Proteins that recognize the drug moiety of antibody-drug conjugates

Abstract

To provide protein that recognizes the drug moiety of an antibody-drug conjugate having a derivative of exatecan as a constituent, a method for measuring the concentration of the antibody-drug conjugate in the plasma of a mammal using the protein, and a method for checking the tissue distribution.SOLUTION: There are provided a protein that recognizes the drug moiety of an antibody-drug conjugate in which a drug represented by the following formula is linked to an antibody via a linker, a method for measuring the concentration of the antibody-drug conjugate in the plasma of a mammal dosed with the antibody-drug conjugate using the protein, and a method for checking the tissue distribution.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a drug moiety of an antibody-drug conjugate that comprises a derivative of exatecan as a constituent element. a protein that recognizes the site, and administering the antibody-drug conjugate using the protein; and a method and composition for quantifying the plasma concentration of said antibody-drug conjugate in a mammal. This article relates to a method for determining the tissue distribution. [Background technology]

[0002] Antibodies that bind to antigens expressed on the surface of cancer cells and can be internalized into the cells are used to treat cancer. Antibody-drug conjugates (Antibody-Drug Conjugates) ADCs (anti-cancer drug delivery systems) are effective in treating cancer by selectively delivering drugs to cancer cells. It is expected that drugs will accumulate inside cells and kill cancer cells (Non-patent documents 1-5). .

[0003] As with small molecule compounds and antibodies, the development of antibody-drug conjugates also requires drug discovery. Conducting pharmacokinetic (PK) studies is essential. PK study results and pharmacological study results in animals and safety test results, and then conduct PK studies in humans. This will enable us to obtain information that is useful for formulating clinical trial designs and considering efficacy and safety in humans. This is because.

[0004] PK studies of antibody-drug conjugates are conducted to evaluate the plasma concentration of antibody-drug conjugates after administration. The plasma concentration of antibody-drug conjugates is determined. For example, (1) the antigen is immobilized on a solid phase. contacting the plate with an antibody-drug conjugate to form a complex; ) a protein capable of recognizing the antibody-drug conjugate and labeled with a marker (3) contacting the complex with the nucleotides to form a further complex, and then subjecting the resulting complex to an enzymatic reaction or the like. and detecting the marker by color development, luminescence, etc., by the antibody-drug interaction. Plasma concentrations of the conjugate can be quantified.

[0005] However, when a protein that recognizes the antibody site of an antibody-drug conjugate is used, the drug is Not only antibody-drug conjugates with the drug in a retained state, but also antibody-drug conjugates with the drug in a free state The plasma concentration of the conjugate (i.e., essentially only the antibody moiety) It is calculated and accurate quantification is not possible.

[0006] By ELISA using a protein that recognizes the drug moiety of the antibody-drug conjugate, Methods for quantifying the plasma concentration of antibody-drug conjugates are known (Non-Patent Documents 6 to 11).

[0007] One of the antibody-drug conjugates is a combination of an antibody and an exon, a topoisomerase I inhibitor. Antibody-drug conjugates containing derivatives of satecan as building blocks are known (see Patent Document 1-7, Non-Patent Documents 12-15). These antibody-drug conjugates have excellent antitumor properties. Because it is effective and safe, clinical trials are currently underway.

[0008] Regarding the antibody-drug conjugates containing the above-mentioned exatecan derivatives as constituents, There was no known method for quantifying plasma concentrations while the substance was being held in place. [Prior art documents] [Chartered documents]

[0009]

Patent Document 1

Patent document 2

Patent Document 3

Patent document 4

Patent document 5

Patent document 6

Patent document 7

Non-licensed literature

[0010]

Non-licensed literature 1

Non-licensed Document 4

Non-licensed Document 5

Non-licensed Document 6

Non-Patent Document 7

Non-Patent Document 8

Non-Patent Document 9

Non-Patent Document 10

Non-Patent Document 11

Non-Patent Document 12

Non-Patent Document 13

Non-Patent Document 14

Non-Patent Document 15

Summary of the Invention

Problems to be Solved by the Invention

[0011] The present invention relates to a drug moiety of an antibody-drug conjugate that comprises a derivative of exatecan as a constituent element. a protein that recognizes the site, and administering the antibody-drug conjugate using the protein; and a method and composition for quantifying the plasma concentration of said antibody-drug conjugate in a mammal. The objective of the present invention is to provide a method for checking the texture distribution. [Means for solving the problem]

[0012] The present inventors have conducted extensive research to solve the above problems and have found that a specific immune screening The protein obtained by the antibody-drug conjugate is composed of a derivative of exatecan. Furthermore, we have found that the above-mentioned protein specifically recognizes the drug site of the adjuvant. A mammal to which the antibody-drug conjugate has been administered, We established methods to quantify plasma concentrations of acetaminophen and to confirm tissue distribution.

[0013] That is, the present invention provides the following [1] to

[51] . [1] formula

[0014] [ka]

[0015] and an antibody-drug conjugate in which the drug represented by the formula (I) is linked to the antibody via a linker, Proteins that recognize drug sites. [2] The drug linker in the antibody-drug conjugate is formula

[0016] [ka]

[0017] (wherein A represents the binding site to the antibody, and the drug linker is bonded to the antibody via a thioether bond.) (bonded together) The protein according to [1], [3] The drug linker in the antibody-drug conjugate is formula

[0018] [ka]

[0019] (wherein A represents the binding site to the antibody, and the drug linker is bonded to the antibody via a thioether bond.) (bonded together) The protein according to [1], [4] The drug linker in the antibody-drug conjugate is formula

[0020] [ka]

[0021] (wherein A represents the binding site to the antibody, and the drug linker is bonded to the antibody via a thioether bond.) (bonded together) The protein according to [1], [5] The antibody-drug conjugate has the formula

[0022] [ka]

[0023] (wherein the formula, the drug linker is bonded to the antibody via a thioether bond, and n is the number of antibodies per (The average number of drug linkers per The protein according to [1], [6] The antibody-drug conjugate has the formula

[0024] [ka]

[0025] (wherein the formula, the drug linker is bonded to the antibody via a thioether bond, and n is the number of antibodies per (The average number of drug linkers per The protein according to [1], [7] The antibody-drug conjugate has the formula

[0026] [ka]

[0027] (wherein the formula, the drug linker is bonded to the antibody via a thioether bond, and n is the number of antibodies per (The average number of drug linkers per The protein according to [1], [8] The average number of drug linkers per antibody in antibody-drug conjugates is between 2 and 1. The protein according to any one of [1] to [7], wherein the protein has a molecular weight of 8. [9] The antibody in the antibody-drug conjugate is an anti-HER2 antibody, an anti-HER3 antibody, an anti-TR antibody, or OP2 antibody, anti-B7-H3 antibody, anti-GPR20 antibody, or anti-CDH6 antibody.[1] The protein according to any one of [8] to [8].

[10] Differences in the average number of drug linkers bound per antibody in antibody-drug conjugates Therefore, there is no substantial difference in the recognition ability of the antibody-drug conjugate, [1] to [9 ] The protein according to any one of the above.

[11] formula

[0028] [ka]

[0029] A protein that recognizes drugs indicated by

[12] formula

[0030] [ka]

[0031] A protein that recognizes drugs indicated by

[13] formula

[0032] [ka]

[0033] A protein that recognizes drugs indicated by

[14] formula

[0034] [ka]

[0035] A protein that recognizes drugs indicated by

[15] The protein according to any one of [1] to

[14] , which is an antibody.

[16] a) CDRH1 consisting of the amino acid sequence shown in SEQ ID NO: 1, and the amino acid sequence shown in SEQ ID NO: 2 CDRH2 consisting of the amino acid sequence shown in SEQ ID NO: 3, and CDR a heavy chain comprising H3, and a CDRL1 consisting of the amino acid sequence shown in SEQ ID NO: 4; CDRL2 consisting of the amino acid sequence shown in SEQ ID NO: 5, and the amino acid sequence shown in SEQ ID NO: 6 an antibody comprising a light chain comprising a CDRL3 consisting of the sequence b) CDRH1 consisting of the amino acid sequence shown in SEQ ID NO: 7, and CDRH2 consisting of the amino acid sequence shown in SEQ ID NO: 8 CDRH2 consisting of the amino acid sequence shown in SEQ ID NO: 3, and CDR a heavy chain comprising H3, and a CDRL1 consisting of the amino acid sequence shown in SEQ ID NO: 4; CDRL2 consisting of the amino acid sequence shown in SEQ ID NO: 5, and the amino acid sequence shown in SEQ ID NO: 6 an antibody comprising a light chain comprising a CDRL3 consisting of the sequence c) CDRH1 consisting of the amino acid sequence shown in SEQ ID NO: 9, CDRH2 consisting of the amino acid sequence shown in SEQ ID NO: 3, and CDRH3 consisting of the amino acid sequence shown in SEQ ID NO: 4. a heavy chain comprising RH3, and CDRL1 consisting of the amino acid sequence shown in SEQ ID NO: 4; CDRL2 consisting of the amino acid sequence shown in SEQ ID NO: 5, and CDRL3 consisting of the amino acid sequence shown in SEQ ID NO: 6. an antibody having a light chain comprising a CDRL3 consisting of the amino acid sequence, d) CDRH1 consisting of the amino acid sequence shown in SEQ ID NO: 11, CDRH2 consisting of the amino acid sequence shown in SEQ ID NO: 13 a heavy chain comprising a CDRH3 and a CDRL consisting of the amino acid sequence shown in SEQ ID NO: 14; 1, CDRL2 consisting of a tripeptide represented by WAS, and an amino acid sequence represented by SEQ ID NO:6

[15] The antibody is characterized in that it has a light chain containing a CDRL3 consisting of the amino acid sequence. The protein described in

[17] A heavy chain consisting of the amino acid sequence set forth in amino acid numbers 20 to 141 in SEQ ID NO: 15. A heavy chain comprising a variable region and an amino acid sequence set forth in amino acid numbers 21 to 127 of SEQ ID NO: 16. The protein described in

[0016] is an antibody comprising a light chain including a light chain variable region consisting of an amino acid sequence.

[18] The protein according to

[17] , which is a mouse antibody.

[19] A heavy chain consisting of the amino acid sequence set forth in amino acid numbers 20 to 477 in SEQ ID NO: 15. and a light chain consisting of the amino acid sequence set forth in amino acid numbers 21 to 234 in SEQ ID NO: 16. The protein according to

[17] , which is an antibody consisting of a chain.

[20] The protein according to

[17] , which is a chimeric antibody. [twenty one] The protein according to

[17] , which is a rabbit chimeric antibody.

[0036] [twenty two] A heavy chain consisting of the amino acid sequence set forth in amino acid numbers 20 to 464 in SEQ ID NO: 19. and a light chain consisting of the amino acid sequence set forth in amino acid numbers 21 to 233 in SEQ ID NO: 20. The protein according to

[17] , which is an antibody consisting of a chain. [twenty three]

[19] or

[22] , wherein the lysine residue at the carboxyl terminal of the heavy chain of the antibody is deleted. The protein according to

[15] , which is an antibody against the antibody. [twenty four]

[19] or

[22] , and have at least 95% identity with the amino acid sequence of the antibody. The protein according to

[15] , which is an antibody consisting of an amino acid sequence. [twenty five]

[19] or

[22] , and have at least 99% identity with the amino acid sequence of the antibody. The protein according to

[15] , which is an antibody consisting of an amino acid sequence.

[26] The antibody is an antibody that competes with the antibody described in

[19] or

[22] in terms of drug recognition ability. The protein according to

[15] , characterized in that:

[27]

[15] to

[26] , which is an antigen-binding fragment of the antibody according to any one of

[15] to

[26] . The protein according to any one of [1] to

[14] .

[28] The antigen-binding fragment of the antibody is Fab, F(ab')2, Fab', or Fv.

[27] The protein described in

[29] [1] to

[28] , and A method for quantifying the plasma concentration of an antibody-drug conjugate in a mammal administered the same. Law.

[30] (1) Plasma antibodies are applied to a plate on which the target antigen of the antibody-drug conjugate is immobilized. (2) contacting the drug conjugate with the marker to form a complex;

[0033] The protein according to any one of [1] to

[28] is contacted with the protein to form a further complex. and then (3) detecting the marker. The method described in

[29] .

[31] (1) Plasma is applied to a plate on which the protein according to any one of [1] to

[28] is immobilized. (2) contacting the antibody-drug conjugate in the A second protein capable of recognizing the antibody portion of the drug conjugate and labeled with a marker (3) contacting the white matter and allowing further complex formation; and then (4) detecting the marker. The method according to

[29] , characterized in that it comprises the step of:

[32] [1] to

[28] , and The plasma concentration of the drug released from the antibody-drug conjugate in a mammal administered the Methods for quantifying concentration.

[33] (1) A plate on which the protein according to any one of [1] to

[28] is immobilized is Release from antibody-drug conjugates in plasma in the presence of a competitor drug labeled with a marker (2) contacting the marker with the drug to form a complex; and (3) detecting the marker. The method according to

[32] , characterized in that it comprises:

[34] [1] to

[28] , and The antibody-drug conjugate and / or the antibody-drug co-conjugate in a mammal to which the antibody-drug conjugate A method for determining the tissue distribution of drug released from conjugates.

[35] (1) Antibody-drug conjugates in tissues and / or from the antibody-drug conjugates The released drug is contacted with the protein according to any one of [1] to

[28] to form a complex. (2) a step of recognizing the protein according to any one of [1] to

[28] . and then contacting a second protein labeled with a marker to form a further complex. The method according to

[0034] , characterized in that it comprises the steps of (1) detecting the marker and (2) detecting the marker.

[0037]

[36] (1) Antibody-drug conjugates in tissues and / or from the antibody-drug conjugates The released drug is treated with a protein according to any one of [1] to

[28] that is labeled with a marker. (2) contacting the marker with the substrate to form a complex; and then (3) detecting the marker. The method according to

[34] , characterized in that it comprises:

[37] The marker is a color-developing reagent, and the detection of the marker is performed by detecting the color development of the marker. The method according to

[30] ,

[31] ,

[33] ,

[35] or

[36] , wherein

[38] The marker is an enzyme, and the detection of the marker is performed by luminescence or the like generated by the reaction of the enzyme with a substrate. The method according to

[30] ,

[31] ,

[33] ,

[35] or

[0036] , wherein the detection is performed by detecting color development.

[39] The marker is a luminescent substance, and the detection of the marker is based on the emission of the marker by an electrochemical reaction. The method according to

[30] ,

[31] ,

[33] ,

[35] or

[0036] , which is carried out by sensing light.

[40] A polynucleotide encoding the protein according to any one of [1] to

[28] .

[41] A vector comprising the polynucleotide described in

[40] .

[42] A transformed host cell containing the polynucleotide described in

[40] .

[43] A transformed host cell containing the vector described in

[41] .

[44]

[42] or

[43] , and then culturing the host cell obtained in the culturing step. and purifying the protein from the culture product. A method for producing the protein described above.

[45] A composition comprising the protein according to any one of [1] to

[28] .

[46] [1] to

[28] , or the composition according to

[45] . kit.

[47] Antibody-drug conjugates in mammals administered the antibody-drug conjugates and / or for quantifying the plasma concentration of the drug released from the antibody-drug conjugate. The kit described in

[46] .

[48] Antibody-drug conjugates in mammals administered the antibody-drug conjugates and / or to confirm the tissue distribution of the drug released from the antibody-drug conjugate.

[46] The kit described in

[46] .

[49] A heavy chain consisting of the amino acid sequence set forth in amino acid numbers 20 to 141 in SEQ ID NO: 15. A heavy chain comprising a variable region and an amino acid sequence set forth in amino acid numbers 21 to 127 of SEQ ID NO: 16. An antibody comprising a light chain having a light chain variable region consisting of the amino acid sequence:

[50] A heavy chain consisting of the amino acid sequence set forth in amino acid numbers 20 to 477 in SEQ ID NO: 15. and a light chain consisting of the amino acid sequence set forth in amino acid numbers 21 to 234 in SEQ ID NO: 16. The antibody described in

[49] , comprising a chain.

[51] A heavy chain consisting of the amino acid sequence set forth in amino acid numbers 20 to 464 in SEQ ID NO: 19. and a light chain consisting of the amino acid sequence set forth in amino acid numbers 21 to 233 in SEQ ID NO: 20. The antibody described in

[49] , comprising a chain. [Effects of the Invention]

[0038] According to the present invention, a drug of an antibody-drug conjugate containing a derivative of exatecan as a component is a protein that recognizes a target site, and a method for administering the antibody-drug conjugate using the protein. and a method for quantifying the plasma concentration of the antibody-drug conjugate in a mammal to which the antibody-drug conjugate has been administered. Methods for determining the tissue distribution and the specificity of the antibody were provided. [Brief explanation of the drawings]

[0039] [Figure 1] 1 shows the amino acid sequence of the mouse antibody 1A3 heavy chain (SEQ ID NO: 15). [Figure 2]1 shows the amino acid sequence of the mouse antibody 1A3 light chain (SEQ ID NO: 16). [Figure 3] The nucleotide sequence (SEQ ID NO: 17) encoding the amino acid sequence of the heavy chain variable region of mouse antibody 1A3 is shown. [Figure 4] The nucleotide sequence (SEQ ID NO: 18) encoding the amino acid sequence of the light chain variable region of mouse antibody 1A3 is shown. [Figure 5] The amino acid sequence of the rabbit chimeric antibody 1A3 heavy chain (SEQ ID NO: 19) is shown. [Figure 6] The amino acid sequence of the rabbit chimeric antibody 1A3 light chain (SEQ ID NO: 20) is shown. [Figure 7] 1 shows the amino acid sequence of the anti-HER2 antibody heavy chain (SEQ ID NO: 21). [Figure 8] 1 shows the amino acid sequence of the anti-HER2 antibody light chain (SEQ ID NO: 22). [Figure 9] 1 shows the amino acid sequence of the anti-HER3 antibody heavy chain (SEQ ID NO: 23). [Figure 10] 1 shows the amino acid sequence of the anti-HER3 antibody light chain (SEQ ID NO: 24). [Figure 11] The amino acid sequence of the anti-TROP2 antibody heavy chain (SEQ ID NO: 25) is shown. [Figure 12] The amino acid sequence of the anti-TROP2 antibody light chain (SEQ ID NO: 26) is shown. [Figure 13] 1 shows the amino acid sequence of the anti-B7-H3 antibody heavy chain (SEQ ID NO: 27). [Figure 14] 1 shows the amino acid sequence of the anti-B7-H3 antibody light chain (SEQ ID NO: 28). [Figure 15] 1 shows calibration curves for HER2-ADC(I)(DAR8) and HER2-ADC(I)(DAR4) when mouse antibody 1A3 was used as a capture reagent. [Figure 16] 1 shows calibration curves for HER2-ADC(I)(DAR8) and HER2-ADC(I)(DAR4) when mouse antibody 8B2 was used as a capture reagent. [Figure 17]1 shows calibration curves for HER2-ADC(I)(DAR8) and HER2-ADC(I)(DAR4) when mouse antibody 11B1 was used as a capture reagent. [Figure 18] 1 shows calibration curves for HER2-ADC(I)(DAR8) and HER2-ADC(I)(DAR4) when mouse antibody 1A3 is used as the detection reagent. [Figure 19] 1 shows calibration curves for HER2-ADC(I)(DAR8) and HER2-ADC(I)(DAR4) when mouse antibody 8B2 is used as the detection reagent. [Figure 20] 1 shows calibration curves for HER2-ADC(I)(DAR8) and HER2-ADC(I)(DAR4) when mouse antibody 11B1 is used as the detection reagent. [Figure 21] 1 shows the calibration curves for B7-H3-ADC(I)(DAR8) and B7-H3-ADC(I)(DAR4) when mouse antibody 1A3 was used as the detection reagent.

[0040] [Figure 22] 1 shows the calibration curves for B7-H3-ADC(I)(DAR8) and B7-H3-ADC(I)(DAR4) when mouse antibody 8B2 was used as the detection reagent. [Figure 23] 1 shows the calibration curves for B7-H3-ADC(I)(DAR8) and B7-H3-ADC(I)(DAR4) when mouse antibody 11B1 was used as the detection reagent. [Figure 24] 1 shows a calibration curve for measuring the plasma concentration of HER2-ADC(I) in mice. [Figure 25] 1 shows a calibration curve for measuring the plasma concentration of HER3-ADC(I) in monkeys. [Figure 26] 1 shows a calibration curve for measuring monkey plasma concentrations of TROP2-ADC(I). [Figure 27] 1 shows a calibration curve for measuring the plasma concentration of B7-H3-ADC(I) in monkeys. [Figure 28] The nucleotide sequence (SEQ ID NO: 29) encoding the amino acid sequence of the human light chain signal sequence and the human kappa chain constant region is shown. [Figure 29] The nucleotide sequence (SEQ ID NO: 30) encoding the amino acid sequence of the heavy chain of rabbit chimerized antibody 1A3 is shown. [Figure 30] The nucleotide sequence (SEQ ID NO: 31) encoding the amino acid sequence of the rabbit chimerized antibody 1A3 light chain is shown. [Figure 31] The amino acid sequence of the anti-GPR20 antibody heavy chain (SEQ ID NO: 32) is shown. [Figure 32] The amino acid sequence of the anti-GPR20 antibody light chain (SEQ ID NO: 33) is shown. [Figure 33] 1 shows the amino acid sequence of the anti-CDH6 antibody heavy chain (SEQ ID NO: 34). [Figure 34] 1 shows the amino acid sequence of the anti-CDH6 antibody light chain (SEQ ID NO: 35). [Figure 35] Immunostaining images using rabbit chimeric antibody 1A3 are shown, comparing the staining images obtained when TROP2-ADC(I) or anti-TROP2 Ab was administered to nude mice subcutaneously implanted with the human head and neck cancer cell line FaDu. [Figure 36] Immunostaining images using rabbit chimeric antibody 1A3 are shown, comparing the staining images obtained when nude mice subcutaneously implanted with the GPR20-overexpressing human gastrointestinal stromal tumor cell line GIST-T1 / GPR20 were administered GPR20-ADC(I) or when not administered. [Figure 37] Immunostaining images using mouse antibody 1A3 are shown, comparing the staining images obtained when tumor tissue collected from a patient with clear cell renal cell carcinoma was subcutaneously implanted in nude mice and CDH6-ADC(I) was administered or not. [Figure 38] Immunostaining images using mouse antibody 1A3 are shown. CDH6-ADC(I) was administered to nude mice subcutaneously implanted with tumor tissue collected from a patient with clear cell renal cell carcinoma. Staining images obtained when mouse antibody 1A3 was mixed with compound (2) or SN-38 before immunostaining were compared with those obtained without mixing. [Figure 39] 1 shows a calibration curve for measuring the plasma concentration of GPR20-ADC(I) in mice. [Figure 40]1 shows a calibration curve for measuring the human serum concentration of HER2-ADC(I). [Figure 41] 1 shows the inhibition rate of competitive inhibitory compounds against the recognition of mouse antibody 1A3 to HER2-ADC(I). DETAILED DESCRIPTION OF THE INVENTION

[0041] A preferred embodiment of the present invention will be described below. The embodiment shows an example of a typical embodiment of the present invention, and the present invention The scope will not be interpreted narrowly.

