JP2017114763A - Anti-CD98 antibody-drug conjugate - Google Patents

Abstract

[PROBLEMS] To provide an antitumor drug having an excellent therapeutic effect and excellent antitumor effect and safety. An anti-CD98 antibody-drug conjugate comprising an anti-CD98 antibody, a linker and a drug that inhibits topoisomerase I and exhibits an antitumor action, wherein the drug is a camptothecin derivative represented by the following formula: Exatecan It is. [Selection figure] None

Description

  The present invention relates to an antibody-drug conjugate in which an anti-CD98 antibody and an antitumor drug having antitumor activity are bound via a linker structure moiety.

  Antibody-Drug Conjugate (ADC) is an antibody-drug conjugate (ADC) that binds to an antibody that binds to an antigen that is expressed on the surface of a cancer cell and can be internalized in the cell. It can be expected that the drug can be delivered to the cancer cell and the drug is accumulated in the cancer cell and the cancer cell is killed (see Non-Patent Documents 1 to 3). As an ADC, for example, Mylotarg (gemtuzumab ozogamicin) obtained by binding calicheamicin to an anti-CD33 antibody is approved as a therapeutic agent for acute myeloid leukemia. Adcetris (brentuximab vedotin) in which auristatin E is bound to an anti-CD30 antibody has been approved as a therapeutic drug for Hodgkin lymphoma and anaplastic large cell lymphoma (see Non-Patent Document 4). Drugs contained in previously approved ADCs target DNA or tubulin.

  As an antitumor low molecular weight compound, a camptothecin derivative which is a compound that inhibits topoisomerase I and expresses an antitumor action is known. The following formula

(Exatecan, chemical name: (1S, 9S) -1-amino-9-ethyl-5-fluoro-2,3-dihydro-9-hydroxy-4-methyl-1H, 12H-benzo [de] pyrano [3 ′, 4 ′: 6,7] indolizino [1,2-b] quinoline-10,13 (9H, 15H) -dione) is a camptothecin derivative (Patent Documents 1 and 2). Unlike irinotecan, which is currently used in clinical practice, activation by an enzyme is unnecessary. In addition, it has stronger topoisomerase I inhibitory activity than SN-38, which is the active body of irinotecan, and topotecan also used in clinical practice, and has stronger cytotoxic activity against various cancer cells in vitro. Yes. In particular, it was effective against cancer cells that were resistant to SN-38, etc. due to the expression of P-glycoprotein. In addition, a mouse subcutaneous tumor transplantation model of mice shows a strong antitumor effect, and clinical trials have been carried out but have not yet been launched (see Non-Patent Documents 5 to 10). It was not clear whether exatecan effectively acts as an ADC.

  DE-310 is a complex in which exatecan is bound to a biodegradable carboxymethyldextran polyalcohol polymer via a GGFG peptide spacer (Patent Document 3). By making exatecan into a polymeric prodrug, it retains high blood retention and passively increases the directivity to the tumor site by using increased permeability of tumor neovascularization and tumor tissue retention It is a thing. In DE-310, exatecan, which is the active body, and exatecan in which glycine is bonded to an amino group are continuously released by cleavage of the peptide spacer by an enzyme. As a result, the pharmacokinetics were improved, and in various tumor evaluation models in non-clinical studies, DE-310 was treated with exatecan even though the dose of exatecan was lower than the dose of exatecan alone. The efficacy was higher than that of single agent administration. While clinical trials have been conducted on DE-310, effective cases have been confirmed in humans, and it has been confirmed that the active substance accumulates in tumors rather than normal tissues, while DE-310 in tumors in humans has been reported. In addition, there is a report that accumulation of active bodies is not much different from accumulation in normal tissues, and passive targeting was not seen in humans (see Non-Patent Documents 11 to 14). As a result, DE-310 also did not go on the market, and it was not clear whether Exatecan effectively functions as a drug aimed at such targeting.

As a related compound of DE-310, a structural portion represented by -NH (CH ₂ ) ₄ C (= O)-is inserted between -GGFG-spacer and exatecan, and -GGFG-NH (CH ₂ ) ₄ C ( A complex having = O)-as a spacer structure is also known (Patent Document 4), but the antitumor effect of the complex is not known at all.

  CD98 is a heterodimer composed of an approximately 80-85 kDa type II single-pass transmembrane heavy chain disulfide-bonded to an approximately 40 kDa multi-pass light chain (Non-patent Document 15). The CD98 heavy chain (known as CD98hc, 4F2 or FRP-1) is encoded by the Slc3a2 gene in mice and the SLC3A2 gene in humans. CD98hc is a type II transmembrane protein and has an extracellular domain, a transmembrane domain, and a cytoplasmic tail. CD98 forms a heterodimer by a disulfide bond between one of at least six CD98 light chains (amino acid transporter, LAT-1, LAT-2, etc.) and the CD98hc extracellular domain. This anti-CD98 antibody targeting CD98 is known to have antitumor activity and immunosuppressive activity (Patent Documents 5 to 11).

JP-A-5-59061 JP-A-8-337584 International Publication WO1997 / 46260 Pamphlet International Publication WO2000 / 25825 Pamphlet International Publication WO2007 / 114496 Pamphlet International Publication WO2008 / 017828 Pamphlet International Publication WO2009 / 043922 Brochure International Publication WO2009 / 090553 Brochure JP 2012-092068 A International Publication WO2011 / 118804 Pamphlet International Publication WO2013 / 078377 Pamphlet

Ducry, L., et al. Bioconjugate Chem. (2010) 21, 5-13. Alley, S. C., et al. Current Opinion in Chemical Biology (2010) 14, 529-537. Damle N.K. Expert Opin. Biol. Ther. (2004) 4, 1445-1452. Senter P. D., et al. Nature Biotechnology (2012) 30, 631-637. Kumazawa, E., Tohgo, A., Exp. Opin. Invest. Drugs (1998) 7, 625-632. Mitsui, I., Kumazawa, E., Hirota, Y., et al. Jpn J. Cancer Res. (1995) 86, 776-786. Takiguchi, S., Tohgo, A., et al. Jpn J. Cancer Res. (1997) 88, 760-769. Joto, N. et al. Int J Cancer (1997) 72, 680-686. Kumazawa, E. et al. Cancer Chemother. Pharmacol. (1998) 42, 210-220. De Jager, R., et al. Ann N Y Acad Sci (2000) 922, 260-273. Inoue, K. et al. Polymer Drugs in the Clinical Stage, Edited by Maeda et al. (2003), 145-153. Kumazawa, E. et al. Cancer Sci (2004) 95, 168-175. Soepenberg, O. et al. Clinical Cancer Research, (2005) 11, 703-711. Wente M. N. et al. Investigational New Drugs (2005) 23, 339-347. Joseph M. Cantor et al. Jornal of Cell Science (2012) 125, 1373-1382

  In the treatment of tumors with antibodies, the anti-tumor effect may not be sufficient even if the antibody recognizes the antigen and binds to the tumor cells, and a more effective anti-tumor antibody is required There is. Many anti-tumor low molecular weight compounds have safety problems such as side effects and toxicity even if they have excellent anti-tumor effects, and they have improved safety and acquired better therapeutic effects. To do is also an issue. That is, an object of the present invention is to obtain and provide an antitumor agent having an excellent therapeutic effect and excellent antitumor effect and safety.

