Dosage of antibody-drug conjugate

Abstract

To provide an antitumor agent having excellent therapeutic efficacy and safety.SOLUTION: The present disclosure relates to the field of pharmaceutical preparations, dosage regimens, and administration of an antibody-drug conjugate (ADC). More specifically, the ADC is composed of an anti-trophoblast cell surface antigen 2 (TROP2) antibody connected via a linker to an anticancer agent such as a topoisomerase I inhibitor.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present disclosure relates to the field of pharmaceutical preparations, dosage regimens and administration of antibody drug conjugates (ADCs). More specifically, the ADCs are compounds that are linked to topoisomers, such as derivatives of exatecan, via a linker. Consisting of an anti-trophoblast cell surface antigen 2 (TROP2) antibody linked to a thrombus I inhibitor It is done.

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

[0003] The following description is provided solely to aid the reader in understanding the present disclosure. , are not admitted to describe or constitute prior art to the present disclosure.

[0004] Trophoblast cell surface antigen 2 (TROP2) is encoded by the Tacstd2 gene It is a 323 amino acid transmembrane glycoprotein that is differentially expressed in many cancers. Expressed intracellular calcium signal transducer (Ripani E et al., Int. J.Cancer, 76(5), 671-676(1998) and El Sewed y T et al., Int. J. Cancer, 75(2), 324-330 (1998) It signals cells for self-renewal, proliferation, invasion, and survival. OP2 is further involved in immune resistance common to human trophoblasts and cancer cells (Fa ulk WP et al., Proc.Natl.Acad.Sci.75(4), 1947~19 51 (1978) and Lipinski M et al., Proc. Natl. Acad. S ci.78(8), 5147-5150 (1981). DNA sequence of human TROP2 and amino acid sequences are available, for example, under accession numbers NM_002353 and NP_002344 ( It is available in public databases at NCBI.

[0005] TROP2 is expressed at low levels in various epithelial cell carcinomas compared with normal epithelial cells. TROP2 expression has been shown to be overexpressed in colorectal cancer (OCR). hmachi T et al., Clin.Cancer Res., 12(10), 3057-3 063 (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 God Glioma (Ning S et al., Neurol. Sci., 34(10), 1745-1750) (2013)) was also reported to be correlated with poor prognosis. Using this method, TROP2 expression enhances anchorage-independent cell growth of tumor cells in immunodeficient mice. and tumorigenesis (Wang J et al., Mol. Ca ncer Ther., 7(2), 280-285(2008)).

[0006] Considering that TROP2 is associated with various cancers, several anti-TROP2 antibodies have been prepared. Among these antibodies, some have been shown to be effective in nude mouse xenograft models. A report of an unconjugated antibody showing antitumor activity (International Patent Publication No. 2008 / 144891, International Patent Publication No. 2011 / 145744, International Patent Publication No. 2011 / 155579 and and International Patent Publication No. 2013 / 077458) and as antibody-drug conjugates (ADCs). Antibodies showing anti-tumor activity have been reported (International Patent Publication No. 2003 / 074566, International Patent Publication No. 2011 / 068845, International Patent Publication No. 2013 / 068946 and U.S. Pat. However, the strength and application of anti-TROP2 antibodies and ADCs are unclear. The coverage has been insufficient to date, and there is a medical need to utilize TROP2 as a therapeutic target. It still remains unfulfilled.

[0007] The present disclosure provides TROP2-specific ADCs and their dosage regimens for treating various cancers. Thus, the present disclosure provides a safe and effective therapeutic agent that targets TROP2. This fulfills the aforementioned need in the art for cancer treatment. Summary of the Invention [Problem to be solved by the invention]

[0008] Anti-tumor antibodies targeting TROP2 have not been successful to date, and many anti-tumor small molecules have not yet been developed. The compounds (even those with excellent antitumor efficacy) have unacceptable side effects and toxicity. Therefore, it is necessary to achieve excellent therapeutic effects while at the same time There remains a need for enhanced safety. Therefore, it is an object of the present disclosure to provide superior therapeutic efficacy. and to provide an antitumor drug that is safe. [Means for solving the problem]

[0009] The antitumor compound exatecan targets tumor cells, recognizes tumor cells, Anti-TROP2 antibodies capable of binding to or internalizing within tumor cells When conjugated to an antibody, the compound is converted into an antibody-drug conjugate via a linker structure moiety. The antibody-based cytocidal activity can be obtained, and the anti-tumor compound is more It is possible for the drug to be delivered more reliably to tumor cells and specifically exhibit antitumor effects. The antitumor effect can be reliably demonstrated, and the antitumor effect can be enhanced compared with the administration of the compound alone. It is possible to reduce the dose of the tumor compound administered, thereby reducing the effect on normal cells. Negative side effects are reduced and safety is increased.

[0010] The present invention relates to novel TROP2 antibodies, including exatecan derivatives and anti-TROP2 antibodies. Dual-targeted ADCs and methods for their use are described.

[0011] In one aspect, the disclosure provides an anti-TROP2 antibody for use in the treatment or prevention of cancer. an antibody-drug conjugate, wherein the antibody-drug conjugate is linked by a linker; The present invention provides an anti-TROP2 antibody-drug conjugate comprising an OP2 antibody and an anti-tumor compound. do.

[0012] In another aspect, the disclosure provides an anti-TROP2 antibody and a and administering to a subject with cancer an anti-TROP2 antibody-drug conjugate comprising an anti-TROP2 antibody and an anti-tumor compound. A method for treating or preventing cancer in elephants is provided.

[0013] In another aspect, the present disclosure provides a method for the manufacture of a medicament for treating or preventing cancer. Use of an anti-TROP2 antibody-drug conjugate in a patient receiving the treatment, wherein the antibody-drug conjugate is linked by a linker. and an anti-TROP2 antibody and an anti-tumor compound linked by be.

[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) (Wherein -(Succinimid-3-yl-N)- is linked to the antibody at its 3-position) and linked to a methylene group in a linker structure containing this structure on the nitrogen atom at position 1. The following formula is used:

[0015] [ka]

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

[0017] [ka]

[0018] wherein the nitrogen atom of the amino group at position 1 is the linking position) It is expressed as follows.

[0019] In some embodiments, the anti-TROP2 antibody has, within its heavy chain variable region, the sequence CDRH1 consisting of the amino acid sequence of SEQ ID NO: 23 and CDRH2 consisting of the amino acid sequence of SEQ ID NO: 24 DRH2 and CDRH3 consisting of the amino acid sequence of SEQ ID NO: 25, and The variable region contains CDRL1 consisting of the amino acid sequence of SEQ ID NO: 26 and 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 anti-tumor compounds bound per antibody is 2 to 100. 8, or in the range of 3 to 8. In some embodiments, one of the anti-tumor compounds The average number of bonds per antibody is in the range of 3.4 to 4.5. Therefore, the average number of anti-tumor compounds bound to one antibody is four.

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

[0022] In some embodiments, the dosage ranges from 2 mg / kg to 10 mg / kg. The antibody-drug conjugate is administered to a subject with cancer. In some embodiments, the antibody-drug conjugate is administered at a dose of about 4 mg / kg. In some embodiments, a dose of 100 mg of the antibody-drug conjugate is administered to a subject with cancer. Thus, a dose of about 6 mg / kg of the antibody-drug conjugate is administered to a subject with cancer. In one embodiment, a dose of about 8 mg / kg of the antibody-drug conjugate is administered to a subject with cancer. will be done.

[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. It is administered once.

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

[0026] In some embodiments, the cancer is resistant or refractory. In this state, the resistance or the unresponsiveness is due to the cancer's acquired resistance to treatment with an anti-cancer drug. In some embodiments, the anticancer drug is an EGFR inhibitor. , ALK inhibitors, platinum-based chemotherapy drugs, or checkpoint inhibitors. In some embodiments, the anticancer drug is gefitinib, erlotinib, osimertinib, afatinib, or rifametinib. tinib, alectinib, crizotinib, ceritinib, cisplatin, carboplatin, Volumab, pembrolizumab, atezolizumab, avelumab, ipilimumab, durval mab, tislelizumab, sintilimab, or cemiplimab.

[0027] In some embodiments, the cancer is a TROP2-expressing cancer. In some embodiments, the TROP2-expressing cancer is In some embodiments, the cancer is a TROP2-overexpressing cancer. are cancers that were given a high score for TROP2 expression by immunohistochemistry. In some embodiments, the TROP2-overexpressing cancer is detected by in situ hybridization. These cancers were given a high score for TROP2 expression in the quantification method.

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

[0029] An antibody-drug conjugate according to any one of the above aspects or embodiments as an active ingredient. A pharmaceutical composition comprising the compound or a salt thereof and a pharmaceutically acceptable formulation component is also referred to herein. Provided.

[0030] The foregoing general description and the following detailed description are exemplary and explanatory only. is intended to provide further explanation of the present disclosure as claimed. Other objects, advantages and novel features are set forth in the following "Brief Description of the Drawings" and "Patent Documents" of this disclosure. This will be readily apparent to those skilled in the art from the detailed description. [Brief explanation of the drawings]

[0031] [Figure 1] 1 shows the structure of a TROP2-targeting antibody-drug conjugate (herein referred to as "antibody-drug conjugate (1)") with a topoisomerase I inhibitor (DXd). The ADC has a tetrapeptide linker that attaches to a cysteine ​​residue on the antibody. The drug-to-antibody ratio of the depicted ADC is 4:1 (i.e., DAR4). [Figure 2] 1 shows the heavy and light chain sequences of anti-TROP2 antibodies that can be incorporated into ADCs of the present disclosure, as well as the structural formulas of cytotoxic agents linked to the antibodies. [Figure 3] 1 shows the antitumor efficacy of antibody-drug conjugates (1) and (2) in a mouse xenograft CFPAC-1 tumor model. [Figure 4] 1 shows an estimate of plasma concentrations during repeated dosing of DS-1062a in humans. [Figure 5] 1 shows a Phase 1 study design for treating patients with non-small cell lung cancer (NSCLC). [Figure 6] 1 shows patient demographics and baseline characteristics for the initial Phase 1 study (Example 5). [Figure 7] The number of patients in the initial Phase 1 study (Example 5) who had a treatment-emergent adverse event (TEAE) occurring in 10% or more of patients, regardless of causality, is shown. [Figure 8] 1 shows tumor responses of subjects (N=35) in an early Phase 1 study (Example 5). [Figure 9-1] Figure 1 shows tumor responses in target (A, B, and C) and non-target (D) lesions following treatment with DS-1062a in an initial Phase 1 study (Example 5). Panel A shows a reduction in target lesion size in a patient treated with 4.0 mg / kg DS-1062a. Panel B shows a reduction in target lesion size in another patient treated with 4.0 mg / kg DS-1062a. Panel C shows a reduction in target lesion size in a patient treated with 2.0 mg / kg DS-1062a. Panel D shows a reduction in the number of non-target lesions in the same patient as Panel C. [Figure 9-2] Same as above. [Figure 10] Figure 1 shows the change in tumor size for subjects in an early Phase 1 study (Example 5). The top panel shows the best percentage change from baseline in the sum of the longest dimension measures of target lesions for subjects in an early Phase 1 study (Example 5). The bottom panel shows a spider plot of the change in tumor size separated by treatment group. [Figure 11] The mean plasma concentrations of DS-1062a in Cycle 1 (PK analysis population) are shown. [Figure 12] 1 shows a summary of the efficacy demonstrated by an early Phase 1 study (Example 5). [Figure 13]1 shows the number of patients in the Phase 1 study (Example 6) at the new cutoff date who had a treatment-emergent adverse event (TEAE) regardless of causality. [Figure 14] 1 shows the best percentage change from baseline in the sum of the longest dimension measures in target lesions for subjects in a Phase 1 study (Example 6) as of the new cutoff date. [Figure 15] A clear dose-effect on the frequency of response is demonstrated by showing the percentage change in tumor size for each dose group over the course of the Phase 1 study (Example 6) as of a new cutoff date. [Figure 16] The study demonstrates durable antitumor responses across multiple dose levels. Most patients experienced partial responses (PR) or stable disease (SD). Only two patients experienced progressive disease (PD) at the end of the study (Example 6). [Figure 17] Figure 1 shows the TROP2 immunohistochemistry H-scores (IHC) based on pretreatment biopsies of patients in a Phase 1 study (Example 6) as of the new cutoff date. IHC scores tended to be higher in those patients who achieved a positive result, such as a partial response (PR). For the purposes of these figures, the following abbreviations were used: anaplastic lymphoma kinase inhibitor (ALKi), baseline (BL), cycle 3 day 1 (C3D1), circulating free DNA (cfDNA), epidermal growth factor receptor inhibitor (EGFRi), end of treatment (EOT), human epidermal growth factor receptor 2 inhibitor (HER2i), immunohistochemistry (IHC), histology score (H-score), immuno-oncology (I / O), not evaluable (NE), partial response (PR), progressive disease (PD), stable disease (SD), patient (Pt), variant allele frequency (VAF). [Figure 18] Preclinical results are shown showing that the antibody-drug conjugate (1) had antitumor activity in lung cancer xenograft mouse models, with stronger antitumor activity in TROP2-positive tumors (NCI-H2170 and HCC827) as opposed to TROP2-negative tumors (Calu-6). [Figure 19]

[0023] Figure 1 shows the change in mutant allele frequency based on cell-free DNA (cfDNA) over the course of treatment. The results show that cfDNA generally decreased as a result of treatment. [Figure 20] 1 shows the overall response rate (ORR) as assessed by change in tumor volume for subjects in various treatment arms of a Phase 1 study (Example 6) as of a new cutoff date. [Figure 21] 1 shows a summary of the efficacy demonstrated by the Phase 1 study (Example 6) as of the new cutoff date. [Figure 22] 1 shows spider plots of the change in tumor size by treatment group in a preliminary efficacy study (Example 7). [Figure 23] 1 shows plasma concentrations of antibody drug conjugate (1), total antibody, and free drug (payload) as determined by pharmacokinetic measurements from a preliminary efficacy study (Example 7). DETAILED DESCRIPTION OF THE INVENTION

[0032] Various embodiments of novel TROP2-targeted ADCs and methods of use thereof are described below with reference to the drawings. The following embodiments are exemplary of the present invention. These are provided as examples and are not intended to limit the scope of the invention.

[0033] The anti-TROP2 antibody-drug conjugate of the present invention is a compound in which the anti-TROP2 antibody is linked via a linker structure. and anti-tumor agents conjugated to anti-tumor compounds, as described in detail below.

[0034] definition The method is not limited to the specific embodiments described and may vary as such. It should be understood that the terms used herein may refer to specific embodiments. For descriptive purposes only and are not intended to be limiting. It should also be understood that the scope of the present technology is limited only by the appended claims. It would be.

[0035] Unless otherwise defined, all technical and scientific terms used herein are , have the same meaning as commonly understood by a person skilled in the art to which this invention pertains. Any methods and materials similar or equivalent to those described herein may be used in the practice of the present invention. Although representative and exemplary methods and materials can be used for any of the above-described tests, they are described herein. do.

[0036] Where a range of values ​​is provided, the range between the upper and lower limits is used unless the context clearly dictates otherwise. Values ​​between each of the values ​​to one-tenth of the unit of the limit, and any other value within that indicated range It is understood that values ​​at or between these are encompassed within the present invention. The upper and lower limits of a range may independently be included in the smaller range, and the stated range Also encompassed within the present invention are any specifically excluded limits within the ranges shown. If the range includes one or both of the limits, exclude either or both of those included limits. The above range is also included in the present invention.

[0037] As used in the specification and claims, the singular forms "a," "one," "an ... "an" and "the" are used in the singular and in the plural unless the context clearly dictates otherwise. and multiple references.

[0038] As used herein, the term "comprising" means The compositions and methods include the recited elements, but do not exclude others. It is intended to mean "consisting essentially" "All of" when used to define compositions and methods or other elements of any substantial importance to said method. "Consisting of" means the claimed composition and a substantial This means excluding more than trace elements of other components of the process step. Embodiments defined by each of these transition terms are within the scope of this disclosure. Thus, the methods and compositions may include (or contain) additional steps and components. or alternatively comprising (consisting essentially of) non-essential steps and compositions; or Alternatively, it is intended to contemplate (consisting of) only the listed method steps or compositions. It is illustrated.

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

[0040] As used herein, "optional" or "Optionally" means that the subsequently described event or circumstance occurs. The description may or may not occur, and the description may include instances in which the event or situation occurs and instances in which the event or situation occurs. This means that it also includes examples where the above situation does not occur.

[0041] "Individual," "Subject," and "Patient" The terms "patient" and "patient" are used interchangeably herein and refer to the methods or or any individual mammal to be treated according to the use, for example, bovine, canine, feline, equine, In a preferred embodiment, the subject is a human. It is.

[0042] As used herein, "effective amount" "therapeutically effective amount" The phrases "therapeutic level" and "therapeutic level" refer to The ADC is administered to a subject in need of such treatment, i.e., a cancer (e.g., lung cancer) , TROP2-expressing cancer or resistant or refractory cancer) A therapeutically effective amount of an ADC refers to the dose or concentration in a subject that provides a pharmacological effect. Alternatively, a therapeutic level refers to a dose that is considered therapeutically effective by those skilled in the art. Even if the compound is not effective in treating the cancers described herein, For convenience only, exemplary doses, drug delivery amounts, therapeutically effective amounts, and Therapeutic levels are provided below. Those skilled in the art will appreciate the level of therapeutic activity required to treat a particular subject and / or condition. Such amounts can be adjusted, if necessary, in accordance with standard practices. The amount will depend on the route of administration and dosage form, the age and weight of the subject, and / or the type and severity of the cancer. The amount of blood flow may vary based on the condition of the subject, including the degree of blood flow.

[0043] "Treatment" or "treatment" as used herein with respect to cancer The term "treating" refers to reducing or suppressing cancer. and / or eliminating cancer cell proliferation. reducing, inhibiting, or eliminating metastasis of cancer, or tumor Treatment and treating refer to the disappearance or death of metastases. Even if cancer cell growth is not inhibited and / or the cancer is not killed, , to improve the subject's quality of life or overall survival rate. It may also mean.

[0044] "Prevent" or "prevent" as used herein with respect to cancer The term "preventing" refers to the prevention of metastasis (i.e., the initiation of treatment). To inhibit or prevent the development of cancer (the growth of cancer at secondary sites where cancer is sometimes not present) and, if the subject achieves remission, or the cancer / tumor is completely destroyed or refers to inhibiting or preventing the recurrence of cancer when killed.

[0045] As used herein, the term "pharmaceutical composition" refers to a pharmaceutical composition comprising an active agent and The invention relates to a method for the preparation of a pharmaceutical composition, particularly one suitable for diagnostic or therapeutic use in vivo or ex vivo. It refers to a complex with an inert or active carrier that allows the compound to be used.

[0046] As used herein, the term "pharmaceutically acceptable carrier" refers to a phosphate Buffered saline, water, emulsions (e.g., oil-in-water emulsions or water-in-oil emulsions), and various The composition may contain any of the standard pharmaceutical carriers, such as humectants, stabilizers, and preservatives. Examples of carriers, stabilizers and adjuvants can be found in, for example, M artin, Remington's Pharmaceutical Science See s, 15th edition, Mack Publ. Co., Easton, PA

[1975] To do.

[0047] As used herein, "parenteral administration" and "parenterally administered" The phrase "administered" refers to modes of administration other than enteral and topical administration, usually by injection, and is not intended to be limiting. It is not intended to be used for intravenous injection and infusion, but for intramuscular injection and infusion, Intra-arterial injection and infusion, intrathecal injection and infusion, intracapsular injection and infusion, orbital Intra- and intra-orbital injection, intracardiac injection, intradermal injection, intraperitoneal injection Intraperitoneal injection, transtracheal injection, subcutaneous injection, epidermal injection subcutaneous injection, intra-articular injection, subcapsular injection, Intrathecal injection and infusion, intrathecal injection and infusion, and intrasternal injection and infusion and intrasternal injection.

[0048] As used herein, "systemic administration," "administered systemically," "peripherally administered" and "administered peripherally" are used interchangeably. The phrases "peripherally administered" and "peripherally administered" refer to the administration of a compound, drug, or other material to the patient. Direct central nervous system activity, such as entering the system and undergoing metabolic and other similar processes. administration of said compound, drug or other material, excluding administration into the body, e.g., subcutaneous administration means.

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

[0050] As used herein, the term "polynucleotide" is synonymous with nucleic acid. used in research, including DNA, RNA, probes, oligonucleotides, and primers .

[0051] The terms "polypeptide" and "protein" as used herein are used interchangeably.

[0052] The term "cell" as used herein refers to a cell within an individual animal and This also includes cultured cells.

[0053] As used herein, the term "TROP2" refers to the TROP2 protein. It is used synonymously with quality.

[0054] As used herein, the term "CDR" refers to a complementarity determining region (CDR Each of the heavy and light chains of an antibody molecule has three complementarity-determining regions (CDRs). CDRs are also called hypervariable domains, and are known to be the domains of the heavy and The variable region of each of the light chains is highly variable within its primary structure. There are three distinct sites within the primary structure of each of the heavy and light polypeptide chains. As used herein, with respect to the CDRs of an antibody, the CDRs of the heavy chain are The amino acid sequence is represented by CDRH1, CDRH2, and CDRH3 from the amino-terminal side. The CDRs of the light chain are CDRL1, CDRL2 and CDRL3 from the amino-terminal side of the light chain amino acid sequence. These sites are located close to each other in the tertiary structure, and the antibody The specificity for the antigen that binds is determined.

[0055] As used herein, "hybridization under stringent conditions" refers to a sequence that hybridizes to a target protein. The phrase "hybridization is performed" refers to the use of commercially available hybridization solutions. essHyb Hybridization Solution (Clontech, I Hybridization was performed at 68°C in a 500-kJ / mL ELISA kit (manufactured by nc.) or The filter having the DNA immobilized thereon is used to perform the filtration of 0.7 to 1.0 M of DNA. Hybridization was performed at 68°C in the presence of NaCl, followed by 0.1–2× SSC solution. Solution (1x SSC solution is made from 150 mM NaCl and 15 mM sodium citrate) Washing at 68°C or equivalent conditions using a It refers to a process in which hybridization occurs under conditions that allow a certain level of hybridization to be achieved.