[0042] 1.Definition In the present invention, the term "protein" is synonymous with "peptide" or "polypeptide."

[0043] The protein of the present invention is preferably an antibody or an antigen-binding fragment of an antibody, but may also be an antibody or an antigen-binding fragment of an antibody. Proteins other than antibodies and antigen-binding fragments of antibodies ( Antibody alternatives may be used. Examples of antibody alternatives include Fibre onectin, ProteinA, Lipocalin, TrxA, A-domain , Ankyrin repeat, APPI, Ras-binding AF-6, etc. Examples include scaffold proteins (Kasper Binz H. et al., Current Opinion n Biotechnology 2005, 16:459-469, Kasper Binz H. et al., Nature Biotechnology 23 (10) 2005, 1257-1268, Skerra A., Current Opinion in Biotechnology 2007, 18:295-3 04, Nygren P., FEBS Journal 275 (2008) 2668-2676, Gronwall C. et al., Journal of Biotechnology 140 (2009) 254-269).

[0044] In the present invention, the term "antibody" refers to a glycoprotein that has the function of recognizing a specific antigen. Specific examples of antibodies include polyclonal antibodies, monoclonal antibodies, chimeric antibodies, and rabbit antibodies. Examples of antibodies include recombinant antibodies, humanized antibodies, and human antibodies.

[0045] In the present invention, the term "antigen" may be used to mean "immunogen."

[0046] In the present invention, the term "antigen-binding fragment of an antibody" refers to an antibody that retains the function of recognizing an antigen. The term "antigen-binding fragment" refers to a partial fragment of an antibody and is synonymous with "functional fragment of an antibody." Examples of the antigen-binding fragment of an antibody include: Examples include Fab, F(ab')2, scFv, Fab', and single-chain immunoglobulins. Such a functional fragment of an antibody can be, but is not limited to, an antibody In addition to those obtained by treating the full-length molecule with enzymes such as papain and pepsin, It may also be a recombinant protein produced in a suitable host cell using a recombinant gene.

[0047] The phrase "recognizes" an antigen by the protein of the present invention means that the protein of the present invention recognizes the antigen by intermolecular forces (electrostatic interactions) The present invention means that the antibody binds to an antigen by means of a force such as van der Waals force or hydrogen bond. The "recognition" of an antigen by a protein can be achieved by, for example, detecting a signal generated by various immunochemical techniques. This can be confirmed by detecting the

[0048] The "recognition ability" of the protein of the present invention to the antigen is different from that of the protein of the present invention. This means that the strength and nature of the binding between the protein of the present invention and the antigen are substantially different. Whether or not "recognition" differs can be determined by, for example, the strength of signals generated by various immunochemical techniques. The results are compared with the standard curve that shows the correlation between antigen concentration and signal strength. This can be confirmed by

[0049] The "site" recognized by the protein of the present invention is a specific site within the target recognized by the protein of the present invention. The target recognized by the protein of the present invention is called an "antigen." The specific site recognized by an antibody is sometimes called an "epitope."

[0050] The heavy and light chains of an antibody each contain three complementarity determining regions (CDRs). It is known that there is a ntarity determining region. The complementarity determining regions are also called hypervariable domains. It is a site within the variable regions of the heavy and light chains of an antibody where the variability of the primary structure is particularly high. In the primary structure of the heavy and light chain polypeptide chains, they are usually separated into three positions. In the present invention, the complementarity determining regions of the heavy chain of the antibody are overlapped. The amino acid sequences of the light chains are designated CDRH1, CDRH2, and CDRH3 from the amino-terminal side. The complementarity determining regions 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 three-dimensional structure, and they exert specificity for the antigen. It has been decided.

[0051] In the present invention, a "gene" refers to a nucleotide sequence that encodes the amino acids of a protein. It refers to the nucleotides contained therein or their complementary chains, e.g., those that code for the amino acids of a protein. a polynucleotide containing a nucleotide sequence of the present invention or a complementary strand thereof; Oligonucleotides, DNA, mRNA, cDNA, cRNA, etc. are included in the meaning of "gene". Such genes may be single-stranded, double-stranded, or triple- or more-stranded nucleotides, and may be expressed as DNA. A complex of ribonucleotides (RNA) and deoxyribonucleotides on a single nucleotide chain. Mixed ribonucleotides (DNA) and duplexes containing such nucleotide chains A strand or triplet or more strands of nucleotides are also included within the meaning of "gene."

[0052] In the present invention, "nucleotide" and "nucleic acid" are synonymous, and include, for example, DNA, RNA, , probes, oligonucleotides, polynucleotides, primers, etc. are also "nucleotides" Such nucleotides are included within the meaning of the term "nucleotides." Such nucleotides may be single-stranded, double-stranded, or triple- or more-stranded. It is a nucleotide, an assembly of DNA and RNA chains, and a ribonucleotide on a single nucleotide chain. Mixtures of nucleotides (RNA) and deoxyribonucleotides (DNA) and such The term "nucleotide" also includes double-stranded or triple-stranded aggregates containing nucleotide chains. It can be enjoyed.

[0053] In the present invention, the term "cells" includes various cells derived from individual animals, subcultured cells, primary cultured cells, and the like. Also included are cultured cells, cell lines, recombinant cells, and microorganisms.

[0054] 2. Antibody-drug conjugates In the present invention, an "antibody-drug conjugate" refers to a conjugate in which an antibody and a drug are bonded via a linker. The term "conjugate" refers to a conjugate formed by bonding via the hydroxyl group.

[0055] In the present invention, the term "drug linker" refers to a linker and and a drug.

[0056] The protein of the present invention has the formula

[0057] [ka]

[0058] (hereinafter referred to as "compound (1)") and an antibody are bound via a linker. The antibody-drug conjugate (hereinafter referred to as "the antibody-drug conjugate of the present invention") It is characterized by recognizing the drug site of the

[0059] Compound (1) is exatecan (IUPAC name: (1S,9S)-1-amino-9-enantiomer). 5-fluoro-1,2,3,9,12,15-hexahydro-9-hydroxy-4-methyl -Methyl-10H,13H-benzo[de]pyrano[3',4':6,7]indolizino [1,2-b]quinoline-10,13-dione (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]chino An antitumor agent called phosphodiesterase (which can also be represented as phosphorus-10,13(9H,15H)-dione) It is an anti-cancer drug and is known to have topoisomerase I inhibitory activity.

[0060] The protein of the present invention can also recognize compound (1) itself.

[0061] The antibody in the antibody-drug conjugate of the present invention is not particularly limited, and may be, for example, Anti-HER2 antibody, anti-HER3 antibody, anti-TROP2 antibody, anti-B7-H3 antibody, anti-CD3 antibody , anti-CD30 antibody, anti-CD33 antibody, anti-CD37 antibody, anti-CD56 antibody, anti-CD98 antibody , anti-DR5 antibody, anti-EGFR antibody, anti-EPHA2 antibody, anti-FGFR2 antibody, anti-FGFR4 Antibodies, anti-FOLR1 antibody, anti-VEGF antibody, anti-CD20 antibody, anti-CD22 antibody, anti-CD7 0 antibody, anti-PSMA antibody, anti-CEA antibody, anti-Mesothelin antibody, anti-A33 antibody, Anti-CanAg antibody, anti-Cripto antibody, anti-G250 antibody, anti-MUC1 antibody, anti-GPNM B antibody, anti-Integrin antibody, anti-Tenascin-C antibody, anti-SLC44A4 antibody , anti-GPR20 antibody, and anti-CDH6 antibody, and preferably anti-HER2 antibody. body, anti-HER3 antibody, anti-TROP2 antibody, anti-B7-H3 antibody, anti-GPR20 antibody, and anti- An example is CDH6 antibody.

[0062] In the present invention, the term "anti-HER2 antibody" refers to an antibody against HER2 (Human Epidermal Growth Factor-2) l Growth Factor Receptor Type 2; ErbB-2) and preferably binds to HER2, thereby being internalized in HER2-expressing cells. The figures show antibodies with localizing activity.

[0063] Examples of anti-HER2 antibodies include trastuzumab (US Patent No. 5821337), Pertuzumab (International Publication No. 01 / No. 00245), and preferably trastuzumab.

[0064] In the present invention, the term "anti-HER3 antibody" refers to an antibody against HER3 (Human Epidermal Growth Factor-3) l Growth Factor Receptor Type 3; ErbB-3) and preferably binds to HER3, thereby being internalized in HER3-expressing cells. The figures show antibodies with localizing activity.

[0065] Examples of anti-HER3 antibodies include patritumab (U3-1 287), U1-59 (International Publication No. 2007 / 077028), MM-121 (Ser ibantumab), the anti-ERBB3 antibody described in WO 2008 / 100624, R G-7116 (Lumretuzumab) and LJM-716 (Elgemtumab) b) and preferably include patritumab and U1-59. do.

[0066] In the present invention, the term "anti-TROP2 antibody" refers to an antibody that specifically binds to TROP2 (TACSTD2: Tumor r-associated calcium signal transducer 2 ; EGP-1) and preferably binds to TROP2, thereby 1 shows antibodies that have the activity of being internalized into ROP2-expressing cells.

[0067] Examples of anti-TROP2 antibodies include hTINA1-H1L1 (International Publication No. 2015 / 003366). No. 098099).

[0068] In the present invention, the term "anti-B7-H3 antibody" refers to a B7-H3 (B cell antibody PD-L3; CD276) and specifically binds to Preferably, the antibody has the activity of being internalized into B7-H3-expressing cells by binding to B7-H3. The antibodies shown are:

[0069] Examples of anti-B7-H3 antibodies include M30-H1-L4 (International Publication No. 2014 / 05 No. 7687).

[0070] In the present invention, the term "anti-GPR20 antibody" refers to an antibody that binds to GPR20 (G Protein-coated Specifically binds to GPR20, preferably GPR20. The figures show antibodies that have the activity of being internalized into GPR20-expressing cells when combined with GPR20.

[0071] Examples of anti-GPR20 antibodies include h046-H4e / L7 (International Publication No. 2018 / 003366). No. 135501).

[0072] In the present invention, the term "anti-CDH6 antibody" refers to an antibody specific to CDH6 (Cadherin-6). and preferably binds to CDH6 and is internalized into CDH6-expressing cells. The antibodies shown have the activity.

[0073] Examples of anti-CDH6 antibodies include H01L02 (International Publication No. 2018 / 212136 No.) can be mentioned.

[0074] The protein of the present invention is preferably formula

[0075] [ka]

[0076] (wherein A represents the binding site to the antibody, and the drug linker is bonded to the antibody via a thioether bond.) (bonded together) An antibody-drug conjugate having a drug linker represented by the formula: (called "Dugate (I)"), formula

[0077] [ka]

[0078] (wherein A represents the binding site to the antibody, and the drug linker is bonded to the antibody via a thioether bond.) (bonded together) An antibody-drug conjugate having a drug linker represented by the formula: (II)), or formula

[0079] [ka]

[0080] (wherein A represents the binding site to the antibody, and the drug linker is bonded to the antibody via a thioether bond.) (bonded together) An antibody-drug conjugate having a drug linker represented by the formula: It is characterized by specifically recognizing the drug site of the nucleotide sequence (referred to as "Dinucleotide (III)").

[0081] The drug linkers of the antibody-drug conjugates (I) to (III) are formed by disulfide bonds between antibody chains. The chimeric proteins formed at the peptide binding sites (two heavy chain-heavy chain and two heavy chain-light chain) It is bound to the ol group (in other words, the sulfur atom of the cysteine ​​residue).

[0082] More preferably, the protein of the present invention specifically binds to the drug moiety of the antibody-drug conjugate (I). It is characterized by its ability to recognize objects.

[0083] The antibody-drug conjugate (I) can also be represented by the following formula:

[0084] [ka]

[0085] Here, the drug linker is bound to the antibody via a thioether bond. The so-called average number of drug-antibody bindings (DAR; Drug-to-Antibody Ratio) It is synonymous with and indicates the average number of drug linkers bound per antibody.

[0086] The average number of drugs bound per antibody molecule in the antibody-drug conjugate of the present invention is The calculation is performed, for example, by measuring the fluorescence intensity of an antibody-drug conjugate at two wavelengths, 280 nm and 370 nm. and its conjugation precursor by measuring the UV absorbance (UV method), or antibody-drug conjugates were treated with a reducing agent and each fragment obtained was analyzed by HPLC. This can be done by a method of quantitatively measuring and calculating (HPLC method).

[0087] The antibody-drug conjugate (I) is transported into cancer cells, and the linker is cleaved. R, formula

[0088] [ka]

[0089] (hereinafter referred to as "compound (2)") is released.

[0090] Compound (2) is a compound that reacts with the antibody-drug conjugate of the present invention by cleaving the linker moiety. It is thought that this is caused by formula

[0091] [ka]

[0092] The aminal structure of the compound represented by the formula (hereinafter referred to as "compound (3)") is decomposed. It is thought that this is more likely to occur.

[0093] Compound (2) is believed to be the main component of the antitumor activity of antibody-drug conjugate (I). It has been confirmed that it has a topoisomerase I inhibitory effect (Ogitani Y. et al., C Clinical Cancer Research, 2016, Oct 15;22(20):5097-5108, Epub 2016 Mar 29).

[0094] The protein of the present invention is a compound (2) released from the antibody-drug conjugate (I). You can also recognize it.

[0095] The antibody-drug conjugate (II) can also be represented by the following formula:

[0096] [ka]

[0097] Here, the drug linker is bound to the antibody via a thioether bond. Synonymous with AR, and indicates the average number of drug linkers bound per antibody.

[0098] The antibody-drug conjugate (II) is translocated into cancer cells and the linker is cleaved. And, formula

[0099] [ka]

[0100] (hereinafter referred to as "compound (4)") is released.

[0101] The protein of the present invention is a compound (4) released from the antibody-drug conjugate (II). ) itself can also be recognized.

[0102] The antibody-drug conjugate (III) can also be represented by the following formula:

[0103] [ka]

[0104] Here, the drug linker is bound to the antibody via a thioether bond. Synonymous with AR, and indicates the average number of drug linkers bound per antibody.

[0105] The antibody-drug conjugate (III) is transported into cancer cells and the linker is cleaved. Rejected, formula

[0106] [ka]

[0107] (hereinafter referred to as "compound (5)") is released.

[0108] The protein of the present invention is a compound ( 5) It is also possible to recognize the object itself.

[0109] In the present invention, the term "anti-HER2 antibody-drug conjugate" refers to an antibody-drug conjugate according to the present invention. The antibody-drug conjugate is an anti-HER2 antibody. vinegar.

[0110] The anti-HER2 antibody preferably comprises the amino acid sequence set forth in amino acid numbers 1 to 449 of SEQ ID NO: 21. and a heavy chain consisting of the amino acid sequence set forth in amino acid numbers 1 to 214 in SEQ ID NO: 22. or an antibody comprising a light chain consisting of the amino acid sequence set forth in SEQ ID NO: 21. and a light chain consisting of the amino acid sequence set forth in SEQ ID NO: 22. be.

[0111] The average number of drug linkers bound per antibody in the anti-HER2 antibody-drug conjugate is Preferably, it is 2 to 8, more preferably 3 to 8, and even more preferably 7 to 8. and even more preferably between 7.5 and 8, and even more preferably about 8.

[0112] The anti-HER2 antibody-drug conjugate is described in WO 2015 / 115091 etc. The manufacturing method can be carried out with reference to the above.

[0113] In the present invention, the term "anti-HER3 antibody-drug conjugate" refers to an antibody-drug conjugate according to the present invention. The antibody-drug conjugate is an anti-HER3 antibody. vinegar.

[0114] The anti-HER3 antibody preferably comprises the amino acid sequence set forth in amino acid numbers 26 to 35 of SEQ ID NO: 23. CDRH1 consisting of the amino acid sequence of amino acid numbers 50 to 65 in SEQ ID NO: 23 and CDRH2 consisting of the amino acid sequence shown in SEQ ID NO: 23, from amino acid number 98 onwards. a heavy chain comprising a CDRH3 consisting of the amino acid sequence set forth in SEQ ID NO: 24; CDRL1 consisting of the amino acid sequence set forth in amino acid numbers 24 to 39 in SEQ ID NO: 2 CDRL2 consisting of the amino acid sequence set forth in amino acid numbers 56 to 62 in 4, and A CDRL consisting of the amino acid sequence set forth in amino acid numbers 95 to 103 in sequence number 24. a light chain comprising 3, More preferably, the amino acid sequence set forth in amino acid numbers 1 to 117 in SEQ ID NO: 23 a heavy chain comprising a heavy chain variable region consisting of amino acid numbers 1 to 113 in SEQ ID NO: 24; an antibody comprising a light chain comprising a light chain variable region consisting of the amino acid sequence set forth in Even more preferably, a heavy chain consisting of the amino acid sequence set forth in SEQ ID NO: 23 and a heavy chain consisting of the amino acid sequence set forth in SEQ ID NO: 24 or a heavy chain carboxyl group of said antibody. An antibody lacking terminal lysine residues.

[0115] The average number of drug linkers bound per antibody in the anti-HER3 antibody-drug conjugate is Preferably, it is 2 to 8, more preferably 3 to 8, and even more preferably 7 to 8. and even more preferably between 7.5 and 8, and even more preferably about 8.

[0116] The anti-HER3 antibody-drug conjugate is described in WO 2015 / 155998 etc. The manufacturing method can be carried out with reference to the above.

[0117] In the present invention, the term "anti-TROP2 antibody-drug conjugate" refers to the antibody of the present invention. -An antibody-drug conjugate, wherein the antibody in the drug conjugate is an anti-TROP2 antibody. Shows.

[0118] The anti-TROP2 antibody preferably has a sequence similar to that shown in amino acids 50 to 54 of SEQ ID NO:25. CDRH1 consisting of the amino acid sequence set forth above, and amino acid numbers 69 to 85 in SEQ ID NO: 25. and CDRH2 consisting of the amino acid sequence set forth in SEQ ID NO: 25, amino acid number 11. a heavy chain comprising a CDRH3 consisting of the amino acid sequence set forth in SEQ ID NO: 2; CDRL1 consisting of the amino acid sequence set forth in amino acid numbers 44 to 54 in 6; CDRL2 consisting of the amino acid sequence set forth in amino acid numbers 70 to 76 in No. 26; and and C consisting of the amino acid sequence set forth in amino acid numbers 109 to 117 in SEQ ID NO: 26. a light chain comprising DRL3, More preferably, the amino acid sequence set forth in amino acid numbers 20 to 140 in SEQ ID NO: 25 a heavy chain comprising a heavy chain variable region consisting of amino acid numbers 21 to 1 in SEQ ID NO: 26; 29. A light chain comprising a light chain variable region consisting of the amino acid sequence set forth in 29. Even more preferably, the amino acids set forth in amino acid numbers 20 to 470 in SEQ ID NO: 25 and a heavy chain consisting of the amino acid sequence set forth in amino acid numbers 21 to 234 in SEQ ID NO: 26. an antibody comprising a light chain consisting of the carboxyl-terminal lysine of said antibody heavy chain, An antibody with deleted residues.

[0119] The average number of drug linkers per antibody in anti-TROP2 antibody-drug conjugates is , preferably 2 to 8, more preferably 3 to 5, and even more preferably 3.5 to 4.5, and even more preferably about 4.

[0120] The anti-TROP2 antibody-drug conjugate may be any of the compounds described in WO 2015 / 098099, etc. The product can be produced by referring to the description.

[0121] In the present invention, the term "anti-B7-H3 antibody-drug conjugate" refers to the antibody of the present invention. -An antibody-drug conjugate, wherein the antibody in the drug conjugate is an anti-B7-H3 antibody Shows.