  The inventors of the present invention are anti-CD98 antibodies that can target tumor cells, that is, antibodies that have the ability to recognize tumor cells, the ability to bind to tumor cells, or the ability to internalize tumor cells. Therefore, by converting exatecan, which is an antitumor compound, into an antibody-drug conjugate conjugated to the same antibody via a linker structure moiety, (1) the exatecan derivative is delivered to tumor cells to be excatecan The ability of the derivative to exert its antitumor effect specifically in tumor cells, (2) the ability to reduce the dose of exatecan derivative as compared to the single administration, as well as the reliable production of the antitumor effect, (3) normal We thought that it was possible to achieve higher safety because the influence of exatecan derivatives on cells could be alleviated.

  For this purpose, the inventors have successfully created a linker of a specific structure and obtained an anti-CD98 antibody-drug conjugate in which an anti-CD98 antibody and exatecan are linked via this linker, and this compound Was found to exhibit an excellent antitumor effect, and the present invention was completed.

That is, the present invention
(1) An anti-CD98 antibody-drug conjugate comprising an anti-CD98 antibody, a linker and a drug, or a salt thereof,
Where the linker is:
- (Succinimid-3-yl- N) -CH 2 CH 2 CH 2 CH 2 CH 2 -C (= O) -GGFG-NH-CH 2 -O-CH 2 -C (= O) -;
- (Succinimid-3-yl- N) -CH 2 CH 2 CH 2 CH 2 CH 2 -C (= O) -GGFG-NH-CH 2 CH 2 -O-CH 2 -C (= O) -; and
-(Succinimid-3-yl-N) -CH ₂ CH ₂ -C (= O) -NH-CH ₂ CH ₂ O-CH ₂ CH ₂ O-CH ₂ CH ₂ -C (= O) -GGFG-NH -CH ₂ CH ₂ CH ₂ -C (= O)-;
A linker selected from the group consisting of
The drug has the following formula:

A compound represented by
The nitrogen atom of the amino group at position 1 of the drug binds to the carbonyl moiety of the linker,
An anti-CD98 antibody binds to the succinimide part of the linker,
An anti-CD98 antibody-drug conjugate;
(2) The antibody-drug conjugate or salt thereof according to (1), wherein the average number of drugs bound per antibody is in the range of 1 to 10;
(3) The antibody-drug conjugate or salt thereof according to (1) above, wherein the average number of drugs bound per antibody ranges from 2 to 8;
(4) The antibody-drug conjugate or salt thereof according to (1) above, wherein the average number of drugs bound per antibody ranges from 3 to 8;
(5) Anti-CD98 antibody
CDRH1 consisting of the amino acid sequence represented by SEQ ID NO: 19 or an amino acid sequence in which one or several amino acid residues are added, deleted or substituted in the amino acid sequence;
CDRH2 consisting of the amino acid sequence represented by SEQ ID NO: 20 or an amino acid sequence in which one or several amino acid residues are added, deleted or substituted in the amino acid sequence;
CDRH3 consisting of the amino acid sequence represented by SEQ ID NO: 21 or an amino acid sequence in which one or several amino acid residues are added, deleted or substituted in the amino acid sequence;
CDRL1 consisting of the amino acid sequence represented by SEQ ID NO: 22 or an amino acid sequence in which one or several amino acid residues are added, deleted or substituted in the amino acid sequence;
CDRL2 consisting of the amino acid sequence represented by SEQ ID NO: 23 or an amino acid sequence in which one or several amino acid residues are added, deleted or substituted in the amino acid sequence; and the amino acid sequence represented by SEQ ID NO: 24, CDRL3 consisting of an amino acid sequence in which one or several amino acid residues are added, deleted or substituted in the amino acid sequence;
The antibody-drug conjugate or the salt thereof according to any one of (1) to (4), which has at least one CDR selected from the group consisting of and binds to a CD98 heavy chain;
(6) The anti-CD98 antibody has at least 80% of the heavy chain variable region having at least 80% sequence identity to the amino acid sequence represented by SEQ ID NO: 2 and / or the amino acid sequence represented by SEQ ID NO: 4 The antibody-drug conjugate or the salt thereof according to any one of (1) to (4), comprising a light chain variable region having% sequence identity;
(7) The anti-CD98 antibody has at least 80% sequence identity to the amino acid sequence represented by SEQ ID NO: 12 or 14, and / or the amino acid sequence represented by SEQ ID NO: 16 or 18 The antibody-drug conjugate or the salt thereof according to any one of (1) to (4), comprising a light chain having at least 80% sequence identity;
(8) The above (1) to (4), wherein the anti-CD98 antibody comprises a heavy chain comprising the amino acid sequence represented by SEQ ID NO: 12 or 14 and a light chain comprising the amino acid sequence represented by SEQ ID NO: 16 or 18. The antibody-drug conjugate or salt thereof according to any one of the above;
(9) Any one of the above (1) to (4), wherein the anti-CD98 antibody comprises a heavy chain comprising the amino acid sequence represented by SEQ ID NO: 12 and a light chain comprising the amino acid sequence represented by SEQ ID NO: 16. Or an antibody-drug conjugate or salt thereof according to
(10) Any one of (1) to (4) above, wherein the anti-CD98 antibody comprises a heavy chain comprising the amino acid sequence represented by SEQ ID NO: 12 and a light chain comprising the amino acid sequence represented by SEQ ID NO: 18. Or an antibody-drug conjugate or salt thereof according to
(11) Any one of the above (1) to (4), wherein the anti-CD98 antibody comprises a heavy chain comprising the amino acid sequence represented by SEQ ID NO: 14 and a light chain comprising the amino acid sequence represented by SEQ ID NO: 16. Or an antibody-drug conjugate or salt thereof according to
(12) Any one of the above (1) to (4), wherein the anti-CD98 antibody comprises a heavy chain consisting of the amino acid sequence represented by SEQ ID NO: 14 and a light chain consisting of the amino acid sequence represented by SEQ ID NO: 18. Or an antibody-drug conjugate or salt thereof according to
(13) CDRH1 consisting of the amino acid sequence represented by SEQ ID NO: 19 or an amino acid sequence in which one or several amino acid residues are added, deleted or substituted in the amino acid sequence;
CDRH2 consisting of the amino acid sequence represented by SEQ ID NO: 20 or an amino acid sequence in which one or several amino acid residues are added, deleted or substituted in the amino acid sequence;
CDRH3 consisting of the amino acid sequence represented by SEQ ID NO: 21 or an amino acid sequence in which one or several amino acid residues are added, deleted or substituted in the amino acid sequence;
CDRL1 consisting of the amino acid sequence represented by SEQ ID NO: 22 or an amino acid sequence in which one or several amino acid residues are added, deleted or substituted in the amino acid sequence;
CDRL2 consisting of the amino acid sequence represented by SEQ ID NO: 23 or an amino acid sequence in which one or several amino acid residues are added, deleted or substituted in the amino acid sequence; and the amino acid sequence represented by SEQ ID NO: 24, CDRL3 consisting of an amino acid sequence in which one or several amino acid residues are added, deleted or substituted in the amino acid sequence;
An anti-CD98 antibody having at least one CDR selected from the group consisting of and binding to a CD98 heavy chain;
(14) A heavy chain variable region having at least 80% sequence identity to the amino acid sequence represented by SEQ ID NO: 2 and / or at least 80% sequence identity to the amino acid sequence represented by SEQ ID NO: 4 An anti-CD98 antibody according to (13), which comprises a light chain variable region having
(15) Heavy chain having at least 80% sequence identity to the amino acid sequence represented by SEQ ID NO: 12 or 14 and / or at least 80% sequence to the amino acid sequence represented by SEQ ID NO: 16 or 18 The anti-CD98 antibody according to (13), which comprises a light chain having identity;
(16) The anti-CD98 antibody according to (13), comprising a heavy chain consisting of the amino acid sequence represented by SEQ ID NO: 12 or 14 and a light chain consisting of the amino acid sequence represented by SEQ ID NO: 16 or 18.
(17) The anti-CD98 antibody according to (13), comprising a heavy chain consisting of the amino acid sequence represented by SEQ ID NO: 12 and a light chain consisting of the amino acid sequence represented by SEQ ID NO: 16;
(18) The anti-CD98 antibody according to (13), comprising a heavy chain consisting of the amino acid sequence represented by SEQ ID NO: 12 and a light chain consisting of the amino acid sequence represented by SEQ ID NO: 18;
(19) The anti-CD98 antibody according to (13), comprising a heavy chain consisting of the amino acid sequence represented by SEQ ID NO: 14 and a light chain consisting of the amino acid sequence represented by SEQ ID NO: 16;
(20) The anti-CD98 antibody according to (13), comprising a heavy chain consisting of the amino acid sequence represented by SEQ ID NO: 14 and a light chain consisting of the amino acid sequence represented by SEQ ID NO: 18;
(21) The antibody-drug conjugate according to any one of (1) to (12) or a salt thereof or the anti-CD98 antibody according to any one of (13) to (20) as an active ingredient A pharmaceutical composition;
(22) The pharmaceutical composition according to the above (21) for antitumor or anticancer;
(23) The tumor or cancer is lung cancer, renal cancer, urothelial cancer, colon cancer, prostate cancer, glioblastoma multiforme, ovarian cancer, pancreatic cancer, breast cancer, melanoma, liver cancer, bladder cancer, stomach cancer, cervical cancer A pharmaceutical composition according to the above (22), which is a head and neck cancer, esophageal cancer, lymphoma, acute myeloid leukemia, acute lymphocytic leukemia, chronic myelogenous leukemia or multiple myeloma;
(24) The antibody-drug conjugate or the pharmaceutically acceptable salt thereof according to any one of (1) to (12) for producing a pharmaceutical composition, or the above (13) to (13) (20) Use of the anti-CD98 antibody according to (20); and (25) the antibody-drug conjugate or salt thereof according to any one of (1) to (12) or any one of (13) to (20) above A method for treating a tumor and / or cancer, comprising administering to the mammal a therapeutically effective amount of the anti-CD98 antibody according to claim;
About.