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

[0057] Conservative amino acid substitutions are preferred as amino acid substitutions herein. Acid substitution refers to a substitution that occurs within the group of an amino acid relative to the amino acid side chain. The acid groups are as follows: acidic groups (aspartic acid and glutamic acid), basic groups (lysine, arginine and histidine), non-polar groups (alanine, valine, leucine, iodopropyl methyl ... soleucine, proline, phenylalanine, methionine and tryptophan) and non Charged polar family (glycine, asparagine, glutamine, cysteine, serine, More preferred amino acid groups are: aliphatic hydrochloride, xyl groups (serine and threonine), amide-containing groups (asparagine and glutamine) , aliphatic groups (alanine, valine, leucine, and isoleucine) and aromatic groups (phenyl Such amino acid substitutions are preferably The modification is carried out within a range that does not impair the properties of the substance having the original amino acid sequence.

[0058] Throughout this specification, compositions are referred to as having or including specific components. or described as comprising or processes and methods The method has, includes, or comprises specific steps When a statement is made that a product is made of a material containing the ingredients, it further means that the product consists essentially of or is made of the listed ingredients. and / or compositions of the present disclosure, consisting essentially of or consisting of the recited processing steps. It is contemplated that there may be processes and methods according to the present disclosure.

[0059] Generally, compositions specifying percentages are by weight unless otherwise specified. Furthermore, if a variable is not defined, then the previous definition of said variable takes precedence.

[0060] TROP2 TROP2 is a member of the TACSTD family expressed in human trophoblast cells. It is a single-pass transmembrane type 1 protein that is involved in immune tolerance common to human trophoblasts and cancer cells. It is a protein.

[0061] For purposes of this disclosure, TROP2 protein refers to a protein derived from a human or non-human mammal (e.g., Is it possible to purify TROP2 directly from rat or mouse TROP2-expressing cells and use it? Alternatively, a cell membrane fraction of the above cells can be prepared and used. can be obtained by its in vitro synthesis or by its production in a host cell by genetic engineering. Specifically, TROP2 cDNA can be expressed in genetically engineered cells. After the TROP2 cDNA was integrated into a vector capable of expressing TROP2, the TROP2 protein was expressed by transcription and and synthesize it in a solution containing the enzymes, substrates, and energy sources required for translation. or by another prokaryotic transformed host cell or a eukaryotic transformed host cell. Alternatively, it can be obtained by expressing TROP2 in a genetically engineered manner. The above-mentioned TROP2-expressing cells or cell lines expressing TROP2 were used to express the TROP2 protein. It may also be used as a quality.

[0062] The DNA and amino acid sequences of TROP2 are available in public databases. See e.g., accession numbers NM_002353 and NP_002344 (NCBI) It can be done.

[0063] Furthermore, one or more amino acids in any of the above amino acid sequences of TROP2 are The amino acid sequence of the protein is substituted, deleted, and / or added. TROP2 also includes proteins that have biological activity equivalent to proteins.

[0064] The human TROP2 protein contains a signal sequence consisting of 26 amino acid residues at the N-terminus. , an extracellular domain consisting of 248 amino acid residues and a membrane domain consisting of 23 amino acid residues. It contains a transmembrane domain 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 is either The species may be derived from any of the following species, preferred examples of which include human, rat, mouse and rabbit. If it is derived from a species other than the human, it is preferably a well-known The antibodies of the present invention may be chimerized or humanized using polyclonal antibody techniques. It may be an antibody or a monoclonal antibody, preferably a monoclonal antibody.

[0066] Anti-TROP2 antibodies target tumor cells, recognize tumor cells, and These antibodies can bind to the target molecules or be internalized in tumor cells, and have antitumor activity. The antibody-drug conjugate can be converted to an antibody-drug conjugate by conjugating the antibody-drug conjugate to a corresponding compound via a linker.

[0067] The binding activity of the antibody to tumor cells can be confirmed using flow cytometry. Examples of methods to confirm antibody internalization into cells include: (1) using a secondary antibody (fluorescent antibody) to the therapeutic antibody; Visualize the intracellularly incorporated antibodies under a fluorescence microscope using a photolabeled conjugation assay ( Cell Death and Differentiation (2008) 15, 751-761), (2) intracellular integration using secondary antibodies (fluorescently labeled) bound to therapeutic antibodies Assay to measure the fluorescence intensity of the incorporated DNA (Molecular Biology of the the Cell, Vol. 15, pp. 5268-5282, December 2004) or (3) Cell Immunotoxins conjugated to therapeutic antibodies that upon incorporation release the toxin and inhibit cell proliferation Mab-ZAP assay (BioTechniques 28:162-165) , January 2000). The catalytic domain and proteins of diphtheria toxin Recombinant complex proteins of G may be used as immunotoxins.

[0068] Since the drug conjugated in the antibody-drug conjugate exerts an antitumor effect, the antibody itself is an antitumor agent. It is preferable, but not essential, for the antitumor compound to have an antitumor effect on tumor cells. Antibodies are translocated into tumor cells for the purpose of specifically and selectively exerting their cell-destroying activity. Therefore, it is both important and desirable to have internalized properties.

[0069] Immunizing an animal with an antigenic polypeptide and collecting and purifying antibodies produced in vivo Anti-TROP2 antibodies are obtained using methods commonly practiced in the art, including The origin of the antigen is not limited to humans, and animals such as mice and rats can also be used. and the like. In this case, the human may be immunized with an antigen derived from a non-human animal, such as a member of the same species. To screen for antibodies applicable to human diseases, the obtained antibodies are bound to the heterologous antigen. The cross-reactivity of the antibody with human antigens can be tested.

[0070] Alternatively, methods known in the art (e.g., Kohler and Milstein, in Nature (1975) 256, pp. 495-497, and Kenne t, R. et al., Monoclonal Antibodies, pp. 365-367, P Produce antibodies against antigens according to the method described in the 1980s by the American Academy of Microbiology. The antibody-producing cells can then be fused with myeloma cells to obtain monoclonal antibodies. A hybridoma is established.

[0071] By genetically engineering host cells to produce genes encoding antigenic proteins. Thus, the antigen can be obtained. Specifically, a vector that enables expression of the antigen gene can be used. A vector is prepared and transferred to a host cell so that the gene is expressed. The antigen expressed by the genetically engineered cells or the pre-expressed cells can be purified. The antibody can also be obtained by immunizing an animal with a cell line expressing the antigen. Cut.

[0072] Anti-TROP2 antibodies can be obtained by techniques known in the art.

[0073] The anti-TROP2 antibody that can be used in the present invention is not particularly limited. However, for example, preferably, those designated by the amino acid sequences shown in the sequence listing of the present application. The anti-TROP2 antibody used in the present invention is preferably It has the properties as described below. (1) The following characteristics: (a) specifically binds to TROP2; and (b) Having the ability to be internalized into TROP2-expressing cells by binding to TROP2 and An antibody having (2) The antibody according to (1), wherein TROP2 is human TROP2. (3) The antibody has a heavy chain complementarity-determining region (CDR) H1, CDRH2 of SEQ ID NO: 45. and CDRH3, and / or CDRL1, CDRL2 and CDRH3 of the light chain of SEQ ID NO: 46 and CDRL3. Alternatively or additionally, the antibody according to (1) or (2) has The antibody comprises a C11111 comprising the amino acid sequence represented by SEQ ID NO: 23 as a heavy chain complementarity determining region. DRH1, CDRH2, which contains the amino acid sequence represented by SEQ ID NO: 24, CDRH3, which contains the amino acid sequence represented by SEQ ID NO: 25 and CDRH3, which contains the amino acid sequence of SEQ ID NO: 1 as the light chain complementarity determining region. CDRL1 comprising the amino acid sequence represented by SEQ ID NO: 26, and CDRL1 comprising the amino acid sequence represented by SEQ ID NO: 27. and CDRL3 comprising the amino acid sequence set forth in SEQ ID NO:28. , (1) or (2). (4) The antibody according to any one of (1) to (3), wherein the constant region is a human-derived constant region. The area is antibodies. (5) The antibody according to any one of (1) to (4), wherein the antibody is a humanized antibody. (6) The antibody comprises: (a) a sequence selected from the group consisting of amino acids 1 to 121 of SEQ ID NO: 45; (b) an amino acid sequence having at least 95% or more homology to (a); and and (c) modifying said sequence (a) or (b) by at least one amino acid deletion, substitution, or addition. or (b), (d) a heavy chain variable region comprising 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) modifying said sequence (d an amino acid sequence selected from the group consisting of: and a light chain variable region comprising a sequence of: The antibody comprises: (a) an amino acid sequence selected from the group consisting of amino acids 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) an 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% or more homology with any one of the sequences (a) to (c); and (e) modifying said sequence by at least one amino acid deletion, substitution, or addition. An amino acid sequence selected from the group consisting of amino acid sequences derived from any of sequences (a) to (c). (f) a heavy chain variable region comprising the amino acid sequence of amino acid positions 21 to 12 in SEQ ID NO: 18; 9, (g) the amino acid sequence of amino acid positions 21 to 129 in SEQ ID NO: 20 (h) the amino acid sequence described in SEQ ID NO: 22, (i) an amino acid sequence that is identical to any one of the sequences (f) to (h) by at least 95% and (j) an amino acid sequence having at least one amino acid deletion, substitution, or or an amino acid sequence derived from any of the sequences (f) to (h) by addition. and a light chain variable region comprising an amino acid sequence selected from the group consisting of: (7) The antibody comprises an amino acid sequence selected from the group consisting of amino acids 1 to 121 in SEQ ID NO: 45. a heavy chain variable region comprising the amino acid sequence set forth in amino acid positions 1 to 109 of SEQ ID NO: 46; and a light chain variable region comprising an amino acid sequence as set forth in (6). Additionally, the antibody may comprise an amino acid sequence selected from the group consisting of amino acids 20 to 140 of SEQ ID NO: 12. The heavy chain variable region containing the amino acid sequence shown at amino acid positions 21 to 129 in SEQ ID NO: 18. a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 12, amino acid positions 20-1 40 and the amino acid sequence of SEQ ID NO: 20. A light chain variable region comprising the amino acid sequence set forth in positions 21 to 129 of SEQ ID NO: 12 A heavy chain variable region comprising the amino acid sequence set forth in amino acid positions 20 to 140 and SEQ ID NO: a light chain variable region comprising the amino acid sequence set forth in amino acid positions 21 to 129 of 22; A heavy chain containing the amino acid sequence set forth in amino acid positions 20 to 140 of SEQ ID NO: 14. The variable region and the amino acid sequence set forth in amino acid positions 21 to 129 of SEQ ID NO: 18 a light chain variable region comprising the amino acids set forth at amino acid positions 20 to 140 in SEQ ID NO: 14; The heavy chain variable region containing the amino acid sequence and the amino acid sequence shown at amino acid positions 21 to 129 in SEQ ID NO: 20. a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 14, amino acid positions 20-1 40 and the amino acid sequence of SEQ ID NO: 22. A light chain variable region comprising the amino acid sequence set forth in positions 21 to 129 of SEQ ID NO: 16 A heavy chain variable region comprising the amino acid sequence set forth in amino acid positions 20 to 140 and SEQ ID NO: a light chain variable region comprising the amino acid sequence set forth in amino acid positions 21 to 129 of FIG. 18; A heavy chain containing the amino acid sequence set forth in amino acid positions 20 to 140 of SEQ ID NO: 16. The variable region and the amino acid sequence set forth in amino acid positions 21 to 129 of SEQ ID NO: 20 and a light chain variable region comprising the amino acid sequence set forth at amino acid positions 20 to 140 in SEQ ID NO: 16. and a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 22 at amino acid positions 21-1. a heavy chain variable region selected from the group consisting of a light chain variable region comprising the amino acid sequence set forth in 29; The antibody according to (6), having a light chain variable region and a light chain variable region. (8) The antibody comprises an amino acid sequence selected from the group consisting of amino acids 20 to 140 in SEQ ID NO: 12. The heavy chain variable region containing the amino acid sequence shown at amino acid positions 21 to 129 in SEQ ID NO: 18. a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 14, amino acid positions 20-1 40 and the amino acid sequence of SEQ ID NO: 18. A light chain variable region comprising the amino acid sequence set forth in positions 21 to 129 of SEQ ID NO: 14 A heavy chain variable region comprising the amino acid sequence set forth in amino acid positions 20 to 140 and SEQ ID NO: a light chain variable region comprising the amino acid sequence set forth in amino acid positions 21 to 129 of and the amino acid sequence set forth in amino acid positions 20 to 140 of SEQ ID NO: 16. A heavy chain variable region comprising the amino acid sequence set forth at amino acid positions 21 to 129 in SEQ ID NO: 22. a heavy chain variable region and a light chain variable region selected from the group consisting of: a heavy chain variable region and a light chain variable region comprising the amino acid sequence The antibody according to (7), having the formula: (9) The antibody comprises an amino acid sequence selected from the group consisting of amino acids 1 to 451 in SEQ ID NO: 45. a heavy chain comprising the amino acid sequence set forth in amino acid positions 1 to 214 of SEQ ID NO: 46; and a light chain comprising a nucleotide sequence. the antibody is a human antibody having an amino acid sequence represented by amino acid positions 20 to 470 in SEQ ID NO: 12; heavy chain containing the sequence set forth in amino acid positions 21-234 in SEQ ID NO: 18; a light chain comprising the amino acid sequence set forth at amino acid positions 20 to 470 in SEQ ID NO: 12; and a heavy chain comprising the amino acid sequence set forth in amino acid positions 21 to 234 in SEQ ID NO: 20. a light chain comprising the amino acid sequence set forth at amino acid positions 20 to 470 in SEQ ID NO: 12; and a heavy chain comprising the amino acid sequence set forth at amino acid positions 21 to 234 in SEQ ID NO: 22. a light chain comprising an amino acid sequence as set forth in amino acid positions 20 to 470 in SEQ ID NO: 14; and a heavy chain comprising the amino acid sequence set forth in amino acid positions 21 to 234 of SEQ ID NO: 18. a light chain comprising the amino acid sequence set forth in amino acid positions 20 to 470 of SEQ ID NO: 14; and a heavy chain comprising the amino acid sequence of SEQ ID NO: 20, a light chain comprising the amino acid sequence set forth in SEQ ID NO: 14, at amino acid positions 20 to 470; A heavy chain comprising the amino acid sequence set forth and amino acid positions 21-23 in SEQ ID NO: 22 4, a light chain comprising the amino acid sequence of SEQ ID NO: 16 at amino acid positions 20 to 47 18. A heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 18, 234, a light chain comprising the amino acid sequence set forth in SEQ ID NO: 16, 470 and amino acid position 2 in SEQ ID NO: 20 1 to 234, and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 16. A heavy chain comprising the amino acid sequence set forth in amino acid positions 20 to 470 and an amino acid sequence set forth in SEQ ID NO: 22. a light chain comprising the amino acid sequence set forth in amino acid positions 21 to 234 of The antibody according to (6) or (7), comprising a heavy chain and a light chain. (10) The antibody comprises a heavy chain comprising the amino acid sequence represented by SEQ ID NO: 45 and a heavy chain comprising the amino acid sequence represented by SEQ ID NO: 4 and a light chain comprising an amino acid sequence represented by SEQ ID NO:6. Alternatively or additionally, the antibody comprises a heavy chain comprising the amino acid sequence set forth in 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: 20, and a light chain comprising the amino acid sequence represented by SEQ ID NO: 12 a heavy chain comprising the amino acid sequence represented by SEQ ID NO: 22 and a light chain comprising the amino acid sequence represented by SEQ ID NO: 23; A heavy chain comprising the amino acid sequence represented by SEQ ID NO: 14 and the amino acid sequence represented by SEQ ID NO: 18 a light chain comprising the amino acid sequence represented by SEQ ID NO: 14; a heavy chain comprising the amino acid sequence represented by SEQ ID NO: 20; a light chain comprising the amino acid sequence represented by SEQ ID NO: 14; a heavy chain comprising the amino acid sequence represented by SEQ ID NO: 15; 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: 18, a light chain comprising the amino acid sequence represented by SEQ ID NO: 16, a heavy chain comprising the amino acid sequence represented by SEQ ID NO: 20 and a light chain comprising the amino acid sequence represented by SEQ ID NO: 21; and a heavy chain comprising the amino acid sequence represented by SEQ ID NO: 16 and a heavy chain comprising the amino acid sequence represented by SEQ ID NO: 22 (6) comprising a heavy chain and a light chain selected from the group consisting of: a light chain comprising the amino acid sequence (7) The antibody according to (7). (11) The antibody is an antibody comprising an amino acid sequence selected from the group consisting of amino acids 20 to 470 in SEQ ID NO: 12. and a heavy chain comprising the amino acid sequence set forth at amino acid positions 21 to 234 in SEQ ID NO: 18. a light chain comprising an amino acid sequence as set forth in amino acid positions 20 to 470 in SEQ ID NO: 14; and a heavy chain comprising the amino acid sequence set forth in amino acid positions 21 to 234 of SEQ ID NO: 18. a light chain comprising the amino acid sequence set forth in amino acid positions 20 to 470 of SEQ ID NO: 14; and a heavy chain comprising the amino acid sequence of SEQ ID NO: 20, A light chain comprising the amino acid sequence set forth herein, as well as amino acid positions 20 to 16 of SEQ ID NO: 16. 470 and amino acid position 2 in SEQ ID NO: 22 a heavy chain and a light chain selected from the group consisting of: a heavy chain and a light chain comprising an amino acid sequence set forth in any one of claims 1 to 234; The antibody according to (8), comprising a chain. (12) The antibody lacks a lysine residue at the carboxyl terminus of the heavy chain. The antibody according to any one of (11) to (11). (13) An antibody obtained by the method for producing an antibody according to any one of (1) to (12). the method comprises the step of: a step of culturing the transformed host cells; and extracting the desired product from the culture obtained in the above step. and recovering said antibody.

[0074] For purposes of this disclosure, the full sequences of SEQ ID NOs: 45 and 46 are set forth below in Table 1 (and Figure 2). Shown below.

[0075] [Table 1]

[0076] Production of anti-TROP2 antibodies The antibodies against TROP2 of the present invention generally bind to TROP2 or the amino acid sequence of TROP2. Immunizing an animal with any polypeptide selected from the series and producing in vivo and recovering and purifying the antibody using methods practiced in the art. The biological species of TROP2 used as an antigen is not limited to humans. Immunizing animals with TROP2 derived from non-human animals such as mice or rats In this case, the cross-reaction between the antibody binding to the heterologous TROP2 and human TROP2 can be observed. By examining differential reactivities, antibodies applicable to human diseases can be selected.

[0077] Additionally, known methods (e.g., Kohler and Milstein, Nature , (1975) 256, pp. 495-497; Kennet, R. (ed.), Monocl onal Antibodies, pp. 365-367, Plenum Press, One that produces antibodies against TROP2 in myeloma cells according to NY (1980) Alternatively, a monoclonal antibody can be produced from a hybridoma established by fusing multiple antibody-producing cells. A monoclonal antibody can be obtained.

[0078] By expressing the TROP2 gene in host cells using genetic engineering, Specifically, the TROP2 gene can be expressed by It is possible to prepare a vector capable of expressing the vector, and the vector obtained can be used to The gene can be transfected into host cells to express the gene, which can then be expressed. The expressed TROP2 can be purified.

[0079] Alternatively, the above-mentioned TROP2-expressing cells or cells expressing TROP2 are genetically engineered. The following describes how to obtain an antibody against TROP2. The law will be explained in detail.

[0080] (1) Antigen preparation

[0081] Examples of antigens that can be used to generate anti-TROP2 antibodies include TROP2, A polypeptide consisting of a partial amino acid sequence containing at least six consecutive amino acids of TROP2. peptide, or by adding a predetermined amino acid sequence or carrier thereto. Derivatives are also included.

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

[0083] Specifically, regarding genetic engineering, a vector capable of expressing TROP2 cDNA was developed. After the TROP2 cDNA is integrated into the target, the antigen is expressed by the enzymes required for transcription and translation. by synthesizing it in a solution containing a substrate and an energy source, or In another transformed prokaryotic or eukaryotic host cell, TROP 2.

[0084] Furthermore, the constant region of the antibody in a suitable host vector system can be used to express the membrane protein TROP2. By expressing the fusion protein obtained by linking the extracellular domains, The antigen can also be obtained as a secretory protein.

[0085] For example, a cDNA library expressing TROP2 cDNA as a template and a TROP2 cDNA library expressing TROP2 cDNA as a template were used. A polymerase chain reaction (hereinafter referred to as "PCR") was performed using primers that specifically amplify P2 cDNA. PCR"; Saiki, RK et al., Science, (1988) 239 TROP2 cD was detected by PCR (see pages 487-489). NA can be obtained.

[0086] In vitro synthesis of the polypeptide can be carried out using, for example, the method described in Roche Diagnostics. Rapid Translation S manufactured by ostics, Inc. An example of such a system is the Real Time Server System (RTS), but it is not limited to this.

[0087] Examples of prokaryotic host cells include Escherichia coli and Bacillus subtilis is an example of a bacterium that transforms host cells with target genes. To transform, a replicon, i.e., an origin of replication derived from a species compatible with the host, is required. and a regulatory sequence. The vector preferably contains a sequence capable of conferring phenotypic selectability to the transformed cell. It has columns.

[0088] Examples of eukaryotic host cells include vertebrate cells, insect cells and yeast cells. Examples of vertebrate cells include monkey COS cells (Gluzman, Y., Cell, ( 1981)23, pp. 175-182, ATCC CRL-1650; ATCC:A American Type Culture Collection), mouse fibroblasts NIH3T3 cells (ATCC No. CRL-1658), Chinese hamster ovary cells A dihydrofolate reductase-deficient strain (Urlau) of CHO cells (ATCC: CCL-61) b, G. and Chasin, L.A., Proc. Natl. Acad. Sci. US A (1980) 77, pp. 4126-4220) and the like are often used. The cells can be, but are not limited to, any of the above.

[0089] The transformants thus obtained are then subjected to a method commonly used in the art. The transformant can be cultured in a suitable medium, and the target polypeptide is expressed in the culture medium. It is produced intracellularly or extracellularly.

[0090] The appropriate medium used for the culture may vary depending on the host cell used. A culture medium can be selected by those skilled in the art from commonly used culture media. For example, LB medium supplemented with antibiotics such as ampicillin or IPMG as needed. It can be used.

[0091] By such culture, recombinant proteins produced intracellularly or extracellularly by the transformants were obtained. Proteins can be synthesized by a variety of known molecules that utilize the physical or chemical properties of the protein. The compound may be isolated and purified by any of a number of isolation techniques.

[0092] Specific examples of such methods include treatment with common protein precipitants, ultrafiltration, and moleculation. Sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography Various types of liquid chromatography, such as chromatographic and affinity chromatography dialysis, and combinations thereof.

[0093] Furthermore, the recombinant protein to be expressed is tagged with six histidine residues. This allows the protein to be efficiently purified on a nickel affinity column. Alternatively, the IgG Fc region can be attached to the recombinant protein to be expressed. The protein can be efficiently purified on a protein A column.