[0122] The anti-B7-H3 antibody preferably comprises the amino acid sequence set forth in amino acid numbers 50 to 54 of SEQ ID NO:27. CDRH1 consisting of the amino acid sequence set forth above, and amino acid numbers 69 to 85 in SEQ ID NO: 27. and CDRH2 consisting of the amino acid sequence set forth in SEQ ID NO: 27, amino acid number 11. a heavy chain comprising a CDRH3 consisting of the amino acid sequence set forth in SEQ ID NO: 2; CDRL1 consisting of the amino acid sequence set forth in amino acid numbers 44 to 53 in 8; CDRL2 consisting of the amino acid sequence set forth in amino acid numbers 69 to 75 in No. 28; and and C consisting of the amino acid sequence set forth in amino acid numbers 108 to 116 in SEQ ID NO: 28. a light chain comprising DRL3, More preferably, the amino acid sequence set forth in amino acid numbers 20 to 141 in SEQ ID NO: 27 a heavy chain comprising a heavy chain variable region consisting of amino acid numbers 21 to 1 in SEQ ID NO: 28; 28. A light chain comprising a light chain variable region consisting of the amino acid sequence of 29. Even more preferably, the amino acids set forth in amino acid numbers 20 to 471 in SEQ ID NO: 27 and a heavy chain consisting of the amino acid sequence set forth in amino acid numbers 21 to 233 in SEQ ID NO: 28. an antibody comprising a light chain consisting of the carboxyl-terminal lysine of said antibody heavy chain, An antibody with deleted residues.

[0123] The average number of drug linkers per antibody in anti-B7-H3 antibody-drug conjugates is , preferably 2 to 8, more preferably 3 to 5, and even more preferably 3.5 to 4.5, and even more preferably about 4.

[0124] The anti-B7-H3 antibody-drug conjugate can be prepared by the method described in WO 2014 / 057687 etc. The product can be produced by referring to the description.

[0125] In the present invention, the term "anti-GPR20 antibody-drug conjugate" refers to the antibody of the present invention. -An antibody-drug conjugate, wherein the antibody in the drug conjugate is an anti-GPR20 antibody Shows.

[0126] The anti-GPR20 antibody preferably comprises the amino acid sequence set forth in amino acid numbers 45 to 54 of SEQ ID NO: 32. CDRH1 consisting of the amino acid sequence set forth above, and amino acid numbers 69 to 78 in SEQ ID NO: 32. and CDRH2 consisting of the amino acid sequence set forth in SEQ ID NO: 32, amino acid number 11. a heavy chain comprising a CDRH3 consisting of the amino acid sequence set forth in SEQ ID NO: 3; CDRL1 consisting of the amino acid sequence set forth in amino acid numbers 44 to 54 in paragraph 3; CDRL2 consisting of the amino acid sequence set forth in amino acid numbers 70 to 76 in No. 33; and and C consisting of the amino acid sequence set forth in amino acid numbers 109 to 117 in SEQ ID NO: 33. a light chain comprising DRL3, More preferably, the amino acid sequence set forth in amino acid numbers 20 to 142 in SEQ ID NO: 32 a heavy chain comprising a heavy chain variable region consisting of amino acid numbers 21 to 1 in SEQ ID NO: 33; 29. A light chain comprising a light chain variable region consisting of the amino acid sequence set forth in 29. Even more preferably, the amino acids set forth in amino acid numbers 20 to 472 in SEQ ID NO: 32 a heavy chain consisting of the amino acid sequence set forth in amino acid numbers 21 to 234 in SEQ ID NO: 33; an antibody comprising a light chain consisting of the carboxyl-terminal lysine of said antibody heavy chain, An antibody with deleted residues.

[0127] The average number of drug linkers per antibody in anti-GPR20 antibody-drug conjugates is , preferably 2 to 8, more preferably 3 to 8, and even more preferably 7 to 8. and even more preferably between 7.5 and 8, and even more preferably about 8.

[0128] The anti-GPR20 antibody-drug conjugate may be any of the compounds described in WO 2018 / 135501, etc. The product can be produced by referring to the description.

[0129] In the present invention, the term "anti-CDH6 antibody-drug conjugate" refers to the antibody- The antibody-drug conjugate is an anti-CDH6 antibody. vinegar.

[0130] The anti-CDH6 antibody preferably comprises the sequence set forth in amino acids 45 to 54 of SEQ ID NO: 34. CDRH1 consisting of the amino acid sequence of amino acid numbers 69 to 78 in SEQ ID NO: 34 CDRH2 consisting of the amino acid sequence shown in SEQ ID NO: 34, and a heavy chain comprising a CDRH3 consisting of the amino acid sequence set forth in any one of SEQ ID NO: 35 to 130; CDRL1 consisting of the amino acid sequence set forth in amino acid numbers 44 to 54 in SEQ ID NO: CDRL2 consisting of the amino acid sequence set forth in amino acid numbers 70 to 76 in 35; and A CD44 comprising the amino acid sequence set forth in amino acid numbers 109 to 116 in SEQ ID NO: 35. a light chain comprising RL3, More preferably, the amino acid sequence set forth in amino acid numbers 20 to 141 in SEQ ID NO: 34 a heavy chain comprising a heavy chain variable region consisting of amino acid numbers 21 to 1 in SEQ ID NO: 35; 28. A light chain comprising a light chain variable region consisting of the amino acid sequence of 29. Even more preferably, the amino acids set forth in amino acid numbers 20 to 471 in SEQ ID NO: 34 a heavy chain consisting of the amino acid sequence set forth in amino acid numbers 21 to 233 in SEQ ID NO: 35; an antibody comprising a light chain consisting of the carboxyl-terminal lysine of said antibody heavy chain, An antibody with deleted residues.

[0131] The average number of drug linkers bound per antibody in the anti-CDH6 antibody-drug conjugate is Preferably, it is 2 to 8, more preferably 3 to 8, and even more preferably 7 to 8. and even more preferably between 7.5 and 8, and even more preferably about 8.

[0132] The anti-CDH6 antibody-drug conjugate is described in WO 2018 / 212136 and the like. The manufacturing method can be carried out with reference to the above.

[0133] 3. Production of the Protein of the Present Invention The protein of the present invention is preferably obtained as an antibody (hereinafter referred to as "antibody of the present invention"). The antibody of the present invention can be prepared by linking compound (1) or a derivative thereof with a carrier protein. Animals are immunized with the antigen protein bound via the , and the antibodies produced in the body are collected and purified. It can be obtained by

[0134] The antigen protein may be, for example, formula

[0135] [ka]

[0136] The compound represented by the formula (hereinafter referred to as "compound (6)") is added with a carrier protein. It can be used.

[0137] As a carrier protein, even small antigens such as low molecular weight compounds and peptides can be stimulated by immunoreactions. There is no particular limitation on the protein as long as it can induce a response. For example, bovine thyroglobulin Phosphorus, bovine serum albumin (BSA), and Keyhole limpet hem Ocyanin (KLH) can be used.

[0138] The obtained antiserum was tested for the desired properties by ELISA using positive and negative controls. You can select what you have.

[0139] Positive controls include, for example, Compound (1), Compound (2), and Compound (6). Antisera that show high inhibition effects by these positive controls were selected. It can be distinguished.

[0140] The lactone ring of compound (1) is in an acidic aqueous solvent (for example, at a pH of about 3). The equilibrium is biased towards the closed ring isomer, whereas in a basic aqueous solvent (e.g., around pH 10), the equilibrium is biased towards the open ring isomer. It is known that the basic skeleton of compound (1) itself is From the viewpoint of selecting an antibody that recognizes the compound, a compound in which the lactone ring is reduced, such as formula

[0141] [ka]

[0142] The compound represented by the formula (hereinafter referred to as "compound (7)") can also be used as a positive control. The antisera that show a high inhibition effect by this positive control are selected. However, from the viewpoint of selecting an antibody that recognizes compound (1) and compound (7) to the same extent, Therefore, it is preferable to exclude antibodies that have a particularly high recognizing ability for compound (7) rather than compound (1). It's nice.

[0143] Furthermore, the antibody-drug conjugate according to the present invention (preferably, The positive controls (I), (II), and (III) can also be used. Antisera that show high inhibition effects by gender control can be selected. .

[0144] On the other hand, from the viewpoint of excluding antibodies that recognize parts distant from the basic skeleton of compound (1), e.g. For example, it is a compound consisting of a cyclohexane ring, which is a partial structure near the linker. formula

[0145] [ka]

[0146] A compound represented by the formula (hereinafter referred to as "compound (8)") can be used as a negative control. By excluding antisera that show a high inhibition effect in this negative control, This can be done.

[0147] The antiserum selected as described above is cloned to produce the antibody of the present invention. It is possible to obtain cells that

[0148] Next, the cleavage was carried out by a known method (for example, Kohler and Milstein, Nature (1975) 256, pp. 495-497, Ken net, R.ed., Monoclonal Antibodies, p.365-367, Plenum Press, NY (1980)). Hybridomas can be produced by fusing cells that produce the antibody of the present invention with myeloma cells. A specific example of such a method is available in the United States. International Publication No. WO09 / 48072 (published April 16, 2009) and WO This is listed in the pamphlet for issue 10 / 117011 (published October 14, 2010).

[0149] The obtained antibodies can be purified to homogeneity. Separation and purification of antibodies are performed in the same way as conventional protein purification. For example, column chromatography, Filtration, ultrafiltration, salting out, dialysis, preparative polyacrylamide gel electrophoresis, isoelectric By appropriately selecting and combining techniques such as spot electrophoresis, antibodies can be separated and purified (Strategy gies for Protein Purification and Characterization:A Laboratory Course Manual, D aniel R. Marshak et al. eds., Cold Spring Harbor Laboratory Press (1996), Antibod ies: A Laboratory Manual. Ed Harlow and David Lane, Cold Spring Harbor Laborator y(1988)), but is not limited to these.

[0150] Chromatography includes affinity chromatography, ion exchange chromatography, chromatograph, hydrophobic chromatography, gel filtration chromatography, reversed phase chromatography Examples of such chromatographs include adsorption chromatography and adsorption chromatography. The analysis can be carried out using liquid chromatography such as HPLC or FPLC. Columns used for affinity chromatography include protein A columns, protein For example, a Protein A column can be a Hyper D column. , POROS, Sepharose FF (Pharmacia), etc. In addition, antibodies can be purified by using a carrier on which an antigen is immobilized, utilizing its binding to the antigen. is also possible.

[0151] Furthermore, the antibody of the present invention is preferably a The antibody-drug conjugates are differentiated depending on the average number of drug linkers attached per antibody (DAR). The feature of this is that the recognition ability for the nucleotides is not substantially different. The antibody has a high DAR (DAR8), for example, in response to an antibody-drug conjugate with a high DAR. The standard curve for the antibody-drug conjugate with low DAR (DAR4) was compared. , can be confirmed by the absence of substantial differences.

[0152] An example of the antibody of the present invention obtained in this manner is the mouse antibody 1A3. The amino acid sequence of the heavy chain variable region of mouse antibody 1A3 is shown in SEQ ID NO: 15. The amino acid sequence shown in amino acid numbers 20 to 141 is a nucleotide sequence encoding the amino acid sequence. The code sequence is the nucleotide sequence from nucleotide numbers 58 to 423 in SEQ ID NO: 17. The amino acid sequence of the light chain variable region of mouse antibody 1A3 is shown in SEQ ID NO: 16. The amino acid sequence shown in amino acid numbers 21 to 127 is a nucleotide sequence encoding the amino acid sequence. The nucleotide sequence is the nucleotide sequence set forth in nucleotide numbers 61 to 381 of SEQ ID NO: 18. It is shown as an array.

[0153] The antibody of the present invention retains all six CDR sequences derived from mouse antibody 1A3, and Any antibody that specifically recognizes the drug moiety of the antibody-drug conjugate of the present invention may be used. There are several known methods for determining CDR sequences, including the definition of Abm, Chot, hia definition, Kabat definition, and Imgt® (The International tional ImMunoGeneTics information system The CDR sequences of the antibodies of the present invention can be determined by any method. It may also be defined by

[0154] According to the definition of Abm, the heavy chain variable region of the antibody of the present invention is the amino acid sequence shown in SEQ ID NO: 1. CDRH1 (GFTFSDYGMV) consisting of the amino acid sequence shown in SEQ ID NO: 2 CDRH2 (YISSGSSAIY) consisting of the sequence shown in SEQ ID NO: 3 The antibody of the present invention has a CDRH3 (PPRYDVYSAWFAY) sequence. The light chain variable region is CDRL1 (KASQDV) consisting of the amino acid sequence shown in SEQ ID NO: 4. GSAVV), CDRL2 (WASTRHT) consisting of the amino acid sequence shown in SEQ ID NO: 5 ), and CDRL3 (QQYSSYPVT) consisting of the amino acid sequence shown in SEQ ID NO: 6 holds the following.

[0155] According to the definition of Chothia, the heavy chain variable region of the antibody of the present invention is shown in SEQ ID NO: 7. CDRH1 (GFTFSDY) consisting of the amino acid sequence shown in SEQ ID NO: 8 CDRH2 (SSGSSA) consisting of the sequence, and the amino acid sequence shown in SEQ ID NO: 3 The light chain of the antibody of the present invention has a CDRH3 (PPRYDVYSAWFAY) sequence. The variable region is CDRL1 (KASQDVGSA) consisting of the amino acid sequence shown in SEQ ID NO: 4. VV), CDRL2 (WASTRHT) consisting of the amino acid sequence shown in SEQ ID NO: 5, and and CDRL3 (QQYSSYPVT) consisting of the amino acid sequence shown in SEQ ID NO: 6. is doing.

[0156] According to the definition of Kabat, the heavy chain variable region of the antibody of the present invention is the amino acid sequence shown in SEQ ID NO: 9. CDRH1 (DYGMV) consisting of the amino acid sequence shown in SEQ ID NO: 10 CDRH2 (YISSGSSAIYYADTVKG) consisting of the following: It has the amino acid sequence CDRH3 (PPRYDVYSAWFAY), The light chain variable region of the antibody of the present invention is CDRL1 (K) consisting of the amino acid sequence shown in SEQ ID NO: 4. ASQDVGSAVV), CDRL2 (WA STRHT), and CDRL3 (QQYSS) consisting of the amino acid sequence shown in SEQ ID NO: 6 YPVT).

[0157] According to the Imgt® definition, the heavy chain variable region of the antibody of the present invention is SEQ ID NO: 11 CDRH1 (GFTFSDYG) consisting of the amino acid sequence shown in SEQ ID NO: 12 CDRH2 (ISSGSSAI) consisting of the amino acid sequence shown in SEQ ID NO: 13 It has CDRH3 (ARPPRYDVYSAWFAY) which consists of the amino acid sequence The light chain variable region of the antibody of the present invention comprises a CDR consisting of the amino acid sequence shown in SEQ ID NO: 14. L1 (QDVGSA), tryptophan-alanine-serine (WAS) CDRL2 consisting of the peptide shown in SEQ ID NO:6, and CDRL3 consisting of the amino acid sequence shown in SEQ ID NO:6. (QQYSSYPVT).

[0158] In addition to the above-mentioned monoclonal antibodies, the antibodies of the present invention have other properties such as reducing heterologous antigenicity. recombinant antibodies artificially modified for the purpose of ) antibody, humanized antibody, or rabbit type These antibodies can be produced using known methods. can.

[0159] Chimeric antibodies include antibodies in which the variable and constant regions of the antibody are heterologous, such as mouse antibodies. Examples of such antibodies include chimeric antibodies in which the variable regions of a mouse or rat-derived antibody are joined to constant regions of human origin. (See Proc. Natl. Acad. Sci. USA, 81, 6851-6855, (1984)). For example, a chimera in which the variable region of an antibody derived from a mouse or rat is joined to the constant region derived from a rabbit is used. Examples of antibodies include rabbit chimeric antibodies.

[0160] A more specific example of a rabbit chimeric antibody is a chimeric antibody having a heavy chain variable region derived from mouse antibody 1A3. A heavy chain comprising the constant region of a rabbit antibody heavy chain, and a heavy chain derived from the mouse antibody 1A3 antibody and a light chain comprising a light chain variable region of a rabbit antibody and a constant region of a rabbit antibody light chain. Examples of the antibody (rabbit chimeric antibody 1A3) include the rabbit chimeric antibody 1A3. The heavy chain consists of the amino acid sequence set forth in amino acid numbers 20 to 464 of SEQ ID NO: 19. The light chain of rabbit chimeric antibody 1A3 is amino acid number 21 to 233 in SEQ ID NO: 20. It consists of the amino acid sequence set forth in

[0161] Mice are often used as non-clinical animal models for cancer research, and mouse antibodies are used If so, its use is limited to avoid competition with endogenous mouse IgG. Therefore, by using rabbit chimeric antibodies, it is possible to differentiate between mouse-derived samples and human-derived samples. This allows for comparative evaluation of both samples on the same platform, making it possible to advance translational research. This could be useful for research.

[0162] As for humanized antibodies, the complementarity determining regions (CDRs) An antibody in which only the terminating region has been incorporated into a human antibody (Nature (1986) 321, pp. 522-525), the amino acid residues of some frameworks in addition to the CDR sequences. Also mentioned are antibodies grafted onto human antibodies (International Publication No. WO90 / 07861). The rabbitized antibody can be prepared by cleaving the complementarity determining region (CDR) of the antibody. Only the ity determining region was incorporated into the rabbit-derived antibody. In addition to the CDR sequence, some of the framework amino acid residues were also transplanted to the rabbit antibody. Examples of such antibodies include:

[0163] In addition, the carboxyl-terminal lysine residue of the heavy chain of the antibody produced in mammalian cultured cells is deleted. It is known that the chromatographic method Similarly, two amino acid residues, glycine and lysine, were deleted from the carboxyl terminal of the heavy chain, resulting in a new carboxyl It is known that the proline residue located at the carboxyl terminus is amidated (Analytical Biochemistry, 360:75-83(2007)). However, these deletions and modifications of the heavy chain sequence are not sufficient for the production of antibodies. The antigen binding ability and effector functions (such as complement activation and antibody-dependent cellular cytotoxicity) of Therefore, the present invention also includes antibodies with such modifications, and the heavy chain carboxyl groups Deletion forms in which one or two amino acids are deleted at the terminal end, and amidated versions of the deletion forms (For example, a heavy chain in which the proline residue at the carboxyl terminal is amidated) However, as long as the antigen-binding ability and effector function are maintained, the antibody of the present invention can be used. The deletion of the carboxyl terminal of the heavy chain of the antibody is not limited to the above types. The two constituting heavy chains are either heavy chains selected from the group consisting of full-length and the above-mentioned deleted heavy chains. It may be one of the two or a combination of two of the two. The ratio may be affected by the type and culture conditions of the cultured mammalian cells that produce the antibody of the present invention. However, the main component of the antibody of the present invention is one amino acid at the carboxyl terminus of each of the two heavy chains. Examples include cases where amino acid residues are deleted.

[0164] The antibody of the present invention may be any antibody derived from mouse antibody 1A3 or rabbit antibody 1A4, provided that the properties of the antibody of the present invention are maintained. At least 95% identity (preferably at least 100%) with the amino acid sequence of chimeric antibody 1A3. The antibody may be an antibody consisting of an amino acid sequence having at least 99% identity with the antibody.

[0165] The identity between two amino acid sequences was determined by Blast (Nucl. Acids Res., 25, pp. 3389-340 2 (1997)) using the default parameters. The last can be found on the internet at www.ncbi.nlm.nih.gov / blast It can also be used.

[0166] The antibody of the present invention can also bind to the drug moiety in the antibody-drug conjugate of the present invention. The antibody competed with mouse antibody 1A3 or rabbit chimeric antibody 1A3 for recognition. That's fine.

[0167] The antibody obtained by the above method is useful in the antibody-drug conjugate of the present invention. The ability to recognize the drug site can be evaluated, and suitable antibodies can be selected. Another example of an index for comparison is antibody stability. DSC (Differential Scanning Calorimeter) is a method to measure the thermal denaturation midpoint (Tm This method allows for quick and accurate measurement of Tm values. By comparing the values, differences in thermal stability can be compared. It is known that the thermal stability of antibodies correlates to some extent with their thermal stability (Lori Burton, et. al., Pharmaceutical Development and Technology (2007) 12, p.265-273), Suitable antibodies can be selected using the qualitative index. The key features of the enzyme are high yield in suitable host cells and low aggregation in aqueous solution. For example, the antibody with the highest yield does not necessarily exhibit the highest thermostability. Therefore, it is necessary to make a comprehensive judgment based on the above-mentioned indicators and select the most suitable antibody. do.

[0168] Alternatively, the full-length sequences of the heavy and light chains of an antibody may be linked using an appropriate linker to form a single-chain immunoglobulin. Those who want to obtain single chain immunoglobulin The method is also known (Lee, HS, et al., Molecular Immunology (1999) 36, pp. 61-71, Sh Irrmann, T. et al., mAbs (2010), 2, (1) p.1-4). By dimerizing, the tetramer retains a structure and activity similar to that of antibodies, which are originally tetramers. Furthermore, the antibody of the present invention has a single heavy chain variable region and a light chain sequence. Such antibodies may be single domain antibodies. These antibodies are called main antibodies (sdAbs) or nanobodies. It has been reported that antigen-binding ability is actually observed in camels and llamas. (Muyldemans S. et al., Protein Eng. (1994) 7(9), 1129-35, Hamers-Casterman (n C. et al., Nature (1993) 363 (6428) 446-8). The above antibody is the antibody of the present invention. It can also be interpreted as a type of antigen-binding fragment.

[0169] When the antibody gene is isolated and then introduced into a suitable host to produce the antibody, A combination of a host and an expression vector can be used. Specific examples of antibody genes include , a gene encoding the heavy chain sequence of the antibody described herein, and a gene encoding the light chain sequence When transforming a host cell, the heavy chain The sequence gene and the light chain sequence gene can be inserted into the same expression vector. Alternatively, they may be inserted into separate expression vectors. When the cells are used, animal cells, plant cells, and eukaryotic microorganisms can be used. (1) Mammalian cells, for example, COS cells, which are monkey cells (Gluzman, Y. Cell (1981) 23 , pp.175-182, ATCC CRL-1650), mouse fibroblast NIH3T3 (ATCC C No. CRL-1658) and Chinese hamster ovary cells (CHO cells, AT A dihydrofolate reductase-deficient strain of the genus CCL-61 (Urlaub, G. and Chasin, L.A. P Proc. Natl. Acad. Sci. USA (1980) 77, pp. 4126-4220). When prokaryotic cells are used, examples thereof include Escherichia coli and Bacillus subtilis. The desired antibody gene is introduced into cells by transformation, and the transformed cells are cultured in vitro. Antibodies are obtained by culturing in vitro. In the above culture methods, the antibody sequence is The yield may vary depending on the antibody, and the antibody with the same binding activity is selected as a pharmaceutical using the yield as an indicator. It is possible to select those that are easy to produce.