  Excellent anti-tumor effects and safety can be achieved by anti-CD98 antibody-drug conjugates in which exatecan is linked to anti-CD98 antibodies via a linker of a specific structure.

FIG. 1 shows the nucleotide sequence (SEQ ID NO: 7) and amino acid sequence (SEQ ID NO: 8) of the chimeric M23 antibody heavy chain. FIG. 2 shows the nucleotide sequence (SEQ ID NO: 9) and amino acid sequence (SEQ ID NO: 10) of the chimeric M23 antibody light chain. FIG. 3 shows the nucleotide sequence (SEQ ID NO: 11) and amino acid sequence (SEQ ID NO: 12) of the hM23-H1 type heavy chain. FIG. 4 shows the nucleotide sequence (SEQ ID NO: 13) and amino acid sequence (SEQ ID NO: 14) of the hM23-H2 type heavy chain. FIG. 5 shows the nucleotide sequence (SEQ ID NO: 15) and amino acid sequence (SEQ ID NO: 16) of the hM23-L1 type light chain. FIG. 6 shows the nucleotide sequence (SEQ ID NO: 17) and amino acid sequence (SEQ ID NO: 18) of the hM23-L2 type light chain. 7 shows the amino acid sequence of CDRH1 of the M23 antibody (SEQ ID NO: 19), the amino acid sequence of CDRH2 (SEQ ID NO: 20), the amino acid sequence of CDRH3 (SEQ ID NO: 21), the amino acid sequence of CDRL1 (SEQ ID NO: 22), and the amino acid of CDRL2 The sequence (SEQ ID NO: 23) and the amino acid sequence of CDRL3 (SEQ ID NO: 24) are shown. FIG. 8 shows the internalization ability of the M23 antibody. FIG. 9 shows the antitumor effect of hM23-H1L1-drug conjugate on human Burkitt lymphoma transplanted mice. FIG. 10 shows the antitumor effect of hM23-H1L2-drug conjugate on human Burkitt lymphoma transplanted mice. FIG. 11 shows the antitumor effect of hM23-H2L2-drug conjugate on mice implanted with human Burkitt lymphoma.

  In the present specification, “cancer” and “tumor” are used interchangeably.

  As used herein, “gene” includes not only DNA but also mRNA, cDNA and cRNA thereof.

  As used herein, “polynucleotide” is used interchangeably with nucleic acid and includes DNA, RNA, probes, oligonucleotides and primers.

  In the present specification, “polypeptide” and “protein” are used interchangeably.

  In the present specification, the “cell” includes a cell in an animal individual and a cultured cell.

  In the present specification, “CD98” is used interchangeably with CD98 protein. Since CD98 consists of a heavy chain and a light chain, “CD98 heavy chain” and “CD98 light chain” are used interchangeably with CD98 heavy chain protein and CD98 light chain protein, respectively. Further, in this specification, “CD98” is interchangeable with “CD98 heavy chain” and “CD98 light chain” or “CD98 heavy chain” or “CD98 light chain” unless otherwise specified. Used for.

  As used herein, “anti-CD98 antibody” refers to an antibody that can bind to the CD98 heavy chain.

  In the present specification, “cytotoxicity” refers to a pathological change in cells in some form, and is not limited to direct trauma, but also includes DNA cleavage, base dimer formation, chromosomal This refers to any structural or functional damage to cells such as cutting, damage to cell division equipment, or reduction of various enzyme activities.

  As used herein, “cytotoxic activity” refers to causing the above cytotoxicity.

  In this specification, “antibody-dependent cellular cytotoxicity” means “antibody dependent cellular cytotoxicity (ADCC) activity”, and NK cells have the activity of damaging target cells such as tumor cells via antibodies. means.

In the present specification, “complement dependent cytotoxicity activity” means “complement dependent cytotoxicity (CDC) activity”, which is an activity in which complement damages target cells such as tumor cells via antibodies. Means.

  In the present specification, the “functional fragment of an antibody” means a partial fragment of an antibody having an antigen-binding activity, and includes Fab, F (ab ′) 2, scFv, and the like. Further, Fab ′, which is a monovalent fragment of the variable region of an antibody obtained by treating F (ab ′) 2 under reducing conditions, is also included in the functional fragment of the antibody. However, the molecule is not limited to these molecules as long as it has the ability to bind to an antigen. These functional fragments include not only those obtained by treating full-length antibody protein molecules with appropriate enzymes, but also proteins produced in appropriate host cells using genetically engineered antibody genes. It is.

  In this specification, “Fab ′” is a monovalent fragment of the variable region of an antibody obtained by treating F (ab ′) 2 under reducing conditions as described above. However, Fab ′ produced using an antibody gene modified by genetic engineering is also included in Fab ′ in the present invention.

  In the present specification, the “epitope” means a partial peptide or partial tertiary structure of CD98 to which a specific anti-CD98 antibody binds. The epitope which is a partial peptide of CD98 can be determined by methods well known to those skilled in the art such as immunoassay, for example, the following method. First, various partial structures of the antigen are prepared. In preparing the partial structure, a known oligopeptide synthesis technique can be used. For example, after preparing a series of polypeptides sequentially shortened by an appropriate length from the C-terminus or N-terminus of CD98 using genetic recombination techniques well known to those skilled in the art, the reactivity of antibodies against them is examined, After determining the recognition site, the epitope can be determined by synthesizing shorter peptides and examining their reactivity with those peptides. In addition, an epitope which is a partial three-dimensional structure of an antigen to which a specific antibody binds can be determined by specifying amino acid residues of the antigen adjacent to the antibody by X-ray structural analysis.