[0094] By combining the above methods, large amounts of target polypeptides can be obtained in high yield and purity. It can be easily produced at low temperatures.

[0095] The transformant itself can also be used as an antigen. Cell lines expressing the antigen may also be used as antigens. Examples of such cell lines include the human lung carcinoma line NCI-H322, PC14, NCIH-H2122 and LCAM1 human prostate cancer lines PC3, human pancreatic cancer line BxPC-3, Capan-1 and PK-1, human ovarian cancer line SKO V3 and the human colorectal cancer line COLO205, The cell lines are not limited to these cell lines as long as they express TROP2.

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

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

[0098] The production of monoclonal antibodies involves the following steps: (a) Purify the biopolymer to be used as an antigen or prepare antigen-expressing cells. Operation steps to be performed, (b) preparing antibody-producing cells by immunizing an animal by injection of said antigen; The procedure involves collecting blood and assaying its antibody titer to determine when to remove the spleen. , (c) preparing myeloma cells (hereinafter referred to as "myeloma"); (d) fusing said antibody-producing cells with said myeloma; (e) screening the population of hybridomas for those producing the desired antibodies; (f) dividing the hybridoma into single cell clones (cloning) , (g) optionally, culturing the hybridoma to produce large amounts of monoclonal antibodies; a manipulation step of culturing or raising the animal into which said hybridoma has been transplanted; (h) The monoclonal antibodies thus produced are analyzed for their biological activity and binding specificity. or use it as a labeling reagent to assay for properties. and the like Generally requires

[0099] The method for producing monoclonal antibodies after the above steps will be described in detail below. The methods include, but are not limited to, spleen cells and non-myeloma antibodies. Somatic producing cells can be used.

[0100] (a) Antigen purification

[0101] As an antigen, TROP2 or a partial peptide thereof prepared by the method described above is used. You can use the

[0102] Furthermore, membrane fractions prepared from recombinant cells expressing TROP2 or cells expressing TROP2 were used. The present invention can be applied to recombinant cells expressing the compounds of the present invention, or to compounds of the present invention chemically synthesized by methods known to those skilled in the art. A partial peptide of the protein can also be used as an antigen.

[0103] Additionally, cell lines expressing TROP2 can be used as antigens.

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

[0105] The antigen obtained in step (a) is mixed with Freund's complete adjuvant or Freund's incomplete adjuvant. Mixed with an adjuvant such as complete adjuvant or an auxiliary agent such as potassium aluminum sulfate and the resulting mixture is used as an immunogen to immunize a laboratory animal. The experimental animal is then immunized with the antigen-expressing cells as an immunogen. Any animal used in the hybridoma production method described above can be used without any problems. Specifically, for example, mice, rats, goats, sheep, cattle, horses, or the like. However, the availability of myeloma cells to be fused with the extracted antibody-producing cells is limited. From the viewpoint of ease of use, mice or rats are preferred as animals to be immunized. is used.

[0106] Furthermore, the strain of mouse or rat used is not particularly limited, but may be any of the following: For example, A, AKR, BALB / c, BDP, BA, CE, C3H, 57BL , C57BL, C57L, DBA, FL, HTH, HT1, LP, NZB, NZW, RF Various strains such as RIII, SJL, SWR, WB, and 129 and the like were used. In the case of rats, for example, Wistar, Low, Lewis , Sprague, Dooley, ACI, BN, Fisher and the like. is possible.

[0107] These mice and rats are used by laboratory animal breeders / distributors, e.g. CLEA Japan, Inc. and Charles River Laboratories ries Japan, Inc.

[0108] Considering the suitability for fusion with myeloma cells described below, animals to be immunized include: The BALB / c strain is particularly preferred for mice, and the Wistar and Rho strains for rats. It's nice.

[0109] Furthermore, considering the antigenic homology between humans and mice, the biology of the autoantibody elimination It is also preferable to use mice with reduced immune function, i.e., mice with autoimmune diseases.

[0110] The age of such mice or rats at the time of immunization is preferably 5 to 12 weeks old, more preferably The age is usually between 6 and 8 weeks.

[0111] To immunize animals with TROP2 or a recombinant form thereof, see, for example, Weir, DM, Handbook of Experimental Immunology Volumes I, II, and III, Blackwell Scientific Pub lications, Oxford (1987); Kabat, E.A. and May er,MM、Experimental Immunochemistry,Cha rles C Thomas Publisher Springfield, Illi Use known methods detailed in Nois (1964) or similar. It is possible.

[0112] Among these immunization methods, the preferred specific method of the present invention is, for example, the following method: It is.

[0113] That is, first, a membrane protein fraction that acts as an antigen or a cell that expresses the antigen is isolated. Animals are administered intradermally or intraperitoneally. However, both routes of administration are used to increase the efficiency of immunization. It is preferable to administer both routes in combination, with intradermal administration in the first half and intraperitoneal administration in the second half or only at the last administration. Immunization efficiency may be particularly increased if the procedure is carried out in a single step.

[0114] The schedule for administering the antigen may vary depending on the type, individual or allogeneic, of the animal being immunized. However, generally, the frequency of antigen administration is 3-6 times, with the interval between administrations being 2-6 weeks. The preferred administration schedule is one in which the frequency of antigen administration is 3 to 4 times and the administration interval is 2 A dosing schedule of 4 weeks is more preferred.

[0115] Furthermore, the dose of the antigen varies depending on the type of animal, individual differences, or the same species, 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, after administration of the antigen as described above. Preferably, the incubation period is 1 to 3 weeks. When the immunogen is a cell, the incubation period is 1×10 6 ~1×10 7 pieces Cells of the type are used.

[0117] The amount of antigen administered for booster immunization depends on the type and size of the animal or species. For example, in the case of mice, the dosage is generally 0.05 to 5 mg, preferably The amount is set to 0.1 to 0.5 mg, more preferably about 0.1 to 0.2 mg. If it is 1×10 6 ~1×10 7 cells are used.

[0118] The spleen cells or lymphocytes containing antibody-producing cells are preferably collected 1 to 10 days after the booster immunization. is aseptically removed from the immunized animal after 2 to 5 days, more preferably after 2 to 3 days. At this time, the antibody titer is measured, and animals with a sufficiently increased antibody titer are selected as a source of antibody-producing cells. If used, subsequent techniques can be performed more efficiently.

[0119] Examples of the method for measuring antibody titer used here include the RIA method and the ELISA method. The methods include, but are not limited to, for example, ELISA. In this case, the antibody titer of the present invention is measured according to the following procedure. It is possible.

[0120] First, purified or partially purified antigen was placed in a 96-well plate for ELISA. The surface of the solid phase, such as bovine serum albumin, is then adsorbed onto the surface of the solid phase, and the surface of the solid phase without the adsorbed antigen is then treated with bovine serum albumin. After cleaning the surface, the surface is covered with a protein unrelated to the antigen, such as serum albumin (BSA). , the surface is contacted with a serially diluted sample (e.g., mouse serum) as the primary antibody. The antibody in the sample is allowed to bind to the antigen.

[0121] Furthermore, as a secondary antibody, an antibody labeled with an enzyme against the mouse antibody was used. After washing, a substrate for the enzyme is added, and the substrate or a cognate thereof is added. The change in absorbance caused by the color development induced by the decomposition of the substance is measured, and the results are The antibody titer is calculated.

[0122] Isolation of antibody-producing cells from spleen cells or lymphocytes of immunized animals has been performed by known methods. method (e.g., Kohler et al., Nature (1975), 256, 495; K Ohler et al., Eur. J. Immunol. (1977), 6, 511; Mi lstein et al., Nature (1977), 266, 550; Walsh, N (1977) 266, 495). In the case of liver cells, antibody-producing cells were isolated by filtration through a stainless steel mesh to obtain cells. To do this, homogenize the spleen and suspend the cells in Eagle's minimum essential medium (MEM). A common method for separating the compounds by

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

[0124] The myeloma cells used for cell fusion are not particularly limited, but may be any of the known Suitable cells can be selected from cell lines. However, for convenience, hybridomas can be selected from When selecting hybridomas from the chromosomes, the selection method is established using HGPRT (hypoxanthine phosphate reductase). It is preferable to use a strain lacking guanine phosphoribosyltransferase.

[0125] More specifically, an example of an HGPRT deletion strain is X63-Ag8 derived from mouse. (X63), NS1-ANS / 1(NS1), P3X63-Ag8.U1(P3U1), X63-Ag8.653(X63.653), SP2 / 0-Ag14(SP2 / 0), M PC11-45.6TG1.7(45.6TG), FO, S149 / 5XXO and BU .1, 210.RSY3.Ag.1.2.3(Y3) derived from rats, and 210.RSY3.Ag.1.2.3(Y3) derived from humans Derived from U266AR (SKO-007), GM1500 GTG-A12 (GM150 0), UC729-6, LICR-LOW-HMy2 (HMy2) and 8226AR / These HGPRT-deficient strains include, for example, ATC C or similar.

[0126] These cell lines were grown in 8-azaguanine medium (glutamine, 2-mercaptoethanol, RPMI supplemented with gentamicin and fetal bovine serum (hereafter referred to as "FBS") Iscove's modified 1640 medium obtained by adding 8-azaguanine Suitable medium such as Integral Molecular Digestion Medium (IMDM) or Dulbecco's Modified Eagle's Medium (DMEM) In this case, the cells are subcultured in normal medium 3 to 4 days before cell fusion. Medium (e.g., ASF104 medium (Ajinomoto Co., Ltd., Japan) containing 10% FCS) On the day of cell fusion, the cells were subcultured in a medium containing 2 × 10 7 More than 100 Secure the cells.

[0127] (d) Cell fusion

[0128] The fusion between the antibody-producing cells and the myeloma cells is carried out in a manner that does not excessively reduce the viability of the cells. Under these conditions, known methods (Weir, D.M., Handbook of Experiments Mental Immunology Volumes I, II, and III, Blackwe ll Scientific Publications,Oxford(1987) ;Kabat, EA and Mayer, MM, Experimental Im munochemistry, Charles C Thomas Publisher , Springfield, Illinois (1964), etc.) It is possible.

[0129] Such methods include, for example, the use of polymers such as polyethylene glycol at high concentrations. A chemical method using a mixture of antibody-producing cells and myeloma cells in a solution containing Among these methods, chemical methods can be used. Specific examples of the method are described below.

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

[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, HAT (hypoxanthine, aminopterin, thymidine) selection method (K Ohler et al., Nature (1975), 256, 495; Milstein et al., Nature (1977), 266, 550) is used.

[0133] This method allows the bone marrow to be isolated from HGPRT-deficient strains that cannot survive in the presence of aminopterin. This is effective when obtaining hybridomas using myeloma cells. By culturing unfused cells and hybridomas in Only hybridomas that are resistant to the cloning process can be selectively allowed to survive and grow.

[0134] (f) division into single-cell clones (cloning)

[0135] Cloning methods for hybridomas include the methylcellulose method and the soft agarose method. Alternatively, known methods such as limiting dilution can be used (see, for example, Barbara, B.M. and Stanley, M.S.: Selected Methods in Cellular Immunology, WHFreeman and Comp (See any, San Francisco (1980). Among these methods, For example, three-dimensional culture methods such as methylcellulose method are preferred. The resulting hybridoma population was selected from the ClonaCell-HY Selection Medium ium D (manufactured by StemCell Technologies, Inc., # The resulting mixture is suspended in a methylcellulose medium such as 03804 and cultured. Obtain monoclonal hybridomas by harvesting hybridoma colonies Each of the collected hybridoma colonies is cultured, and the resulting hybridomas are Hybridomas that were confirmed to have stable antibody titers in the culture supernatant were designated T A hybridoma line producing ROP2 monoclonal antibody is selected.

[0136] Examples of hybridoma lines established in this manner include the TROP2 hybridoma TINA1. In the present specification, the TROP2 hybridoma TINA1 The antibodies produced thereby are referred to as "TINA1 antibodies," or simply "TINA1."

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

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

[0139] By culturing the hybridomas selected in this way, monoclonal antibodies However, before culturing, the target monoclonal antibody-producing cells are isolated. It is preferable to screen hybridomas that contain the desired gene.

[0140] Known methods can be used in such screening.

[0141] The antibody titer in the present invention can be measured, for example, by the ELI method described in the above item (b). This can be done by the SA method.

[0142] The hybridomas obtained by the above method can be stored in liquid nitrogen or frozen at -80°C or below. It can be stored frozen in the sinter.

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

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

[0145] Furthermore, the hybridomas were cultured in the same strain as the hybridomas (e.g., BALB / c as mentioned above). The hybridomas were grown by intraperitoneal injection into mice or Nu / Nu mice. As a result, ascites containing a large amount of the monoclonal antibody of the present invention can be obtained.

[0146] When hybridomas are administered intraperitoneally, 2, 6, 10, 14 -If mineral oils such as tetramethylpentadecane (pristane) are administered, a larger amount of abdominal Water can be obtained.

[0147] For example, an immunosuppressant was injected into the abdominal cavity of a mouse of the same strain as the hybridoma. T cells were inactivated. 20 days later, 10 6 ~10 7 Hybridoma clone cells The cells are suspended in serum-free medium (0.5 mL) and the suspension is administered intraperitoneally to mice. When the abdomen becomes distended and filled with ascites, the ascites is collected from the mouse. Therefore, monoclonal antibodies are present at concentrations approximately 100 times or much higher than those in the culture medium. It can be obtained in low concentrations.

[0148] The monoclonal antibodies obtained by the above method are, for example, those prepared by Weir, DM:H andbook of Experimental Immunology, Vol. I, No. Volumes II and III, Blackwell Scientific Publicati The purified product can be purified by the method described in Ons, Oxford (1978).

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

[0150] (h) Monoclonal antibody assay

[0151] The isotypes and subclasses of the monoclonal antibodies thus obtained are as follows: It can be determined as follows:

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

[0153] The Ouchterlony method is simple, but when the concentration of monoclonal antibodies is low, the concentration process is difficult. It is necessary.

[0154] On the other hand, when ELISA or RIA is used, the culture supernatant is directly mixed with the antigen-adsorbed solid phase. The secondary antibodies react with various immunoglobulin isotypes and subclasses. By using the corresponding antibody, the isotype and subclass of the monoclonal antibody can be determined. can be identified.

[0155] Furthermore, as a more convenient method, commercially available identification kits (e.g., Bio-R Mouse Typer manufactured by AD Laboratories, Inc. Kit) or the like may also be used.

[0156] Furthermore, protein quantification was performed using the Folin-Lowry method and absorbance at 280 nm. The calculation was based on the following formula: 1.4 (OD280) = 1 mg / mL of immunoglobulin. It can be applied.

[0157] Furthermore, by repeating steps (a) to (h) of (2), monoclonal antibodies were isolated. Even when obtained separately and independently, they have cytotoxic activity equivalent to that of TINA1 antibodies. or an antibody comprising a heavy chain comprising SEQ ID NO: 45 and a light chain comprising SEQ ID NO: 46. An example of such an antibody is a TINA1 antibody that binds to the same epitope as the TINA1 antibody. or an antibody comprising a heavy chain comprising SEQ ID NO: 45 and a light chain comprising SEQ ID NO: 46. The monoclonal antibody produced is a TINA1 antibody or a heavy chain comprising SEQ ID NO: 45. Binds to a partial peptide or partial tertiary structure to which an antibody containing a light chain containing sequence number 46 binds. If so, it can be determined that the monoclonal antibodies bind to the same epitope. Additionally, the monoclonal antibody is selected from the group consisting of TINA1 antibody and SEQ ID NO: 1 for binding to TROP2. 45 and a light chain comprising SEQ ID NO: 46 (i.e., monoclonal antibody The clonal antibody comprises a TINA1 antibody or a heavy chain comprising SEQ ID NO: 45 and a heavy chain comprising SEQ ID NO: 46. by confirming that the antibody inhibits the binding between the antibody and TROP2, and the antibody containing the light chain Even if the specific epitope sequence or structure has not been determined, monoclonal antibodies It can be determined that the antibody binds to the same epitope as the anti-TROP2 antibody. If the antibody is confirmed to bind to the same epitope as the anti-TROP2 antibody, The null antibody may be a TINA1 antibody or a TINA2 antibody having a heavy chain comprising SEQ ID NO: 45 and a light chain comprising SEQ ID NO: 46. It is strongly expected that the antibody will have antigen-binding affinity and biological activity equivalent to that of an antibody comprising

[0158] (3) Other antibodies

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

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

[0161] Humanized antibodies are created by integrating only the complementarity-determining regions (CDRs) into a human-derived antibody. The resulting antibody (see Nature (1986) 321, 522-525) and a part of the amino acid residues of the framework and the CDR sequences by the CDR grafting method. Antibodies obtained by grafting sequences onto human antibodies (WO 90 / 07861) ) can be exemplified.

[0162] However, the humanized antibody derived from the TINA1 antibody has six TINA1 antibody fragments. The present invention is not limited to specific humanized antibodies, as long as they have all of the CDR sequences of the above type. The heavy chain variable region of the NA1 antibody is C DRH1 (TAGMQ) and a CD consisting of the amino acid sequence represented by SEQ ID NO: 24 in the sequence listing. RH2 (WINTHSGVPKYAEDFKG) and the amplified fragment represented by SEQ ID NO: 25 in the sequence listing. It has a CDRH3 (SGFGSSYWYFDV) consisting of the amino acid sequence. The light chain variable region of the NA1 antibody is C DRL1 (KASQDVSTAVA) and the amino acid sequence represented by SEQ ID NO: 27 in the sequence listing and CDRL2 (SASYRYT) consisting of the amino acid sequence represented by SEQ ID NO: 28 in the sequence listing. and CDRL3 (QQHYITPLT) consisting of a sequence.

[0163] Examples of humanized antibodies of the mouse antibody TINA1 include (1) SEQ ID NOs: 12 and 14 in the Sequence Listing. or amino acid residues 20 to 140 of SEQ ID NO: 16 or amino acid residues 1 to 121 of SEQ ID NO: 45 (2) an amino acid sequence having at least 95% homology with the amino acid sequence (1) above; and (3) one or more of the amino acid sequences in (1) above. Any of the amino acid sequences in which amino acids are deleted, substituted, or added (4) a heavy chain comprising a heavy chain variable region consisting of one of SEQ ID NOs: 18, 20, or 2 2 or amino acid residues 1 to 109 of SEQ ID NO: 46 (5) an amino acid sequence having at least 95% homology with the amino acid sequence (4) above. (6) one or more amino acids in the amino acid sequence (4) above. is any one of the amino acid sequences in which deletions, substitutions, or additions are made. Examples of such a light chain include any combination of a light chain comprising a light chain variable region consisting of:

[0164] An antibody having the above-mentioned preferred combination of heavy chain and light chain is an antibody having an amino acid sequence of SEQ ID NO: 45 a heavy chain comprising a variable region comprising amino acids 1-121 of SEQ ID NO:46 and a variable region comprising amino acids 1-109 of SEQ ID NO:46; an antibody comprising a light chain containing an amino acid sequence consisting of amino acid positions 20 to 140 of SEQ ID NO: 12; a heavy chain comprising a variable region consisting of the amino acid sequence of SEQ ID NO: 18 and a an antibody comprising a light chain containing a variable region consisting of the amino acid sequence of SEQ ID NO: 12; A heavy chain comprising a variable region consisting of an amino acid sequence of 20 to 140 and an amino acid sequence of SEQ ID NO: 20 an antibody comprising a light chain including a variable region having an amino acid sequence consisting of amino acid positions 21 to 129; Contains a variable region consisting of an amino acid sequence consisting of amino acid positions 20 to 140 of SEQ ID NO: 12 A heavy chain and a variable region consisting of an amino acid sequence consisting of amino acid positions 21 to 129 of SEQ ID NO: 22 an antibody comprising a light chain comprising amino acids at positions 20 to 140 of SEQ ID NO: 14; a heavy chain comprising a variable region consisting of the sequence of SEQ ID NO: 18 and an amino acid sequence consisting of amino acid positions 21 to 129 of SEQ ID NO: 18; an antibody comprising a light chain containing a variable region consisting of the amino acid sequence of amino acid position 2 of SEQ ID NO: 14; a heavy chain comprising a variable region consisting of an amino acid sequence of 0 to 140 and an amino acid sequence of SEQ ID NO: 20 an antibody comprising a light chain containing a variable region consisting of an amino acid sequence of positions 21 to 129; A variable region consisting of an amino acid sequence consisting of amino acid positions 20 to 140 of sequence number 14 a variable region consisting of an amino acid sequence consisting of amino acid positions 21 to 129 of SEQ ID NO: 22; an antibody comprising a light chain containing an amino acid sequence consisting of amino acid positions 20 to 140 of SEQ ID NO: 16; a heavy chain comprising a variable region consisting of amino acid positions 21 to 129 of SEQ ID NO: 18; an antibody comprising a light chain containing a variable region consisting of the amino acid sequence of amino acid position 20 of SEQ ID NO: 16; a heavy chain comprising a variable region consisting of an amino acid sequence of 140 to 140 and an amino acid sequence of SEQ ID NO: 20; an antibody comprising a light chain including a variable region consisting of an amino acid sequence of positions 21 to 129; and a variable region consisting of an amino acid sequence consisting of amino acid positions 20 to 140 of SEQ ID NO: 16. a heavy chain comprising a variable region consisting of an amino acid sequence consisting of amino acid positions 21 to 129 of SEQ ID NO: 22; An example of such an antibody is an antibody comprising a light chain containing the IL-1 region.

[0165] Furthermore, as an antibody having the above-mentioned more preferable combination of heavy chains and light chains, the antibody represented by SEQ ID NO: an antibody comprising a heavy chain comprising SEQ ID NO: 45 and a light chain comprising SEQ ID NO: 46, amino acid position 2 of SEQ ID NO: 12; a heavy chain consisting of an amino acid sequence consisting of amino acid positions 0 to 470 of SEQ ID NO: 18 and amino acid positions 21 to 23 of SEQ ID NO: 18; an antibody comprising a light chain consisting of an amino acid sequence consisting of amino acid position 20 of SEQ ID NO: 12; a heavy chain consisting of an amino acid sequence of amino acids 21 to 234 of SEQ ID NO: 20; an antibody comprising a light chain having an amino acid sequence consisting of amino acid positions 20 to 29 of SEQ ID NO: 12; a heavy chain consisting of an amino acid sequence of 470 and a an antibody comprising a light chain having an amino acid sequence comprising: a heavy chain consisting of an amino acid sequence of 70 and a heavy chain consisting of amino acid positions 21 to 234 of SEQ ID NO: 18; and a light chain comprising an amino acid sequence selected from the group consisting of amino acid positions 20 to 47 of SEQ ID NO: 14. a heavy chain consisting of an amino acid sequence of SEQ ID NO: 20; and a heavy chain consisting of an amino acid sequence of SEQ ID NO: 21 to SEQ ID NO: 234. an antibody having a light chain consisting of an amino acid sequence represented by the amino acid sequence of SEQ ID NO: 14; a heavy chain consisting of an amino acid sequence comprising: an antibody having a light chain consisting of an amino acid sequence from amino acid positions 20 to 470 of SEQ ID NO: 16; and a heavy chain consisting of an amino acid sequence comprising amino acid positions 21 to 234 of SEQ ID NO: 18. an antibody having a light chain consisting of the amino acid sequence from amino acid positions 20 to 470 of SEQ ID NO: 16; and a heavy chain consisting of an amino acid sequence of amino acid positions 21 to 234 of SEQ ID NO: 20. and a light chain consisting of the amino acid sequence of amino acid positions 20 to 470 of SEQ ID NO: 16. a heavy chain consisting of an amino acid sequence comprising: An example of such an antibody is one that comprises a light chain consisting of an amino acid sequence.