[0170] The isotype of the antibody of the present invention is not limited, and may be, for example, IgG (IgG1, IgG2 , IgG3, IgG4), IgM, IgA (IgA1, IgA2), IgD or Ig Examples of antibodies include IgG and IgM, but preferably IgG or IgM, and more preferably IgG1 Alternatively, IgG2 can be mentioned.

[0171] The antibody of the present invention is also an antigen-binding fragment of the antibody having the antigen-binding site thereof, or a modified fragment thereof. The antibody may be treated with a proteolytic enzyme such as papain or pepsin, or By modifying the antibody gene by genetic engineering techniques and expressing it in appropriate cultured cells, Thus, fragments of the antibody can be obtained. Among such antibody fragments, the full-length antibody molecule A fragment that retains all or part of the function of an antibody can be called an antigen-binding fragment of an antibody. do.

[0172] For example, antibody fragments include Fab, F(ab'), Fv, or F(ab') of heavy and light chains. single-chain Fv (scFv) in which Fv is linked with an appropriate linker; diabody ( Examples include antibodies, diabodies, linear antibodies, and multispecific antibodies formed from antibody fragments. In addition, monovalent fragments of the variable region of an antibody obtained by treating F(ab')2 under reducing conditions can be obtained. The fragment Fab' is also included in the antibody fragments.

[0173] Furthermore, the antibody of the present invention is a multispecific antibody having specificity for at least two different antigens. Such molecules usually bind to two different antigens (i.e. That is, bispecific antibodies), "Multispecific antibodies" include antibodies with specificity for more than one antigen (e.g., three types). It includes.

[0174] The multispecific antibodies of the present invention may be full-length antibodies or fragments of such antibodies (e.g., F (ab')2 bispecific antibodies) are also possible. Bispecific antibodies are antibodies that combine two types of antibody heavy and light chains ( They can be made by combining two different monoclonal antibodies (HL pairs), or by combining two different HL pairs that produce different monoclonal antibodies. Bispecific antibody-producing hybridomas can also be fused to generate hybridomas. It can be produced by the method described in Millstein et al., Nature (1983) 305, pp. 537-539.

[0175] The antibody of the present invention may be a single-chain antibody (also referred to as scFv). A heavy chain variable region and a light chain variable region are linked by a polypeptide linker. (Pluckthun, The Pharmacology of Monoclonal Antibodies, 113 (Rosenberg and Moore ed., Springer Verlag, New York, p.269-315(1994), Nature Biotechnology (2005), 23, pp.1126-1136). In addition, two scFvs are linked together with a polypeptide linker to form BiscFv fragments can also be used as bispecific antibodies.

[0176] Methods for producing single chain antibodies are well known in the art (see, e.g., U.S. Patent No. 4, 946,778, U.S. Patent No. 5,260,203, U.S. Patent No. 5,091,513 (See, for example, U.S. Patent No. 5,455,030.) In this scFv, the heavy chain variable region and The light chain variable region is preferably linked to a linker, preferably a polypeptide linker, that does not form a conjugate. The nucleotides are linked via a linker (Huston, JS et al., Proc. Natl. Acad. Sci. USA (19 88), 85, pp. 5879-5883). The heavy chain variable region and the light chain variable region of the scFv are the same antigen. The polypeptide linking the variable regions may be derived from the body or from separate antibodies. As the anchor, for example, any single-chain peptide consisting of 12 to 19 residues can be used.

[0177] The DNA encoding the scFv is DNA encoding the heavy chain or heavy chain variable region of the antibody. and DNA encoding the light chain or light chain variable region, the whole or The DNA portion encoding the desired amino acid sequence is used as a template, and primers that define both ends of the template are used. The pair is then amplified by PCR, and a polypeptide linker portion is further added. The DNA is then combined with a primer pair that specifies that both ends of the DNA are to be linked to the heavy and light chains, respectively. It is obtained by combining and amplifying the

[0178] Once the DNA encoding the scFv is prepared, expression vectors containing the DNA can be used. and a host transformed with the expression vector can be obtained by a conventional method. By using the host, scFv can be obtained according to a conventional method. The fragments can be produced by the host by obtaining and expressing the genes in the same manner as described above. do.

[0179] The antibody of the present invention may be polymerized to increase its affinity for the antigen. Even one type of antibody may recognize multiple epitopes of the same antigen. It may be a plurality of antibodies. As a method for multimerizing antibodies, an IgG CH3 domain and Binding to two scFvs, binding to streptavidin, helix-turn-helix Examples include the introduction of a motif.

[0180] The antibody of the present invention is a polyclonal antibody, which is a mixture of multiple types of antibodies with different amino acid sequences. An example of a polyclonal antibody is a polyclonal antibody having multiple types of antibodies with different CDRs. Such polyclonal antibodies may contain a mixture of antibodies. A mixture of cells producing the antibody can be cultured and the antibody purified from the culture can be used. (See WO2004 / 061104).

[0181] Antibodies conjugated with various molecules such as polyethylene glycol (PEG) as modified antibodies can also be used.

[0182] The antibodies of the present invention may also be conjugated with other drugs ( Examples of such antibodies include antibodies that are capable of releasing Examples include those bound to radioactive substances or compounds with pharmacological properties (Nature Bio technology (2005) 23, p.1137-1146).

[0183] 4. Use of the Protein of the Present Invention The protein of the present invention can be used in ELISA (Enzyme-Linked ImmunoSorb ent Assay) method, ECL (Electrochemiluminescence) method ) method, RIA (Radio Immunoassay) method, ELISPOT (Enzyme e-Linked ImmunoSpot) method, dot blot method, Ouchterlony method, C IE (Counterimmunoelectrophoresis) method, CLIA (C hemiluminescent immunoassay), and FCM (Flow detection methods such as cytometry, as well as immunohistochemistry It can be used in IHC (Immunochromatography) methods, and preferably in ELISA, EC It can be used in the L method and IHC method.

[0184] In the ELISA and ECL methods, for example, The mammal to which the antibody is administered ("mammal" in the present invention includes, for example, humans, mice, rats, etc.) Examples of mammals include, but are not limited to, dogs, monkeys, rabbits, etc. ) can be used in a method for quantifying the plasma concentration of the antibody-drug conjugate in can.

[0185] Specifically, (1) the target antigen of the antibody-drug conjugate is immobilized on a plate, and the blood is then transferred to the plate. (2) contacting the antibody-drug conjugate in plasma to form a complex; contacting the protein of the present invention, labeled with α-amino-2-hydroxybenzoates, with the protein of the present invention to form a further complex; and then (3) detecting the marker.

[0186] In addition, (1) the antibody-drug conjugate in plasma is immobilized on a plate on which the protein of the present invention is immobilized. (2) contacting the antibody of the antibody-drug conjugate with the antibody to form a complex; A second protein capable of recognizing the site and labeled with a marker is contacted, and a further (3) allowing a complex to form, and then (4) detecting the marker. It is also possible.

[0187] In addition, the ELISA method and the ECL method were carried out using the antibody-drug conjugate of the present invention. The drug (e.g., compound (1)) released from the antibody-drug conjugate in a mammal ), Compound (2), Compound (4), and Compound (5)) in plasma. It can also be used.

[0188] Specifically, (1) a plate on which the protein of the present invention is immobilized is coated with a marker-labeled antibody. The drug released from the antibody-drug conjugate in plasma was contacted with the antibody-drug conjugate in the presence of a competing drug. (2) allowing a complex to form; and (3) detecting the marker.

[0189] For example, the IHC method may involve administering the antibody-drug conjugate of the present invention to a mammal. the antibody-drug conjugate and / or the antibody-drug conjugate This method can be used to quantify the tissue distribution of drugs.

[0190] Specifically, (1) antibody-drug conjugates in tissues and / or the antibody-drug conjugates contacting the drug released from the conjugate with the protein of the present invention to form a complex; 2) contacting a second protein that can recognize the protein of the present invention and is labeled with a marker; allowing the formation of additional complexes, and then (3) detecting the marker. Includes:

[0191] In addition, (1) antibody-drug conjugates in tissues and / or the antibody-drug conjugates The drug released from the target is brought into contact with the protein of the present invention labeled with a marker to form a complex. and then (2) detecting the marker.

[0192] In the present invention, the term "marker" refers to a substance that generates a detectable signal or other group. The term "marker" refers to any substance that induces a substance to produce a detectable signal. Examples of the carrier include fluorescent substances, enzymes, enzyme fragments, enzyme substrates, enzyme inhibitors, coenzymes, catalysts, etc. Examples of suitable photocatalysts include solvents, dyes, luminescent materials, sensitizers, and radioactive materials.

[0193] In the present invention, "labeled with a marker" means that the marker is directly or in any partial structure. It means that biotin and avidin (or This also includes binding via interactions with streptavidin.

[0194] To label the protein of the present invention with a marker, for example, A reagent (having an active ester group) is reacted with the lysine residue of the protein of the present invention to form an amide. It is sufficient to form a bond, but the present invention is not limited to this.

[0195] When the marker is a fluorescent substance, the marker is detected by sensing the fluorescence of the marker. This is done by:

[0196] Examples of such fluorescent materials include DyLight (registered trademark) 350 and DyLi ght(registered trademark) 405, DyLight(registered trademark) 488, DyLight(registered trademark) DyLight® 550, DyLight® 594, DyLight® 633 , DyLight® 650, DyLight® 680, DyLig ht(registered trademark) 747, DyLight(registered trademark) 755, DyLight(registered trademark) Alexa Fluor® 800, Alexa Fluor® 350, Alexa Fluor® Alexa Fluor® 405, Alexa Fluor® 488, Alexa Fluor® (registered trademark) 532, Alexa Fluor (registered trademark) 546, Alexa Flu or® 555, Alexa Fluor® 568, Alexa F Fluor® 594, Alexa Fluor® 647, Alexa Fluor® 680, Alexa Fluor® 750, BOD IPY® FL, Coumarin, Cy® 3, Cy® 5 , Cy (registered trademark), Fluorescein (FITC), Oregon Green (registered trademark), Pacific Blue, Pacific Green, Pacifi c Orange, Tetramethylrhodamine (TRITC), Tex as Red (registered trademark).

[0197] Also, Qdot (registered trademark) 525, Qdot (registered trademark) 565, Qdot (registered trademark) Qdot® 605, Qdot® 655, Qdot® 705, and Qdot Nanocrystals such as 800 (registered trademark) and Allophycocyanin (AP C), R-Phycoerythrin (R-PE), Cyan Fluorescen t Protein(CFP), Green Fluorescent Protein (GFP), and Red Fluorescent Protein (RFP) Fluorescent proteins can also be used as fluorescent substances.

[0198] When the marker is an enzyme, the detection of the marker occurs by the reaction of the enzyme with a substrate. This is done by sensing the emission of light or color.

[0199] Such enzymes include, for example, peroxidase (e.g., horseradish peroxidase) In this case, examples of the enzyme substrate include, for example, For example, TMB (3,3',5,5'-tetramethylbenzidine), DA B(3,3'-Diaminobenzidine tetrahydrochlori de), OPD (o-Phenylenediamine), and ABTS (3-Eth ylbenzothiazoline-6-sulfonic acid) can be done.

[0200] In addition, other enzymes include alkaline phosphatase, in which case, The substrate of the enzyme is, for example, BCIP (5-bromo-4-chloro-3-in dolyl phosphate), and PNPP (ρ-nitrophenyl ph phosphate).

[0201] Furthermore, the other enzyme may be luciferase, in which case the substrate of the enzyme Examples of such compounds include Luciferin and Coelenterazines. It is possible.

[0202] Further, other enzymes include β-galactosidase, in which case The substrate of the enzyme is, for example, o-nitrophenyl-β-D-galactose. pyranoside (ONPG) can be exemplified.

[0203] When the marker is a luminescent material, the detection of the marker is based on an electrochemical reaction. This is done by detecting the light emitted by the sensor.

[0204] Such a luminescent material can include, for example, a ruthenium complex, and is preferably Ruthenium-pyridine complexes are preferred, and ruthenium (II) trioxides are more preferred. Mention may be made of lith(bipyridyl) complexes.

[0205] The above electrochemical reaction can be carried out in the presence of, for example, tripropylamine (TPA). Specifically, the electrode reaction can be carried out by reacting the TPA cation radical with a trivalent ruthenium complex. After generation, the TPA cation radical immediately loses a hydrogen ion and exhibits strong reducing properties. This forms a TPA radical, which reacts with a trivalent ruthenium complex to produce light. .

[0206] When the marker is a radioactive substance, the detection of the marker is carried out by detecting the radiation emitted by the marker. This is done by sensing the

[0207] Such radioactive materials include, for example, tritium ( 3 H), carbon-14( 14 C) , nitrogen-15( 15 N), sulfur-35( 35 S), Yttrium-90( 90 Y), Tech Netium-99( 99 Tc), Indium-111( 111 In), iodine-125( 1 25 I), and iodine-131( 131 I) and the like can be mentioned.

[0208] The protein of the present invention may contain a pH buffer, an osmotic pressure regulator, a salt, a stabilizer, a preservative, a color developer, an enhancer, By incorporating a sensitizer, an anti-aggregating agent, etc., the composition (hereinafter referred to as "the composition of the present invention") ) can be used as.

[0209] The proteins of the invention (or compositions of the invention) may be used in the materials and reagents used to perform the assays. It can be used as a kit in combination with a drug (hereinafter referred to as the "kit of the present invention"). Reagents may be liquid or lyophilized in the same or separate containers depending on their stability. The amounts and proportions of reagents provided in the kits of the present invention may vary depending on the particular application. The kit of the present invention may contain the protein (or In addition to the composition of the present invention, for example, a reagent for labeling with a marker, a substrate for an enzyme, a bromine Coding reagent, polymer reagent, antigen retrieval solution, calibrator, dilution buffer, washing buffer It includes a buffer solution, a buffer solution for immobilization, an immobilized antibody, an antibody for detection, and a microtiter well. The kit of the present invention may also include instructions for using the kit. The antibody-drug conjugate and / or the antibody-drug conjugate are released using a This makes it possible to confirm the tissue distribution of the drug and quantify its plasma concentration. [Example]

[0210] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples. These are not intended to be limiting in any way. Unless otherwise specified, each genetic manipulation procedure is performed under the heading "Molecular Cloning (MCL)." olecular Cloning)” (Sambrook, J., Fritsch, E. .F. and Maniatis, T., Cold Spring Harbor Lab or by the method described in the publication of the International Journal of Clinical Chemistry, Vol. 1, No. 1, 1989, by the Department ... When commercially available reagents or kits were used, they were used according to the instructions of the commercially available products. In the above, reagents, solvents, and starting materials not specifically mentioned are readily available from commercial sources. is.

[0211] [Example 1] Synthesis of Compound i) 8-[(2,5-dioxopyrrolidin-1-yl)oxy]-N-(4-{[(1 S,9S)-9-Ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-di Oxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de] Pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl]amino Synthesis of}-4-oxobutyl-8-oxooctaneamide (compound (6))

[0212] [ka]

[0213] 4-amino-N-[(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4 -Methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H ,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]ky [Norin-1-yl]butanamide (0.032 g, 0.050 mmol) (International Publication No. 2002 / 0022666). N,N-dimethylformamide (the compound obtained in Step 2 of Example 1 described in Patent Publication No. 014 / 057687) To a solution of 1,000 mL of dimethylformamide (0.5 mL), triethylamine (7 μL, 0.050 mmol) ), and di(N-succinimidyl) suberate (20.4 mg, 0.055 mmol) was added. The solvent was evaporated under reduced pressure, and the resulting residue was purified by silica gel column chromatography. Chromatography [chloroform to chloroform:methanol = 9:1 (v / v)] Purification gave the title compound (16.0 mg, 41%) as a pale yellow solid. 1 H-NMR (400MHz,DMSO-d6) δ:0.87(3H,t,J=7. 6Hz),1.18-1.37(4H,m),1.40-1.50(2H,m),1.5 4-1.64(2H,m),1.65-1.74(2H,m),1.78-1.93(2 H,m),2.02(2H,t,J=7.6Hz),2.09-2.20(4H,m), 2.40(3H,s),2.60-2.68(2H,m),2.80(4H,s),3. 00-3.08(2H,m),3.13-3.21(2H,m),5.19(2H,dd ,J =32.0,18.0Hz),5.37-5.47(2H,m),5.53-5. 60(1H,m),6.52(1H,s),7.30(1H,s),7.74-7.82 (2H,m), 8.44 (1H,d,J=8.5Hz). MS(APCI)m / z:774(M+H) +

[0214] ii) 4-amino-N-[(1S,9S)-9-ethyl-5-fluoro-9,10-di Hydroxy-4-methyl-13-oxo-2,3,9,10,13,15-hexahydro -1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2- Synthesis of b]quinolin-1-yl]butanamide trifluoroacetate (compound (7))

[0215] [ka]

[0216] Step 1: tert-Butyl (4-{[(1S,9S)-9-ethyl-5-fluoro-9-hydro Oxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydride 1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2 -b]quinolin-1-yl]amino}-4-oxobutyl)carbamate (0.092g , 0.148mmol) (Example 1, Step 1 described in WO 2014 / 057687 The compound obtained in (1.6 mL) was dissolved in methanol and cooled on ice. Sodium (0.028 g, 0.740 mmol) was added and the mixture was stirred for 20 minutes under ice cooling. Dilute with ethanol (10 mL) and chloroform (50 mL), add 10% aqueous citric acid solution, and The resulting organic layer was extracted with chloroform. The mixture was washed with water, dried over sodium sulfate, and filtered. The solvent was evaporated under reduced pressure, and the resulting residue was Silica gel column chromatography [chloroform:methanol=100:0-95: 5 (v / v)] to obtain a pale yellow solid, tert-butyl (4-{[(1S,9S)- 9-Ethyl-5-fluoro-9-hydroxy-4-methyl-13-oxo-2,3,9, 10,13,15-Hexahydro-1H,12H-benzo[de]pyrano[3',4': 6,7]indolizino[1,2-b]quinolin-1-yl]amino}-4-oxobutyl ) carbamate (0.078 g, 85%). 1 H-NMR (400MHz,DMSO-d6) δ:0.89(3H,t,J=7. 4Hz),1.31(9H,s),1.62-1.73(4H,m),2.08-2.1 8(4H,m), 2.39(3H,s),2.91(2H,q,J=6.5Hz),3 .12-3.20(2H,m),4.49(1H,d,J=17.2Hz),4.61( 1H,d,J=17.2Hz),4.97(1H,s),4.99(1H,d,J=4. 7Hz),5.10(2H,d,J=18.8Hz),5.21(2H,d,J=18. 8Hz),5.54-5.58(1H,m),6.74-6.82(2H,m),7.3 3(1H,s),7.78(1H,d,J=11.0Hz),8.40(1H,d,J= 8.6Hz). Step 2: The compound obtained in step 1 above (0.078 g, 0.125 mmol) was dissolved in dichloromethane (2 The mixture was cooled on ice, trifluoroacetic acid (2 mL) was added, and the mixture was stirred for 40 minutes. The residue was evaporated under reduced pressure and purified by silica gel column chromatography [chloroform - The mixture was purified by partitioning with chloroform:methanol:water=7:3:1 (v / v / v) organic layer. The obtained solid was dissolved in methanol, and ether was added to the solution. The precipitate was collected by filtration and dried under vacuum. This afforded the title compound (0.045 g, 56%) as a yellow solid. 1 H-NMR (400MHz,DMSO-d6) δ:0.89(3H,t,J=7. 4Hz),1.71(2H,q,J=7.4Hz),1.80-1.88(2H,m), 2.09-2.19(2H,m),2.27(2H,t,J=7.0Hz),2.40( 3H,s),2.84(2H,t,J=7.6Hz),3.13-3.20(2H,m) ,4.50(1H,d,J=17.2Hz),4.62(1H,d,J=17.2Hz) ,4.97-5.02(2H,m),5.09(1H,d,J=18.8Hz),5.2 1(1H,d,J=18.8Hz),5.55-5.59(1H,m),6.79(1H ,d,J=4.7Hz),7.34(1H,s),7.64-7.75(3H,m),7 .79(1H,d,J=11.3Hz),8.53(1H,d,J=8.6Hz). MS(APCI)m / z:523(M+H) +

[0217] iii) Synthesis of 4-amino-N-cyclohexyl-butanamide hydrochloride (compound (8)) Growth

[0218] [ka]

[0219] Step 1: 4-(tert-butoxycarbonylamino)butanoic acid (0.492 g, 2.42 mm 0L) was dissolved in dichloromethane (15 mL) and N,N-dimethylformamide (2 mL). N-hydroxysuccinimide (0.279 g, 2.42 mmol) and EDC I(1-Ethyl-3-(3-dimethylaminopropyl)carbo diimide Hydrochloride) (0.464 g, 2.42 mmol) The reaction solution was added with cyclohexylamine (0.200 g, The mixture was added dropwise to a dichloromethane solution (2 mL) of 2.02 mmol of ethanol and stirred at room temperature for 20 minutes. The reaction mixture was diluted with dichloromethane, and a 10% aqueous solution of citric acid was added. The resulting organic layer was washed with saturated aqueous sodium bicarbonate, dried over sodium sulfate, and filtered. The solvent was evaporated under reduced pressure, and the resulting residue was purified by silica gel column chromatography [hexane: ethyl acetate = 50:50 to 25:75 (v / v)] to give a colorless oily tert-butyl ether. ethyl N-[4-(cyclohexylamino)-4-oxo-butyl]carbamate (0.3 08g, 54%). 1 H-NMR (400MHz,CDCl3) δ:1.08-1.23(4H,m), 1.29-1.42(2H,m),1.44(9H,s),1.66-1.75(2H, m),1.77-1.83(2H,m),1.86-1.95(2H,m),2.17( 2H,t, J=7.0Hz),3.11-3.21(2H,m),3.69-3.82 (1H,m),4.75(1H,brs),5.89(1H,brs). Step 2: The compound obtained in step 1 above (0.200 g, 0.703 mmol) was dissolved in ethyl acetate (50 ml) The mixture was dissolved in 4N hydrochloric acid and dioxane (10 mL). The resulting precipitate was collected by filtration and dried in vacuo to give the title compound (0.098 g) as a colorless solid. , 63%). 1 H-NMR (400MHz,DMSO-d6) δ:1.06-1.30(5H,m ),1.50-1.58(1H,m),1.62-1.82(6H,m),2.16(2 H,t,J= 7.2Hz),2.69-2.79(2H,m),3.45-3.58( 1H,m),4.73(1H,brs),7.89(1H,d,J=7.8Hz),7. 96-8.10(2H,m). MS(APCI)m / z:185(M+H) +

[0220] [Example 2] Preparation of antibody-drug conjugate i) Preparation of anti-B7-H3 antibody-drug conjugate (1) According to the production method described in WO 2014 / 057687, an anti-B7-H3 antibody ( A heavy chain consisting of the amino acid sequence set forth in amino acid numbers 20 to 471 in SEQ ID NO: 27, and and a light chain consisting of the amino acid sequence set forth in amino acid numbers 21 to 233 in SEQ ID NO: 28. an antibody comprising the formula

[0221] [ka]

[0222] (wherein A represents the binding site to the antibody) and an anti-B7-H3 antibody bound via a thioether bond. B7-H3 antibody-drug conjugate (referred to as "B7-H3-ADC(I)" in the present invention) (hereinafter referred to as "the 'product') was manufactured.