  As used herein, “an antibody that binds to the same epitope” means a different antibody that binds to a common epitope. If the second antibody binds to the partial peptide or the three-dimensional structure to which the first antibody binds, it can be determined that the first antibody and the second antibody bind to the same epitope. In addition, by confirming that the second antibody competes for the binding of the first antibody to the antigen (that is, the second antibody prevents the binding of the first antibody and the antigen), a specific epitope is determined. Even if the sequence or structure of is not determined, it can be determined that the first antibody and the second antibody bind to the same epitope. Furthermore, when the first antibody and the second antibody bind to the same epitope and the first antibody has a special effect such as antitumor activity, the second antibody is expected to have the same activity. it can. Therefore, if the second anti-CD98 antibody binds to the partial peptide to which the first anti-CD98 antibody binds, it can be determined that the first antibody and the second antibody bind to the same epitope of CD98. An antibody in which the first antibody and the second antibody bind to the same epitope of CD98 by confirming that the second anti-CD98 antibody competes for the binding of the first anti-CD98 antibody to CD98. Can be determined.

  In this specification, “CDR” refers to a complementarity determining region (CDR). It is known that there are three CDRs in each of the heavy and light chains of the antibody molecule. CDRs, also called hypervariable domains, are sites in the variable region of the heavy and light chains of an antibody that have particularly high primary structure variability and are heavy and light chain polypeptide chains. In the primary structure, each is separated into three locations. In the present specification, for the CDR of an antibody, the CDR of the heavy chain is represented as CDRH1, CDRH2, CDRH3 from the N-terminal side of the heavy chain amino acid sequence, and the CDR of the light chain is represented by CDRL1 from the N-terminal side of the light chain amino acid sequence. , CDRL2, and CDRL3. These sites are close to each other on the three-dimensional structure and determine the specificity for the antigen to be bound.

  In the present specification, “several” means 2 to 10, 2 to 9, 2 to 8, 2 to 7, 2 to 6, 2 to 5, 2 to 4, 2 to 3 Means two or two.

(CD98)
CD98 is a heterodimer composed of an approximately 80-85 kDa type II single-pass transmembrane heavy chain disulfide-bonded to an approximately 40 kDa multi-pass light chain (Non-patent Document 15). The CD98 heavy chain (known as CD98hc, 4F2 or FRP-1) is encoded by the Slc3a2 gene in mice and the SLC3A2 gene in humans. CD98hc is a type II transmembrane protein and has an extracellular domain, a transmembrane domain, and a cytoplasmic tail. CD98 forms a heterodimer by a disulfide bond between one of at least six CD98 light chains (LAT-1, LAT-2, etc.) and the CD98hc extracellular domain. The CD98 heavy chain is involved in integrin signaling and the CD98 light chain is involved in amino acid transport.

  The DNA sequence and amino acid sequence of CD98 heavy chain are published on public databases. For example, the DNA sequence can be referred to by NM_001013251 (GenBank), the amino acid sequence can be referred to by NP_001013269 (GenBank), and the like.

  CD98 can be used by directly purifying from CD98-expressing cells of humans and non-human mammals (rats, mice, etc.) or by preparing a cell membrane fraction of the cells. It can be obtained by synthesis in vitro or production in a host cell by genetic manipulation. Specifically, in genetic manipulation, CD98 cDNA is incorporated into a vector capable of expression and then synthesized in a solution containing enzymes, substrates and energy substances necessary for transcription and translation, or other prokaryotic or eukaryotic organisms. The protein can be obtained by expressing CD98 by transforming the host cell. In addition, CD98-expressing cells obtained by the above-described genetic manipulation or cell lines expressing CD98 can be used as CD98.

  In addition, the CD98 heavy chain includes a protein having an amino acid sequence in which one or several amino acids are substituted, deleted, and / or added in the amino acid sequence of the CD98 heavy chain, and having biological activity equivalent to that protein. .

(Anti-CD98 antibody)
The anti-CD98 antibody used in the present invention is not particularly limited as long as it can bind to the CD98 heavy chain. Such antibodies have the property of recognizing tumor cells expressing CD98, the property of being able to bind to such tumor cells, and the property of being taken up and internalized within such tumor cells.

  The anti-CD98 antibody used in the present invention immunizes an animal with a CD98 heavy chain or any polypeptide selected from the amino acid sequence of CD98 heavy chain using a conventional method, and collects an antibody produced in vivo, It can be obtained by purification. The species of CD98 as an antigen is not limited to humans, and animals can be immunized with CD98 derived from animals other than humans such as mice and rats. In this case, an antibody applicable to a human disease can be selected by examining the cross-reactivity between the obtained antibody that binds to heterologous CD98 and human CD98.

  Further, according to a known method (for example, Kohler and Milstein, Nature (1975) 256, p.495-497, Kennet, R. ed., Monoclonal Antibodies, p.365-367, Plenum Press, NY (1980)) A hybridoma can be established by fusing an antibody-producing cell that produces an anti-CD98 antibody and a myeloma cell to obtain a monoclonal antibody.

  The CD98 heavy chain used as an antigen can be obtained by causing a host cell to produce a CD98 heavy chain gene by genetic manipulation.

  Specifically, a vector capable of expressing the CD98 heavy chain gene is prepared, introduced into a host cell to express the gene, and the expressed CD98 heavy chain is purified. Hereinafter, a method for obtaining an anti-CD98 antibody will be specifically described.

(1) Preparation of antigen Antigen for producing anti-CD98 antibody includes CD98 heavy chain or polypeptide consisting of at least 6 consecutive partial amino acid sequences, and derivatives in which any amino acid sequence or carrier is added to these. And cells expressing CD98 heavy chain.

  CD98 can be used by directly purifying from human tumor tissue or tumor cells, and can be obtained by synthesizing CD98 in vitro or producing it in a host cell by genetic manipulation.

  Specifically, in genetic manipulation, CD98 cDNA is incorporated into an expressible vector and then synthesized in a solution containing enzymes, substrates and energy substances necessary for transcription and translation, or other prokaryotic organisms or true organisms. An antigen can be obtained by expressing CD98 by transforming a nuclear host cell.

  It is also possible to obtain an antigen as a secreted protein by expressing a fusion protein in which an extracellular region of CD98, which is a membrane protein, and an antibody constant region are linked in an appropriate host / vector system.

  The CD98 cDNA is, for example, a polymerase chain reaction (hereinafter referred to as “PCR”) using a cDNA library expressing CD98 cDNA as a template and a primer that specifically amplifies the CD98 cDNA (Saiki, RK, et al. Science (1988) 239, p. 487-489).

  Examples of in vitro synthesis of a polypeptide include, but are not limited to, a rapid translation system (RTS) manufactured by Roche Diagnostics.

  Examples of prokaryotic cell hosts include Escherichia coli and Bacillus subtilis. To transform a gene of interest into these host cells, the host cell is transformed with a plasmid vector containing a replicon or origin of replication from a species compatible with the host and regulatory sequences. The vector preferably has a sequence capable of imparting phenotypic (phenotypic) selectivity to transformed cells.

