3, wherein the non-small cell lung cancer is metastatic. **Claim 5** The pharmaceutical composition according to any one of claims 1 to 4, wherein the cancer is resistant or refractory. **Claim 6** The pharmaceutical composition according to claim 5, wherein the resistance or the refractoriness is resistance or refractoriness acquired by the cancer for treatment with an anticancer drug. **Claim 7** The pharmaceutical composition according to claim 6, wherein the anticancer drug is an EGFR inhibitor, an ALK inhibitor, a platinum-based chemotherapeutic agent or a checkpoint inhibitor. **Claim 8** The pharmaceutical composition according to claim 6, wherein the anticancer drug is gefitinib, erlotinib, osimertinib, afatinib, alectinib, crizotinib, ceritinib, cisplatin, carboplatin, nivolumab, pembrolizumab, atezolizumab, avelumab, ipilimumab, durvalumab, tislelizumab, sintilimab or semaprilimab. **Claim 9** The pharmaceutical composition according to any one of claims 1 to 8, wherein the cancer is a TROP2-expressing cancer.

10. The pharmaceutical composition according to claim 9, wherein the TROP2-expressing cancer is a TROP2-overexpressing cancer.

11. The pharmaceutical composition according to any one of claims 1 to 10, wherein the cancer is an inoperable cancer or a recurrent cancer.

12. The pharmaceutical composition according to any one of claims 1 to 11, further comprising a pharmaceutically acceptable formulation ingredient.

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