[0223] ii) Preparation of anti-B7-H3 antibody-drug conjugate (2) According to the production method described in WO 2014 / 057687, an anti-B7-H3 antibody ( A heavy chain consisting of the amino acid sequence set forth in amino acid numbers 20 to 471 in SEQ ID NO: 27, and and a light chain consisting of the amino acid sequence set forth in amino acid numbers 21 to 233 in SEQ ID NO: 28. an antibody comprising the formula

[0224] [ka]

[0225] (wherein A represents the binding site to the antibody) and an anti-B7-H3 antibody bound via a thioether bond. B7-H3 antibody-drug conjugate (referred to as "B7-H3-ADC(II)" in the present invention) (hereinafter referred to as "the '

[0226] iii) Preparation of anti-B7-H3 antibody-drug conjugate (3) According to the production method described in WO 2014 / 057687, an anti-B7-H3 antibody ( A heavy chain consisting of the amino acid sequence set forth in amino acid numbers 20 to 471 in SEQ ID NO: 27, and and a light chain consisting of the amino acid sequence set forth in amino acid numbers 21 to 233 in SEQ ID NO: 28. an antibody comprising the formula

[0227] [ka]

[0228] (wherein A represents the binding site to the antibody) and an anti-B7-H3 antibody bound via a thioether bond. B7-H3 antibody-drug conjugate (referred to as "B7-H3-ADC(III)" in the present invention) ") was manufactured.

[0229] iv) Production of anti-HER2 antibody-drug conjugates (1) An anti-HER2 antibody (synthetic compound) was prepared according to the production method described in WO 2015 / 115091. A heavy chain and a sequence comprising the amino acid sequence set forth in amino acid numbers 1 to 449 in sequence number 21. A light chain consisting of the amino acid sequence set forth in amino acid numbers 1 to 214 in sequence number 22. Using an antibody comprising

[0230] [ka]

[0231] (wherein A represents the binding site to the antibody) and an anti-HER2 antibody linked via a thioether bond. HER2 antibody-drug conjugate (referred to as "HER2-ADC(I)" in the present invention) ) was manufactured.

[0232] v) Production of anti-HER3 antibody-drug conjugates (1) An anti-HER3 antibody (synthetic compound) was prepared according to the production method described in WO 2015 / 155998. A heavy chain consisting of the amino acid sequence set forth in SEQ ID NO: 23 and a an antibody comprising a light chain comprising:

[0233] [ka]

[0234] (wherein A represents the binding site to the antibody) and an anti-HER3 antibody linked via a thioether bond. HER3 antibody-drug conjugate (referred to as "HER3-ADC(I)" in the present invention) ) was manufactured.

[0235] vi) Preparation of anti-TROP2 antibody-drug conjugates (1) According to the production method described in WO 2015 / 098099, an anti-TROP2 antibody ( A heavy chain consisting of the amino acid sequence set forth in amino acid numbers 20 to 470 in SEQ ID NO: 25, and and a light chain consisting of the amino acid sequence set forth in amino acid numbers 21 to 234 in SEQ ID NO: 26. an antibody comprising the formula

[0236] [ka]

[0237] (wherein A represents the binding site to the antibody) and an anti-TROP2 antibody via a thioether bond. TROP2 antibody-drug conjugate (referred to as "TROP2-ADC(I)" in the present invention) (hereinafter referred to as "the 'product') was manufactured.

[0238] [Example 3] Production of monoclonal antibodies i) Preparation of antigen protein The antigen protein is compound (6) with bovine thyroglobulin as a carrier protein. (hereinafter referred to as "antigen protein (1)") and BSA-attached (hereinafter referred to as "antigen protein (2)") (2)) was used.

[0239] ii) immunity For immunization, BALB / cAnNCrlCrlj (BALB / c) mice (4 individuals) and B Four female 6D2F1 / Crlj(BDF1) mice (Charles River Japan Co., Ltd.) were used. The first time, antigen protein (1) and Freund's Complete A From the second round onwards, the mixture was mixed with antigen protein (1) and F Reund's Incomplete Adjuvant (Wako Pure Chemical Industries, Ltd.) The drugs were administered subcutaneously and intradermally every seven days for a total of four times.

[0240] iii) Antiserum antibody titer evaluation BALB / c mice (4 mice) and BDF1 mice (4 mice) before immunization and after four doses The serum was diluted from 200 to 204,800 times and used as a positive control. (2) and B7-H3-ADC(II), and the antibody titer against BSA was confirmed as a negative control. The antigen protein (2), B7-H3-ADC (II), and BSA were used as immunoassays for ELISA. The plate was immobilized and diluted pre-immunization and post-fourth-administration mouse sera were incubated at 37°C for 30 minutes. After washing, horseradish peroxidase-conjugated ed anti-mouse IgG (anti-mouse IgG-HRP) for 37 The mixture was incubated at 5°C for 30 minutes. After washing, o-phenylenediamine dihydr After adding OPD solution and stopping the color development, the absorbance at 490 nm was measured. It was confirmed that antibody titers against the positive control were elevated in all individuals.

[0241] iv) Inhibition ELISA After four doses, the serum of BALB / c mice (4 mice) and BDF1 mice (4 mice) Compounds (6), (7) and (1) were used as positive controls, and compound ( 8) was added, and the unabsorbed rate of antigen protein (2) was calculated. Mouse serum and compound (6) prepared at 12.5, 25, 50, and 100 μg / mL. (8), compound (7) and compound (1) were mixed and reacted at 4°C overnight. The substance (2) was added to an ELISA immunoplate solid-phased and reacted at 37°C for 30 minutes. After washing, the plate was reacted with anti-mouse IgG-HRP at 37°C for 30 minutes. After washing, OPD solution was added, and the absorbance at 490 nm was measured after the color development was stopped. The unabsorbed rate was calculated. Unabsorbed rate (%) = (a / b) × 100 a: Absorbance of the positive control or negative control at an added concentration of 12.5 μg / mL b: Absorbance without adding positive or negative control All mouse sera reacted to the positive control compounds (6) and (1), and some mice reacted to the compounds. Compound (7) showed a high inhibitory effect. No significant inhibitory effect was observed in any of the plasma samples. Compound (7) showed a high inhibitory effect, and compound (8) showed a high inhibitory effect. BALB / c mice and BDF1 mice with serum in which the bition effect was not observed One individual was selected from each group, and the lymph nodes and spleen were collected and used to prepare hybridomas. .

[0242] v) Hybridoma production iv) Lymph node cells and spleen cells from the individuals selected in step iv) were transfected with mouse myeloma cells using the PEG method. Cell fusion was performed. The culture supernatant of the resulting hybridoma was used to identify antibody-producing hybridomas. I did the cleaning.

[0243] vi) Evaluation of specific binding to antigen protein (2) The antigen protein (2) was immobilized on an immunoplate for ELISA, and the antibody-producing antibody was diluted 2-fold. The hybridoma supernatant was incubated at 37°C for 120 minutes. After washing, anti-mouse Ig G-HRP was reacted for 30 minutes at 37°C. After washing, OPD solution was added and color development was stopped. The absorbance at 490 nm was measured. Culture supernatants with an OD value of 0.2 or higher were considered to be producing haploids. Eleven hybridoma strains were selected as positive.

[0244] vii) Cross-check For the 11 strains selected as positive in vi), compound (6) and compound ( 1), B7-H3-ADC(II), B7-H3-ADC(I) and B7-H3-ADC (III), and compounds (8) and (7) were used as reactivity confirmation compounds. Inhibition ELISA was performed. 2-fold dilution of vi) was selected as positive. The positive control and reactivity confirmation compounds were added to the culture supernatant of the 11 strains at a final concentration of 25 μg / mL. After overnight reaction at 4°C, the antigen protein (2) was immobilized on an ELISA plate. The mixture was added to an immunoplate for use in the immunoassay and reacted at 37°C for 30 minutes. e IgG-HRP was reacted for 30 minutes at 37°C. After washing, OPD solution was added and color development was performed. After stopping the reaction, the absorbance at 490 nm was measured. The reaction was highly reactive to antigen protein (2), and the result was positive. Eight strains that showed inhibition effects by the control were selected and subjected to primary cloning. did.

[0245] viii) Primary cloning and primary screening The eight strains selected in vii) were cloned by limiting dilution. Mouse thymocytes were seeded at 5 x 10 per well of a 96-well plate. 6 Add cells / well The cells were cultured using TIL (Immuno-Biological Laboratories Co., Ltd.) containing 10% FBS. Various inhibition ELISAs were performed and specificity was confirmed for 8 strains x 6 subclones. I selected the option.

[0246] ix) Primary Cross-Check B7-H3-ADC(II) and B7- Reactivity to H3-ADC(I) was confirmed. B7-H3 C1 domain Lot B7_OmJ1 was diluted to 1 μg / mL with immobilized buffer and 100 μL of each solution was added to an xi-Sorp plate and immobilized overnight at 4°C. The medium was removed, 180 μL of 5% BSA-containing PBS was added to each well, and the mixture was left to stand at room temperature for 3 hours. After washing twice with PBS containing 0.05% Tween 20, the cells were treated with B7 at a concentration of 0.1 μg / mL. 100 μL each of B7-H3-ADC(II) and B7-H3-ADC(I) was added, and the mixture was incubated at room temperature. After washing twice with PBS containing 0.05% Tween 20, 50 μL of hybridoma supernatant diluted in 7 steps from 100 μg / mL to 100 μg / mL at a common ratio of 3 was added to each well, and then the mixture was incubated at room temperature. After washing twice with PBS containing 0.05% Tween 20, the plate was diluted 5000 times. Peroxidase AffiniPure Goat Anti-Mouse Ig G, Fcγ Fragment Specific(Jackson Immuno R 100 μL of HCl (Research Laboratories, Inc.) was added to each well and incubated at room temperature. After washing three times with PBS containing 0.05% Tween 20, the plate was incubated with HRP substrate (0 100 μL of 1M HCl was added to each well to develop a color reaction. After stopping the color reaction by adding 4 μL of each solution, the mixture was incubated with ARVO (PerkinElmer) for 4 h. The absorbance at 50 nm was measured. B7-H3-ADC was detected in all 8 strains x 6 subclones. Reactivity to B7-H3-ADC (II) and B7-H3-ADC (I) was confirmed. The 4 strains x 6 subclones with the highest activity were subjected to primary calibration curve confirmation.

[0247] x) Primary calibration curve confirmation B7-H3-ADC(I)(DAR7) and B Reactivity to 7-H3-ADC(I)(DAR5) was confirmed by ELISA. Pure Goat Anti-Mouse IgG,Fcγ fragment sp Coat ecific(Jackson ImmunoResearch Inc.) Dilute to 1µg / mL in Infusion Buffer and inject 100µL into a Maxi-Sorp plate. After adding 1 L of each, the plate was left to solidify overnight at 4°C. The next day, the medium was removed from the plate, and the plate was washed with PBS. 100 μL of PBS containing 10% BSA was added to each well, and the plate was left to stand at 37°C for 2.5 hours. The medium was removed and 4 hybridoma strains x 6 subclones were cultured in 2% BSA, 0.2% Poly sorbate 20 (hereinafter referred to as "PS20")-containing PBS at 10, 25 and 100 nM The plate was diluted to 0.0 μg / mL, and 100 μL of each was added to the plate and left to stand at 37°C for 1 hour. After washing four times with 5% PS20-containing PBS, the cells were washed with 2% BSA and 0.2% Tween 20-containing PBS. B7-H3-ADC(I)(DAR7) and B7-H3-ADC(I)(DAR5) Dilute to 1, 2.5, 10, 25, 100, and 250ng / mL and apply 100µL to the plate. After washing four times with PBS containing 0.05% PS20, 2% Goat Anti-Hum diluted 7500-fold with BSA and 0.2% PS20 in PBS an kappa-HRP(Southern Biotechnology Asso. 100 μL of HCl (Prod., Inc.) was added to each well and the mixture was left to stand at room temperature for 1 hour. After washing four times with PBS containing 100% PS20, the cells were treated with TMB soluble reagent (Scy Tek Laboratories) at 100 μL / well to develop a color reaction. TMB stop buffer (ScyTek Laboratories) 100 The color reaction was stopped by adding 1 μL / well of VersaMax (Molecula r Devices) and measure the absorbance at 450 nm (reference 650 nm). The relative error was calculated using the following formula, and clones with the smallest value were selected. Relative error (%) = {(measured value of DAR7) / (measured value of DAR5)-1} x 100 From these, 4 strains x 2 subclones were subjected to secondary cloning.

[0248] xi) Secondary cloning and screening Secondary cloning of the 4 strains x 2 subclones selected in x) was performed, and positive results were obtained from each clone. A total of 24 clones were selected by selecting 3 wells from each well. The antibody titer against compound (6) was confirmed in the same manner as in vi). , compound (1), B7-H3-ADC(II), B7-H3-ADC(I) and B7-H 3-ADC(III), and compounds (8) and (7) as compounds for confirming reactivity Inhibition ELISA was performed using the above in the same manner as in vii).

[0249] xii) Secondary calibration curve confirmation Among the 24 clones selected in xi), the response to compound (7) was higher than that to compound (1). For the 18 clones excluding 6 clones with particularly high efficacy, HER2-ADC(I)(DA R8), HER2-ADC(I)(DAR4), and HER2-ADC(I)(DAR2 ) and the calibration curve were measured using the Gyrolab xP workstation (GYR The capture reagent was B iotin-SP-conjugated AffiniPure Goat Anti -Mouse IgG, Fcγ specific(Jackson Immuno Research Laboratories, Inc.) and 0.01% PS20 The cell culture supernatant was prepared using Rexxip CCS (GYR The concentration was adjusted to 200 ng / mL using the ELISA kit (OS PROTEIN Technologies). ER2-ADC(I)(DAR8), HER2-ADC(I)(DAR4), and HER 2-ADC(I)(DAR2) was administered at 1000ng in PBS containing 0.01% PS20. The detection reagent was Goat Anti-Hum an kappa(Southern Biotechnology Associat es, Inc.) was used with the Alexa Fluor® 647 labeling kit (Th The R The reagents, cell culture supernatant, and calibration curve test solution were prepared at 10 nM using exxip F. The mixture was placed in a 96-well PCR plate and incubated with Gyrolab Bioaffy 200. Set it on the Gyrolab xP workstation and run it in 4-Step (2x C) Measurement was performed using the wizard method. The calibration curve was obtained using Gyrolab E A four-parameter logistic model (weighted) was calculated using the Evaluator Software. All 18 clones evaluated were HER2-A positive. The reactivity was independent of the DAR of DC(I), and no difference in reactivity between strains was observed. Three clones (1A3, 8B2, and 11B1) with high IgG concentrations were selected from a single strain and then produced in small quantities. was carried out.

[0250] xiii) Preparation and cross-check of purified Protein A antibodies The three clones (1A3, 8B2, and 11B1) selected in step xii were cultured in serum-free medium ( ASF104(N)) was cultured in a roller bottle, and the culture supernatant was collected and filtered through a 0.45 μm filter. The antigen protein (2) was filtered through a filter and purified on a Protein A column. 50µL of Protein A was added to the ELISA immunoplate. Add 50 μL of a solution of 3 purified clones diluted 10 times in 2-fold serial dilutions starting from 5 μg / mL. The plate was then incubated at 37°C for 30 minutes. After washing, anti-mouse IgG-HRP was added to the plate. The reaction was carried out at 7°C for 30 minutes. After washing, OPD solution was added, and the absorbance at 490 nm was measured after the color development was stopped. The luminescence intensity was measured. All Protein A purified antibodies showed good reactivity with antigen protein (2). showed.

[0251] xiv) Confirmation of the standard curve for Protein A purified antibodies Regarding the three clones (1A3, 8B2, and 11B1) purified with Protein A in (xiii), HER2-ADC(I)(DAR8) and HER2-ADC(I)(DAR4) , and B7-H3-ADC(I)(DAR8), and B7-H3-ADC(I)(D Reactivity to AR4) was confirmed using the Gyrolab xP workstation did.

[0252] The capture reagent was Protein A purified from three clones and then transferred to EZ-Link NHS. -LC-Biotin(Thermo Fisher Scientific Inc. ) was used and adjusted to 700 nM in PBS containing 0.01% PS20. HER2-ADC(I)(DAR8) and HER2-ADC(I)(DAR4) were xxip HN, all diluted in six steps starting from 1000ng / mL at a 4-fold common ratio. The detection reagent was Goat Anti-Human kappa (Southern Bio technology Associates, Inc.) to Alexa Fluor( Labeled with the 647 Labeling Kit and diluted with Rexxip F at 10 nM The prepared reagents and antibody-drug conjugates (standard curve samples) were placed in a 96-well plate. Place in a CR plate and mix with Gyrolab Bioaffy 200. b Set it in the XP workstation and The calibration curve was measured using the Gyrolab Evaluator So A 4-parameter logistic model (weighting; Respon The standard curve for clone 1A3 is shown in Figure 15, and the standard curve for clone 8B2 is shown in Figure 1 The standard curve for clone 11B1 is shown in Figure 17. -ADC(I) showed DAR-independent reactivity.

[0253] Furthermore, EZ-Link NHS-LC-Biotin was used as a capture reagent. Fluorescently labeled Human Her2 / ErbB2 Protein (ACROBiosy) stems), and similarly biotin-labeled B7-H3 C1 domain were used. The concentration was adjusted to 700 nM in PBS containing 0.1% PS20. HER2-ADC(I) (DAR8 ) and HER2-ADC(I)(DAR4), and B7-H3-ADC(I)(DAR 8) and B7-H3-ADC(I)(DAR4) are listed in Rexxip HN. The detection reagent was prepared by diluting the sample in six steps starting from 1000 ng / ml at a 4-fold common ratio. After inA purification, three clones (1A3, 8B2, and 11B1) were incubated with DyLight650 ( Trademark) Labeling Kit (Thermo Fisher Scientific Inc. The reagents were labeled with .) and prepared at 10 nM with Rexxip F. Standard curve samples of antibody-drug conjugates were placed in a 96-well PCR plate and analyzed by Gyrol ab Bioaffy 200 with Gyrolab xP workstation Set it to n and measure it with 200-3W-001-A (wizard method). The calibration curve was generated using Gyrolab Evaluator Software. Regression was performed using a logistic model (weighted; response). HER2-A Regarding the reactivity with DC(I), the standard curve for clone 1A3 is shown in Figure 18, and that for clone 8B2 is shown in Figure 19. The standard curve for clone 11B1 is shown in Figure 19, and the standard curve for B7-H3-AD is shown in Figure 20. Regarding the reactivity with C(I), the calibration curve for clone 1A3 is shown in Figure 21, and the calibration curve for clone 8B2 is shown in Figure 22. The calibration curve for clone 11B1 is shown in Figure 22, and the calibration curve for clone 11B1 is shown in Figure 23. Furthermore, DAR-independent reactions were observed between HER2-ADC(I) and B7-H3-ADC(I). showed responsiveness.

[0254] From the above results, it was found that the DAR and antibody moiety were independent of each clone. The antibody-drug conjugate is reactive to the capture reagent and the detection reagent. It was confirmed that both can be used. Lone 1A3 was selected for use in detecting the antibody-drug conjugates of the present invention. did.

[0255] xv) Isotyping of mouse monoclonal antibodies The mouse monoclonal antibody (hereinafter referred to as "mouse monoclonal antibody") related to clone 1A3 obtained in The isotype of the antibody (referred to as "antibody 1A3") is Mouse monoclonal isot The results were determined using a typing test kit (AbD Serotec). As a result, the isotype was confirmed to be IgG2b, κ chain.