  Eukaryotic host cells include cells such as vertebrates, insects, and yeasts. Examples of vertebrate cells include COS cells (Gluzman, Y. Cell (1981) 23, p. 175-182, ATCC CRL-1650), mouse fibroblast NIH3T3 (ATCC No. CRL-1658) and Chinese hamster ovary cells (CHO cells, ATCC CCL-61) dihydrofolate reductase deficient strains (Urlaub, G. and Chasin, LAProc. Natl. Acad. Sci. USA (1980) 77, p. 4126-4220), etc. are often used, but are not limited thereto.

  The transformant obtained as described above can be cultured according to a conventional method, and the target polypeptide is produced intracellularly or extracellularly by the culture.

  As the medium used for the culture, various commonly used media can be appropriately selected according to the employed host cells. For E. coli, for example, an antibiotic such as ampicillin or IPMG can be added to the LB medium as necessary. It can be added and used.

  Recombinant protein produced inside or outside of the transformant by the above culture can be separated and purified by various known separation procedures utilizing the physical and chemical properties of the protein. it can.

  Specific examples of the method include various liquid chromatography such as treatment with a normal protein precipitating agent, ultrafiltration, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, affinity chromatography, and the like. , Dialysis methods, combinations thereof, and the like.

  Moreover, by connecting a histidine tag consisting of 6 residues to the recombinant protein to be expressed, it can be efficiently purified with a nickel affinity column. Alternatively, it can be efficiently purified on a protein A column by linking the IgG Fc region to the recombinant protein to be expressed.

  By combining the above methods, the desired polypeptide can be easily produced in large quantities with high yield and high purity.

(2) Production of anti-CD98 monoclonal antibody
As an example of an antibody that specifically binds to CD98, a monoclonal antibody that specifically binds to CD98 can be mentioned. The method for obtaining the antibody is as described below.

  In producing a monoclonal antibody, the following work steps are generally required. That is,

(A) preparing an antigen;
(B) a step of preparing antibody-producing cells after immunization by injecting an antigen into an animal, collecting blood and determining its antibody titer by determining the timing of splenectomy;
(C) a step of preparing myeloma,
(D) a step of fusing antibody-producing cells and myeloma,
(E) a step of selecting a hybridoma group that produces the target antibody;
(F) dividing (cloning) into single cell clones;
(G) In some cases, a step of culturing a hybridoma to produce a monoclonal antibody, or a step of raising an animal transplanted with a hybridoma to produce a monoclonal antibody,
(H) A step of examining the physiological activity and the binding specificity of the monoclonal antibody thus produced, or testing the characteristics as a labeling reagent.

  Hereinafter, although the production method of a monoclonal antibody is explained in full detail along the said process, the production method of this antibody is not restricted to this, For example, antibody producing cells other than a spleen cell and myeloma can also be used.

(A) Step of preparing antigen Antigen includes CD98 prepared by the method as described above or a part thereof, membrane fraction prepared by CD98-expressing recombinant cell, CD98-expressing recombinant cell, CD98-expressing cell line Using a method well known to those skilled in the art, a chemically synthesized CD98 partial peptide and the like can be used.

(B) Step of preparing antibody-producing cells The antigen obtained in step (a) is mixed with Freund's complete or incomplete adjuvant, or an auxiliary agent such as potassium alum, and an experimental animal is immunized as an immunogen. . Alternatively, experimental animals are immunized with CD98-expressing cells as an immunogen. As the experimental animal, an animal used in a known hybridoma production method can be used without any problem. Specifically, for example, mouse, rat, goat, sheep, cow, horse and the like can be used. However, from the viewpoint of easy availability of myeloma cells to be fused with the extracted antibody-producing cells, it is preferable to use mice or rats as immunized animals.

  In addition, there are no particular restrictions on the mouse and rat strains actually used, and in the case of mice, for example, each strain A, AKR, BALB / c, BDP, BA, CE, C3H, 57BL, C57BL, C57L, DBA , FL, HTH, HT1, LP, NZB, NZW, RF, R III, SJL, SWR, WB, 129, etc., and in the case of rats, for example, Wistar, Low, Lewis, Sprague, Dawley, ACI, BN Fischer or the like can be used.

  These mice and rats can be obtained, for example, from laboratory animal breeders such as Japan Claire and Japan Charles River.

  Among these, in consideration of fusion compatibility with the myeloma cells described later, the BALB / c strain is more preferable as the immunized animal in mice and the Wistar and Low strains in rats.

  In consideration of the homology of antigens between humans and mice, it is also preferable to use mice with reduced biological mechanisms for removing autoantibodies, that is, autoimmune disease mice.

  In addition, the age at the time of immunization of these mice or rats is preferably 3 to 12 weeks old, more preferably 4 to 6 weeks old.

  Methods for immunizing animals include, for example, Weir, DM, Handbook of Experimental Immunology Vol.I.II.III., Blackwell Scientific Publications, Oxford (1987), Kabat, EAand Mayer, MM, Experimental Immunochemistry, Charles C Thomas Known methods described in Publisher Springfield, Illinois (1964) and the like can be used.

  Of these immunization methods, the method of administering an antigen subcutaneously to an animal is preferred. In order to increase immune efficiency, it is more preferable to administer intradermally in the first half and intravenously only in the latter half or the final round.

  The antigen administration schedule varies depending on the type of animal to be immunized, individual differences, etc., but in general, the number of antigen administrations is preferably 3 to 6 times, the administration interval is 1 to 3 weeks, the administration frequency is 4 to 5 times, the administration interval is 1 to 2 weeks is more preferred.

  The dose of the antigen varies depending on the kind of animal and individual differences, but is generally 0.05 to 5 mg, preferably about 0.1 to 0.5 mg.

The booster immunization is performed 1 to 6 weeks after the antigen administration as described above, preferably 1 to 4 weeks, and more preferably 1 to 3 weeks. When the immunogen is a cell, 1 × 10 ⁶ to 1 × 10 ⁷ cells are used.

The dose of antigen for booster immunization varies depending on the type and size of the animal, but generally 0.05 to 5 mg, preferably 0.1 to 0.5 mg, more preferably 0.1 to 0.2 mg in the case of mice, for example. To the extent. When the immunogen is a cell, 1 × 10 ⁶ to 1 × 10 ⁷ cells are used.

  Spleen cells or lymphocytes containing antibody-producing cells are aseptically removed from the immunized animal 1 to 10 days after the booster, preferably 2 to 5 days, and more preferably 2 to 3 days later. In this case, if the antibody titer is measured and an animal having a sufficiently high antibody titer is used as a source of antibody-producing cells, the efficiency of subsequent operations can be increased.

  Examples of the antibody titer measurement method used here include, but are not limited to, the RIA method and the ELISA method. In the case of ELISA method, it can be carried out by the procedure as described below.

  First, the purified or partially purified antigen is adsorbed on a solid phase surface such as a 96-well plate for ELISA, and a solid phase surface on which no antigen is adsorbed is separated from a protein unrelated to the antigen, such as bovine serum albumin (in the present specification, “ After the surface is washed, it is brought into contact with a serially diluted sample (eg, mouse serum) as the first antibody, and the antibody in the sample is bound to the antigen.

  Furthermore, an antibody against a mouse antibody that is enzyme-labeled as a second antibody is added and bound to the mouse antibody. After washing, the substrate of the enzyme is added, and the change in absorbance due to color development based on substrate degradation is measured, thereby determining the antibody titer. calculate.

  Separation of antibody-producing cells from the spleen cells or lymphocytes of the immunized animal can be performed by a known method (for example, Kohler et al., Nature (1975) 256, p.495, Kohler et al., Eur. J. Immunol. (1977) 6, p. 511, Milstein et al., Nature (1977), 266, p. 550, Walsh, Nature, (1977) 266, p. 495). For example, in the case of spleen cells, a general method of separating antibody-producing cells by chopping the spleen and filtering the cells through a stainless mesh and then suspending them in Eagle's minimum essential medium (MEM) can be employed. .