[0166] As an antibody having a more preferable combination of the heavy chain and the light chain, SEQ ID NO: 4 a heavy chain comprising a variable region comprising amino acids 1 to 121 of SEQ ID NO: 5 and amino acids 1 to 109 of SEQ ID NO: 46; an antibody comprising a light chain containing a variable region comprising amino acid residues 20 to 140 of SEQ ID NO: 12; a heavy chain comprising a variable region consisting of an amino acid sequence comprising amino acid residues 21-1 of SEQ ID NO: 18; an antibody comprising a light chain containing a variable region consisting of an amino acid sequence of SEQ ID NO: 14; A heavy chain containing a variable region consisting of an amino acid sequence of amino acid residues 20 to 140 and SEQ ID NO: a light chain containing a variable region consisting of an amino acid sequence of 18 amino acid residues 21 to 129; an antibody comprising the amino acid sequence of amino acid residues 20 to 140 of SEQ ID NO: 14; A heavy chain containing a variable region and an amino acid sequence consisting of amino acid residues 21 to 129 of SEQ ID NO: 20 and a light chain comprising a variable region comprising amino acid residues 20 to 14 of SEQ ID NO: 16. a heavy chain comprising a variable region consisting of an amino acid sequence consisting of amino acid residue 21 of SEQ ID NO: 22; Examples of antibodies include antibodies having a light chain containing a variable region consisting of an amino acid sequence of 1 to 129. This can be done.

[0167] Furthermore, as an antibody having another more preferable combination of the above-mentioned heavy chain and light chain, An antibody consisting of a heavy chain comprising SEQ ID NO: 45 and a light chain comprising SEQ ID NO: 46, an amino acid sequence of SEQ ID NO: 12 an antibody comprising a heavy chain consisting of the amino acid sequence of SEQ ID NO: 18 and a light chain consisting of the amino acid sequence of SEQ ID NO: 19; It consists 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 having a heavy chain consisting of the amino acid sequence of SEQ ID NO: 12 and an amino acid sequence of SEQ ID NO: 22; an antibody comprising a light chain having the amino acid sequence of SEQ ID NO: 14 and a heavy chain having the amino acid sequence of SEQ ID NO: 18; an antibody comprising a light chain consisting of the amino acid sequence of SEQ ID NO: 14; a heavy chain consisting of the amino acid sequence of SEQ ID NO: 15; an antibody having a light chain consisting of the amino acid sequence of SEQ ID NO: 20; an antibody having a light chain consisting of the amino acid sequence of SEQ ID NO: 14; an antibody comprising a heavy chain having the amino acid sequence of SEQ ID NO: 22 and a light chain having the amino acid sequence of SEQ ID NO: 16; an antibody comprising a heavy chain consisting of the amino acid sequence of SEQ ID NO: 18 and a light chain consisting of the amino acid sequence of SEQ ID NO: 19; 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 comprising a heavy chain consisting of the amino acid sequence of SEQ ID NO: 16 and an amino acid sequence of SEQ ID NO: 22; An example of such an antibody is an antibody comprising a light chain comprising:

[0168] As an antibody having a more preferable combination of the heavy chain and the light chain, SEQ ID NO: 4 an antibody comprising a heavy chain comprising SEQ ID NO: 5 and a light chain comprising SEQ ID NO: 46, ... a heavy chain consisting of an amino acid sequence of amino acids 21 to 234 of SEQ ID NO: 18; an antibody comprising a light chain having an amino acid sequence consisting of amino acid positions 20 to 29 of SEQ ID NO: 14; a heavy chain consisting of an amino acid sequence of 470 and a an antibody comprising a light chain having an amino acid sequence comprising: a heavy chain consisting of an amino acid sequence of 70 and a and a light chain comprising an amino acid sequence comprising: a heavy chain consisting of an amino acid sequence of amino acids 21 to 234 of SEQ ID NO: 22; and a light chain consisting of an amino acid sequence consisting of:

[0169] Furthermore, antibodies having even better preferred combinations of the above heavy chains and light chains include and a heavy chain consisting of an amino acid sequence consisting of amino acid positions 20 to 469 of SEQ ID NO: 12. an antibody comprising a light chain having an amino acid sequence consisting of amino acid positions 21 to 234 of No. 18; a heavy chain consisting of an amino acid sequence consisting of amino acid positions 20 to 469 of SEQ ID NO: 14; an antibody comprising a light chain having an amino acid sequence consisting of amino acid positions 21 to 234 of No. 18; a heavy chain consisting of an amino acid sequence consisting of amino acid positions 20 to 469 of SEQ ID NO: 14; an antibody comprising a light chain having an amino acid sequence consisting of 20 amino acid positions 21 to 234; and a heavy chain consisting of an amino acid sequence consisting of amino acid positions 20 to 469 of SEQ ID NO: 16. an antibody having a light chain consisting of an amino acid sequence consisting of amino acid positions 21 to 234 of No. 22; Examples include:

[0170] A sequence having high homology with the heavy chain amino acid sequence is selected from the group consisting of a sequence having high homology with the light chain amino acid sequence. By combining with a sequence having the same sequence, it is possible to obtain a biological activity equivalent to that of each of the above antibodies. Such homology is generally 80% or more. Homologous, preferably 90% or more homologous, more preferably 95% or more homologous and most preferably 99% or more homology. residues are substituted or deleted in the heavy or light chain amino acid sequence; Alternatively, by combining the added amino acid sequences, it is possible to obtain antibodies equivalent to each of the above antibodies. It is also possible to select antibodies that have biological activity.

[0171] The homology between two amino acid sequences was calculated using the Blast algorithm version 2.2.2. The fault parameters can be determined using Altschul, Stephen F. Thomas L. Madden, Alejandro A. Schaeffer, Ji nghui Zhang, Zheng Zhang, Webb Miller, and D avid J. Lipman (1997), “Gapped BLAST and PS I-BLAST: a new generation of protein data base search programs”, Nucleic Acids Res. 25:3389-3402). The Blast algorithm is available at the site ncbi.nlm.n It can be used over the internet by visiting ih.gov / blast It is possible.

[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, and amino acid residues 20 to 140 are The amino acid sequence consisting of amino acid residues 141 to 470 is a variable region. The sequence 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, and amino acid residue 21 The amino acid sequence consisting of ~129 is the variable region, and the amino acid sequence consisting of 130 to 234 is The amino acid sequence is a constant region.

[0174] Furthermore, the antibodies of the present invention include human antibodies that bind to TROP2. The term "human" refers to an antibody that has only antibody sequences derived from human chromosomes. The antibody is a human antibody having a human chromosome fragment containing human antibody heavy and light chain genes. It can be obtained by a method using live-born mice (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 (Kitag awa, Y., Matuda, T. and Iijima, S. (Eds.), Kluwer Ac Academic Publishers, 1999; Tomizuka, K. et al., Pro c. Natl. Acad. Sci. USA (2000) 97, pp. 722-727 (See also:

[0175] Specifically, such a human antibody-producing mouse can be produced as follows. The immunoglobulin heavy chain locus and the endogenous immunoglobulin light chain locus are disrupted, and Instead, the human immunoglobulin heavy chain locus and the human immunoglobulin light chain locus are Genetically modified animals introduced via YAC vectors or similar By creating knockout and transgenic animals and mating these animals, and is produced by.

[0176] Furthermore, each of such heavy and light chains of a human antibody can be cloned in accordance with recombinant DNA techniques. By using a cDNA to be loaded and preferably a vector containing such a cDNA, The recombinant human monoclonal antibody is then transformed into a eukaryotic cell. The cells can also be cultured, thereby obtaining antibodies from the culture supernatant.

[0177] Here, examples of the host include eukaryotic cells, preferably mammalian cells, such as CH O cells, lymphocytes or myeloma cells can be used.

[0178] Furthermore, a phage display-derived human antibody selected from a human antibody library is obtained. Methods (Wormstone, IM et al., Investigative Ophthalmology mology & Visual Science.(2002)43(7), 230 pp. 1-2308; Carmen, S. et al., Briefings in Functio onal Genomics and Proteomics (2002), 1(2) , pp. 189-203; Siriwardena, D. et al., Ophthalmolog y (2002) 109(3), pp. 427-431) is also known.

[0179] For example, the variable regions of human antibodies are expressed on the surface of phages as single-chain fragments (scFv). The phage display method (Nature Biology) is used to select phages that bind to the antigen. otechnology (2005), 23, (9), 1105-1116) It is possible.

[0180] By analyzing the genes of phages selected based on their binding to antigens, The DNA sequences encoding the variable regions of the binding human antibodies can be determined.

[0181] When determining the DNA sequence of an scFv that binds to an antigen, an expression vector containing the sequence is and then introducing the vector into a suitable host to express it, thereby producing a human antibody. The body can be obtained (WO 92 / 01047, WO 92 / 20791 , International Publication No. 93 / 06213, International Publication No. 93 / 11236, International Publication No. 93 / 1 9172, WO 95 / 01438, WO 95 / 15388;Annu .Rev.Immunol.(1994)12, pp.433-455;Nature Biotechnology (2005) 23(9), pp. 1105-1116 .

[0182] The newly produced human antibodies target the partial peptide or partial triplicate to which the TINA1 antibody binds. When the human antibody binds to the 2D structure, it is determined that the human antibody binds to the same epitope as the TINA1 antibody. Furthermore, human antibodies can compete with TINA1 antibodies for binding to TROP2. (i.e., the human antibody inhibits the binding between the TINA1 antibody and TROP2). By confirming the sequence and structure of the specific epitope, We determined that the human antibody binds to the same epitope as the TINA1 antibody even when the antibody is not present. It has been confirmed that the human antibody binds to the same epitope as the TINA1 antibody. When human antibodies are used, they are strongly expected to have biological activity equivalent to that of TINA1 antibodies. can be.

[0183] The chimeric, humanized, or human antibodies obtained by the above method can be produced by known methods. or the like, it is possible to evaluate the binding properties to the antigen, and it is preferred. A new antibody can be selected.

[0184] Another example of an index for use in comparing antibody properties is antibody stability. Differential scanning calorimetry (DSC) can be used to measure the relative conformational stability of proteins. The thermal denaturation midpoint (Tm), which is used as a good indicator, can be measured quickly and accurately. This is a possible measure. By measuring the Tm value using DSC and comparing the values, it is possible to determine the thermal stability. The qualitative differences can be compared. The storage stability of antibodies is somewhat correlated with the thermal stability of antibodies. It is known that the drug exhibits anti-inflammatory properties (Lori Burton et al., Pharmaceutica l Development and Technology (2007) 12, 26 5-273), and the thermal stability is used as an index to select a preferred antibody. Other examples of criteria for selecting antibodies include the following characteristics: High yield in suitable host cells and low aggregation in aqueous solution. For example, the antibody with the highest yield does not necessarily have the highest thermostability. Therefore, a comprehensive evaluation based on the above indicators will be conducted to determine the most suitable drug for human administration. It is necessary to select the antibody.

[0185] The present invention also includes modified variants of antibodies. Modified variants are chemically modified versions of the antibodies of the present invention. It refers to a variant obtained by chemical modification or biological modification. Examples include those in which chemical modifications have been made by linking chemical moieties to the amino acid backbone. Variants, N-linked or O-linked carbohydrate chains and chemically modified variants Examples of biologically modified variants include post-translational modifications (e.g., N-linked glutamic acid residues). Glycosylation or O-linked glycosylation, N-terminal processing or C-terminal processing (aspartic acid decomposition, deamidation, isomerization of aspartic acid, or oxidation of methionine) A mutant in which a methionine residue is added to the N-terminus by expression in a prokaryotic host cell Examples of such mutants include:

[0186] Additionally, antibodies that are labeled to allow for detection or isolation of the antibody or antigen of the present invention. Also included within the meaning of modified variants are, for example, enzyme-labeled antibodies, fluorescent-labeled antibodies, and affinity-labeled antibodies. Such modified variants of the antibodies of the present invention may improve the stability and blood pool of the antibody. reducing its antigenicity, detecting or isolating antibodies or antigens, etc. It is useful for

[0187] Furthermore, modifications of glycans linked to the antibodies of the present invention (glycosylation, defucosylation, By adjusting the expression level of the antibody, antibody-dependent cellular cytotoxicity activity can be enhanced. As a method for controlling the modification of antibody glycans, WO 1999 / 54342 , International Publication No. 2000 / 61739, International Publication No. 2002 / 31140, etc. However, the above techniques are not limited to these. Also included are antibodies in which glycan modifications are modulated.

[0188] First, antibody genes are isolated, and then the genes are introduced into a suitable host to produce antibodies. When producing the organism, a combination of a suitable host and a suitable expression vector can be used. Specific examples of antibody genes include those encoding the heavy chain sequences of the antibodies described herein. The combination of the gene and the gene encoding the light chain sequence is also included. In the conversion, the heavy chain sequence gene and the light chain sequence gene are inserted into the same expression vector. It is also possible to insert them separately into different expression vectors.

[0189] When eukaryotic cells are used as hosts, animal cells, plant cells, and eukaryotic microorganisms are used. As animal cells, mammalian cells, for example, monkey COS cells (Gluz man, Y., Cell, (1981) 23, pp. 175-182, ATCC CR L-1650), mouse fibroblast NIH3T3 (ATCC number CRL-1658), and and Chinese hamster ovary cells (CHO cells; ATCC: CCL-61) dihydroxide. Folate reductase-deficient strain (Urlaub, G. and Chasin, L.A., Proc. Natl. Acad. Sci. USA (1980) 77, pp. 4126-4220 Examples include:

[0190] When prokaryotic cells are used, examples include Escherichia coli and Bacillus subtilis.

[0191] The desired antibody genes are introduced into these cells by transformation, and the transformed cells are The antibodies can be obtained by culturing the cells in vitro. In this method, the yield may vary depending on the antibody sequence. By using the yield as an index for antibodies with binding activity such as Therefore, it is possible to select antibodies that are easily produced. a step of culturing the transformed host cells; and extracting a desired product from the culture product obtained in the culturing step. and recovering the antibody. Antibodies that have been produced by the method of the present invention are also included.

[0192] The heavy chain of an antibody produced in cultured mammalian cells lacks a carboxyl-terminal lysine residue. It is known to cause loss of A, 705:129-134 (1995)), antibodies produced in cultured mammalian cells Two amino acid residues (glycine and lysine) were deleted from the carboxyl terminus of the heavy chain of the IgG1 gene. It is also known that proline residues located at the carboxyl terminus are amidated. (Analytical Biochemistry, 360:75~83 (2007) However, such deletions and modifications of the heavy chain sequence may affect the antigen-binding affinity and endogenous activity of the antibody. It does not affect effector functions (complement activation, antibody-dependent cellular cytotoxicity, etc.). The antibodies of the present invention may also be modified in such a manner as to produce functional fragments of the antibodies. a deletion mutant in which one or two amino acids are deleted at the carboxyl terminus of the heavy chain; A mutant obtained by amidation of the deletion mutant (e.g., a mutant having a carboxy-terminal proline residue) The heavy chains of the antibodies of the present invention also include those in which the heavy chains are amidated. Deletion mutant forms with deletions at the carboxyl terminus have been shown to affect antigen binding affinity and effector functions. The present invention is not limited to the above-mentioned variants, as long as the function is preserved. The two heavy chains constituting the antibody are one selected from the group consisting of a full-length heavy chain and the deletion mutants described above. or two types in combination selected therefrom The ratio of the amounts of each deletion mutant may be adjusted depending on the amount of the cultured mammal that produces the antibody of the present invention. Although it may be affected by mammalian cell type and culture conditions, one of the carboxyl termini The two heavy chains of the antibody of the present invention contain the amino acid residues An example is when both are deleted.

[0193] The isotype of the antibody of the present invention may be, for example, IgG (IgG1, IgG2, IgG3 Examples of the antibody include IgG1 and IgG2. It is possible to do this.

[0194] The biological activity of antibodies generally includes antigen-binding activity, which expresses the antigen by binding to the antigen. The activity of internalizing the antigen in cells, the activity of neutralizing the activity of the antigen, the activity of enhancing the activity of the antigen, Antibody-dependent cytotoxicity (ADCC) activity, complement-dependent cytotoxicity (CDC) activity and antibody-dependent Examples of the functions of the antibodies of the present invention include: Binding activity to TROP2, preferably TROP2-expressing cells by binding to TROP2 Furthermore, the antibody of the present invention has ADCC activity in addition to the cell-internalizing activity. The antibody may have CDC activity and / or ADCP activity.

[0195] The resulting antibodies may be purified to homogeneity. Antibody isolation and purification can be performed using conventional techniques. Protein separation and purification may be performed using methods such as column chromatography. -, filtration, ultrafiltration, salt precipitation, dialysis, preparative polyacrylamide gel electrophoresis By appropriately selecting and combining chromatographic techniques, isoelectric focusing, and the like, antibodies can be Protein Peptides can be isolated and purified (Strategies for Protein Peptides urification and Characterization:A Labor atory Course Manual, edited by Daniel R. Marshak et al., C old Spring Harbor Laboratory Press (1996) );Antibodies:A Laboratory Manual.Ed Harl ow and David Lane, Cold Spring Harbor Lab oratory (1988), but the methods are not limited thereto.

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

[0197] Such chromatography is performed using a liquid chromatograph such as HPLC or FPLC. This can be done using a

[0198] The columns used in affinity chromatography are protein A columns and protein B columns. For example, a column using a protein G column can be used. Hyper D, POROS, Sepharose FF (Pharmacia) and the like.

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

[0200] anti-cancer compounds In this section, anti-TROP2 Anti-tumor compounds conjugated to antibodies are described.

[0201] The antitumor compound used in the present invention has an antitumor effect and a substituent or linker. The compound having a partial structure that allows linkage to the structure is not particularly limited. If a part or all of the linker is cleaved in the tumor cell, the antitumor compound portion is released. The linker is cleaved at the site of attachment to the drug, releasing the compound's antitumor activity. Upon administration, the antitumor compound is released in its unmodified form to exert its intrinsic antitumor effect. .

[0202] The antitumor compound used in the present invention is one of camptothecin derivatives. Exatecan (shown in the formula below (1S,9S)-1-amino-9-ethyl-5- Fluoro-2,3-dihydro-9-hydroxy-4-methyl-1H,12H-benzo[d e]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-10,13( 9H,15H)-dione) can be preferably used. Exatecan is represented by the following formula 1: Shown below.

[0203] [ka]

[0204] Although exatecan has excellent antitumor effects, it has not been commercialized as an antitumor drug. The compound can be easily obtained by known methods, and preferably has an amino group at the 1-position. The alkyl group can be used as a linking point to the linker structure. It is also possible to release exatecan within tumor cells while the portion remains attached. However, even with such a structure, it remains an excellent anticancer compound that exhibits excellent antitumor activity. be.

[0205] Since exatecan has a camptothecin structure, the equilibrium is in an aqueous acidic medium (e.g., p In aqueous basic media, the structure with a closed lactone ring (closed ring) is transformed into (e.g., pH 10) to move to a structure with an open lactone ring (open ring). It is known that when the exatecan residue corresponding to the closed ring structure is introduced into the drug conjugate, In this case, the open ring structure is expected to have the same antitumor effect, and both of these situations are within the scope of the present invention. is within the range.

[0206] Other examples of antitumor compounds include doxorubicin, daunorubicin, and mitomycin C. , bleomycin, cyclocytidine, 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) anti-cancer drugs).

[0207] For antibody-drug conjugates, the number of drug molecules conjugated per antibody molecule affects efficacy and This is a major factor affecting safety. The production of antibody-drug conjugates requires the production of a certain number of conjugated drug molecules. Clarify the reaction conditions, including the amounts of raw materials and reagents used, so that the reaction has a The antibody-drug conjugates are generally different from the chemical reactions of small molecules. The resulting mixture contains a number of conjugated drug molecules. The number of entities is expressed or specified as an average, i.e., the average number of conjugated drug molecules. Unless specifically stated as a principle, the number of conjugated drug molecules may vary depending on the number of conjugates. The antibody-drug conjugate mixture having a specific number of conjugated drug molecules is The average value is used except when referring to an antibody-drug conjugate containing exonuclease conjugated to an antibody molecule. The number of satecan molecules can be controlled, with the average number of conjugated drug molecules per antibody being: Approximately 1 to 10 exatecans can be linked. In some embodiments, 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 exatecans can be linked together Preferably, it is 2 to 8, more preferably 3 to 8, more preferably On the other hand, a person skilled in the art would be able to determine the following based on the description of the examples of the present application: Reactions for conjugating the required number of drug molecules to an antibody molecule can be designed and controlled. It is possible to obtain an antibody drug conjugate with a limited number of exatecan molecules.

[0208] Linker Structure Regarding the anti-TROP2 antibody-drug conjugate of the present invention, an antitumor compound is conjugated to an anti-TROP2 antibody. The linker structure for conjugation is described below: The linker has the 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 L 1 The end of (L 2 the end opposite to the linkage to the antitumor The compound is -L a -(CH2)n 2 It is linked to the carbonyl group of the -C(=O)- moiety.

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

[0211] L 1

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

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

[0214] [ka]

[0215] Position 3 of the above partial structure is the linking position to the anti-TROP2 antibody. The binding to the P2 antibody is characterized by thioether formation. The nitrogen atom at position 1 of the moiety is connected to the carbon atom of a methylene present in a linker containing the structure. Specifically, -(Succinimid-3-yl-N)-(CH2)n 3 -C(=O)-L 2 - is represented by the following formula (in the present specification, "antibody-S-" refers to an antibody-derived This is a structure expressed as follows:

[0216] [ka]

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

[0218] L 1 Specific examples include: -(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)- Examples include:

[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, in which case L 2 is a single bond. n 4 is an integer of 1 to 6, preferably 2 to 4. 2 is L at its terminal amino group. 1 In The carbonyl group at the other end of the P is linked to.