[0256] xvi) Gene cloning and N-terminal amino acid sequence analysis of monoclonal antibodies From the hybridoma producing mouse antibody 1A3, TRIzol Reagent (LIFE Total RNA was prepared using purified mouse The N-terminal amino acid sequence of antibody 1A3 was analyzed (Edman degradation method). The nucleotide sequence of clone 1A3 was analyzed by subcloning. As a result of the screening, one sequence each for the heavy chain variable region and the light chain variable region was obtained, and the refined The results of the N-terminal amino acid sequence analysis of the mouse antibody 1A3 and the antibody gene cloning It was confirmed that the N-terminal amino acid sequence of the nucleotide sequence of the obtained clone 1A3 was identical. It was recognized.

[0257] The amino acid sequence of the heavy chain of mouse antibody 1A3 is shown in SEQ ID NO: 15. The amino acid sequence of amino acid numbers 1 to 19 represents a signal sequence, and amino acid number 20 The amino acid sequence of 142 to 141 represents the heavy chain variable region, and the amino acid sequence of 142 to 477 represents the heavy chain variable region. The amino acid sequence shown in represents the amino acid sequence of the heavy chain constant region.

[0258] The amino acid sequence of the mouse antibody 1A3 light chain is shown in SEQ ID NO: 16. The amino acid sequence of amino acid numbers 1 to 20 represents a signal sequence, and amino acid number 21 The amino acid sequence from 128 to 234 represents the light chain variable region. The amino acid sequence shown in represents the amino acid sequence of the light chain constant region.

[0259] The nucleotide sequence encoding the amino acid sequence of the heavy chain of mouse antibody 1A3 is set forth in SEQ ID NO: 17. The nucleotide sequence from nucleotide numbers 1 to 57 in SEQ ID NO: 17 is The nucleotide sequence from nucleotide numbers 58 to 423 represents the heavy chain signal sequence. It encodes the amino acid sequence of the variable region.

[0260] The nucleotide sequence encoding the amino acid sequence of the mouse antibody 1A3 light chain is set forth in SEQ ID NO: 18. The nucleotide sequence from nucleotide numbers 1 to 60 in SEQ ID NO: 18 is The nucleotide sequence from nucleotide numbers 61 to 459 represents the heavy chain signal sequence. It encodes the amino acid sequence of the variable region.

[0261] Additionally, CDR analysis was performed.

[0262] According to the definition of Abm, the heavy chain variable region of mouse antibody 1A3 is the amino acid sequence shown in SEQ ID NO: 1. CDRH1 (GFTFSDYGMV) consisting of the amino acid sequence shown in SEQ ID NO: 2 CDRH2 (YISSGSSAIY) consisting of the sequence, and the amino acid sequence shown in SEQ ID NO: 3 It has the CDRH3 (PPRYDVYSAWFAY) sequence, which is related to mouse antibody 1 The A3 light chain variable region is CDRL1 (KASQ) consisting of the amino acid sequence shown in SEQ ID NO: 4. DVGSAVV), CDRL2 (WASTR) consisting of the amino acid sequence shown in SEQ ID NO: 5 HT), and CDRL3 (QQYSSYPV) consisting of the amino acid sequence shown in SEQ ID NO: 6 It was found that he possesses T.

[0263] According to Chothia's definition, the heavy chain variable region of murine antibody 1A3 is shown in SEQ ID NO:7. CDRH1 (GFTFSDY) consisting of the amino acid sequence shown in SEQ ID NO: 8 CDRH2 (SSGSSA) consisting of the amino acid sequence shown in SEQ ID NO: 3 It contains the CDRH3 (PPRYDVYSAWFAY) and the mouse antibody 1A3. The chain variable region is CDRL1 (KASQDVG) consisting of the amino acid sequence shown in SEQ ID NO: 4. SAVV), CDRL2 (WASTRHT) consisting of the amino acid sequence shown in SEQ ID NO:5 and CDRL3 (QQYSSYPVT) consisting of the amino acid sequence shown in SEQ ID NO: 6. It was found that he possessed it.

[0264] According to Kabat's definition, the heavy chain variable region of murine antibody 1A3 is shown in SEQ ID NO: 9. CDRH1 (DYGMV) consisting of the amino acid sequence shown in SEQ ID NO: 10 CDRH2 (YISSGSSAIYYADTVKG) consisting of It has CDRH3 (PPRYDVYSAWFAY) consisting of the amino acid sequence The mouse antibody 1A3 light chain variable region contains CDRL1 consisting of the amino acid sequence shown in SEQ ID NO:4. (KASQDVGSAVV), CDRL2 consisting of the amino acid sequence shown in SEQ ID NO: 5 ( WASTRHT), and CDRL3 (QQY) consisting of the amino acid sequence shown in SEQ ID NO: 6 It was discovered that he holds the SSYPVT.

[0265] According to the Imgt® definition, the murine antibody 1A3 heavy chain variable region is represented by SEQ ID NO: 1 CDRH1 (GFTFSDYG) consisting of the amino acid sequence shown in SEQ ID NO: 1; CDRH2 (ISSGSSAI) consisting of the amino acid sequence shown in SEQ ID NO: 13 It has CDRH3 (ARPPRYDVYSAWFAY) consisting of the amino acid sequence The light chain variable region of mouse antibody 1A3 is C1A ... DRL1 (QDVGSA), WAS (tryptophan-alanine-serine) CDRL2 consisting of a polypeptide, and CDR consisting of the amino acid sequence shown in SEQ ID NO: 6 It was discovered that he possesses L3 (QQYSSYPVT).

[0266] [Example 4] Plasma concentration measurement in non-clinical trials Using the mouse antibody 1A3 obtained in Example 2, mouse blood samples of HER2-ADC(I) were analyzed. Plasma concentrations and HER3-ADC(I), TROP2-ADC(I), and B7-H3 We developed a method for measuring the plasma concentration of -ADC(I) in monkeys. Mouse antibody 1A3 was used as a detection reagent. DyLight650® or Alexa Fluor® 647 labeling Either method can be used to produce the antibody-drug conjugate of the present invention. Furthermore, the plasma concentration of the antibody can be measured without depending on the DAR and the antibody moiety. A dose curve can be prepared and used to measure the plasma concentration of the antibody-drug conjugate of the present invention. be.

[0267] i) HER2-ADC(I) Mouse plasma concentration measurement of HER2-ADC(I) was performed using Gyrolab xP work The capture reagent was biotinylated MoI. se Anti-(Anti-HER2 Ab) idiotype Ab (where, "( "Anti-HER2 Ab" is a sequence represented by amino acid numbers 1 to 449 in SEQ ID NO: 21. and a heavy chain consisting of the amino acid sequence set forth in SEQ ID NO: 22, ... from amino acid number 1 to 214. (13C1) (IBL) is used to express an antibody having a light chain consisting of the amino acid sequence described above. The standard curve sample was prepared using Rexxip. Mouse plasma diluted 100 times with HN at HER2-ADC(I) concentrations of 0 and 0.150 , 0.200, 0.600, 1.60, 4.00, 16.0, 40.0, 100, 140 The detection reagent was prepared by conjugating mouse antibody 1A3 to DyLight65. Labeled with the 0 (registered trademark) labeling kit and then 10n These prepared reagents and calibration curve samples were placed in a 96-well PCR plate. Gyrolab xP work with Gyrolab Bioaffy 200 The measurement wizard was set to 200-3W-002-A (PMT1 ) was used. Regression analysis was performed using Gyrolab Evaluator 3.3.9.175. A 5-parameter logistic model was used. The calibration curve is shown in Figure 24.

[0268] Measurement of mouse plasma concentrations of total antibodies (anti-HER2 antibodies and HER2-ADC(I)) The standard method was established on a Gyrolab xP workstation. Biotinylated Mouse Anti-(Anti-HER2 Ab) Idiotype Ab (13C1) (IBL) was used, and PB containing 0.1% PS20 was used. The standard curve sample was prepared by diluting 100 times with Rexxip HN. Plasma HER2-ADC(I) concentrations were 0, 0.150, 0.200, 0.600, and 1 Prepare to 0.60, 4.00, 16.0, 40.0, 100, and 140 μg / mL The detection reagent was Alexa Fluor® 647 anti-human I. gG, Fcγ Antibody (Jakckson Immno Research L (Laboratories, Inc.) was prepared at 10 nM with Rexxip F. The prepared reagents and standard curve samples were placed in a 96-well PCR plate and Set up on a Gyrolab xP workstation with oaffy 200 The measurement wizard used was 200-3W-002-A (PMT1). Regression analysis The five-parameter log was obtained using Gyrolab Evaluator 3.3.9.175. It was done using the gistic model.

[0269] The concentration measurement results are shown in Table 1.

[0270] [Table 1]

[0271] ii) HER3-ADC(I) Gyrolab xP works for measuring HER3-ADC(I) plasma concentrations in monkeys The system was constructed and validated using EZ-Li as the capture reagent. nk Sulfo-NHS-LC-Biotin(Thermo Fisher Sci Biotin-labeled HER3 (Recombinant H) uman ErbB-3 / HER3 Protein, ACRObiosystems) The standard curve sample was monkey plasma. At HER3-ADC(I) concentrations of 0, 0.0750, 0.100, 0.250, and 0.7 Prepared to give 50, 2.25, 6.75, 20.0, 38.0, and 48.0 μg / mL The resulting solution was further mixed with PBS containing Polysorbate 20 and Rexxip A. The sample was diluted 100-fold with GYROS PROTEIN Technologies. The reagent used was mouse antibody 1A3 labeled with Alexa Fluor (registered trademark) 647 labeling kit. The resulting solution was prepared at 10 nM with Rexxip F. The reagents and standard curve samples were placed in a 96-well PCR plate and analyzed using the Gyrolab xP wo The measurement wizard was set to rkstation 200-3W-002-A (PM T5) was used. Regression analysis was performed using Gyrolab Evaluator 3.3.7.17 A 4-parameter logistic model (weights: response) was performed using 1. The calibration curve is shown in FIG.

[0272] iii) TROP2-ADC(I) The plasma concentration of TROP2-ADC(I) in monkeys was measured using Gyrolab xP work The system was constructed on a station and validated. EZ-L was used as the capture reagent. Ink: New hTro labeled with biotin using Sulfo-NHS-LC-Biotin p2 (Lot No. V35, Daiichi Sankyo Co., Ltd.) was diluted with 0.1% PS20 in PBS for 35 min. The standard curve sample was prepared in monkey plasma at concentrations of 0, 0. 00750, 0.0100, 0.0250, 0.0750, 0.250, 0.750, 2 The solutions were prepared to 0.50, 7.50, and 10.0 μg / mL, and then Rex Dilute 10 times with xip HN (GYROS PROTEIN Technologies) The detection reagent was mouse antibody 1A3 labeled with Alexa Fluor (registered trademark) 647. The antibody was labeled using a labeling kit and adjusted to 10 nM with Rexxip F. The reagents and calibration curve samples prepared from the above were placed in a 96-well PCR plate and then placed in a Gyrolab x The measurement wizard was 200-3W-002- A (PMT1) was used. Regression analysis was performed using Gyrolab Evaluator 3.3. 7.171 with a 4-parameter logistic model (weights: Response) The calibration curve is shown in Figure 26.

[0273] iv) B7-H3-ADC(I) The plasma concentration of B7-H3-ADC(I) in monkeys was measured using Gyrolab xP work The system was constructed on a station and validated. EZ-L was used as the capture reagent. ink Sulfo-NHS-LC-Biotin(Thermo Fisher Sc Biotin-labeled B7-H3 C1 domain (L (product number B7_OmJ1, Daiichi Sankyo Co., Ltd.) was used, and the cells were incubated for 70 minutes in PBS containing 0.1% PS20. The standard curve sample was prepared using monkey plasma containing B7-H3-ADC(I) at concentrations of 0, 0. 0750, 0.l00, 0.250, 0.750, 2.25, 6.75, 20.0, 38 The solution was prepared to 0.0, 48.0 μg / mL, and then further treated with Polysorbate PBS containing te 20 and Rexxip HN (GYROS PROTEIN Tech The detection reagent was mouse antibody 1A3 diluted 50-fold with Alexa FI The product was labeled with the uor® 647 labeling kit and then used in Rexxip These prepared reagents and the standard curve samples were placed in a 96-well PCR plate. The measurement was performed in a Gyrolab xP workstation. The ard used was 200-3W-001-A (PMT5). Regression analysis was performed using Gyrolab A 4-parameter logistic model was used with Evaluator 3.3.7.171. The calibration curve is shown in Figure 27.

[0274] [Example 5] Immunostaining using mouse antibody 1A3 i) Confirmation of staining using human-derived subcutaneously transplanted tumors Human-derived tumors were subcutaneously transplanted into severely immunodeficient mice (NOG mice). After administration of the body-drug conjugate, the tumor tissue is collected and a paraffin-embedded specimen is prepared. The staining of mouse antibody 1A3 was examined. Tumor tissue collected from NOG mice was used as a negative control. Deparaffinization and antigen retrieval were performed using Au Tostainer Link pre-processing system (PT Link: manufactured by DAKO) and Target Retrieval Solution Low pH: The subsequent staining process is carried out using an automatic staining device (DAKO Autos) This is carried out using EnVision F (Dako tainer Link48). After washing with LEX WASH BUFFER (manufactured by DAKO), Add Block 3% H2O2 (DAKO) and incubate. Wash with FLEX WASH BUFFER. Protein Blocks Add erum free (DAKO), incubate, and blow air into the liquid. Mouse antibody 1A3 was removed using REAL Antibody Diluent (DAK EnVision FLEX WASH BUFFER After washing with EnVision+ System-HRP Labeled Polymer Add er Anti-Mouse #K4000 (DAKO) and incubate. Then wash with EnVision FLEX WASH BUFFER.

[0275] DAKO Liquid DAB+Substrate Chromogen Sys After adding tem and incubating, EnVision FLEX WASH BUFF Wash in ER. Add EnVision FLEX Hematoxylin and incubate. After washing, wash with EnVision FLEX WASH BUFFER and ion-exchanged water. Wash with.

[0276] Mouse antibody against NOG mice administered the antibody-drug conjugate of the present invention 1A3 showed good staining, and the staining intensity increased with increasing concentration of mouse antibody 1A3. Furthermore, we confirmed that NOG mice not administered the antibody-drug conjugate of the present invention We confirmed that mouse antibody 1A3 does not stain for B. Because the cells are deficient, background staining by endogenous IgG from the mouse is not observed. It is known that this is not possible (Ito M, et al. Blood 100(9):3175-3182, 2002).

[0277] ii) Confirmation of the specificity of mouse antibody 1A3 Mouse antibody 1A3 was mixed with compound (2) or SN-38 in advance and then subjected to immunostaining. The mixing ratio based on molecular weight was antibody 1A3: compound (2): SN-38 = 0.1:0 .04:0.03. Staining is carried out in the same manner as in i).

[0278] It is confirmed that the staining ability of mouse antibody 1A3 disappears upon mixing with compound (2). We also confirmed that the staining of mouse antibody 1A3 does not disappear when mixed with SN-38. do.

[0279] [Example 6] Preparation of rabbit chimeric antibody i) Design of rabbit chimeric antibody of mouse antibody 1A3 A rabbit chimeric antibody of mouse antibody 1A3 (hereinafter referred to as "rabbit chimeric antibody 1A3") The sequence of the rabbit chimeric antibody was designed as follows: HG*02 and rabbit light chain constant region IGKC2*01 from IMGT® The design was based on the following:

[0280] The amino acid sequence of the heavy chain of rabbit chimeric antibody 1A3 is shown in SEQ ID NO: 19. The amino acid sequence of amino acid numbers 1 to 19 in the formula (I) represents a signal sequence, and The amino acid sequence of numbers 20 to 141 represents the heavy chain variable region, and amino acid numbers 142 to 143 represent the heavy chain variable region. The amino acid sequences shown in Figures 1 to 464 represent the amino acid sequences of the heavy chain constant region.

[0281] The amino acid sequence of the rabbit chimeric antibody 1A3 light chain is shown in SEQ ID NO: 20. The amino acid sequence of amino acid numbers 1 to 20 in the formula (I) represents a signal sequence, and The amino acid sequence of numbers 21 to 127 represents the light chain variable region, and the amino acid sequence of numbers 128 to The amino acid sequences from 2 to 233 represent the amino acid sequences of the light chain constant region.

[0282] ii) Construction of antibody expression vector pCMA-LK Restriction of the plasmid pcDNA3.3-TOPO / LacZ (Invitrogen) A fragment of approximately 5.4 kb obtained by digestion with the enzymes XbaI and PmeI and a human light chain Nucleotide sequence encoding the signal sequence and the amino acid sequence of the human kappa chain constant region (sequence The DNA fragment containing the nucleotide sequence (number 29) was subjected to In-Fusion Advantage PCR cloning. The DNA was ligated using a DNA ligation kit (CLONTECH) to create pcDNA3.3 / LK. Made.

[0283] pCMA was created by removing the neomycin expression unit from pcDNA3.3 / LK. -LK was built.

[0284] iii) Construction of rabbit chimeric antibody 1A3 heavy chain expression vector A nucleotide sequence encoding the amino acid sequence of the rabbit chimeric antibody 1A3 heavy chain (SEQ ID NO: 19) A DNA fragment having the sequence (SEQ ID NO: 30) was synthesized (GENEART). The nucleotide sequence set forth in nucleotide numbers 26 to 82 in No. 30 is a signal sequence The nucleotide sequence from nucleotide numbers 83 to 448 represents the heavy chain variable region The nucleotide sequence encoding the amino acid sequence is set forth in nucleotide numbers 449 to 1417. encodes the amino acid sequence of the constant region.

[0285] In-Fusion HD PCR cloning kit (CLONTECH) was used. The synthesized DNA fragment and pCMA-LK were digested with XbaI and PmeI to obtain the light chain signal. and the nucleotide sequence encoding the amino acid sequence of the human κ chain constant region (SEQ ID NO:2) 9) By ligating the DNA fragment from which the nucleotide sequence has been removed, the rabbit chimeric antibody 1A3 heavy chain is expressed. A vector was constructed.

[0286] iv) Construction of rabbit chimeric antibody 1A3 light chain expression vector A nucleotide sequence encoding the amino acid sequence of the rabbit chimeric antibody 1A3 light chain (SEQ ID NO: 20) A DNA fragment having the sequence (SEQ ID NO: 31) was synthesized (GENEART). The nucleotide sequence set forth in nucleotide numbers 26 to 85 in No. 31 is a signal sequence The nucleotide sequence from nucleotide numbers 86 to 406 represents the amino acid sequence of the light chain variable region. The nucleotide sequence from nucleotide numbers 407 to 724 encodes the amino acid sequence The amino acid sequence of the constant region is encoded by the method similar to that of iii). Three light chain expression vectors were constructed.

[0287] v) Production of rabbit chimeric antibody 1A3 FreeStyle 293F cells (Invitrogen) were cultured according to the manufacturer's instructions. Subculture and culture were performed. 2.4 × 109 FreeStyle 293 cells in the logarithmic growth phase were used. F cells (Invitrogen) were cultured in an Optimum Growth 5L flask. (Thomson) and FreeStyle293 expression Dilute with edium (Invitrogen) to a concentration of 1.88 x 106 cells / mL 40 mL of Opti-Pro SFM medium (Invitrogen) was added to 0.48 mg of rabbit chimeric antibody 1A3 heavy chain expression vector and 0.72 mg of rabbit chimeric antibody 1A3 light chain expression vector and 3.6 mg of Polyethyleneimine (Poly Science #24765) and stir gently, then let stand for another 5 minutes. The cells were incubated at 37°C in an 8% CO2 incubator for 4 h. After shaking culture at 90 rpm for 2 hours, 1200 mL of EX-CELL VPRO medium (SA FC Biosciences), 18 mL of GlutaMAX I (GIBCO) , and 60 mL of Yeastolate Ultrafiltrate (GIBCO) The mixture was added and incubated at 37°C in an 8% CO2 incubator with shaking at 90 rpm for 7 days. The obtained culture supernatant was filtered through a Disposable Capsule Filter (Adva Rabbit chimeric antibody 1A was isolated by filtration through a filtration filter (NTEC #CCS-045-E1H). The culture supernatant containing 3 was obtained.

[0288] vi) Purification of rabbit chimeric antibody 1A3 The culture supernatant obtained in v) was subjected to rProtein A affinity chromatography. The culture supernatant was filled with MabSelectSuRe equilibrated with PBS. The column was packed with 1000 sachets (GE Healthcare Bioscience). After that, the column was washed with PBS, which was twice the volume of the column. The antibody was eluted with a phosphate solution (pH 4.0) and the fractions containing the antibody were collected. ermo Scientific, Slide-A-Lyzer Dialysis The buffer was replaced with PBS using a Centrifugal Cassette. UF Filter Device VIVASPIN20 (molecular weight cutoff UF10K, The antibody was concentrated using a centrifuge (Sartorius) to adjust the IgG concentration to 2 mg / mL or higher. Afterwards, it was filtered through a Minisart-Plus filter (Sartorius) and A purified sample of the chimeric antibody 1A3 was obtained.

[0289] [Example 7] Preparation of antibody-drug conjugate i) Preparation of anti-GPR20 antibody-drug conjugate (1) According to the production method described in WO 2018 / 135501, an anti-GPR20 antibody ( A heavy chain consisting of the amino acid sequence set forth in amino acid numbers 20 to 472 in SEQ ID NO: 32, and and a light chain consisting of the amino acid sequence set forth in amino acid numbers 21 to 234 in SEQ ID NO: 33. an antibody comprising the formula

[0290] [ka]

[0291] (wherein A represents the binding site to the antibody) and an anti-GPR20 antibody linked via a thioether bond. GPR20 antibody-drug conjugate (referred to as "GPR20-ADC(I)" in the present invention) (hereinafter referred to as "the 'product') was manufactured.