(C) Process for preparing myeloma The myeloma used for cell fusion is not particularly limited, and can be appropriately selected from known cell lines. However, in consideration of convenience when selecting hybridomas from the fused cells, it is preferable to use a HGPRT (Hypoxanthine-guanine phosphoribosyl transferase) deficient strain in which the selection procedure has been established.

  That is, X63-Ag8 (X63), NS1-ANS / 1 (NS1), P3X63-Ag8.U1 (P3U1), X63-Ag8.653 (X63.653), SP2 / 0-Ag14 (SP2 / 0) derived from mouse ), MPC11-45.6TG1.7 (45.6TG), FO, S149 / 5XXO, BU.1, etc. Rat-derived 210.RSY3.Ag.1.2.3 (Y3) etc. Human-derived U266AR (SKO-007) GM1500 / GTG-A12 (GM1500), UC729-6, LICR-LOW-HMy2 (HMy2), 8226AR / NIP4-1 (NP41), etc. These HGPRT-deficient strains can be obtained from, for example, the American Type Culture Collection (ATCC).

These cell lines were supplemented with a suitable medium, such as 8-azaguanine medium (RPMI-1640 medium with glutamine, 2-mercaptoethanol, gentamicin, and fetal calf serum (sometimes referred to herein as “FBS”). Subculture in medium supplemented with 8-azaguanine], Iscove's Modified Dulbecco's Medium (hereinafter referred to as “IMDM”), or Dulbecco's Modified Eagle Medium (hereinafter referred to as “DMEM”) However, 3-4 days before cell fusion, subculture in normal medium (for example, ASF104 medium (Ajinomoto Co., Inc.) containing 10% FBS), and secure 2 × 10 ⁷ or more cells on the day of fusion. Keep it.

(D) Cell fusion step Fusion of antibody-producing cells and myeloma cells is performed by a known method (Weir, DM, Handbook of Experimental Immunology Vol. I. II. III., Blackwell Scientific Publications, Oxford (1987), Kabat, EAand. Mayer, MM, Experimental Immunochemistry, Charles C Thomas Publisher Springfield, Illinois (1964), etc.) and can be appropriately performed under conditions that do not extremely reduce the cell viability.

  As such a method, for example, a chemical method of mixing antibody-producing cells and myeloma cells in a high-concentration polymer solution such as polyethylene glycol, a physical method using electrical stimulation, or the like can be used. Of these, specific examples of the chemical method are as follows.

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

(E) Step of selecting a hybridoma group The method of selecting a hybridoma obtained by cell fusion is not particularly limited, but is usually a HAT (hypoxanthine / aminopterin / thymidine) selection method (Kohler et al., Nature (1975) 256). , p. 495, Milstein et al., Nature (1977) 266, p. 550).

  This method is effective when hybridomas are obtained using myeloma cells of HGPRT-deficient strains that cannot survive with aminopterin.

  That is, by culturing unfused cells and hybridomas in a HAT medium, only hybridomas having resistance to aminopterin can selectively remain and grow.

(F) Step of dividing (cloning) into single cell clones As a method for cloning a hybridoma, a known method such as a methyl cellulose method, a soft agarose method, or a limiting dilution method can be used (for example, Barbara, BMand Stanley, MS). : Selected Methods in Cellular Immunology, WHFreeman and Company, San Francisco (1980)). Among these methods, a three-dimensional culture method such as a methyl cellulose method is particularly suitable. For example, a hybridoma group formed by cell fusion is suspended and cultured in a methylcellulose medium such as ClonaCell-HY Selection Medium D (manufactured by StemCell Technologies # 03804), and the formed hybridoma colonies are recovered to recover the monoclonal hybridoma. Acquisition is possible. Each of the collected hybridoma colonies is cultured, and the one in which the antibody titer is stably recognized in the obtained hybridoma culture supernatant is selected as a CD98 monoclonal antibody-producing hybridoma strain.

  As an example of the hybridoma strain established in this way, CD98 hybridoma M23 can be mentioned. In the present specification, an antibody produced by CD98 hybridoma M23 is referred to as “M23 antibody” or simply “M23”.

  The heavy chain variable region of the M23 antibody has the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing. The light chain variable region of the M23 antibody has the amino acid sequence shown in SEQ ID NO: 4 in the sequence listing.

(G) Step of producing monoclonal antibody The hybridoma selected in this manner can be obtained efficiently by culturing the hybridoma. Prior to the culturing, the hybridoma that produces the target monoclonal antibody can be obtained. It is desirable to screen.

  For this screening, a method known per se can be employed.

  The antibody titer can be measured, for example, by the ELISA method described in the item (b) above.

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

  The hybridoma that has been cloned is cultured by changing the medium from the HAT medium to the normal medium.

  Mass culture is performed by rotary culture using a large culture bottle or spinner culture. A monoclonal antibody that specifically binds to the protein of the present invention can be obtained from the supernatant in this large-scale culture by purification using a method well known to those skilled in the art, such as protein A column purification.

  In addition, ascites containing a large amount of the monoclonal antibody of the present invention is obtained by injecting a hybridoma into the abdominal cavity of the same strain of mice (for example, BALB / c as described above) or Nu / Nu mouse and growing the hybridoma. be able to.

  When administered intraperitoneally, mineral oil such as 2,6,10,14-tetramethylpentadecane (2,6,10,14-tetramethylpentadecane; pristane) is administered in advance (3-7 days ago). A large amount of ascites is obtained.

For example, after injecting an immunosuppressant into the abdominal cavity of a mouse of the same strain as that of the hybridoma to inactivate T cells, 20 ⁶ days later, 10 ⁶ to 10 ⁷ hybridoma clonal cells were added to serum-free medium. Float (0.5 mL) and administer into the abdominal cavity. Ascites is collected from the mouse when the abdomen usually swells and ascites accumulates. By this method, a monoclonal antibody having a concentration of about 100 times or more compared with that in the culture solution can be obtained.

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

  The monoclonal antibody thus obtained has a high antigen specificity for CD98.

(H) Assay of monoclonal antibody The isotype and subclass of the monoclonal antibody thus obtained can be determined as follows.

  First, examples of the identification method include octterlony method, ELISA method, and RIA method.

  The octerulony method is simple, but concentration is necessary when the concentration of the monoclonal antibody is low.

  On the other hand, when the ELISA method or the RIA method is used, the culture supernatant is directly reacted with the antigen-adsorbed solid phase, and further, antibodies corresponding to various immunoglobulin isotypes and subclasses are used as secondary antibodies. Isotypes and subclasses can be identified.

  Further, as a simpler method, a commercially available identification kit (for example, mouse typer kit; manufactured by Bio-Rad) or the like can be used.

Furthermore, protein quantification can be performed by the Foreign Lowry method and the method of calculating from the absorbance at 280 nm (1.4 (OD ₂₈₀ ) = immunoglobulin 1 mg / mL).