[0222] L 2 Specific examples include: -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)- Examples include:

[0223] L P

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

[0225] In the linker, L P The amino acids constituting the Examples include L-amino acids or D-amino acids, preferably L-amino acids. And it contains, in addition to α-amino acids, β-alanine, ε-aminocaproic acid or γ- It can be an amino acid having a structure such as N-aminobutyric acid, and further, it can be an amino acid having a structure such as N-aminobutyric acid, It can be an unnatural amino acid, such as a methylated amino acid.

[0226] L P The amino acid sequence of is not particularly limited, but examples of constituent amino acids include: Phenylalanine (Phe; F), tyrosine (Tyr; Y), leucine (Leu; L), Glycine (Gly; G), alanine (Ala; A), valine (Val; V), lysine (L ys;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, These include citrulline, serine, glutamic acid, and aspartic acid. The drug release pattern can be controlled accordingly. It is possible.

[0228] L P Specific examples include: -GGF-, -DGGF-, -(D-)D-GGF-, -EGGF-, -GGFG-, -SGGF-, -KGGF-, -DGGFG-, -GGFGG-, -DDGGFG-, -KDGGFG-, -GGFGGGF- Examples include:

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

[0230] L for the antibody-drug conjugate of the present invention P A particularly preferred example is the tetracycline -GGFG-. Examples include peptide residues.

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

[0232] L a -(CH2)n 2 L in -C(=O)- a is the structure of -O- or a single bond. n 2 is an integer of 0 to 5, more preferably 0 to 3, and 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)- Examples of the compound include those having the following structure:

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

[0235] -NH-(CH2)n in the linker 1 -L a -(CH2)n 2 -C(=O)- Specific examples of the structures represented include: -NH-CH2-C(=O)-, -NH-CH2CH2-C(=O)-, -NH-CH2-O-CH2-C(=O)-, -NH-CH2CH2-OC(=O)-, -NH-CH2CH2-O-CH2-C(=O)-, -NH-CH2CH2CH2-C(=O)-, -NH-CH2CH2CH2CH2-C(=O)-, -NH-CH2CH2CH2CH2CH2-C(=O)- The following can be mentioned: -NH-CH2CH2CH2-C(=O)-, -NH-CH2-O-CH2-C(=O) - or -NH-CH2CH2-OC(=O)- is preferred.

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

[0237] Regarding the anti-TROP2 antibody-drug conjugate of the present invention, When transferred to the interior of the cell, the linker is cleaved, and NH2-(CH2)n 1 -L a -(CH2)n 2 -C(=O)-(NH-DX) It is believed that the conductor is released from the antibody-drug conjugate of the present invention, thereby exerting an antitumor effect. Examples of antitumor derivatives that exhibit antitumor effects by releasing -NH-(CH 2)n 1 -L a -(CH2)n 2 The structure represented by -C(=O)- has a terminal amino group Examples of antitumor derivatives include those having the structural moiety, and particularly preferred ones include the following: It can be obtained. 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 minar structure is unstable, it will undergo autolysis again to form the following HO-CH2-C(=O)-(NH-DX) Preferably, these compounds are used in the antibody-drug conjugates of the present invention. It can also be used as a production intermediate.

[0239] For the antibody-drug conjugate of the present invention in which exatecan is used as the drug, the following structure is a drug-linker structural moiety [-L 1 -L 2 -L P -NH-(CH2)n 1 -L a -( CH2)n 2 -C(=O)-(NH-DX)] is preferably linked to the antibody. The average number of the drug-linker structural moieties conjugated per antibody is 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, more preferably 3.5 to 4.5, is 4. -(Succinimid-3-yl-N)-CH2CH2-C(=O)-GGFG-N H-CH2CH2-C(=O)-(NH-DX), -(Succinimid-3-yl-N)-CH2CH2-C(=O)-GGFG-N H-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-CH2 CH2-O-CH2CH2-O-CH2CH2-C(=O)-GGFG-NH-CH2C H2-C(=O)-(NH-DX), -(Succinimid-3-yl-N)-CH2CH2-C(=O)-NH-CH2 CH2-O-CH2CH2-O-CH2CH2-C(=O)-GGFG-NH-CH2C H2CH2-C(=O)-(NH-DX), -(Succinimid-3-yl-N)-CH2CH2-C(=O)-NH-CH2 CH2-O-CH2CH2-O-CH2CH2-O-CH2CH2-O-CH2CH2- C(=O)-GGFG-NH-CH2CH2-C(=O)-(NH-DX), -(Succinimid-3-yl-N)-CH2CH2-C(=O)-NH-CH2 CH2-O-CH2CH2-O-CH2CH2-O-CH2CH2-O-CH2CH2- C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-(NH-DX)

[0240] Among them, the following are more preferred: -(Succinimid-3-yl-N)-CH2CH2-C(=O)-GGFG-N H-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-CH2 CH2-O-CH2CH2-O-CH2CH2-C(=O)-GGFG-NH-CH2C H2CH2-C(=O)-(NH-DX), -(N-Succinimid-3-yl)-CH2CH2-C(=O)-NH-CH2 CH2-O-CH2CH2-O-CH2CH2-O-CH2CH2-O-CH2CH2- C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-(NH-DX)

[0241] Particularly preferred are the following. -(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-CH2 CH2-O-CH2CH2-O-CH2CH2-C(=O)-GGFG-NH-CH2C H2CH2-C(=O)-(NH-DX)

[0242] In the antibody-drug conjugate of the present invention, a phosphorus for conjugating the anti-TROP2 antibody and the drug Regarding the linker structure, preferred linkers are shown for each of the linker moieties described above. The linker structure can be constructed by linking the preferred structures. The following structure can be used: The right end is the linking position to the drug. -(Succinimid-3-yl-N)-CH2CH2-C(=O)-GGFG-N H-CH2CH2-C(=O)-, -(Succinimid-3-yl-N)-CH2CH2-C(=O)-GGFG-N H-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)-, -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(= O)-GGFG-NH-CH2CH2CH2CH2CH2-C(=O)-, -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(= O)-GGFG-NH-CH2-O-CH2-C(=O)-, -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(= O)-GGFG-NH-CH2CH2-O-CH2-C(=O)-, -(Succinimid-3-yl-N)-CH2CH2-C(=O)-NH-CH2 CH2-O-CH2CH2-O-CH2CH2-C(=O)-GGFG-NH-CH2C 6 H2-C(=O)-、 7 -(Succinimid-3-yl-N)-CH2CH2-C(=O)-NH-CH2 6 CH2-O-CH2CH2-O-CH2CH2-C(=O)-GGFG-NH-CH2C 7 H2CH2-C(=O)-、 7 -(Succinimid-3-yl-N)-CH2CH2-C(=O)-NH-CH2 6 CH2-O-CH2CH2-O-CH2CH2-O-CH2CH2-O-CH2CH2- 7 C(=O)-GGFG-NH-CH2CH2-C(=O)-、 1 -(Succinimid-3-yl-N)-CH2CH2-C(=O)-NH-CH2 2 CH2-O-CH2CH2-O-CH2CH2-O-CH2CH2-O-CH2CH2- P C(=O)-GGFG-NH-CH2CH2CH2-C(=O)- 1 a 2 Among them, the more preferred ones are as follows. 1 -(Succinimid-3-yl-N)-CH2CH2-C(=O)-GGFG-N 2 H-CH2CH2CH2-C(=O)-、 a -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(= 2 O)-GGFG-NH-CH2CH2CH2-C(=O)-、 1 -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(= 1 O)-GGFG-NH-CH2-O-CH2-C(=O)-、 1 -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(= 3 O)-GGFG-NH-CH2CH2-O-CH2-C(=O)-、 3 -(Succinimid-3-yl-N)-CH2CH2-C(=O)-NH-CH2 3CH2-O-CH2CH2-O-CH2CH2-C(=O)-GGFG-NH-CH2C H2CH2-C(=O)-, -(Succinimid-3-yl-N)-CH2CH2-C(=O)-NH-CH2 CH2-O-CH2CH2-O-CH2CH2-O-CH2CH2-O-CH2CH2- C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-

[0244] Particularly preferred are the following: -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(= O)-GGFG-NH-CH2-O-CH2-C(=O)-, -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(= O)-GGFG-NH-CH2CH2-O-CH2-C(=O)-, -(Succinimid-3-yl-N)-CH2CH2-C(=O)-NH-CH2 CH2O-CH2CH2O-CH2CH2-C(=O)-GGFG-NH-CH2CH2 CH2-C(=O)-

[0245] Regarding the anti-TROP2 antibody-drug conjugate used in the present invention, When the linker moiety is transferred to the hydroxyl group, the linker moiety is cleaved to form a hydroxyl group having the formula: NH2-CH2-O-CH2-C(=O)-(NH-DX) A drug derivative having a structure represented by the formula: If the aminal structure in the molecule of the drug derivative is unstable, it will undergo autolysis again, It was confirmed that the compound represented by the formula: HO-CH2-C(=O)-(NH-DX) was released. was done.

[0246] The compound has the following formula:

[0247] [ka]

[0248] (hereinafter also referred to as "Compound 1" in the present invention).

[0249] Compound 1 is the main pharmaceutical agent responsible for the antitumor activity of the antibody-drug conjugate used in the present invention. It is believed to be a pharmacologically active substance and has been confirmed to have topoisomerase I inhibitory effects. (Ogitani Y. et al., Clinical Cancer Research, 20 October 15, 2016;22(20):5097~5108, Epub March 2016 9th).

[0250] Generation method Next, representative methods for producing the antibody-drug conjugate of the present invention or its intermediates will be described. On the other hand, the compounds are described below in the present specification as being those represented by the respective chemical reaction formulas. They are listed together with the compound number. Specifically, they are "compound of formula (1)", "compound ( 1)" or the like. Compounds with other numbers are similarly described. .

[0251] Generation method A

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

[0253] [ka]

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

[0255] [ka]

[0256] In the above formula, the nitrogen atom is a linking point, and specifically, L 1 No-(Succini mid-3-yl-N)-(CH2)n 3 -C(=O)- in Succinim The -(NH-DX)- group represents a group in which the -(NH-DX)- group is a maleimidyl group. ) is expressed as:

[0257] [ka]

[0258] It represents a structure represented by the formula: represents a group derived by removing an atom.

[0259] Furthermore, the compound of formula (1) in the above chemical reaction formula has a phase structure from the drug to the linker terminal. It is understood that one corresponding structural part is linked to one antibody. This description is provided for convenience only; in reality, multiple structural moieties may be linked to one antibody molecule. The same applies to the generation method described below.

[0260] The antibody-drug conjugate (1) can be prepared by reacting compound (2), which can be obtained by the method described below, with It can be generated by reacting with an antibody (3a) that has a sulfhydryl group.

[0261] The antibody (3a) having sulfhydryl groups can be prepared by methods well known in the art (H ermanson, GT, Bioconjugate Techniques, 56~ 136, pp. 456-493, Academic Press (1996) Examples include: reacting Traut's reagent with the amino groups of the antibody; Succinimidyl S-acetylthioalkanoate was reacted with the amino groups of the antibody, followed by Reaction with hydroxylamine; N-succinimidyl 3-(pyridyldithio)propionate After reaction with thionate, the antibody is reacted with a reducing agent; Dicaptoethanol and tris(2-carboxyethyl)phosphine hydrochloride (TCEP ) to reduce disulfide bonds within the antibody to sulfhydryl groups. Examples include, but are not limited to, forming

[0262] Specifically, 0.3 to 3 molar equivalents of reducing agent per disulfide in the antibody. TCEP is used to react with the antibody in a buffer solution containing a chelating agent, and Antibodies can also be obtained with partially or fully reduced disulfides. Examples of anti-inflammatory agents include ethylenediaminetetraacetic acid (EDTA) and diethylenetriaminepentaacetic acid (DTA). acetic acid (DTPA). It can be used at a concentration of 1 mM to 20 mM. Examples of buffer solutions that can be used include sodium phosphate, sodium borate, or acetic acid. Specifically, the antibody is incubated at 4°C to 37°C for 1 to 4 hours in a solution of T Partially or completely reduced sulfhydryl groups can be converted to hydroxybenzoates by reaction with CEP. Antibody (3a) having the above structure can be obtained.

[0263] Alternatively, a reaction can be carried out to add a sulfhydryl group to the drug-linker moiety. Thus, the drug-linker moiety can be conjugated via a thioether bond.

[0264] 2 to 20 molar equivalents of compound (2) are added to one sulfhydryl-containing antibody (3a). This method is used to generate antibody-drug conjugates (1) in which 2 to 8 drug molecules are conjugated to one antibody. Specifically, a solution containing the compound (2) dissolved therein can be used for the reaction. It is sufficient to add the antibody (3a) containing sulfhydryl groups for the purpose of Examples of buffer solutions that can be used herein include sodium acetate solutions. The pH for the reaction is: The pH of the reaction is preferably 5 to 9, and more preferably, the reaction is carried out at about pH 7. Examples of solvents for dissolving include dimethyl sulfoxide (DMSO), dimethylform amide (DMF), dimethylacetamide (DMA) and N-methyl-2-pyridone ( Examples of suitable organic solvents include NMP.

[0265] The organic solvent solution containing compound (2) dissolved therein is treated with a sulfhydryl compound for the reaction. It is sufficient to add 1 to 20% v / v to a buffer solution containing an antibody (3a) having an alkyl group. The reaction temperature is 0 to 37°C, preferably 10 to 25°C, and the reaction time is 0.5 The reaction time is about 2 hours. This is to deactivate the reactivity of unreacted compound (2) with the thiol-containing reagent. The reaction can be terminated by adding thiol-containing reagents such as cysteine, methyl ... These include N-acetyl-L-cysteine ​​and N-acetyl-L-cysteine ​​(NAC). Add 1 to 2 molar equivalents of NAC to the compound (2) to be used and incubate at room temperature for 10 to 30 minutes. The reaction can be terminated by bating.

[0266] The resulting antibody-drug conjugate (1) was concentrated, buffer exchanged, purified, and then purified as described below. The antibody concentration and the average number of drug molecules conjugated per antibody molecule were determined by a common method Measurement and identification of the antibody-drug conjugate (1) can be performed.

[0267] General Method A: Concentration of aqueous solutions of antibodies or antibody-drug conjugates

[0268] Amicon Ultra (50,000MWCO, Millipore Corp. Add the antibody or antibody-drug conjugate solution to the centrifuge (Alleg ra X-15R, Beckman Coulter, Inc.) and centrifugation ( Antibody or antibody-drug conjugate by centrifugation at 2000G to 3800G for 5 to 20 minutes The solution was concentrated.

[0269] General Method B: Measurement of antibody concentration

[0270] UV detector (Nanodrop1000, Thermo Fisher Scientific Antibody concentrations were measured using a ELISA kit (Diagnostic Inc.) according to the method specified by the manufacturer. At that time, a different 280 nm absorption coefficient (1.3 mL mg) was measured for each antibody. -1 c m -1 ~1.8mLmg -1 cm -1 ) was used.

[0271] General Method C-1: Buffer Exchange for Antibodies

[0272] NAP-25 column (Cat. No. 17-) using Sephadex G-25 carrier 0852-02, GE Healthcare Japan Corporation) The solution was diluted with sodium chloride (137 mM) and ethanol according to the method specified by the manufacturer. Phosphate buffer (10 mM, pH 6.0) containing ethylenediaminetetraacetic acid (EDTA, 5 mM) (which is referred to herein as PBS 6.0 / EDTA). The solution was applied to a single NAP-25 column in a volume of 2.5 mL, followed by 3.5 mL of PBS. The fractions (3.5 mL) eluted with 6.0 / EDTA were collected. The antibody was concentrated by method A. The antibody concentration was measured using general method B, and then diluted with PBS 6.0 / The antibody concentration was adjusted to 10 mg / mL with EDTA.

[0273] General Method C-2: Buffer Exchange for Antibodies

[0274] NAP-25 column (Cat. No. 17-) using Sephadex G-25 carrier 0852-02, GE Healthcare Japan Corporation) was diluted with sodium chloride (50 mM) and EDTA according to the method specified by the manufacturer. Phosphate buffer (50 mM, pH 6.5; herein referred to as P The antibody solution was equilibrated with 2.5 mL of PBS (referred to as BS6.5 / EDTA). Apply to a NAP-25 column and then elute with 3.5 mL of PBS 6.5 / EDTA. The fractions (3.5 mL) were collected and concentrated by General Method A. After measuring the antibody concentration using method B, the antibody concentration was measured using PBS6.5 / EDTA. The concentration was adjusted to 20 mg / mL.

[0275] General Method D: Purification of Antibody-Drug Conjugates

[0276] The NAP-25 column was filled with commercially available phosphate buffer (PBS 7.4, Cat. N o.10010-023, Invitrogen), sodium chloride (137 mM) sodium phosphate buffer (10 mM, pH 6.0; it is called PBS6.0), and acetate buffer (10 mM, pH 5.5; as used herein) containing sorbitol (5%). The antibody-drug conjugate was equilibrated with any buffer selected from the group consisting of: The aqueous solution of the reaction was applied to a NAP-25 column in a volume of approximately 1.5 mL, and then eluted with 10 mL of ethanol as per the manufacturer's instructions. The antibody fraction was collected by elution with a specified amount of buffer. The gel filtration purification process was repeated on the 25 column for elution with buffer for a total of 2 By repeating three times, the unconjugated drug linker and small molecule compound (tris(2-carboxymethyl)-2-pyridinyl) carboxyethyl)phosphine hydrochloride (TCEP), N-acetyl-L-cysteine ​​(NA C) and dimethyl sulfoxide) were excluded to obtain antibody-drug conjugates.

[0277] General Method E: Measurement of antibody concentration in antibody-drug conjugates and single antibody molecules (1) Average number of conjugated drug molecules per

[0278] The UV absorbance of the antibody-drug conjugate solution was measured at two wavelengths: 280 nm and 370 nm. The conjugated drug concentration in the antibody-drug conjugate was calculated by determining the It is possible to perform calculations that

[0279] The total absorbance at any wavelength is equal to the sum of the absorbances of all light-absorbing species present in the system. (additivity of absorbance), the molar absorption coefficients of the antibody and drug are If they are unchanged before and after, the antibody concentration and drug concentration in the antibody-drug conjugate are calculated using the following formula: It is expressed as: A 280 =A D,280 +A A,280 =ε D,280 C D +ε A,280 C A formula( I) A 370 =A D,370 +A A,370 =ε D,370 C D +ε A,370 C A formula( II)

[0280] In the above, A 280 represents the absorbance of an aqueous solution of the antibody-drug conjugate at 280 nm. And A 370 represents the absorbance of an aqueous solution of the antibody-drug conjugate at 370 nm, and A A,2 80 represents the absorbance of the antibody at 280 nm, and A A,370 is at 370 nm represents the absorbance of the antibody, A D,280 represents the absorbance of the complex precursor at 280 nm , A D,370 represents the absorbance of the complex precursor at 370 nm, and ε A,280 teeth, represents the molar absorption coefficient of the antibody at 280 nm, ε A,370 is the anti- represents the molar absorption coefficient of the D,280 is the molar absorption of the complex precursor at 280 nm represents the coefficient, and ε D,370 represents the molar absorption coefficient of the complex precursor at 370 nm, C A represents the antibody concentration in the antibody-drug conjugate, and C D is the amount of Represents drug concentration.

[0281] In the above, ε A,280 , ε A,370 , ε D,280 and ε D,370 Regarding , based on pre-prepared values ​​(calculated values ​​obtained by UV measurement of compounds or measured values) For example, ε is calculated from the amino acid sequence of an antibody using a known calculation method. A, 280 can be estimated (Protein Science, 1995, Vol. 4) , 2411~2423). ε A,370 is generally zero. By measuring the absorbance of a solution in which a certain molar concentration of Based on the law (absorbance = molar concentration × molar absorption coefficient × cell path length), ε D,280 and ε D,370 The A of the aqueous solution of the antibody-drug conjugate can be obtained. 280 and A 370 of and using the measured values ​​to solve the coupled equations (I) and (II), C A Oh and C D Furthermore, C D C A By dividing by (diving) The average number of drugs conjugated per antibody can be obtained.

[0282] General Method F: Average number of drug molecules conjugated per antibody molecule within an antibody-drug conjugate Measurement of numbers - (2).

[0283] In addition to the general procedure E described above, the following methods can be used to identify the nucleotide sequence within an antibody-drug conjugate: The average number of drug molecules conjugated per antibody molecule was determined by high performance liquid chromatography (HPLC). It can also be determined by LC analysis.

[0284] [F-1. Sample Preparation for HPLC Analysis (Reduction of Antibody-Drug Conjugate)]

[0285] Dissolve the antibody-drug conjugate solution (approximately 1 mg / mL, 60 μL) in dithiothreitol (DTT) water. The mixture was then mixed with 100 mM NaCl solution (15 μL). The mixture was then incubated at 37°C for 30 minutes. The disulfide bond between the L chain and H chain of the antibody-drug conjugate is cleaved. is used in the HPLC analysis.

[0286] [F-2.HPLC analysis]

[0287] HPLC analysis is performed under the following measurement conditions.

[0288] HPLC system: Agilent 1290 HPLC system (Agilent Te Technologies, Inc.

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

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

[0291] Column temperature: 80℃

[0292] Mobile phase A: 0.04% trifluoroacetic acid (TFA) in water

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

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

[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 unconjugated antibody, L chain (L chain linked to one drug molecule: L1) and H chain (H chain linked to one drug molecule: L2) H chain linked to two drug molecules: H1; H chain linked to three drug molecules: H2; The heavy chain (H3) exhibits higher hydrophobicity in proportion to the number of conjugated drug molecules and therefore Therefore, these chains are divided into L0 and L1, or H0, H1, H2 and The peaks detected are identified by comparing the retention times with L0 and H0. It can be assigned to any of L0, L1, H0, H1, H2 and H3.

[0298] [F-3-2] Because the drug linker has UV absorption, the peak area values ​​are and the molar absorption coefficient of the drug linker, the conjugated drug linker molecule is calculated according to the following equation: is corrected according to the number of

[0299]

number

[0300]

number

[0301] Here, the molar extinction coefficient (280 nm) of each antibody's L chain or H chain is calculated using known methods. Extraction method (Protein Science, 1995, Vol. 4, 2411-2423) The value can be estimated from the amino acid sequence of the L chain or H chain of each antibody by In the case of hTINA, the molar extinction coefficient (molar) is 34690 according to its amino acid sequence. r extinction coefficient) and a molar extinction coefficient of 95,000 The molar extinction coefficients of the drug linkers ( 280 nm), mercaptoethanol or N-acetyl cis- Compounds in which the maleimide group has been converted to a succinimide thioether by reaction with thiol The measured molar extinction coefficient (280 nm) was used.