[0292] ii) Preparation of anti-CDH6 antibody-drug conjugate (1) An anti-CDH6 antibody (synthetic compound) was prepared according to the production method described in WO 2018 / 212136. A heavy chain consisting of the amino acid sequence set forth in amino acid numbers 20 to 471 in sequence number 34; A light chain consisting of the amino acid sequence set forth in amino acid numbers 21 to 233 in SEQ ID NO: 35. an antibody comprising the formula

[0293] [ka]

[0294] (wherein A represents the binding site to the antibody) and an anti-CDH6 antibody linked via a thioether bond. CDH6 antibody-drug conjugate (referred to as "CDH6-ADC(I)" in the present invention) ) was manufactured.

[0295] [Example 8] Immunostaining using rabbit chimeric antibody 1A3 i) Confirmation of staining after administration of TROP2-ADC(I) using human-derived subcutaneously transplanted tumors The human head and neck cancer cell line FaDu was subcutaneously transplanted into immunodeficient mice (nude mice). After administration of ROP2-ADC(I), tumor tissue was collected and paraffin-embedded. The staining ability of the rabbit chimeric antibody 1A3 was examined. a heavy chain consisting of the amino acid sequence set forth in amino acid numbers 20 to 470 in SEQ ID NO: 2; 6, comprising a light chain consisting of the amino acid sequence set forth in amino acid numbers 21 to 234. From nude mice administered with the antibody (hereinafter referred to as "Anti-TROP2 Ab"). The collected tumor tissue was used as a negative control. Deparaffinization and antigen retrieval were performed using Autostain. Antigen retrieval was performed using the er Link pretreatment system (PT Link: manufactured by DAKO). Target Retrieval Solution Low pH: DAKO The staining was carried out at 97°C for 40 minutes using an automatic staining device (Dako A The assay was carried out at room temperature using a cytostainer Link48 (manufactured by DAKO). After washing once with Vision FLEX WASH BUFFER (manufactured by DAKO), EAL Peroxidase-Blocking Solution (DAKO) ) and incubate for 5 minutes. The mixture was washed once with serum-free Protein Block (DAKO). ) was added, and the mixture was incubated for 30 minutes, after which the liquid was removed with an air blower. The antibody 1A3 was diluted to 0.1 with REAL Antibody Diluent (DAKO). The solution was diluted to 1 μg / mL and allowed to react for 30 minutes. EnVision FLEX WASH B After washing three times with UFFER, EnVision+ System-HRP Labelle d Polymer Anti-Rabbit (manufactured by DAKO) was added and the ink was left for 30 minutes. After incubation, the samples were washed twice with EnVision FLEX WASH BUFFER. .

[0296] DAKO Liquid DAB+Substrate Chromogen Sys Add tem and incubate for a total of 10 minutes, then wash with EnVision FLEX WASH Washed once with BUFFER. EnVision FLEX Hematoxylin Add and incubate for 5 minutes, then wash with EnVision FLEX WASH BUFFE The mixture was washed three times with IR and ion-exchanged water.

[0297] Typical staining images are shown in Figure 35. Subcutaneous tumors in animals administered TROP2-ADC(I). The rabbit chimeric antibody 1A3 showed good staining ability in tumor tissue, while Anti-TRO The rabbit chimeric antibody 1A3 stained subcutaneous tumor tissues from animals administered P2 Ab. did not show any.

[0298] ii) Confirmation of staining after administration of GPR20-ADC(I) using human-derived subcutaneously transplanted tumors GPR20-overexpressing human gastrointestinal stromal tumor cell line, GI, was transfected into immunodeficient mice (nude mice). ST-T1 / GPR20 tumors were subcutaneously implanted. After administration of GPR20-ADC(I), the tumor tissue The tissue was collected and paraffin-embedded, and staining with rabbit chimeric antibody 1A3 was examined. In addition, tumor tissue collected from mice not administered GPR20-ADC(I) was used as a negative control. Staining was carried out in the same manner as in i).

[0299] Typical staining images are shown in Figure 36. In contrast, rabbit chimeric antibody 1A3 showed good staining, while GPR20-ADC(I) The rabbit chimeric antibody 1A3 showed no staining in the subcutaneous tumor tissue of untreated animals.

[0300] [Example 9] Immunostaining using mouse antibody 1A3 i) Confirmation of staining after administration of CDH6-ADC(I) using human-derived subcutaneously transplanted tumors Tumor tissue collected from a patient with clear cell renal cell carcinoma was injected into severely immunodeficient mice (NOG mice). After administration of CDH6-ADC(I), the tumor tissue was collected and paraffin-transferred. Embedded specimens were prepared and staining with mouse antibody 1A3 was examined. Tumor tissue collected from untreated NOG mice was used as a negative control. The raw material is pre-processing system for Autostainer Link (PT Link:DAK Target Retrieval Solution (manufactured by Company O) was used. Low pH (manufactured by DAKO) was used for 40 minutes at 97°C. The subsequent staining process was carried out automatically. The staining was performed at room temperature using a staining device (DAKO Autostainer Link48: manufactured by DAKO). The test was carried out using EnVision FLEX WASH BUFFER (manufactured by DAKO). After washing once, Peroxidase Block 3% H2O2 (DAKO) Add EnVision FLEX WASH BUFFER and incubate for 5 minutes. Washed once with Protein Block serum-free (DAKO) The mixture was incubated for 30 minutes and the liquid was removed with an air blower. 3 in REAL Antibody Diluent (DAKO) at 0.03 μg / m The EnVision FLEX W was diluted to 0.3 μg / mL and allowed to react for 60 minutes. After washing three times with ASH BUFFER, EnVision+ System-HRP La Belled Polymer Anti-Mouse #K4000 (DAKO) Add EnVision FLEX WASH BUF and incubate for 30 minutes. Washed twice with FER.

[0301] DAKO Liquid DAB+Substrate Chromogen Sys Add tem and incubate for a total of 10 minutes, then wash with EnVision FLEX WASH Washed once with BUFFER. EnVision FLEX Hematoxylin Add and incubate for 5 minutes, then wash with EnVision FLEX WASH BUFFE The mixture was washed three times with IR and ion-exchanged water.

[0302] Typical staining images are shown in Figure 37. 1A3 showed good staining, and the staining intensity increased with increasing concentration of mouse antibody 1A3. On the other hand, mouse antibody 1A3 showed no staining in animals not administered CDH6-ADC(I). In addition, because NOG mice lack B cells, endogenous IgG derived from mice No background staining was observed (Ito M, et al. Blood 100(9):3175-31 82, 2002).

[0303] ii) Confirmation of the specificity of mouse antibody 1A3 Mouse antibody 1A3 was mixed with compound (2) or SN-38 in advance and then subjected to immunostaining. Used.

[0304] The compound (2) is represented by the formula

[0305] [ka]

[0306] It is a compound represented by the formula: SN-38 is a formula

[0307] [ka]

[0308] It is a compound represented by the formula:

[0309] The mixing ratio based on molecular weight was mouse antibody 1A3: compound (2): SN-38 = 0.1:0 The ratio was 0.04:0.03. Staining was carried out in the same manner as in i).

[0310] A typical staining image is shown in Figure 38. The staining of mouse antibody 1A3 was improved by mixing with compound (2). The staining of mouse antibody 1A3 was lost when mixed with SN-38, but the staining of mouse antibody 1A3 was not lost when mixed with SN-38. This indicates that mouse antibody 1A3 specifically recognizes compound (2) in tissues. was shown.

[0311] [Example 10] Plasma concentration measurement in non-clinical trials Mouse plasma concentration measurement of GPR20-ADC(I) was performed using Gyrolab xP work Built with kstation (GYROS PROTEIN Technologies) Mouse antibody 1A3 was used as a capture reagent using a labeling kit (ChromaLin k Biotin Protein Labeling Kit (Solulink) Biotin-labeled antibody was used and adjusted to 700 nM in PBS containing 0.1% PS20. The calibration curve sample was mouse plasma with GPR20-ADC(I) at concentrations of 0, 5, 10, 20, 50, l00, 200, 500, 1000, 2000, 5000, 10000ng / mL The resulting solution was used in Rexxip AN (GYROS PROTEIN Tech) The detection reagent was Mouse Anti-(Anti -GPR20 Ab) (wherein "(Anti-GPR20 Ab)" is SEQ ID NO: 32 a heavy chain consisting of the amino acid sequence set forth in amino acid numbers 20 to 472 in SEQ ID NO: 3; 3, comprising a light chain consisting of the amino acid sequence set forth in amino acid numbers 21 to 234. Idiotype Ab(71C1) (IBL) was used with DyLight650 ( The product was labeled with the Rexxip F (GYROS) labeling kit. These were prepared at 10 nM by Protein Technologies. The reagents and standard curve samples were placed in a 96-well PCR plate and analyzed using the Gyrolab xP wo The Bioaffy200 was used and the wizard was set to 20 Measurements were performed using 0-3W-002-A (PMT1). Regression analysis was performed using Gyrolab Evalu A four-parameter logistic model (weights: R) was run using ator 3.3.9.175. The calibration curve is shown in Figure 39.

[0312] [Example 11] Serum concentration measurement in clinical trials We established a method for measuring human serum concentrations of HER2-ADC(I) using ECL. Antibody 1A3 was placed on a High Bind plate (Meso Scale Diagnostics After coating with blocking buffer (MRSA, Inc., MSD), the plate was washed. After blocking with PBS containing PS20 containing BSA, the following were measured: 0, 200, 400, 800 , 1600, 3200, 6400, 12800, 20000 and 25600ng / mL The HER2-ADC(I) prepared in the above was diluted 1000-fold with assay diluent to prepare the standard curve sample. Furthermore, EZ-Link Sulfo-NHS-LC-LC Biotin (T Biotin-labeled Bis-Peptide (Biotin-Peptide) otinylated Mouse Anti-(Anti-HER2)idiotyp e Ab(13C1) (IBL) and sulfo-tagged streptavidin (MSD) were pre-treated. The detection solution was prepared by incubation. After adding the calibration curve sample, the plate was incubated and washed. The detection solution was added to the plate to allow the complex to form. SD) was diluted 2-fold with purified water and added to the washed plate. ger 6000 (control software: MSD Discovery Workbench Measurements were performed using a 4-parameter logistic regression model (Version 3.0.18). Model (weight: 1 / Response 2 The calibration curve is shown in Figure 40.

[0313] [Example 12] Verification of the chemical structure recognized by mouse antibody 1A3 The chemical structure recognized by mouse antibody 1A3 was analyzed on a Gyrolab xP workstation. HER2-AD using GYROS PROTEIN Technologies The competitive inhibition of C(I) was verified. The compounds used for the competitive inhibition were Compound (1), Compound (2), and Compound (3). (2), compound (7), compound (8), compound (9), compound (10), compound (11) , Topotecan, and Rubitecan were selected.

[0314] The compound (1) is represented by the formula

[0315] [ka]

[0316] It is a compound represented by the formula: Compound (2) is represented by the formula

[0317] [ka]

[0318] It is a compound represented by the formula: Compound (7) is represented by the formula

[0319] [ka]

[0320] It is a compound represented by the formula: Compound (8) is represented by the formula

[0321] [ka]

[0322] It is a compound represented by the formula: Compound (9) is represented by the formula

[0323] [ka]

[0324] (Sugimori M. et al., J. Med. Chem. 1994, 3033-3039). Compound (10) is represented by the formula

[0325] [ka]

[0326] It is a compound represented by the formula (U.S. Pat. No. 5,834,476). Compound (11) is represented by the formula

[0327] [ka]

[0328] (Atsumi R. et al., Arzneimittel-Forschung 2001, 253-257) . Topotecan is a formula

[0329] [ka]

[0330] It is a compound represented by the formula: Rubitecan is a formula

[0331] [ka]

[0332] It is a compound represented by the formula:

[0333] Mouse antibody 1A3 was used as the capture reagent in a labeling kit (ChromaLink Biotin Protein Labeling Kit (Solulink) The thiazolinone-labeled antibody was used and adjusted to 700 nM in PBS containing 0.1% PS20. Compounds (1), (2), (7), and (8), which are compounds for competitive inhibition; Compound (9), Compound (10), Compound (11), Topotecan, and Rubit After dissolving ecan in DMSO, 10% or 20% DMSO in Rexxi Diluted with pHN (GYROS PROTEIN Technologies) at 0, 1 After adjusting the concentration to 100 μg / mL, add an equal volume of 700 nM mouse antibody 1A3 solution and mix. The mixture was incubated at room temperature in the dark for at least 1 hour. HER2-ADC(I) was prepared by Rexxip H In N, 0, 0.244, 0.977, 3.91, 15.6, 62.5, 250, 1000 The detection reagent was prepared at 250 ng / mL. ) Idiotype Ab(13C1)(IBL) was used for immunofluorescence analysis using DyLight650® The cells were labeled with a labeling kit and then transfected with Rexxip F (GYROS PROTECT The above solution was prepared at 10 nM using Gyrol (EIN Technologies). Place it on the ab xP workstation and use the Gyrolab Bioaffy 2 Measurements were performed using 200-3W-002-A (PMT5) with Gyrol A 4-parameter logistic regression model was performed using ab Evaluator 3.4.0.24. The results were analyzed using a weighted response (weight: Response) model. A standard curve for DC(I) was prepared, and the Re at a HER2-ADC(I) concentration of 250 ng / mL was calculated. The inhibition rate (%) was calculated by comparing the results with the absence of the compound (concentration 0 ng / mL).

[0334] The inhibition rate graph is shown in Figure 41. Compound (1), Compound (2), Compound (7), and Compound (8) Compound (9) showed strong competitive inhibition. On the other hand, compounds (8), (10), and (11) ), Topotecan, and Rubitecan did not show competitive inhibition. Therefore, the mouse antibody 1A3 has the basic structure of compound (1) and retains the methyl group at the 4-position. It was shown that the compound specifically recognizes the chemical structure. [Sequence List Free Text]

[0335] SEQ ID NO: 1: CDRH1 SEQ ID NO: 2: CDRH2 SEQ ID NO: 3: CDRH3 SEQ ID NO: 4: CDRL1 SEQ ID NO: 5: CDRL2 SEQ ID NO: 6: CDRL3 SEQ ID NO: 7: CDRH1 SEQ ID NO: 8: CDRH2 SEQ ID NO: 9: CDRH1 SEQ ID NO: 10: CDRH2 SEQ ID NO: 11: CDRH1 SEQ ID NO: 12: CDRH2 SEQ ID NO: 13: CDRH3 SEQ ID NO: 14: CDRL1 SEQ ID NO: 15: Amino acid sequence of the heavy chain of mouse antibody 1A3 SEQ ID NO: 16: Amino acid sequence of the light chain of mouse antibody 1A3 SEQ ID NO: 17: Nucleotide sequence encoding the amino acid sequence of the heavy chain variable region of mouse antibody 1A3 array SEQ ID NO: 18: Nucleotide sequence encoding the amino acid sequence of the light chain variable region of mouse antibody 1A3 array SEQ ID NO: 19: Amino acid sequence of the heavy chain of rabbit chimeric antibody 1A3 SEQ ID NO: 20: Amino acid sequence of the rabbit chimeric antibody 1A3 light chain SEQ ID NO: 21: Amino acid sequence of anti-HER2 antibody heavy chain SEQ ID NO: 22: Amino acid sequence of the anti-HER2 antibody light chain SEQ ID NO: 23: Amino acid sequence of the anti-HER3 antibody heavy chain SEQ ID NO: 24: Amino acid sequence of the anti-HER3 antibody light chain SEQ ID NO: 25: Amino acid sequence of the anti-TROP2 antibody heavy chain SEQ ID NO: 26: Amino acid sequence of the anti-TROP2 antibody light chain SEQ ID NO: 27: Amino acid sequence of the anti-B7-H3 antibody heavy chain SEQ ID NO: 28: Amino acid sequence of the anti-B7-H3 antibody light chain SEQ ID NO: 29: Encoding the amino acid sequence of the human light chain signal sequence and the human kappa chain constant region Nucleotide sequence SEQ ID NO: 30: Nucleotide sequence encoding the amino acid sequence of the heavy chain of rabbit chimeric antibody 1A3 array SEQ ID NO: 31: Nucleotide sequence encoding the amino acid sequence of the rabbit chimeric antibody 1A3 light chain array SEQ ID NO: 32: Amino acid sequence of the anti-GPR20 antibody heavy chain SEQ ID NO: 33: Amino acid sequence of the anti-GPR20 antibody light chain SEQ ID NO: 34: Amino acid sequence of the anti-CDH6 antibody heavy chain SEQ ID NO: 35: Amino acid sequence of the anti-CDH6 antibody light chain

Claims

1. A method for quantifying the plasma concentration of an antibody-drug conjugate in a mammal to which the antibody-drug conjugate has been administered, using an antibody, comprising: The antibody formula 【Chemistry 1】 and an antibody conjugate formed by linking the antibody to a drug represented by the formula (I) via a linker, wherein the antibody recognizes the drug moiety of the antibody-drug conjugate; a) an antibody comprising a heavy chain comprising a CDRH1 consisting of the amino acid sequence set forth in SEQ ID NO: 1, a CDRH2 consisting of the amino acid sequence set forth in SEQ ID NO: 2, and a CDRH3 consisting of the amino acid sequence set forth in SEQ ID NO: 3, and a light chain comprising a CDRL1 consisting of the amino acid sequence set forth in SEQ ID NO: 4, a CDRL2 consisting of the amino acid sequence set forth in SEQ ID NO: 5, and a CDRL3 consisting of the amino acid sequence set forth in SEQ ID NO: 6; b) an antibody comprising a heavy chain comprising a CDRH1 consisting of the amino acid sequence set forth in SEQ ID NO: 7, a CDRH2 consisting of the amino acid sequence set forth in SEQ ID NO: 8, and a CDRH3 consisting of the amino acid sequence set forth in SEQ ID NO: 3, and a light chain comprising a CDRL1 consisting of the amino acid sequence set forth in SEQ ID NO: 4, a CDRL2 consisting of the amino acid sequence set forth in SEQ ID NO: 5, and a CDRL3 consisting of the amino acid sequence set forth in SEQ ID NO: 6; c) an antibody comprising a heavy chain comprising a CDRH1 consisting of the amino acid sequence set forth in SEQ ID NO: 9, a CDRH2 consisting of the amino acid sequence set forth in SEQ ID NO: 10, and a CDRH3 consisting of the amino acid sequence set forth in SEQ ID NO: 3, and a light chain comprising a CDRL1 consisting of the amino acid sequence set forth in SEQ ID NO: 4, a CDRL2 consisting of the amino acid sequence set forth in SEQ ID NO: 5, and a CDRL3 consisting of the amino acid sequence set forth in SEQ ID NO: 6; or d) an antibody comprising a heavy chain comprising a CDRH1 consisting of the amino acid sequence set forth in SEQ ID NO: 11, a CDRH2 consisting of the amino acid sequence set forth in SEQ ID NO: 12, and a CDRH3 consisting of the amino acid sequence set forth in SEQ ID NO: 13, and a light chain comprising a CDRL1 consisting of the amino acid sequence set forth in SEQ ID NO: 14, a CDRL2 consisting of the tripeptide WAS, and a CDRL3 consisting of the amino acid sequence set forth in SEQ ID NO: 6, the antibody in the antibody-drug conjugate is not an anti-HER2 antibody or an anti-TROP2 antibody; method.

2. The drug linker in the antibody-drug conjugate is formula 【Chemistry 2】 (wherein A represents the binding site to the antibody, and the drug linker is bound to the antibody via a thioether bond). The method of claim 1 , wherein

3. The drug linker in the antibody-drug conjugate is formula 【Transformation 3】 (wherein A represents the binding site to the antibody, and the drug linker is bound to the antibody via a thioether bond). The method of claim 1 , wherein

4. The drug linker in the antibody-drug conjugate is formula 【Chemistry 4】 (wherein A represents the binding site to the antibody, and the drug linker is bound to the antibody via a thioether bond). The method of claim 1 , wherein

5. The method according to any one of claims 1 to 4, wherein the average number of drug linkers bound per antibody in the antibody-drug conjugate is in the range of 2 to 8.

6. The method according to any one of claims 1 to 5, wherein the recognition ability for the antibody-drug conjugate does not substantially differ depending on the average number of drug linkers bound per antibody in the antibody-drug conjugate.