  Furthermore, even when the steps (a) to (h) of (2) are performed again and a monoclonal antibody is obtained separately, an antibody having cytotoxic activity equivalent to that of the M23 antibody can be obtained. Is possible. An example of such an antibody is an antibody that binds to the same epitope as the M23 antibody. If the newly produced monoclonal antibody binds to the partial peptide or partial conformation to which the M23 antibody binds, it can be determined that the monoclonal antibody binds to the same epitope as the M23 antibody. In addition, by confirming that the monoclonal antibody competes for binding of M23 antibody to CD98 (that is, the monoclonal antibody prevents binding of M23 antibody to CD98), the sequence or structure of a specific epitope can be determined. Even if not determined, it can be determined that the monoclonal antibody binds to the same epitope as the CD98 antibody. When it is confirmed that the epitope is the same, it is strongly expected that the monoclonal antibody has the same antigen binding ability or biological activity as the M23 antibody.

(3) Other antibodies In addition to the monoclonal antibody against CD98, the antibodies used in the present invention include genetically engineered antibodies that have been artificially modified for the purpose of reducing heteroantigenicity against humans, such as chimeras. (Chimeric) antibodies, humanized antibodies, human antibodies and the like are also included. These antibodies can be produced using known methods.

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

  A chimeric antibody derived from mouse anti-human CD98 antibody M23 is an antibody comprising a heavy chain comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 2 and a light chain comprising a light chain variable region comprising the amino acid sequence of SEQ ID NO: 4. Any human-derived constant region may be included.

  As an example of such a chimeric antibody, a heavy chain having an amino acid sequence consisting of amino acid residues 1 to 465 of SEQ ID NO: 8 and a light chain having an amino acid sequence consisting of amino acid residues 1 to 240 of SEQ ID NO: 10 The antibody which consists of can be mentioned. In the heavy chain sequence shown in SEQ ID NO: 8, the amino acid sequence consisting of the 1st to 19th amino acid residues is a signal sequence, the amino acid sequence consisting of the 20th to 135th amino acid residues is a variable region, The amino acid sequence consisting of the 136th to 465th amino acid residues is a constant region. In the light chain sequence shown in SEQ ID NO: 10 in the sequence listing, the amino acid sequence consisting of amino acid residues 1 to 20 is a signal sequence, and the amino acid sequence consisting of amino acid residues 21 to 135 is a variable region. The amino acid sequence consisting of the 136th to 240th amino acid residues is a constant region.

  The heavy chain amino acid sequence shown in SEQ ID NO: 8 is encoded by the nucleotide sequence shown in SEQ ID NO: 7. The nucleotide sequence consisting of nucleotides 1 to 57 of the nucleotide sequence shown in SEQ ID NO: 7 encodes the heavy chain signal sequence of the antibody, and the nucleotide sequence consisting of nucleotides 58 to 405 represents the heavy chain variable region of the antibody. The nucleotide sequence consisting of nucleotides 406 to 1395 encodes the heavy chain constant region of the antibody.

  The light chain amino acid sequence shown in SEQ ID NO: 10 is encoded by the nucleotide sequence shown in SEQ ID NO: 9. The nucleotide sequence consisting of nucleotides 1 to 60 of the nucleotide sequence shown in SEQ ID NO: 9 encodes the light chain signal sequence of the antibody, and the nucleotide sequence consisting of nucleotides 61 to 405 represents the light chain variable region of the antibody. The nucleotide sequence consisting of nucleotides 406 to 720 encodes the light chain constant region of the antibody.

  The sequences of SEQ ID NOs: 7 and 8 are shown in FIG. 1, and the sequences of SEQ ID NOs: 9 and 10 are shown in FIG.

  Humanized antibodies include antibodies in which only CDRs are incorporated into human-derived antibodies (see Nature (1986) 321, pp.522-525), and by CDR grafting, in addition to CDR sequences, the amino acid residues of some frameworks An example is an antibody (international pamphlet WO 90/07861) transplanted to a human antibody.

  The humanized antibody derived from the M23 antibody (in this specification, sometimes referred to as “humanized M23 antibody”, “humanized M23”, “hM23 antibody”, or “hM23”) includes all six types of M23 antibodies. As long as it retains the CDR sequence, it is not limited to a specific humanized antibody. The heavy chain variable region of the M23 antibody consists of CDRH1 (NYLIE) consisting of the amino acid sequence shown in SEQ ID NO: 19, CDRH2 (VINPGSGVTNYNEKFKG) consisting of the amino acid sequence shown in SEQ ID NO: 20, and the amino acid sequence shown in SEQ ID NO: 21 We have CDRH3 (AEAWFAY) consisting of The light chain variable region of the M23 antibody is represented by CDRL1 (KSSQSLLYSSNQKNYLA) consisting of the amino acid sequence shown in SEQ ID NO: 22 in the sequence listing, CDRL2 (WASTRES) consisting of the amino acid sequence shown in SEQ ID NO: 23, and SEQ ID NO: 24. Possesses CDRL3 (QRYYGYPWT) consisting of the amino acid sequence.

As an example of the humanized antibody of mouse antibody M23,
(1) an amino acid sequence consisting of amino acid residues 20 to 135 of SEQ ID NO: 12 or 14,
(2) an amino acid sequence having at least 95% homology to the amino acid sequence of (1) above, and (3) one or several amino acids in the amino acid sequence of (1) above deleted, substituted or added A heavy chain comprising a heavy chain variable region selected from the group consisting of: and (4) an amino acid sequence consisting of amino acid residues 21 to 135 of SEQ ID NO: 16 or 18;
(5) an amino acid sequence having at least 95% homology with the amino acid sequence of (4) above, and (6) one or several amino acids in the amino acid sequence of (4) above deleted, substituted or added A light chain comprising a light chain variable region selected from the group consisting of:
Any combination of these can be mentioned.

As the amino acid substitution, conservative amino acid substitution is preferred. Conservative amino acid substitutions are those that take place within a group of amino acids that are related to an amino acid side chain. Suitable amino acid groups are as follows:
Acidic group = aspartic acid and glutamic acid; basic group = lysine, arginine and histidine;
Nonpolar group = alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine and tryptophan; and uncharged polar family = glycine, asparagine, glutamine, cysteine, serine, threonine and tyrosine.

Other suitable amino acid groups are:
Aliphatic hydroxy group = serine and threonine;
Amide-containing group = asparagine and glutamine;
Aliphatic group = alanine, valine, leucine and isoleucine; and aromatic group = phenylalanine, tryptophan and tyrosine.

  Such amino acid substitution is preferably performed within a range that does not deteriorate the properties of the substance having the original amino acid sequence.

As an antibody of a suitable combination of the above heavy chain and light chain,
A heavy chain having a heavy chain variable region consisting of an amino acid sequence consisting of 20th to 135th amino acid residues of SEQ ID NO: 12 and a light chain variable region consisting of an amino acid sequence consisting of 21st to 135th amino acid residues of SEQ ID NO: 16; An antibody comprising a light chain having,
A heavy chain having a heavy chain variable region consisting of an amino acid sequence consisting of 20th to 135th amino acid residues of SEQ ID NO: 12 and a light chain variable region consisting of an amino acid sequence consisting of 21st to 135th amino acid residues of SEQ ID NO: 18; An antibody comprising a light chain having,
A heavy chain having a heavy chain variable region consisting of an amino acid sequence consisting of amino acid residues 20 to 135 of SEQ ID NO: 14 and a light chain variable region consisting of an amino acid sequence consisting of 21st to 135th amino acid residues of SEQ ID NO: 16 An antibody comprising a light chain having, and
A heavy chain having a heavy chain variable region consisting of an amino acid sequence consisting of 20th to 135th amino acid residues of SEQ ID NO: 14 and a light chain variable region consisting of an amino acid sequence consisting of 21st to 135th amino acid residues of SEQ ID NO: 18; An antibody comprising a light chain having,
Can be mentioned.