[0302] [F-3-3] The peak area of ​​each chain is calculated based on the sum of the corrected peak areas according to the following formula: Calculate the area ratio (%).

[0303]

number

[0304] [F-3-4] Average number of drug molecules conjugated per antibody molecule in antibody-drug conjugates is calculated according to the following formula:

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

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

[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 is -NH-(CH2CH2-O)n 4 -CH2CH2-C(=O)- or single bond In the formula, n 4 represents an integer from 1 to 6, L P It contains phenylalanine, glycine, valine, lysine, citrulline, serine, and glutamic acid. a peptide consisting of 2 to 7 amino acid residues selected from cis- and aspartic acid; Represents, n 1 represents an integer from 0 to 6, n 2 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),

[0309] [ka] (wherein the nitrogen atom is the linking point)

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

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

[0312] L 2 is a single bond or -NH-(CH2CH2-O)n 4 -CH2CH2-C(=O)- If n 4 Compounds in which is an integer of 2 to 4 are preferred as production intermediates.

[0313] L P Regarding the peptide residues, phenylalanine, glycine, valine, lysine, citric acid A peptide containing an amino acid selected from phosphorus, serine, glutamic acid, and aspartic acid. Compounds having a peptide residue are preferred as intermediates. P A compound in which the residue is a peptide consisting of four amino acids is preferred as a production intermediate. Specifically, L P A compound in which the tetrapeptide residue of -GGFG- is present is preferred as a production intermediate. I wish.

[0314] Furthermore, -NH-(CH2)n 1 -L a -(CH2)n 2 - with respect to -NH-CH2 CH2-, -NH-CH2CH2CH2-, -NH-CH2CH2CH2CH2-, -N H-CH2CH2CH2CH2CH2-, -NH-CH2-O-CH2-, or -NH A compound having -CH2CH2-O-CH2- is preferred as a production intermediate. H2CH2CH2-, -NH-CH2-O-CH2-, or -NH-CH2CH2-O Compounds with -CH2 are more preferred.

[0315] Furthermore, in the compound represented by formula (2), n 3 is an integer between 2 and 5, and L 2is one-line where -NH-(CH2)n 1 -L a -(CH2)n 2 -NH-CH2CH2- , -NH-CH2CH2CH2-, -NH-CH2CH2CH2CH2-, -NH-CH 2CH2CH2CH2CH2-, -NH-CH2-O-CH2-, or -NH-CH2 The compound CH2-O-CH2- is preferred as a production intermediate. 1 -L a -(CH2)n 2 - is -NH-CH2CH2-, -NH-CH2CH2CH2- , -NH-CH2-O-CH2-, or -NH-CH2CH2-O-CH2- A mixture is more preferred. 3 Compounds in which is an integer of 2 or 5 are more preferred.

[0316] Furthermore, in the compound represented by formula (2), n 3 is an integer between 2 and 5, and L 2 Ga-N H-(CH2CH2-O)n 4 -CH2CH2-C(=O)-, n 4 is 2 to 4 is the number, -NH-(CH2)n 1 -L a -(CH2)n 2 -NH-CH2CH2- , -NH-CH2CH2CH2-, -NH-CH2CH2CH2CH2-, -NH-CH 2CH2CH2CH2CH2-, -NH-CH2-O-CH2-, or -NH-CH2 The compound CH2-O-CH2- is preferred as a production intermediate. 4 integers of 2 or 4 More preferred are compounds in which the number is —NH—(CH2)n. 1 -L a -(CH2)n 2 -teeth- NH-CH2CH2CH2-, -NH-CH2-O-CH2-, or -NH-CH2C More preferred is the compound H2-O-CH2-.

[0317] Preferred such intermediates useful in producing the compounds of the present invention include: Examples can be given. (Maleimid-N-yl)-CH2CH2-C(=O)-GGFG-NH-CH2 CH2-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)-GG FG-NH-CH2CH2-C(=O)-(NH-DX), (Maleimid-N-yl)-CH2CH2-C(=O)-GGFG-NH-CH2 CH2CH2-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)-GG FG-NH-CH2CH2CH2-C(=O)-(NH-DX), (Maleimid-N-yl)-CH2CH2-C(=O)-GGFG-NH-CH2 CH2CH2CH2-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)-GG FG-NH-CH2CH2CH2CH2-C(=O)-(NH-DX)、 (Maleimid-N-yl)-CH2CH2-C(=O)-GGFG-NH-CH2 CH2CH2CH2CH2-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)-GG FG-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)-GG FG-NH-CH2-O-CH2-C(=O)-(NH-DX), (Maleimid-N-yl)-CH2CH2-C(=O)-GGFG-NH-CH2 CH2-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)-GG FG-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] A drug-linker compound selected from the above group of intermediate compounds is used to produce an anti-TROP2 antibody or or its reactive derivative to bind to the disulfide bond present in the anti-TROP2 antibody. By forming a thioether bond with the In this case, a reactive derivative of an anti-TROP2 antibody is preferably used. In particular, reactive derivatives obtained by reducing anti-TROP2 antibodies are preferred. .

[0319] The following are more preferred compounds as production intermediates: (Maleimid-N-yl)-CH2CH2-C(=O)-GGFG-NH-CH2 CH2-C(=O)-(NH-DX), (Maleimid-N-yl)-CH2CH2-C(=O)-GGFG-NH-CH2 CH2CH2-C(=O)-(NH-DX), (Maleimid-N-yl)-CH2CH2CH2CH2CH2-C(=O)-GG FG-NH-CH2CH2-C(=O)-(NH-DX), (Maleimid-N-yl)-CH2CH2CH2CH2CH2-C(=O)-GG FG-NH-CH2CH2CH2-C(=O)-(NH-DX), (Maleimid-N-yl)-CH2CH2CH2CH2CH2-C(=O)-GG FG-NH-CH2CH2CH2CH2CH2-C(=O)-(NH-DX), (Maleimid-N-yl)-CH2CH2CH2CH2CH2-C(=O)-GG FG-NH-CH2-O-CH2-C(=O)-(NH-DX), (Maleimid-N-yl)-CH2CH2CH2CH2CH2-C(=O)-GG FG-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 group of intermediate compounds, compounds of the 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)-GG FG-NH-CH2-O-CH2-C(=O)-(NH-DX), or (Maleimid-N-yl)-CH2CH2CH2CH2CH2-C(=O)-GG Compound represented by FG-NH-CH2CH2-O-CH2-C(=O)-(NH-DX) is a more preferred compound.

[0321] To ensure the amount of the conjugate is equivalent (e.g., approximately ±1) to the number of drugs. Multiple conjugates obtained under similar production conditions can be mixed to prepare new lots. In this case, the average number of drugs is set to be equal to or smaller than the average number of drugs in the complex before the mixing. be.

[0322] Generation method 2

[0323] The compound of formula (2) and its pharmacological properties are obtained as intermediates in the above-mentioned production method. Acceptable salts can be prepared, for example, by the following method.

[0324] [ka]

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

[0326] Compound (6) can be prepared by converting carboxylic acid (5) into an active ester, a mixed acid anhydride, an acid halide, or or a similar compound, which is then derivatized in the presence of a base to form NH2-DX(4) or its pharmacologically active compound. NH2-DX(4) can be produced by reacting it with a biologically acceptable salt. Xatecan (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)- represents Zeon.

[0327] The reaction reagents and reaction conditions generally used for peptide synthesis were used for the reaction. There are various types of activated esters. For example, it is N,N'-dicyclohexane. xylcarbodiimide or 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide p-Nitrophenol, N-hydroxybenzotriazole, etc. can be obtained by using a condensing agent such as imide hydrochloride. phenols such as azoles, N-hydroxysuccinimides or the like, The activated ester can be produced by reacting it with an acid (5). Reaction of carboxylic acid (5) with pentafluorophenyl trifluoroacetate or the like, carboxylic acid (5) and 1-benzotriazolyloxytripyrrolidinophosphonium hexafluoride Reaction of carboxylic acid (5) with diethyl cyanophosphonate (chloride) method), carboxylic acid (5) with triphenylphosphine and 2,2'-dipyridyldisulfide Reaction of carboxylic acid (5) with sulfide (Mukaiyama's method) and 4-(4,6-dicarboxylic acid Methoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride ( It can also be produced by reaction with triazine derivatives such as DMTMM) or the like. Furthermore, the reaction can be carried out, for example, by reacting carboxylic acid (5) with thionyl chloride and It can also be carried out by the acid halide method, which involves treating with an acid halide such as oxalyl chloride. .

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

[0329] Specific examples of the base used for each of the above steps include sodium carbonate, calcium carbonate, Sodium, sodium ethoxide, potassium butoxide, sodium hydroxide, potassium hydroxide of alkali or alkaline earth metals, including ammonium, sodium hydride and potassium hydride carbonates, alkoxides, hydroxides or hydrides, n-butyllithium, lithium Representative alkyllithiums include dialkylaminolithiums including mu-diisopropylamide. Organometallic bases, including bissilylamines, including lithium bis(trimethylsilyl)amide and tertiary amines or nitrogen-containing heterocyclic compounds, such as pyridine, 2,6-lutidine, collidine, 4-dimethylaminopyridine, triethylamine, N-methyl ethylmorpholine, diisopropylethylamine and diazabicyclo[5.4.0]un Organic bases including dec-7-ene (DBU) may be mentioned.

[0330] Examples of the inert solvent used for the reaction of the present invention include dichloromethane, chloroform, halogenated hydrocarbon solvents such as fluorine and carbon tetrachloride, tetrahydrofuran, 1,2-dichloromethane, Ether solvents such as methoxyethane and dioxane, aromatic solvents such as benzene and toluene Aromatic hydrocarbon solvents, as well as N,N-dimethylformamide, N,N-dimethylacetone Other examples include amide solvents such as methylpyrrolidin-2-one and N-methylpyrrolidin-2-one. In addition, sulfoxide solvents such as dimethyl sulfoxide and sulfolane may also be used. Ketone solvents such as acetone and methyl ethyl ketone, as well as methanol and ethanol Alcoholic solvents such as alcohol may also be used. Furthermore, these solvents may be mixed for use. Good too.

[0331] Protecting group P for the terminal amino group of compound (6) 1 Regarding peptide synthesis, Protective groups for amino groups used in the synthesis of hydroxybenzoates, such as tert-butyloxycarbonyl groups, Using 9-fluorenylmethyloxycarbonyl and benzyloxycarbonyl groups Other examples of protecting groups for amino groups include alkanoyl groups such as acetyl groups. alkoxycarbonyl groups such as alkoxycarbonyl groups, methoxycarbonyl groups, and ethoxycarbonyl groups , para-methoxybenzyloxycarbonyl group and para-(or ortho-)nitrobenzyl Oxycarbonyl group (para (or ortho)nitroybenzyloxy arylmethoxycarbonyl groups such as benzyl groups, and arylmethyl groups such as triphenylmethyl groups, aroyl groups such as benzoyl groups, etc. and 2,4-dinitrobenzenesulfonyl and orthonitrobenzenesulfonyl groups Examples of the protecting group P include arylsulfonyl groups such as 1 For example, The selection can be made depending on the properties of the compound having an amino group.

[0332] A protecting group P for the terminal amino group of the obtained compound (6) 1 By deprotecting Compound (7) can be obtained. For this deprotection, the reagents and You can select the conditions.

[0333] P 2 The peptide carboxylic acid (8) having the N-terminus protected by the active ester, by derivatizing it into a hydrate or similar product and reacting it with the resulting compound (7). Compound (9) can be produced by the reaction of peptide carboxylic acid (8) and compound ( 7) Reaction conditions, reagents, bases and inerts used to form peptide bonds between The solvent can be appropriately selected from those described for the synthesis of compound (6). The protecting group P can be selected from those described for the protecting group of compound (6). 2 Select and use For example, it is possible to select a suitable compound based on the properties of the compound having the amino group to be protected. As commonly used in peptide synthesis, the selection can be made as follows: The reaction and deprotection of the amino acid or peptide constituting the peptide carboxylic acid (8) are carried out successively. Compound (9) can also be produced by repeating the above procedure.

[0334] A protecting group P for the amino group of the obtained compound (9) 2 By deprotecting the compound The deprotection can be carried out using a variety of reagents and conditions depending on the protecting group. You can select the item.

[0335] Carboxylic acid (11) is converted into an activated ester, mixed acid anhydride, acid halide, or the like. and reacting it with the resulting compound (10), thereby obtaining compound (2). It is possible to produce a peptide between carboxylic acid (11) and compound (10). The reaction conditions, reagents, bases and inert solvents used to form the bond are described in connection with compound (6). ) can be appropriately selected and used from those described for the synthesis of

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

[0337] P 2 The peptide carboxylic acid (8) having the N-terminus protected by the active ester, derivatized to a hydrate or the like, which is then reacted with P 3 Protected by Carbo By reacting with an amine compound (12) having an oxy group, compound (13) is produced. The peptide bond between the peptide carboxylic acid (8) and the compound (12) The reaction conditions, reagents, bases and inert solvents used to form compound (6) The synthesis can be appropriately selected from those described and used.

[0338] A protecting group P for the amino group of compound (13) 2 is protected with a commonly used protecting group That's fine.

[0339] Specifically, examples of protecting groups for hydroxyl groups include alkyl groups such as methoxymethyl groups. oxymethyl group, benzyl group, 4-methoxybenzyl group, and triphenylmethyl group arylmethyl groups, alkanoyl groups such as acetyl groups, and aroyl groups such as benzoyl groups and silyl groups such as tert-butyldiphenylsilyl. The groups include, for example, alkyl groups such as methyl, ethyl and tert-butyl groups, aryl groups, or as an ester with an arylmethyl group such as a benzyl group. Examples of protecting groups for amino groups include, for example, tert-butyloxycarbonyl, methyl ... alkyloxycarbonyl groups such as ethoxycarbonyl and alkyloxycarbonyl groups; methyloxycarbonyl group, 9-fluorenylmethyloxycarbonyl group, benzyl oxycarbonyl group, para-methoxybenzyloxycarbonyl group and para (or ol) g) arylmethoxycarbonyl groups such as nitrobenzyloxycarbonyl groups, acetyl alkanoyl groups such as the group, arylmethyl groups such as the benzyl group and triphenylmethyl group aroyl groups such as benzoyl groups, and 2,4-dinitrobenzenesulfonyl groups or an arylsulfonyl group such as an ortho-nitrobenzenesulfonyl group.

[0340] Protecting group P for carboxy group 3 In organic synthetic chemistry, especially peptide synthesis, A protecting group generally used as a protecting group for a carboxy group can be used. Examples include esters with alkyl groups such as methyl, ethyl, or tert-butyl. ester, allyl ester and benzyl ester, and the protecting group is selected from the above protecting groups. In such a case, a protecting group for the amino group and a protecting group for the carboxy group can be appropriately selected. The protecting groups are preferably removed by different methods or under different conditions. It is preferable to be able to do this. For example, a typical example is P 2 tert-butyloxycarbonyl is a carbonyl group, and P 3 is a benzyl group. Depending on the characteristics of the compound having the amino and carboxy groups to be protected, For the removal of the protecting group, the reagent and conditions are selected depending on the protecting group. It is possible.

[0341] A protecting group P for the carboxy group of the resulting compound (13) 3 By deprotecting For this deprotection, the reagents and conditions are the same as for the protection group. The selection is made depending on the protecting group.

[0342] The resulting compound (14) is converted into an active ester, a mixed acid anhydride, an acid halide, or the like. and reacting it with compound (4) in the presence of a base to give compound (9). ) can be produced. For this reaction, The reaction reagents and reaction conditions can also be used, and the reaction conditions used for the reaction The conditions, reagents, bases and inert solvents are selected from those described for the synthesis of compound (6). It is possible to select the appropriate one.

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

[0344] A protecting group P for the amino group of compound (13) 2 By deprotecting the compound (1 5) can be produced. For this deprotection, the reagents and conditions are selected depending on the protecting group. You can choose.

[0345] The carboxylic acid derivative (11) is converted into an active ester, a mixed acid anhydride, an acid halide, or the like. and reacting it with the resulting compound (15) in the presence of a base. Compound (16) can be produced by the reaction of peptide carboxylic acid (11) and The reaction conditions, reagents, bases and and an inert solvent are appropriately selected from those described for the synthesis of compound (6). can be done.

[0346] The protecting group for the carboxy group of the obtained compound (16) is removed to obtain the compound (16). This deprotection can be used to form compound (17). This can be carried out in the same manner as deprotection of the carboxy group.

[0347] Compound (17) is derivatized to an active ester, mixed acid anhydride, acid halide, or the like. and reacting it with compound (4) in the presence of a base to obtain compound (2). For this reaction, a reaction generally used for peptide synthesis can be used. The reaction reagents and reaction conditions can also be used, and the reaction conditions used for the reaction The reagents, bases and inert solvents are selected from those described for the synthesis of compound (6). It is possible to select.

[0348] Generation method 3

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

[0350] [ka]

[0351] In the above formula, L 1’ is converted to a maleimidyl group at the end, and P 4 represents a protecting group L with structure 1 is equivalent to

[0352] Compound (11) is derivatized to an active ester, mixed anhydride, or the like, and then , in the presence of a base, P 4 Reaction with peptide carboxylic acid (18) with a protected C-terminus Compound (19) can be produced by reacting the peptide carboxylic acid (1 The reaction conditions used to form the peptide bond between compound (8) and compound (11), The reagent, base and inert solvent are appropriately selected from those described for the synthesis of compound (6). The protecting group P for the carboxy group of compound (18) can be selected from the group 4 The above protection The alkyl group may be suitably selected from the group consisting of aryl, ... and aryl.

[0353] The protecting group for the carboxy group of the obtained compound (19) is removed to obtain the compound (19). This deprotection can be used to form compound (20). This can be carried out in the same manner as deprotection of the carboxy group.

[0354] The resulting compound (20) is derivatized to an active ester, mixed anhydride, or the like. , which can be reacted with compound (7) to produce compound (2). For the reaction, the reaction reagents and reaction conditions generally used for peptide synthesis are used. The reaction conditions, reagents, bases and inerts used for the reaction may also be used. The solvent can be appropriately selected from those described for the synthesis of compound (6). be.

[0355] Generation method 4

[0356] Hereinafter, in the present specification, n 1 = 1. L a A method for producing compound (10b) where =O is described in detail below. For example, A compound represented by formula (10b), a salt or a solvate thereof is prepared according to the following method: It is possible.

[0357] [ka]

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

[0359] Japanese Patent Application Laid-Open No. 2002-60351 or the literature (J. Org. Chem., Vol. 51, p. 319 6, 1986) and, if necessary, By removing the protecting group or modifying the functional group, a compound represented by formula (21) is obtained. Alternatively, the amino acid can be prepared by treating the amino acid with a protected terminal amino group. or of an oligopeptide with a protected amino group bearing an aldehyde or ketone It can also be obtained by treating the acid amide.

[0360] Compound (21) is reacted with 2-chloro-2-methyl-2-propanol in an inert solvent in the presence of an acid or a base under cooled temperature conditions. By reacting with a compound (22) having a hydroxyl group at temperatures ranging up to room temperature, Compound (23) can be produced by the reaction of

[0361] Examples of the acid that can be used here include hydrofluoric acid, hydrogen chloride, sulfuric acid, nitric acid, phosphoric acid, and the like. Acid and inorganic acids such as boric acid, acetic acid, citric acid, paratoluenesulfonic acid and methane Organic acids such as sulfonic acid, as well as tetrafluoroborates, zinc chloride, tin chloride, chloride Lewis acids such as aluminum and iron chlorides can be mentioned. As regards the base, any one of the bases mentioned above is preferred. Suitable species can be selected and used. Preferred examples thereof include potassium tert-butyl phosphate. Alkali metal alkoxides such as t-butoxide, sodium hydroxide and potassium hydroxide alkali metal hydroxides such as sodium hydride and potassium hydride; Metal hydrides, dialkylaminolithium compounds such as lithium diisopropylamide Representative organometallic bases and bis(trimethylsilyl)amides such as lithium bis(trimethylsilyl)amide Examples of solvents that can be used for the reaction include organometallic bases such as methyl methyl amine. ether solvents such as tetrahydrofuran and 1,4-dioxane, and benzene and aromatic hydrocarbon solvents such as toluene. Furthermore, P 5 Protection for the amino group as exemplified by The protecting group is not particularly limited as long as it is a group generally used for protecting an amino group. Representative examples include the protecting groups for amino groups described in Production Method 2. However, in this reaction, P 5 A protecting group for the amino group is developed as exemplified by In such cases, it may be necessary to reintroduce the protecting group. Therefore, it is necessary to carry out the reaction with a suitable reagent to protect the amino group. is.

[0362] Protecting group P of compound (23) 6 Compound (24) can be obtained by removing In this specification, P 6 Protecting groups for carboxy groups as exemplified by Representative examples are listed in Generation Method 2, and an appropriate one can be selected from them. In compound (23), the protecting group P for the amino group 5 and protection for the carboxyl group Mamoru P 6 is preferably a protecting group that can be removed by different methods or under different conditions. For example, a typical example is P 5is a 9-fluorenylmethyloxycarbonyl group Ri, P 6 is a benzyl group. The protecting group can be selected depending on the properties of the compound having the carboxyl group. For the removal of the protecting group, the reagents and conditions are selected depending on the protecting group.

[0363] Carboxylic acid (24) as an active ester, mixed acid anhydride, acid halide, or the like and reacting it with compound (4) or a pharmaceutically acceptable salt thereof to form a compound. After forming compound (25), the protecting group P of the resulting compound (25) is 5 By removing The reaction between compound (4) and carboxylic acid (24) can produce compound (26). Reaction and Protecting Group P 6 The reaction for removing the The same reagents and reaction conditions can be used as in the previous example.

[0364] Compound (26) is reacted with an amino acid having a protected terminal amino group or a protected amino acid. The resulting compound (9b) is reacted with an oligopeptide (27) having a hydroxyl group. The protecting group P of compound (9b) 7 By removing the compound (10b), P 7 The protecting group for the amino group represented by the formula: There are no particular limitations on the method used. A typical example is the method described in Production Method 2. The protecting groups for the amino groups are included in the amine groups. The reagents and conditions for removing the protecting groups are as follows: , selected depending on the protecting group. For the reaction between compound (26) and compound (27), Reaction reagents and reaction conditions generally used for peptide synthesis can be used. Compound (10b) produced by the above method can be used to prepare the compound of the present invention according to the above method. It can be derivatized to (1).