7. A method for quantifying the plasma concentration of an antibody-drug conjugate in a mammal to which the antibody-drug conjugate has been administered, using an antibody, comprising: The antibody formula 【Transformation 5】 an antibody that recognizes a drug represented by a) an antibody comprising a heavy chain comprising a CDRH1 consisting of the amino acid sequence set forth in SEQ ID NO: 1, a CDRH2 consisting of the amino acid sequence set forth in SEQ ID NO: 2, and a CDRH3 consisting of the amino acid sequence set forth in SEQ ID NO: 3, and a light chain comprising a CDRL1 consisting of the amino acid sequence set forth in SEQ ID NO: 4, a CDRL2 consisting of the amino acid sequence set forth in SEQ ID NO: 5, and a CDRL3 consisting of the amino acid sequence set forth in SEQ ID NO: 6; b) an antibody comprising a heavy chain comprising a CDRH1 consisting of the amino acid sequence set forth in SEQ ID NO: 7, a CDRH2 consisting of the amino acid sequence set forth in SEQ ID NO: 8, and a CDRH3 consisting of the amino acid sequence set forth in SEQ ID NO: 3, and a light chain comprising a CDRL1 consisting of the amino acid sequence set forth in SEQ ID NO: 4, a CDRL2 consisting of the amino acid sequence set forth in SEQ ID NO: 5, and a CDRL3 consisting of the amino acid sequence set forth in SEQ ID NO: 6; c) an antibody comprising a heavy chain comprising a CDRH1 consisting of the amino acid sequence set forth in SEQ ID NO: 9, a CDRH2 consisting of the amino acid sequence set forth in SEQ ID NO: 10, and a CDRH3 consisting of the amino acid sequence set forth in SEQ ID NO: 3, and a light chain comprising a CDRL1 consisting of the amino acid sequence set forth in SEQ ID NO: 4, a CDRL2 consisting of the amino acid sequence set forth in SEQ ID NO: 5, and a CDRL3 consisting of the amino acid sequence set forth in SEQ ID NO: 6; or d) an antibody comprising a heavy chain comprising a CDRH1 consisting of the amino acid sequence set forth in SEQ ID NO: 11, a CDRH2 consisting of the amino acid sequence set forth in SEQ ID NO: 12, and a CDRH3 consisting of the amino acid sequence set forth in SEQ ID NO: 13, and a light chain comprising a CDRL1 consisting of the amino acid sequence set forth in SEQ ID NO: 14, a CDRL2 consisting of the tripeptide WAS, and a CDRL3 consisting of the amino acid sequence set forth in SEQ ID NO: 6, the antibody in the antibody-drug conjugate is not an anti-HER2 antibody or an anti-TROP2 antibody; method.

8. A method for quantifying the plasma concentration of an antibody-drug conjugate in a mammal to which the antibody-drug conjugate has been administered, using an antibody, comprising: The antibody formula 【Transformation 6】 an antibody that recognizes a drug represented by a) an antibody comprising a heavy chain comprising a CDRH1 consisting of the amino acid sequence set forth in SEQ ID NO: 1, a CDRH2 consisting of the amino acid sequence set forth in SEQ ID NO: 2, and a CDRH3 consisting of the amino acid sequence set forth in SEQ ID NO: 3, and a light chain comprising a CDRL1 consisting of the amino acid sequence set forth in SEQ ID NO: 4, a CDRL2 consisting of the amino acid sequence set forth in SEQ ID NO: 5, and a CDRL3 consisting of the amino acid sequence set forth in SEQ ID NO: 6; b) an antibody comprising a heavy chain comprising a CDRH1 consisting of the amino acid sequence set forth in SEQ ID NO: 7, a CDRH2 consisting of the amino acid sequence set forth in SEQ ID NO: 8, and a CDRH3 consisting of the amino acid sequence set forth in SEQ ID NO: 3, and a light chain comprising a CDRL1 consisting of the amino acid sequence set forth in SEQ ID NO: 4, a CDRL2 consisting of the amino acid sequence set forth in SEQ ID NO: 5, and a CDRL3 consisting of the amino acid sequence set forth in SEQ ID NO: 6; c) an antibody comprising a heavy chain comprising a CDRH1 consisting of the amino acid sequence set forth in SEQ ID NO: 9, a CDRH2 consisting of the amino acid sequence set forth in SEQ ID NO: 10, and a CDRH3 consisting of the amino acid sequence set forth in SEQ ID NO: 3, and a light chain comprising a CDRL1 consisting of the amino acid sequence set forth in SEQ ID NO: 4, a CDRL2 consisting of the amino acid sequence set forth in SEQ ID NO: 5, and a CDRL3 consisting of the amino acid sequence set forth in SEQ ID NO: 6; or d) an antibody comprising a heavy chain comprising a CDRH1 consisting of the amino acid sequence set forth in SEQ ID NO: 11, a CDRH2 consisting of the amino acid sequence set forth in SEQ ID NO: 12, and a CDRH3 consisting of the amino acid sequence set forth in SEQ ID NO: 13, and a light chain comprising a CDRL1 consisting of the amino acid sequence set forth in SEQ ID NO: 14, a CDRL2 consisting of the tripeptide WAS, and a CDRL3 consisting of the amino acid sequence set forth in SEQ ID NO: 6, the antibody in the antibody-drug conjugate is not an anti-HER2 antibody or an anti-TROP2 antibody; method.

9. A method for quantifying the plasma concentration of an antibody-drug conjugate in a mammal to which the antibody-drug conjugate has been administered, using an antibody, comprising: The antibody formula 【Transformation 7】 an antibody that recognizes a drug represented by a) an antibody comprising a heavy chain comprising a CDRH1 consisting of the amino acid sequence set forth in SEQ ID NO: 1, a CDRH2 consisting of the amino acid sequence set forth in SEQ ID NO: 2, and a CDRH3 consisting of the amino acid sequence set forth in SEQ ID NO: 3, and a light chain comprising a CDRL1 consisting of the amino acid sequence set forth in SEQ ID NO: 4, a CDRL2 consisting of the amino acid sequence set forth in SEQ ID NO: 5, and a CDRL3 consisting of the amino acid sequence set forth in SEQ ID NO: 6; b) an antibody comprising a heavy chain comprising a CDRH1 consisting of the amino acid sequence set forth in SEQ ID NO: 7, a CDRH2 consisting of the amino acid sequence set forth in SEQ ID NO: 8, and a CDRH3 consisting of the amino acid sequence set forth in SEQ ID NO: 3, and a light chain comprising a CDRL1 consisting of the amino acid sequence set forth in SEQ ID NO: 4, a CDRL2 consisting of the amino acid sequence set forth in SEQ ID NO: 5, and a CDRL3 consisting of the amino acid sequence set forth in SEQ ID NO: 6; c) an antibody comprising a heavy chain comprising a CDRH1 consisting of the amino acid sequence set forth in SEQ ID NO: 9, a CDRH2 consisting of the amino acid sequence set forth in SEQ ID NO: 10, and a CDRH3 consisting of the amino acid sequence set forth in SEQ ID NO: 3, and a light chain comprising a CDRL1 consisting of the amino acid sequence set forth in SEQ ID NO: 4, a CDRL2 consisting of the amino acid sequence set forth in SEQ ID NO: 5, and a CDRL3 consisting of the amino acid sequence set forth in SEQ ID NO: 6; or d) an antibody comprising a heavy chain comprising a CDRH1 consisting of the amino acid sequence set forth in SEQ ID NO: 11, a CDRH2 consisting of the amino acid sequence set forth in SEQ ID NO: 12, and a CDRH3 consisting of the amino acid sequence set forth in SEQ ID NO: 13, and a light chain comprising a CDRL1 consisting of the amino acid sequence set forth in SEQ ID NO: 14, a CDRL2 consisting of the tripeptide WAS, and a CDRL3 consisting of the amino acid sequence set forth in SEQ ID NO: 6, the antibody in the antibody-drug conjugate is not an anti-HER2 antibody or an anti-TROP2 antibody; method.

10. A method for quantifying the plasma concentration of an antibody-drug conjugate in a mammal to which the antibody-drug conjugate has been administered, using an antibody, comprising: The antibody formula 【Transformation 8】 an antibody that recognizes a drug represented by a) an antibody comprising a heavy chain comprising a CDRH1 consisting of the amino acid sequence set forth in SEQ ID NO: 1, a CDRH2 consisting of the amino acid sequence set forth in SEQ ID NO: 2, and a CDRH3 consisting of the amino acid sequence set forth in SEQ ID NO: 3, and a light chain comprising a CDRL1 consisting of the amino acid sequence set forth in SEQ ID NO: 4, a CDRL2 consisting of the amino acid sequence set forth in SEQ ID NO: 5, and a CDRL3 consisting of the amino acid sequence set forth in SEQ ID NO: 6; b) an antibody comprising a heavy chain comprising a CDRH1 consisting of the amino acid sequence set forth in SEQ ID NO: 7, a CDRH2 consisting of the amino acid sequence set forth in SEQ ID NO: 8, and a CDRH3 consisting of the amino acid sequence set forth in SEQ ID NO: 3, and a light chain comprising a CDRL1 consisting of the amino acid sequence set forth in SEQ ID NO: 4, a CDRL2 consisting of the amino acid sequence set forth in SEQ ID NO: 5, and a CDRL3 consisting of the amino acid sequence set forth in SEQ ID NO: 6; c) an antibody comprising a heavy chain comprising a CDRH1 consisting of the amino acid sequence set forth in SEQ ID NO: 9, a CDRH2 consisting of the amino acid sequence set forth in SEQ ID NO: 10, and a CDRH3 consisting of the amino acid sequence set forth in SEQ ID NO: 3, and a light chain comprising a CDRL1 consisting of the amino acid sequence set forth in SEQ ID NO: 4, a CDRL2 consisting of the amino acid sequence set forth in SEQ ID NO: 5, and a CDRL3 consisting of the amino acid sequence set forth in SEQ ID NO: 6; or d) an antibody comprising a heavy chain comprising a CDRH1 consisting of the amino acid sequence set forth in SEQ ID NO: 11, a CDRH2 consisting of the amino acid sequence set forth in SEQ ID NO: 12, and a CDRH3 consisting of the amino acid sequence set forth in SEQ ID NO: 13, and a light chain comprising a CDRL1 consisting of the amino acid sequence set forth in SEQ ID NO: 14, a CDRL2 consisting of the tripeptide WAS, and a CDRL3 consisting of the amino acid sequence set forth in SEQ ID NO: 6, the antibody in the antibody-drug conjugate is not an anti-HER2 antibody or an anti-TROP2 antibody; method.

11. The method according to any one of claims 1 to 10, wherein the antibody comprises a heavy chain comprising a heavy chain variable region consisting of the amino acid sequence set forth in amino acid numbers 20 to 141 of SEQ ID NO: 15, and a light chain comprising a light chain variable region consisting of the amino acid sequence set forth in amino acid numbers 21 to 127 of SEQ ID NO:

16.

12. 12. The method of claim 11, wherein the antibody is a mouse antibody.

13. The method according to claim 11, characterized in that the antibody is an antibody comprising a heavy chain consisting of the amino acid sequence set forth in amino acid numbers 20 to 477 of SEQ ID NO: 15 and a light chain consisting of the amino acid sequence set forth in amino acid numbers 21 to 234 of SEQ ID NO:

16.

14. The method of claim 11, wherein the antibody is a chimeric antibody.

15. The method of claim 11, wherein the antibody is a rabbit chimerized antibody.

16. The method according to claim 11, characterized in that the antibody is an antibody comprising a heavy chain consisting of the amino acid sequence set forth in amino acid numbers 20 to 464 of SEQ ID NO: 19 and a light chain consisting of the amino acid sequence set forth in amino acid numbers 21 to 233 of SEQ ID NO:

20.

17. the antibody lacks a lysine residue at the carboxyl terminus of the heavy chain, A heavy chain consisting of the amino acid sequence set forth in amino acid numbers 20 to 477 in SEQ ID NO: 15 and a light chain consisting of the amino acid sequence set forth in amino acid numbers 21 to 234 in SEQ ID NO: 16, or A heavy chain consisting of the amino acid sequence set forth in amino acid numbers 20 to 464 of SEQ ID NO: 19, and a light chain consisting of the amino acid sequence set forth in amino acid numbers 21 to 233 of SEQ ID NO:

20. The method according to any one of claims 1 to 10, characterized in that it comprises:

18. The antibody has a heavy chain consisting of the amino acid sequence set forth in amino acid numbers 20 to 477 of SEQ ID NO: 15 and a light chain consisting of the amino acid sequence set forth in amino acid numbers 21 to 234 of SEQ ID NO: 16, or A heavy chain consisting of the amino acid sequence set forth in amino acid numbers 20 to 464 of SEQ ID NO: 19, and a light chain consisting of the amino acid sequence set forth in amino acid numbers 21 to 233 of SEQ ID NO:

20. The method according to any one of claims 1 to 10, wherein the antibody comprises an amino acid sequence having at least 95% identity with the amino acid sequence of an antibody comprising the amino acid sequence:

19. The antibody has a heavy chain consisting of the amino acid sequence set forth in amino acid numbers 20 to 477 of SEQ ID NO: 15 and a light chain consisting of the amino acid sequence set forth in amino acid numbers 21 to 234 of SEQ ID NO: 16, or A heavy chain consisting of the amino acid sequence set forth in amino acid numbers 20 to 464 of SEQ ID NO: 19, and a light chain consisting of the amino acid sequence set forth in amino acid numbers 21 to 233 of SEQ ID NO:

20. The method according to any one of claims 1 to 10, wherein the antibody comprises an amino acid sequence having at least 99% identity with the amino acid sequence of an antibody comprising the amino acid sequence:

20. The antibody has the ability to recognize a drug, A heavy chain consisting of the amino acid sequence set forth in amino acid numbers 20 to 477 in SEQ ID NO: 15 and a light chain consisting of the amino acid sequence set forth in amino acid numbers 21 to 234 in SEQ ID NO: 16, or A heavy chain consisting of the amino acid sequence set forth in amino acid numbers 20 to 464 of SEQ ID NO: 19, and a light chain consisting of the amino acid sequence set forth in amino acid numbers 21 to 233 of SEQ ID NO:

20. The method according to any one of claims 1 to 10, characterized in that the antibody is an antibody that competes with an antibody comprising:

21. A method for quantifying the plasma concentration of an antibody-drug conjugate in a mammal to which the antibody-drug conjugate has been administered, using an antibody, comprising: The antibody an antibody comprising a heavy chain comprising a heavy chain variable region consisting of the amino acid sequence set forth in amino acid numbers 20 to 141 of SEQ ID NO: 15, and a light chain comprising a light chain variable region consisting of the amino acid sequence set forth in amino acid numbers 21 to 127 of SEQ ID NO: 16, the antibody in the antibody-drug conjugate is not an anti-HER2 antibody or an anti-TROP2 antibody; method.

22. The method of claim 21, wherein the antibody comprises a heavy chain consisting of the amino acid sequence set forth in amino acid numbers 20 to 477 of SEQ ID NO: 15 and a light chain consisting of the amino acid sequence set forth in amino acid numbers 21 to 234 of SEQ ID NO:

16.

23. The method of claim 21, wherein the antibody comprises a heavy chain consisting of the amino acid sequence set forth in amino acid numbers 20 to 464 of SEQ ID NO: 19 and a light chain consisting of the amino acid sequence set forth in amino acid numbers 21 to 233 of SEQ ID NO:

20.

24. The method according to any one of claims 1 to 23, comprising the steps of: (1) contacting an antibody-drug conjugate in plasma with a plate on which a target antigen of the antibody-drug conjugate has been immobilized to form a complex; (2) contacting the complex with an antibody labeled with a marker to form a further complex; and (3) detecting the marker.

25. The method according to any one of claims 1 to 23, comprising: (1) a step of contacting an antibody-drug conjugate in plasma with a plate on which the antibody has been immobilized to form a complex; (2) a step of contacting the complex with a second protein that is capable of recognizing the antibody site of the antibody-drug conjugate and that is labeled with a marker to form a further complex; and (3) a step of detecting the marker.

26. A method for quantifying the plasma concentration of a drug released from an antibody-drug conjugate in a mammal to which the antibody-drug conjugate has been administered, using an antibody, comprising: The antibody formula 【Chemistry 9】 and an antibody conjugate formed by linking the antibody to a drug represented by the formula (I) via a linker, wherein the antibody recognizes the drug moiety of the antibody-drug conjugate; a) an antibody comprising a heavy chain comprising a CDRH1 consisting of the amino acid sequence set forth in SEQ ID NO: 1, a CDRH2 consisting of the amino acid sequence set forth in SEQ ID NO: 2, and a CDRH3 consisting of the amino acid sequence set forth in SEQ ID NO: 3, and a light chain comprising a CDRL1 consisting of the amino acid sequence set forth in SEQ ID NO: 4, a CDRL2 consisting of the amino acid sequence set forth in SEQ ID NO: 5, and a CDRL3 consisting of the amino acid sequence set forth in SEQ ID NO: 6; b) an antibody comprising a heavy chain comprising a CDRH1 consisting of the amino acid sequence set forth in SEQ ID NO: 7, a CDRH2 consisting of the amino acid sequence set forth in SEQ ID NO: 8, and a CDRH3 consisting of the amino acid sequence set forth in SEQ ID NO: 3, and a light chain comprising a CDRL1 consisting of the amino acid sequence set forth in SEQ ID NO: 4, a CDRL2 consisting of the amino acid sequence set forth in SEQ ID NO: 5, and a CDRL3 consisting of the amino acid sequence set forth in SEQ ID NO: 6; c) an antibody comprising a heavy chain comprising a CDRH1 consisting of the amino acid sequence set forth in SEQ ID NO: 9, a CDRH2 consisting of the amino acid sequence set forth in SEQ ID NO: 10, and a CDRH3 consisting of the amino acid sequence set forth in SEQ ID NO: 3, and a light chain comprising a CDRL1 consisting of the amino acid sequence set forth in SEQ ID NO: 4, a CDRL2 consisting of the amino acid sequence set forth in SEQ ID NO: 5, and a CDRL3 consisting of the amino acid sequence set forth in SEQ ID NO: 6; or d) an antibody comprising a heavy chain comprising a CDRH1 consisting of the amino acid sequence set forth in SEQ ID NO: 11, a CDRH2 consisting of the amino acid sequence set forth in SEQ ID NO: 12, and a CDRH3 consisting of the amino acid sequence set forth in SEQ ID NO: 13, and a light chain comprising a CDRL1 consisting of the amino acid sequence set forth in SEQ ID NO: 14, a CDRL2 consisting of the tripeptide WAS, and a CDRL3 consisting of the amino acid sequence set forth in SEQ ID NO: 6, the antibody in the antibody-drug conjugate is not an anti-HER2 antibody or an anti-TROP2 antibody; method.

27. The method according to claim 26, comprising: (1) a step of contacting a drug released from an antibody-drug conjugate in plasma with a plate on which the antibody has been immobilized in the presence of a competing drug labeled with a marker to form a complex; and (2) a step of detecting the marker.

28. A method for determining tissue distribution of an antibody-drug conjugate and / or a drug released from the antibody-drug conjugate in a mammal to which the antibody-drug conjugate has been administered, using an antibody, comprising: The antibody formula 【Chemistry 10】 and an antibody conjugate formed by linking the antibody to a drug represented by the formula (I) via a linker, wherein the antibody recognizes the drug moiety of the antibody-drug conjugate; a) an antibody comprising a heavy chain comprising a CDRH1 consisting of the amino acid sequence set forth in SEQ ID NO: 1, a CDRH2 consisting of the amino acid sequence set forth in SEQ ID NO: 2, and a CDRH3 consisting of the amino acid sequence set forth in SEQ ID NO: 3, and a light chain comprising a CDRL1 consisting of the amino acid sequence set forth in SEQ ID NO: 4, a CDRL2 consisting of the amino acid sequence set forth in SEQ ID NO: 5, and a CDRL3 consisting of the amino acid sequence set forth in SEQ ID NO: 6; b) an antibody comprising a heavy chain comprising a CDRH1 consisting of the amino acid sequence set forth in SEQ ID NO: 7, a CDRH2 consisting of the amino acid sequence set forth in SEQ ID NO: 8, and a CDRH3 consisting of the amino acid sequence set forth in SEQ ID NO: 3, and a light chain comprising a CDRL1 consisting of the amino acid sequence set forth in SEQ ID NO: 4, a CDRL2 consisting of the amino acid sequence set forth in SEQ ID NO: 5, and a CDRL3 consisting of the amino acid sequence set forth in SEQ ID NO: 6; c) an antibody comprising a heavy chain comprising a CDRH1 consisting of the amino acid sequence set forth in SEQ ID NO: 9, a CDRH2 consisting of the amino acid sequence set forth in SEQ ID NO: 10, and a CDRH3 consisting of the amino acid sequence set forth in SEQ ID NO: 3, and a light chain comprising a CDRL1 consisting of the amino acid sequence set forth in SEQ ID NO: 4, a CDRL2 consisting of the amino acid sequence set forth in SEQ ID NO: 5, and a CDRL3 consisting of the amino acid sequence set forth in SEQ ID NO: 6; or d) an antibody comprising a heavy chain comprising a CDRH1 consisting of the amino acid sequence set forth in SEQ ID NO: 11, a CDRH2 consisting of the amino acid sequence set forth in SEQ ID NO: 12, and a CDRH3 consisting of the amino acid sequence set forth in SEQ ID NO: 13, and a light chain comprising a CDRL1 consisting of the amino acid sequence set forth in SEQ ID NO: 14, a CDRL2 consisting of the tripeptide WAS, and a CDRL3 consisting of the amino acid sequence set forth in SEQ ID NO: 6, the antibody in the antibody-drug conjugate is not an anti-HER2 antibody or an anti-TROP2 antibody; method.

29. The method of claim 28, comprising: (1) contacting an antibody-drug conjugate in a tissue and / or a drug released from the antibody-drug conjugate with the antibody to form a complex; (2) contacting the complex with a second protein that can recognize the antibody and is labeled with a marker to form a further complex; and (3) detecting the marker.

30. The method of claim 28, comprising: (1) contacting an antibody-drug conjugate in a tissue and / or a drug released from the antibody-drug conjugate with the antibody labeled with a marker to form a complex; and (2) detecting the marker.

31. 31. The method of claim 24, 25, 27, 29 or 30, wherein the marker is a fluorescent substance and the marker is detected by detecting the fluorescence emitted by the marker.

32. 31. The method according to claim 24, 25, 27, 29 or 30, wherein the marker is an enzyme, and the marker is detected by detecting the luminescence or color produced by the reaction of the enzyme with a substrate.

33. 31. The method of claim 24, 25, 27, 29 or 30, wherein the marker is a luminescent substance and the marker is detected by sensing the luminescence of the marker based on an electrochemical reaction.

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