[0365] The anti-TROP2 antibody-drug conjugate of the present invention may remain in the air or may be, for example, purified. When recrystallized to form a hydrate, it absorbs moisture and has adsorbed water or changes to a hydrate. Such compounds and water containing salts are also included in the present invention.

[0366] Compounds labeled with various radioactive or non-radioactive isotopes are also included in the present invention. One or more atoms constituting the antibody-drug conjugate of the invention contain atomic isotopes in unnatural ratios. Examples of atomic isotopes include deuterium ( 2 H), tritium ( 3 H), Yo Elementary 125( 125 I) and carbon-14 ( 14 Furthermore, the compound of the present invention is tritium ( 3 H), iodine-125( 125 I), carbon-14 ( 14 C), copper 64( 6 4 Cu), Zirconium 89 ( 89 Zr), Iodine-124 ( 124 I), fluorine 18( 1 8 F), Indium 111 ( 111 I), carbon-11 ( 11 C) and iodine-131( 13 1 The compound may be radiolabeled with a radioisotope such as I). are used as therapeutic or prophylactic agents, research reagents such as assay reagents, and in vivo diagnostic imaging agents. The antibody-drug conjugate of the present invention is useful as a diagnostic agent for the following diseases: Any isotopic variants of the isomer are within the scope of the present invention.

[0367] Antibody-drug conjugates (ADCs) The present disclosure relates to a combination of anti-TROP2 antibodies and anti-cancer compounds such as topoisomerase I inhibitors (DXd). The present invention provides a TROP2-targeting antibody-drug conjugate (ADC) comprising: In some embodiments, the TROP2-targeted ADC has Formula 13, as shown below: may include:

[0368] [ka]

[0369] In some embodiments, the heavy chain of the ADC is QVQLVQSGAEVKKPGASVKVSCKASGYTFTTAGMQWVRQAPGQGLEWMGWINTHSGVPKYAEDFKGRVTISADTSTSTAY LQLSSLKSEDTAVYYCARSGFGSSYWYFDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTV SWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLG GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNG KEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 45) may include:

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

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

[0372] That is, the anti-TROP2 antibody-drug conjugate of the present invention is a major method for treating cancer. It can be selectively used as a drug for certain chemotherapy, thereby slowing the growth of cancer cells. This can inhibit the proliferation of cancer cells and even kill them. The patient will be free of symptoms caused by cancer or the quality of life of the cancer patient will be improved. It is possible to achieve a therapeutic effect by prolonging the lifespan of cancer patients. The anti-TROP2 antibody-drug conjugate of the present invention has the following properties: , inhibiting or controlling the proliferation of cancer cells, thereby promoting longer survival of cancer patients. It is possible that cancer patients will achieve a higher quality of life while achieving a longer treatment period.

[0373] In such drug treatments, it is used both as a sole drug and as an adjuvant treatment. It can be used as a drug in combination with additional treatments in It can be used in combination with other treatments, hormone therapy or the like. It can also be used as a drug for drug therapy in bunt therapy.

[0374] In addition to the therapeutic uses mentioned above, it is also effective in inhibiting the growth of micrometastatic cancer cells and in preventing the proliferation of these cancer cells. The ability to bind to and kill cells is also determined by the binding properties of antibodies to antigens. This is especially expected when TROP2 expression is confirmed in primary cancer cells. inhibiting cancer metastasis by administering the anti-TROP2 antibody-drug conjugate of the present invention, or For example, the prevention of cancer cells in body fluids during the metastasis process can be expected. Inhibiting and killing effects, or for example, micro-cancer cells immediately after implantation in any tissue. Furthermore, it is expected to have the effect of inhibiting and killing the cells, especially after surgical removal of cancer. Therefore, it is expected that the inhibitory or preventive effect of cancer metastasis can be achieved. It is possible to expect this to have a beneficial effect.

[0375] The anti-TROP2 antibody-drug conjugate of the present invention can be administered to a patient as a systemic treatment, and Additionally, it is expected that local administration to cancer tissue will have a therapeutic effect.

[0376] Examples of cancer types to which the anti-TROP2 antibody-drug conjugate of the present invention can be applied include lung cancer, kidney cancer, Urothelial cancer, colorectal cancer, prostate cancer, glioblastoma multiforme, ovarian cancer, pancreatic cancer, breast cancer, black Cancers to be treated include liver cancer, bladder cancer, stomach cancer, cervical cancer, head and neck cancer, and esophageal cancer. The antibody in the antibody-drug conjugate recognizes a protein in the cancer cell. There is no limitation thereto, so long as it is a cancer cell that expresses the gene.

[0377] The anti-TROP2 antibody-drug conjugate of the present invention can be preferably administered to mammals, but more preferably Preferably, it is administered to a human.

[0378] Pharmaceutical Compositions and Modes of Administration The substances used in the pharmaceutical compositions containing the anti-TROP2 antibody-drug conjugates of the present invention are In view of the amount or administration concentration, formulation additives or the like generally used in the art may be used. The above-mentioned can be appropriately selected and applied.

[0379] The anti-TROP2 antibody-drug conjugate of the present invention comprises at least one pharmaceutically suitable component. For example, the pharmaceutical composition typically contains at least one In some embodiments, the liquid may contain a pharmaceutical carrier (e.g., a sterile liquid). The body contains, for example, water and oil (petroleum and oils of animal, plant or synthetic origin). The oil may be, for example, peanut oil, soybean oil, mineral oil, or sesame oil. When the pharmaceutical composition is administered intravenously, water is the more typical carrier. As liquid carriers for the preparation of the drug, saline solution, aqueous dextrose solution, and aqueous glycerol solution were used. Suitable pharmaceutical vehicles are known in the art. The compositions may also contain trace amounts of humectants, emulsifiers, or pH buffering agents. Examples of drug carriers are given in "Remington's Pharmac" by E.W. Martin. The formulation will correspond to the mode of administration.

[0380] Pharmaceutically 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, isotonicity agents, buffers and soothing agents for the preparation are known. In some embodiments, the pharmaceutical composition contains one or more preservatives, antioxidants, The composition may comprise one or more additional ingredients such as anti-inflammatory agents, stabilizers, and the like.

[0381] Additionally, the disclosed pharmaceutical compositions are suitable for high drug concentrations, such as solutions, microemulsions, and the like. The carrier may be, for example, water, ethanol, or other ordered structures. alcohols, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene) solvents or dispersion media, including polyethylene glycols and the like, and suitable mixtures thereof For example, by using a coating such as lecithin, the dispersion maintains proper fluidity by maintaining the required particle size and by the use of surfactants In some embodiments, the composition may contain an isotonicity agent, such as sugars, polyhydric alcohols, e.g., mannitol, sorbitol, or sodium chloride It is preferable to include agents that delay absorption, for example, monostearate salts and gelatin. By including it in the composition, sustained absorption of the injectable composition can be achieved. Cut.

[0382] in a suitable solvent, optionally with one or a combination of the ingredients listed above. A sterile injectable solution is prepared by incorporating the required amount of active compound and then sterile microfiltration. Generally, a basic dispersion medium and the necessary components from those listed above can be used to prepare a dispersion medium. Dispersions are prepared by incorporating the active compound into a sterile vehicle containing the other ingredients. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred method of preparation is to mix the active ingredient with the powder in advance. The resulting powder is then sterile filtered from the solution with any additional desired ingredients and vacuum dried. and freeze-drying (lyophilization).

[0383] Various delivery systems are known and can be used to administer the anti-TROP2 antibody-drug conjugates of the present invention. Examples of routes of administration include intradermal, intramuscular, intraperitoneal, intravenous, and The administration may be by, but not limited to, intravenous and subcutaneous routes. In a particularly preferred embodiment, the antibody drug Administration of the compound conjugate is by injection, with parenteral administration being the preferred route of administration.

[0384] According to an exemplary embodiment, the pharmaceutical composition is administered intravenously to humans according to conventional techniques. Compositions for intravenous administration are typically defined as pharmaceutical compositions suitable for , a solution in a sterile and isotonic aqueous buffer. Optionally, the drug may be solubilized. and a local anesthetic (e.g., lignocaine) to relieve pain at the injection site. Generally, the above ingredients are contained in an ampoule or sachet containing a certain amount of active agent. Either the freeze-dried powder or the anhydrous concentrate contained in the container obtained by sealing in The drugs are provided individually as one of the drugs or as a mixture in a unit dosage form. If the dosage form is via injection, administer it from a syringe containing sterile pharmaceutical grade water or saline. When the drug is administered by injection, the above components may be mixed together before administration. An ampoule of sterile water or saline for injection may be provided as needed.

[0385] The pharmaceutical composition of the present invention includes a pharmaceutical composition containing only the anti-TROP2 antibody-drug conjugate of the present application; or a composition comprising an 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 may be used in combination with other cancer treatment agents. They can be administered to an individual together, simultaneously, or sequentially. Another anti-cancer drug used for such a purpose is the antibody drug The antibody-drug conjugate may be administered simultaneously with the antibody-drug conjugate, separately from the antibody-drug conjugate, or subsequently to the antibody-drug conjugate. The administration intervals for each of the drugs may be varied. These include Abraxane, paclitaxel, cisplatin, gemcitabine, and irinotecan ( CPT-11), paclitaxel, pemetrexed, sorafenib, vinorelbine, international Drugs described in Publication No. 2003 / 038043, LH-RH analogs (Lupril, estramustine phosphate, estrogen antagonists steroids (tamoxifen, raloxifene, or similar) and aromatase inhibitors ( anastrozole, letrozole, exemestane or similar), There is no limitation on the drug as long as it has antitumor activity.

[0386] The pharmaceutical composition may be a lyophilized formulation or a pharmaceutical composition having the desired composition and required purity. It can be formulated into a liquid or lyophilized formulation. The formulation may contain suitable formulation additives used in the field. However, it is formulated as a liquid formulation containing various formulation additives used in the art. It can be done.

[0387] The composition and concentration of the pharmaceutical composition may vary depending on the method of administration. The anti-TROP2 antibody-drug conjugate contained in the pharmaceutical composition of the present invention is an antibody-drug conjugate comprising an antigen higher affinity for the antigen, i.e., in terms of the dissociation constant (i.e., Kd value) for the antigen If a compound has a higher affinity (= a lower Kd value), it will exhibit a medicinal effect even at a lower dose. Therefore, in order to determine the dose of the antibody-drug conjugate, The dosage can be determined taking into consideration the affinity between the complex and the antigen. When the antibody-drug conjugate of the present invention is administered to a human, for example, about 0.001 to 100 mg / kg It can be administered as a single dose or as multiple doses with an interval of 1 to 180 days. This can be done.

[0388] TROP2-expressing cancer TROP2 is highly expressed in epithelial cancers and its expression is associated with poor survival. Examples of current cancers include lung cancer, kidney cancer, urothelial cancer, colorectal cancer, prostate cancer, and glioblastoma multiforme. carcinoma, ovarian cancer, pancreatic cancer, breast cancer, melanoma, liver cancer, bladder cancer, stomach cancer, cervical cancer, head and neck cancer, and Any of these cancers may be caused by: However, cancer cells may be treated with the disclosed ADCs and ADC dosage regimens. The present study aims to identify patients with cancers that do not fall into the above-listed categories, as long as they express TROP2. It should be understood that any of these may also be treated according to the disclosed methods.

[0389] Non-small cell lung cancer (NSCLC) is a potential target for treatment utilizing the disclosed ADCs and dosage regimens. For example, in Examples 5-7, the disclosed ADCs are used in the treatment of NSCLC. A phase 1 clinical trial in subjects with SCLC is detailed.

[0390] To treat any of the above TROP2-expressing cancers, the disclosed TROP2 targets An ADC can be used.

[0391] Treatment Methods and Uses The present disclosure provides a method for administering an anti-TROP2 antibody-drug conjugate as disclosed herein. The present invention provides a method for treating cancer, comprising administering to a subject a therapeutically effective amount of the disclosed anti-TRO antibody for use in treating cancer. Any of the PADCs are provided herein.

[0392] In some embodiments, the cancer is a TROP2-expressing cancer. Cancers include lung cancer (e.g., non-small cell lung cancer or NSCLC), kidney cancer, urothelial cancer, and colon cancer. Rectal cancer, prostate cancer, glioblastoma multiforme, ovarian cancer, pancreatic cancer, breast cancer, melanoma, liver cancer, bladder cancer Cancers include, but are not limited to, gastric cancer, cervical cancer, head and neck cancer, and esophageal cancer. It's not something like that.

[0393] For purposes of this disclosure, the term "TROP2-overexpressing cancer" refers to a cancer that is characterized by a TROP2-overexpressing gene that is expressed by a person skilled in the art. There are no particular limitations as long as it is recognized as a TROP2-overexpressing cancer. Preferred examples of hyper-expressing cancers include those identified by immunohistochemistry (IHC) or in situ hybridization. High scores were given for TROP2 expression in the in situ hybridization (ISH) method. The in situ hybridization method of the present invention includes the use of fluorescent Light in situ hybridization (FISH) and two-color in situ hybridization Examples include the dish-in-situ hybridization (DISH) method.

[0394] a method for scoring the degree of TROP2 expression by immunohistochemistry; or Positive or negative for TROP2 expression by in situ hybridization The method for determining is not particularly limited, as will be appreciated by those skilled in the art.

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

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

[0397] In some embodiments, the ADCs and treatment methods and uses of the present invention are directed to existing Cancers that show resistance to anticancer drugs (i.e., resistant cancers), especially those that show resistance to existing anticancer drugs It exhibits excellent antitumor activity against acquired cancers (i.e., secondary resistant cancers). The ADC for treatment of the present invention is useful for cancer patients with cancers that are resistant to existing anticancer drugs. When applied to a group of patients (patients with a history of treatment with existing anticancer drugs), In particular, the cancer to be treated is one that has been treated with an EGFR inhibitor (i.e., ritinib, erlotinib, osimertinib, afatinib), ALK inhibitor treatment (i.e. alectinib, crizotinib, ceritinib), platinum-based chemotherapy (i.e., cisplatin, tin, carboplatin) and / or checkpoint inhibitor treatment (i.e., nivolumab, Mab, pembrolizumab, atezolizumab, avelumab, ipilimumab, durvalumab , tislelizumab, sintilimab, cemiplimab) There may be.

[0398] The ADCs for treatment of the present invention may be used in place of existing anti-cancer drugs or as a treatment for these existing anti-cancer drugs. By administering it to cancer patients in combination with existing anti-cancer drugs, This can be highly effective against cancers that have acquired resistance to the drug.

[0399] Thus, in some embodiments of the disclosed methods and uses, the cancer to be treated 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 , resistant forms of cervical cancer, head and neck cancer and esophageal cancer.

[0400] The ADC and the method for treatment or use of the present invention may slow the growth of cancer cells. It is possible to inhibit the proliferation and even kill cancer cells. These effects allow cancer patients to eliminate or eliminate symptoms caused by cancer. It has the potential to improve the quality of life (QOL) of cancer patients and prolong their lifespan. The anti-TROP2 antibody-drug conjugate of the present invention is effective in treating cancer cells. Inhibiting or controlling the growth of cancer cells, even if it does not achieve cell death This will help achieve longer survival times and provide a higher quality of life for cancer patients. There is a possibility.

[0401] In some embodiments of the disclosed methods and uses, the ADC is administered in combination with the drug alone. It can be used as a treatment for rheumatoid arthritis or in combination with additional treatments in adjuvant therapy. It can be used as a drug and can be used in conjunction with surgery, radiation therapy, hormone therapy or the like. Furthermore, it can be used in combination with other drugs in neoadjuvant therapy. In some embodiments, it can also be used as a drug for AD. C is, for example, Abraxane, paclitaxel, cisplatin, carboplatin, gemcitabine Tabine, irinotecan (CPT-11), pemetrexed, sorafenib, vinorelbine , drugs described in WO 2003 / 038043, LH-RH analogs (LH-RH analogs) purine, goserelin or similar), estramustine phosphate, estrogen an agonists (tamoxifen, raloxifene, or similar), aromatase inhibitors (anastrozole, letrozole, exemestane, or similar), EGFR inhibitors Drug treatment (gefitinib, erlotinib, osimertinib, afatinib), ALK inhibitors Treatment (alectinib, crizotinib, ceritinib) and / or checkpoint inhibition Drug treatment (nivolumab, pembrolizumab, atezolizumab, avelumab, ipilimumab, durvalumab, tislelizumab, sintilimab, and cemiplimab), It may be used in combination with anticancer drugs, including but not limited to:

[0402] In addition to the above-mentioned methods and uses, the present invention also inhibits the growth of small metastatic cancer cells and kills them. In particular, the expression of TROP2 in primary cancer cells can be expected to have a preventive effect. If this is confirmed, cancer metastasis can be prevented by administering the anti-TROP2 antibody-drug conjugate of the present invention. For example, the effect of inhibiting or preventing the metastasis of the tumor in body fluids can be expected. The effect of inhibiting and killing cancer cells in the tissue or, for example, implantation in any tissue. Immediately after the administration, it is expected that the effect of inhibiting and killing small cancer cells can be expected. After surgical removal of the cancer, inhibition of cancer metastasis or a preventive effect can be expected.

[0403] In some embodiments of the methods and uses, cancer (e.g., TROP2-expressing For patients with cancer, about 0.1 to about 15 mg / kg, about 0.5 to about 12 mg / kg, and about 1.0 to It may be administered at about 10 mg / kg, or at about 4 to about 8 mg / kg. In one embodiment, the dose of the ADC administered to the subject is 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 0.5mg / kg or more, approximately 0.6mg / kg or more, approximately 0.7mg / kg or more, approximately 0.8m g / kg or more, approximately 0.9 mg / kg or more, approximately 1.0 mg / kg or more, approximately 1.25 mg / k g 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 3.0mg / kg or more, about 3.25mg / kg or more, about 3.5mg / kg or more, about 3.7 5mg / kg or more, approximately 4.0mg / kg or more, approximately 4.25mg / kg or more, approximately 4.5mg / kg or more, approximately 4.75mg / kg or more, approximately 5.0mg / kg or more, approximately 5.25mg / k g 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 7.0mg / kg or more, about 7.25mg / kg or more, about 7.5mg / kg or more, about 7.7 5mg / kg or more, about 8.0mg / kg or more, about 8.25mg / kg or more, about 8.5mg / kg or more, approximately 8.75mg / kg or more, approximately 9.0mg / kg or more, approximately 9.25mg / k g or more, about 9.5 mg / kg or more, about 9.75 mg / kg or more, about 10.0 mg / kg or more Above, about 10.25mg / kg or more, about 10.5mg / kg or more, about 10.75mg / 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 dose of the ADC administered to the subject is 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 In some embodiments, the dose is about 2 mg / kg or more. mg / kg ~ approx. 10 mg / kg, approx. 2 mg / kg ~ approx. 8 mg / kg, approx. 4 mg / kg ~ approx. 10mg / kg, about 4mg / kg to about 8mg / kg, about 6mg / kg to about 10mg / kg In a preferred embodiment, the dose may be about 6 mg / kg to about 8 mg / kg. The dosages are 2 mg / kg, 3 mg / kg, 4 mg / kg, 5 mg / kg, 6 mg / kg g, 7 mg / kg, 8 mg / kg, 9 mg / kg, or 10 mg / kg , and more preferably 4 mg / kg, 6 mg / kg, or 8 mg / kg.

[0404] In some embodiments of the methods and uses, an anti-TROP2 ADC or The pharmaceutical composition is administered to a subject with cancer via parenteral administration. The routes of administration include injections such as intravenous injection, intramuscular injection, and subcutaneous injection. The anti-TROP2 antibody-drug conjugate used in the present invention is not limited to the following: By application as a systemic treatment to the affected body and, additionally, by local application to the cancerous tissue. Therefore, it is expected that the therapeutic effect will be exerted.

[0405] The timing or method of administration is once a week (q1w) or once every two weeks (q2w) ), once every 3 weeks (q3w), once every 4 weeks (q4w), once every 5 weeks (q5w) , once every 6 weeks (q6w), once every 7 weeks (q7w), once every 8 weeks (q8w), It may be administered once every 9 weeks (q9w) or once every 10 weeks (q10w), but is preferred. Or once every three or four weeks.

[0406] The dosage regimen is determined based on the 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 adjusted to provide: 2 mg / kg once every 3 weeks (q3w), 4 mg / kg once every 3 weeks (q3w), 3 weeks 6 mg / kg once every 3 weeks (q3w), 8 mg / kg once every 3 weeks (q3w), or every 4 weeks 2 mg / kg once every 4 weeks (q4w), 4 mg / kg once every 4 weeks (q4w), May be administered as 6 mg / kg once every 4 weeks (q4w) or 8 mg / kg once every 4 weeks (q4w). And, in some embodiments, a single bolus may be administered, while in some In some embodiments, several divided doses may be administered over time, or The dosage may be proportionally reduced or increased as indicated by the circumstances.

[0407] Furthermore, the subject of the method and the use is generally a cancer patient, but the age of the patient may also be There is no age limitation. The disclosed methods and uses are suitable for all age groups and age cohorts. Treating cancer, malignancies or cancer cell proliferation with a range of recurrence and prognosis outcomes across a range of Thus, in some embodiments, the subject is a pediatric subject. In other embodiments, the subject may be an adult subject.

[0408] The following examples are provided to illustrate the present invention. However, the present invention is not limited to these examples. It should be understood that no limitation to the specific conditions or details set forth is intended. [Example] [Example]

[0409] Generation of antibody-drug conjugates The compounds disclosed in International Publication Nos. 2015 / 098099 and 2017 / 002776 According to the methods shown, an anti-TROP2 antibody (e.g., the amino acid sequence in SEQ ID NO: 45) is prepared. a heavy chain consisting of the amino acid sequence at amino acid positions 1 to 451 of SEQ ID NO: 46; and a light chain consisting of an amino acid sequence at amino acid positions 1 to 214) The anti-TROP2 antibody is linked via a thioether bond to the following formula:

[0410] [ka]

[0411] where n represents the average drug-to-antibody ratio (DAR) per single antibody molecule, The value of n of the antibody-drug conjugate (1) is in the range of 3.5 to 4.5, and 2) The value of n is in the range of 6.5 to 8.0) and is linked to a drug linker represented by Antibody-drug conjugate (1) and antibody-drug conjugate (2) (hereinafter referred to as "antibody-drug conjugate (1)" and and "antibody-drug conjugate (2)").

[0412] The schematic structures and sequences of antibody-drug conjugate (1) and antibody-drug conjugate (2) are shown in Figure 1 and and Fig. 2. [Example]

[0413] Testing the antitumor effects of antibody-drug conjugates For the purposes of this experiment, 5-6 week old female BALB / c nude mice (Charles The experiments were conducted at ATCC (Atlantic River Laboratories Japan). Human pancreatic adenocarcinoma cell line (CFPAC-1 cells) purchased from Iwate University Hospital was suspended in saline and diluted to 4 × 10 6 The cells were transplanted into the right side of each of the female nude mice. 14 days after transplantation, The mice were divided into groups (Day 0). In the single-dose administration group (once every 3 weeks), Conjugates (1) and (2) were administered at a dose of 0.3 mg / kg or 1 mg / kg on Day 0. In the frequent administration group (once a week for 3 weeks), antibody-drug conjugate (1) and (2) were administered at a dose of 0.3 mg / kg on Day 0, Day 8, and Day 14. The vehicle-administered group was determined as the control group. Tumor growth inhibition (T) on Day 22 In any of the treatment groups, there were no particularly significant findings such as weight loss. was not confirmed.

[0414] Measurement / calculation method: Electronic digital caliper (CD-15CX, Mitutoyo Corporation) The long and short axes of the tumor were measured twice weekly, and tumor volume (mm 3 ) was calculated. is as shown below. Tumor volume (mm 3 )=1 / 2×long axis(mm)×[short axis(mm)] 2

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

[0416] Antibody-drug conjugates (1) and (2) were dissolved in acetate-buffered saline (pH 5.5) (Nacalai Tesque) The diluted solutions were administered via the tail vein. The solution (10 mL / kg) was administered.

[0417] The antitumor effects of antibody-drug conjugates (1) and (2) are shown in Figure 3. In (1), the TGI of the single-dose group of 0.3 mg / kg was 15%, and the TGI of the 1 m The TGI in the single-dose group at a dose of 0.3 mg / kg was 86%, but The TGI in the frequent administration group of antibody-drug conjugate (2) was 34%. The TGI in the single-dose group at 1 mg / kg was 43%, and the TGI in the single-dose group at 1 mg / kg was 43%. The TGI of the group was 94%, but the TGI of the group administered frequently at a dose of 0.3 mg / kg was 8. The rate was 0%.

[0418] From the above results, in both antibody-drug conjugates (1) and (2), 1 mg / When comparing a single dose of 0.1 mg / kg with a multiple dose of 0.3 mg / kg, both Although both groups provided almost the same total dose, the TGI in the single-dose group was significantly higher than that in the multiple-dose group. Therefore, the single-dose method, in which the total dose is administered only once every three weeks, was This has been shown to be more effective than a multiple-dose regimen in which the dose is repeated three times a week. In the comparison between antibody-drug conjugates (1) and (2), a dose of 1 mg / kg The TGI of the single-dose antibody-drug conjugate (1) group was 0.3 mg / kg. The TGI was higher than that of the single-dose group of conjugate (2), and the antibody-drug conjugate ( This indicates that the TGI of antibody-drug conjugates (1) and (2) is lower than that of the single-dose group. The difference in therapeutic dose between the two groups was shown to be within a three-fold range. [Example]

[0419] Safety evaluation of antibody-drug conjugates Antibody-drug conjugates (1) and (2) prepared according to Example 1 were separately analyzed for cross-reactive species ( More specifically, the antibody-drug conjugate (1) was administered to cynomolgus monkeys (Macaca fascicularis) for 3 weeks. The antibody-drug conjugate (2) was administered once a week for a total of three doses. In the case of antibody-drug conjugate (1), observation was continued until the day after the final administration. In the case of antibody-drug conjugate (2), observation was continued until the week following the final administration. The maximum non-toxic dose (HNSTD) of antibody-drug conjugate (2) is 10 mg. / kg, whereas the HNSTD of antibody-drug conjugate (1) was 30 mg / kg. Therefore, antibody-drug conjugate (1) has better safety than antibody-drug conjugate (2). It was shown that... [Example]

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

[0421] Antibody-drug conjugate (1) (i.e., DS-1062a) was administered to cynomolgus monkeys at a dose of 0.2 ml. 0.6 mg / kg, 2 mg / kg, or 6 mg / kg as a single intravenous dose Subsequently, a target-mediated pharmacokinetic model was developed based on the plasma concentration of the antibody-drug conjugate (1). Pharmacokinetic parameters were calculated using the pharmacokinetic model. The changes in plasma concentrations of the antibody-drug conjugate (1) were estimated. 3 times (q3w × 3), 0.27 mg / kg, 0.54 mg / kg, 0.81 mg / kg , and a dosing cycle consisting of 1.6 mg / kg, 3.2 mg / kg, and 6.4 mg / kg. The results are shown in Figure 4. Antibody-drug complex predicted in humans The time course of plasma concentration of the compound (1) was measured in a CFPAC-1 tumor-bearing mouse model. The results were compared with the plasma concentration of ay21. The estimated plasma concentrations at the time of administration of The minimum concentration showing tumor regression in mice (1 mg / kg administration group, 0.312 μg / mL ) were 0.27 mg / kg and 0.81 mg / kg, respectively. Therefore, the effective dose / administration of antibody-drug conjugate (1) in humans is A single dose was estimated to be more than 0.27 mg / kg. [Example]

[0422] Early Phase 1 Clinical Trials

[0423] preface DS-1062a is a novel topoisomerase I inhibitor (exatecan derivative; DXd ) is a trophoblast cell surface antigen 2 (TROP2)-targeted antibody-drug conjugate. 062a binds to TROP2 on the cell surface, inhibits topoisomerase I, and induces target cell death. After enzymatic processing that leads to apoptosis, DXd is internalized and released into the cytoplasm.

[0424] TROP2 is highly expressed in epithelial cancers, including lung cancer, and is associated with poor survival. In this study, DS-1062a showed promising antitumor activity in xenograft mouse models. Ta.

[0425] the purpose

[0426] The purpose of this study was to evaluate the safety and tolerability of DS-1062a and to determine the maximum tolerated dose (MT D) and the recommended dose (RDE) in the dose development part (cli (See nicaltrials.gov identifier NCT03401385).

[0427] Study design and methods

[0428] The Phase 1 study is a multicenter, multicenter trial of DS-1062a that enrolled subjects in the United States and Japan. This was an open-label, repeated-dose, first-in-human study. The dose-escalation group included a dose-escalation group and a dose-expansion group, as shown in Figure 1. The dose-escalation group was Cycle 1 included a single intravenous infusion of 100 mg ... The dose expansion group will administer the DS-1062a dose in the RDE to NSCLC patients. This included:

[0429] The primary objective of the dose escalation group was to The objectives were to identify the TD and to assess the safety and tolerability of said doses.

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

[0431] Secondary objectives include evaluating the pharmacokinetic (PK) properties of DS-1062a, the total TROP2 antibody, and the drug Exploratory objectives included determining the antitumor activity of DS1062a, its components, and DS1062a. The study included assessing biomarkers that correlate with response to I-1062a.

[0432] Inclusion criteria were patients with pathologically confirmed metastatic NSCLC with no standard treatment options. Patients aged 0 years or older (Japan) or 18 years or older (USA), Eastern Oncology Collaborative Group Performance status 0 or 1, measured according to RECIST version 1.1 possible disease, life expectancy of ≥3 months, and recent measurement of TROP2 levels by immunohistochemistry Available tumor tissue for determination was included.

[0433] Exclusion criteria were multiple primary malignancies (adequately resected non-melanoma skin cancer, curatively treated In situ disease or curatively treated without evidence of disease for 3 years or more Patients with other solid tumors (excluding other solid tumors) or clinically significant / suspected lung disease were included.

[0434] Patient assessment consisted of echocardiogram or MUGA scan at pre-specified visits, 1 These included 2-lead ECG, AE, PK, human anti-human antibodies, biomarkers, and tumor assessments. Demographic and baseline characteristics of patients enrolled in this early Phase 1 study The characteristics are shown in FIG.

[0435] Dose escalation of DS-1062a to determine the MTD was performed using escalation Bayesian logistic regression model following the principle of overdose control Objective response rates (ORR) were determined by a modified serial review method using the Progression-free survival was summarized by 95% confidence intervals (CI) using the Lopper-Pearson method. Progression-free survival (PFS) / overall survival (OS) rates were summarized using the Kaplan-Meier method. Safety endpoints, PK parameters of DS-1062a, anti-TROP2 antibody, DXd and blood Plasma anti-drug antibodies were summarized using descriptive statistics.

[0436] result

[0437] Thirty-nine patients were divided into seven DS-1062a treatment groups, as well as patient demographic characteristics. Gender and baseline characteristics were included at the cutoff point. Patients (N=39) , over a median (range) period of 8.86 (3.0-31.1) weeks, DS-1062 Patients were exposed to a median (range) of 3.0 (1-10) treatment cycles of a.

[0438] Two patients required interruption of DS-1062a administration (1 in the 4 mg / kg group). One patient in the 8 mg / kg group and one in the 6.0 mg / kg group Overall, 23 (54.8%) patients required dose reduction. Treatment with 2a was discontinued. The primary reason for discontinuation was failure per RECIST in 13 patients. The PD was 0.5 mg / kg and 2.0 mg / kg (n=4 for each). ;[1.0mg / kg], n=3;[0.27mg / kg and 4.0mg / kg], n=1 for each. Two patients discontinued due to clinical progression (0.27m For 6.0 mg / kg and 6.0 mg / kg, one patient each), two patients discontinued (0 One patient each for 0.5 mg / kg and 4.0 mg / kg, and one patient was diagnosed with a condition that was not treated by physician decision. The study was discontinued based on the clinical trial (1.0 mg / kg group). Five patients (in the 1.0 mg / kg group) In the 0.27 mg / kg group (n=3) and the 0.27 mg / kg group (n=2), patients were discontinued for "other" reasons. did.

[0439] Overall, 87.2% (34 / 39) of patients reported one or more TEAEs; All but one of the reported TEAEs were considered grade 3 or less (Figure 7). The most common TEAE was fatigue, reported in 13 (33.3%) of the patients. All grade 3 TEAEs occurred in two patients (0.5 mg / kg and Except for grade 3 fatigue reported in 1 patient each in the 2.0 mg / kg and 2.0 mg / kg groups, Each was reported in only one patient.

[0440] Drug-related TEAEs were reported in 23 / 39 (59.0%) patients. Of the patients with TEAEs, 21 / 23 (91.3%) of these patients had a severity of Grade 1 or 2. Most frequent (in ≥3 affected patients) The most common TEAEs were nausea (n=10), infusion-related reactions (n=8), and fatigue (n=7). , alopecia (n=6), vomiting (n=5), anemia and rash (n=4 each), and decreased appetite The infusion-related reactions were all grade 1 or 2. The event was manageable / reversible.

[0441] Serious TEAEs were reported in 10 / 39 (25.6%) patients, with the majority ( n=8) were grade 3, and one each was grade 2 and grade 5 (grade 5 loss). Serious T There were no EAEs. Only one serious TEAE was considered drug-related (pyrexia, glaucoma, nausea, vomiting, dizziness, dizziness, dizziness). Grade 2: 4.0 mg / kg administration group).

[0442] One dose-limiting toxicity (DLT) (maculopapular rash) was observed in the 6.0 mg / kg group. A rash (grade 3; resolved) occurred, but the MTD was not reached.

[0443] As shown in Figure 8, among 35 tumor-evaluable patients, there were 7 PRs (RECIS T-based, including a single PR, but no confirmed response) After data cut, there were 3 additional PRs (8.0%) for a total of 10 PRs. (All cases in the mg group) were observed.

[0444] In three patients, the computerized emission (Figures 9A, 9C, and 9D) and positive Two patients in the 4.0 mg / kg group were observed to have D Four and a half months after the start of treatment with S-1062a, tumor size increased by up to 36.6% ( 9A) and 38.4% (Fig. 9B). Patients had tumors that increased in size by up to 65% three months after initiating treatment with DS-1062a. showed a 0.5% reduction in pulmonary metastases at 3 and 7 months after treatment initiation (Figure 9C). There was a significant reduction in the number of metastases (non-target lesions) (Figure 9D).

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

[0446] Regarding pharmacokinetics, as shown in Figure 11, the systemic exposure to DS-1062a was Plasma levels of DS-1062a and total anti-T increased in an almost dose-proportional manner. This may explain why DS-1062a remains stable in the circulation, whereas the ROP2 antibody did similarly. The exposure to DXd was lower than that to DS-1062a.

[0447] summary

[0448] At the time of data cut, DS-1062a was well tolerated. There was one DLT in the 200mg / kg group, a grade 3 skin rash that was transient and reversible. DS-1062a resulted in 10 PRs and 16 stable disease. Two of the R patients received conventional EGFR inhibitor treatment or ALK inhibitor treatment (i.e. , alectinib, crizotinib, ceritinib, and osimertinib). The physical effectiveness is provided in FIG. [Example]

[0449] Phase 1 clinical trials as of the new cutoff date After initial data cut, additional patients will be enrolled in the Phase 1 trial, bringing the overall number of subjects to 5. 9 (N=59). All patients had relapsed / refractory to standard of care (SOC). Patients had unresectable advanced NSCLC tumors. 57.7% were male, 8 8.5% had stage IV disease, 73.1% had adenocarcinoma histology, and 80.8 % had an Eastern Cooperative Oncology Group Performance Status (ECOG PS) of 1 86.5% of patients were refractory to conventional immune checkpoint inhibitor treatment. and a dose-expansion study design was used.

[0450] New cups with non-causal treatment-emergent adverse events (TEAEs) The number of cases in the Phase 1 study at the time of cut-off is shown in Figure 13. Briefly, Dose-limiting toxicity (DLT) reached 10 mg / kg, and the maximum tolerated dose (MTD) is expected to be reached by future doses. The recommended dose (RDE) in the expanded dose part was 8 mg / kg. The median duration of exposure for patients was 10.6 (range 3.0-43 Serious TEAEs occurred in 14 patients (26.9%), with 3 patients experiencing severe TEAEs. Death occurred in 5.8% of patients, but the deaths were not related to the study drug. TEAEs related to treatment or discontinuation were 5 (9.6%), 5 (9.6%), and 10 (10%), respectively. The incidence of rheumatoid arthritis was 1.2% in 12 patients treated with 6.0 mg / kg of rheumatoid arthritis and 2 (3.8%) in 12 patients treated with 6.0 mg / kg of rheumatoid arthritis. One patient (1.9%) with disease progression was determined not to have interstitial lung disease (ILD). One patient developed a pulmonary adverse event of particular interest: respiratory failure (grade 5). There were four reported cases of potential ILD that remain undetermined, including a post-throw-off case. (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 [disease progression] Respiratory failure in patients with rheumatoid arthritis; 8.0 mg / kg].

[0451] Twelve partial responses (at least 12) were observed across all doses in the dose-escalation arms of the study. At 8 mg / kg, 5 / 7 patients had a partial response. In this group, 6 / 7 patients showed stable disease (SD) and 2 / 7 patients showed positive response (PR). Figure 14 shows the change in the sum of the longest dimension measures from baseline in the target lesions of interest. Figure 15 shows that those affected in the higher dose groups More consistent and significant tumor size reductions were observed, demonstrating a clear dosing effect on the frequency of responses. Figure 16 shows the dose-effect and Figure 17 shows the antitumor activity observed in the various treatment groups (previously E Patients treated with GFR-, ALK-, and HER2-targeted therapies were included. will be done.

[0452] Pretreatment tumor biopsies were evaluated by immunohistochemistry to determine TROP2 expression. Patient responses are shown in Figure 17. As shown in Figures 12, 17, 21 and 26, several Patients were either previously treated with EGFR inhibitors or ALK inhibitors, or received immuno-oncology treatment. Six of the eight patients achieved a partial response (PR). H-scores were greater than the median, whereas 8 / 15 with stable disease (SD) and 8 / 15 with progressive disease 4 / 12 patients had an H-score greater than the median. This suggests that antibody-drug conjugates (1) (i.e., DS-1062a) in contrast to TROP2-negative tumors (Calu-6). Stronger antitumor activity in TROP2-positive tumors (NCI-H2170 and HCC827) Preclinical studies have demonstrated antitumor activity in a lung cancer xenograft mouse model. This is consistent with the data (see Figure 18).

[0453] Variable allele frequency (VAF) by assessing cell-free DNA (cfDNA) Changes in Cycle 3, Day 1 (C3D1) and End of Treatment (EOT) were also determined. These results, shown in Figure 19, confirm that DS-1062a is a It was shown that cfDNA was reduced in patients who achieved D and PR.

[0454] In summary, DS-1062a was administered at doses up to 8 mg / day, which is the established MTD and RDE. The 10 mg / kg dose was well tolerated in two subjects with grade 3 mucosal ulcers. Both 8 mg / kg and 6 mg / kg were well tolerated. Although the efficacy was good, the 8 mg / kg group showed a significant improvement compared with the 6 mg / kg group. It showed better preliminary efficacy signals with a higher overall response rate (ORR). Figure 20 shows that the ORR was best in the 8 mg / kg dose group.

[0455] A dose-dependent effect on antitumor activity was observed over the range of 2.0 to 8.0 mg / kg. relapsed or progressed from standard of care (SOC), including immune checkpoint inhibitors. In addition, 12 partial responses were observed during dose escalation in heavily pretreated unselected NSCLC patients. A summary of the efficacy results is provided in Figure 21. [Example]

[0456] Preliminary efficacy of antibody-drug conjugates As of November 16, 2019, 8 of 95 subjects treated with DS-1062a Eight patients were evaluable for response.

[0457] Investigator-assessed overall response rate (ORR; unconfirmed) was (5 / 18 subjects responded, all PR) 27.8% (95% CI: 9.7, 53. 5) and 8 mg / kg groups (13 / 34, all PR) were 38.2% (95% CI The disease control rate (DCR) was 22.2, 56.4 (Table 2 and Figure 22). The mean CR+PR+SD was 72.2% at 6 mg / kg and 8 mg / kg. The figure was 79.4%.

[0458] At the data cutoff date, all 5 subjects in the 6 mg / kg dose group had a PR. Treatment was ongoing without disease progression or death.

[0459] In the 8 mg / kg dose group, 6 of 13 subjects with PR experienced disease progression or death. Treatment is ongoing without any adverse events, 2 developed progressive disease, 1 died, and 4 progressed beyond the initial stage. Discontinued DS-1062a for reasons other than death or stroke.

[0460] [Table 2]

[0461] Efficacy-evaluable subjects were assessed by both baseline and post-baseline tumor assessments. Subjects either underwent a steroid or discontinued study treatment.

[0462] Pharmacokinetics

[0463] 61 patients who received DS-1062a (0.27 mg / kg to 10 mg / kg) Exploratory single-dose PK and exploratory multiple-dose PK studies were performed using non-compartmental analysis in rats. The penalty kick was evaluated.

[0464] Figure 23 shows the effect of DS-1062a on the responsiveness of DS-1062a to repeated administration of 8 mg / kg. Plasma concentrations, total antibody and free drug (referred to as payload in the figure) are shown. The mean UClast, Cmax, and elimination half-life (t1 / 2) were 914 μg, respectively. g·d / mL, 196 μg / mL, and 5.45 days.

[0465] Plasma levels of DS-1062a and total anti-TROP2 antibodies were similar, with The exposure was lower than that of DS-1062a, which suggests that DS-1062a is more circulating. This suggests that the drug was stable at this dose.

[0466] conclusion

[0467] DS-1062a was well tolerated at doses up to 8 mg / kg in a Phase 1 study. It was shown to be effective and safe.

[0468] Among 88 efficacy-evaluable subjects, DS-1062a demonstrated no significant improvement at doses of 2 mg / kg or higher. Although it was effective, the ORR was 38.2% (13 / 34 subjects) in the 8 mg / kg group and A DCR of 79.4% (27 / 34 subjects) was achieved.

[0469] Results show that after immune checkpoint inhibitors and platinum-based chemotherapy in NSCLC It is superior to docetaxel, which is used as the standard treatment (Table 3).

[0470] Furthermore, 90.9% (20 / 22) of PR subjects had prior immune checkpoint inhibitors (e.g., nivolumab, pembrolizumab, atezolizumab, avelumab, ipilimumab) All subjects were treated with platinum-based chemotherapy (e.g., rituximab, durvalumab) and all subjects had not received prior platinum-based chemotherapy (e.g., , cisplatin, and carboplatin). in subjects with NSCLC, including those who are refractory to or intolerant of these standard therapies This suggests the possibility of replacing docetaxel.

[0471] Furthermore, a competitive antibody-drug conjugate targeting TROP2, developed in the United States, Sactizumab govitecan is a standard of care In a phase 2 study of patients receiving rituximab, the ORR in NSCLC was 19%, This suggests that DS-1062a may be more effective than the competing drugs. do.

[0472] Therefore, the therapeutic agent and therapeutic medicine containing DS-1062a used in the present invention Pharmaceutical compositions and therapeutic methods comprising administering DS-1062a of the present invention are also provided. refractory to standard treatment, or relapses, or is not amenable to standard treatment has been shown to be superior for the treatment of patients with unresectable advanced non-small cell lung cancer Ta.

[0473] Safety and preliminary efficacy of 4 mg, 6 mg, and 8 mg doses were evaluated in a Phase I study. This project continues to be carried out.

[0474] Additionally, several Phase II trials are planned to begin in 2020.

[0475] [Table 3] 1. Borghaei H, Paz-Ares L, Horn L, et al. Nivolumab versus Docetaxel in advanced nons quamous non-small-cell lung cancer.N Engl J Med.2015;373(17):1627~39 2. Garon EB, Ciuleanu TE, Arrieta O, et al. Ramucirumab plus docetaxel versus placebo plus docetaxel for second-line treatment of stage IV non-small-cell lung cancer after disease progression on platinum-based therapy(REVEL):a multicentre,double -blind,randomised phase 3 trial Lancet.2014;384(9944):665~73, Suppl.:3

[0476] All patents and publications mentioned in this specification are indicative of the levels of those skilled in the art to which this disclosure pertains. All patents and publications are incorporated by reference unless each individual publication is specifically and individually indicated to be incorporated by reference. to the same extent as if each of the preceding claims were expressly incorporated by reference herein.

[0477] Furthermore, those skilled in the art will appreciate that the present disclosure will enable one to carry out the objects and achieve the objects and advantages described and those therein. Those skilled in the art will readily recognize that the method is well suited to obtaining a unique Modifications therein and other uses will occur to those skilled in the art, and these modifications are within the spirit and scope of this disclosure. The present disclosure is defined by the scope of the claims, which are encompassed within and represent non-limiting embodiments of the present disclosure.

Claims

[Claim 1] The invention described herein.

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