Further suitable antibody combinations include
A heavy chain having a heavy chain variable region consisting of an amino acid sequence consisting of 20th to 135th amino acid residues of SEQ ID NO: 12 and a light chain variable region consisting of an amino acid sequence consisting of 21st to 135th amino acid residues of SEQ ID NO: 16; An antibody comprising a light chain having,
A heavy chain having a heavy chain variable region consisting of an amino acid sequence consisting of 20th to 135th amino acid residues of SEQ ID NO: 12 and a light chain variable region consisting of an amino acid sequence consisting of 21st to 135th amino acid residues of SEQ ID NO: 18; An antibody comprising a light chain having, and
A heavy chain having a heavy chain variable region consisting of an amino acid sequence consisting of 20th to 135th amino acid residues of SEQ ID NO: 14 and a light chain variable region consisting of an amino acid sequence consisting of 21st to 135th amino acid residues of SEQ ID NO: 18; An antibody comprising a light chain having,
Can be mentioned.

Further, as another suitable combination of antibodies,
An antibody comprising a heavy chain consisting of the amino acid sequence set forth in SEQ ID NO: 12 and a light chain consisting of the amino acid sequence set forth in SEQ ID NO: 16,
An antibody consisting of a heavy chain consisting of the amino acid sequence set forth in SEQ ID NO: 12 and a light chain consisting of the amino acid sequence set forth in SEQ ID NO: 18,
An antibody consisting of a heavy chain consisting of the amino acid sequence set forth in SEQ ID NO: 14 and a light chain consisting of the amino acid sequence set forth in SEQ ID NO: 16, and
An antibody consisting of a heavy chain consisting of the amino acid sequence set forth in SEQ ID NO: 14 and a light chain consisting of the amino acid sequence set forth in SEQ ID NO: 18,
Can be mentioned.

Further suitable antibody combinations include
An antibody comprising a heavy chain consisting of the amino acid sequence set forth in SEQ ID NO: 12 and a light chain consisting of the amino acid sequence set forth in SEQ ID NO: 16,
An antibody consisting of a heavy chain consisting of the amino acid sequence set forth in SEQ ID NO: 12 and a light chain consisting of the amino acid sequence set forth in SEQ ID NO: 18, and
An antibody consisting of a heavy chain consisting of the amino acid sequence set forth in SEQ ID NO: 14 and a light chain consisting of the amino acid sequence set forth in SEQ ID NO: 18,
Can be mentioned.

  It is possible to select an antibody having biological activity equivalent to that of each of the above antibodies by combining the heavy chain amino acid sequence and the light chain amino acid sequence having high homology. Such homology is generally 80% or more, preferably 90% or more, more preferably 95% or more, most preferably 99% or more. Homology. In addition, an antibody having the same biological activity as each of the above antibodies can be selected by combining an amino acid sequence in which one to several amino acid residues are substituted, deleted or added to the amino acid sequence of the heavy chain or light chain. Is possible.

  The homology between the two amino acid sequences is the Blast algorithm version 2.2.2 (Altschul, Stephen F., Thomas L. Madden, Alejandro A. Schaffer, Jinghui Zhang, Zheng Zhang, Webb Miller, and David J. Lipman (1997). ), “Gapped BLAST and PSI-BLAST: a new generation of protein database search programs”, Nucleic Acids Res. 25: 3389-3402). The Blast algorithm can also be used by accessing www.ncbi.nlm.nih.gov/blast on the Internet.

  In the heavy chain amino acid sequence shown in SEQ ID NO: 12 or 14, the amino acid sequence consisting of amino acid residues 1 to 19 is a signal sequence, and the amino acid sequence consisting of amino acid residues 20 to 135 is a variable region. The amino acid sequence consisting of the 136th to 465th amino acid residues is a constant region. The heavy chain amino acid sequence shown in SEQ ID NO: 12 or 14 is encoded by the nucleotide sequence shown in SEQ ID NO: 11 or 13, respectively. The nucleotide sequence consisting of the 1st to 57th nucleotides of each nucleotide sequence encodes the antibody heavy chain signal sequence, the nucleotide sequence consisting of the 58th to 405th nucleotides encodes the antibody heavy chain variable region, The nucleotide sequence consisting of nucleotides 406 to 1395 encodes the heavy chain constant region of the antibody. The sequences of SEQ ID NOs: 11 and 12 are shown in FIG. 3, and the sequences of SEQ ID NOs: 13 and 14 are shown in FIG.

  In the light chain amino acid sequence shown in SEQ ID NO: 16 or 18, the amino acid sequence consisting of amino acid residues 1 to 20 is a signal sequence, and the amino acid sequence consisting of amino acid residues 21 to 135 Is a variable region, and the amino acid sequence consisting of amino acid residues 136 to 240 is a constant region. The light chain amino acid sequence shown in SEQ ID NO: 16 or 18 is encoded by the nucleotide sequence shown in SEQ ID NO: 15 or 17, respectively. The nucleotide sequence consisting of the 1st to 60th nucleotides of each nucleotide sequence encodes the antibody light chain signal sequence, the nucleotide sequence consisting of the 61st to 405th nucleotides encodes the antibody light chain variable region, The nucleotide sequence consisting of nucleotides 406 to 720 encodes the light chain constant region of the antibody. The sequences of SEQ ID NOs: 15 and 16 are shown in FIG. 5, and the sequences of SEQ ID NOs: 17 and 18 are shown in FIG.

  Homology between these nucleotide sequences and other antibody nucleotide sequences can be determined by the Blast algorithm.

  The antibody used in the present invention can further include a human antibody that binds to the same epitope as the M23 antibody. An anti-CD98 human antibody means a human antibody having only the gene sequence of an antibody derived from a human chromosome. The anti-CD98 human antibody is obtained by a method using a human antibody-producing mouse having a human chromosome fragment containing heavy and light chain genes of a human antibody (Tomizuka, K. et al., Nature Genetics (1997) 16, p.133). -143; Kuroiwa, Y. et.al., Nucl. Acids Res. (1998) 26, p. 3447-3448; Yoshida, H. et.al., Animal Cell Technology: Basic and Applied Aspects vol. 10, p. .69-73 (Kitagawa, Y., Matsuda, T. and Iijima, S. eds.), Kluwer Academic Publishers, 1999 .; Tomizuka, K. et.al., Proc. Natl. Acad. Sci. USA (2000) ) See 97, p.722-727, etc.).

  Specifically, in such a human antibody-producing mouse, endogenous immunoglobulin heavy chain and light chain loci are disrupted, and instead, human immunoglobulins are transmitted via a yeast artificial chromosome (YAC) vector or the like. Genetically modified animals introduced with heavy and light chain loci can be created by creating knockout and transgenic animals and crossing these animals together.

  In addition, by means of gene recombination technology, eukaryotic cells are transformed with cDNA encoding each heavy chain and light chain of such a human antibody, preferably a vector containing the cDNA, to produce a gene recombinant human monoclonal antibody. This antibody can also be obtained from the culture supernatant by culturing the transformed cells.

  Here, for example, eukaryotic cells, preferably CHO cells, mammalian cells such as lymphocytes and myeloma can be used as the host.

  In addition, a method for obtaining a human antibody derived from phage display selected from a human antibody library (Wormstone, IMet.al, Investigative Ophthalmology & Visual Science. (2002) 43 (7), p.2301-2308; Carmen, S .et.al., Briefings in Functional Genomics and Proteomics (2002), 1 (2), p.189-203; Siriwardena, D. et.al., Ophthalmology (2002) 109 (3), p.427-431 Etc.) are also known.

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

  By analyzing the gene of the phage selected by binding to the antigen, the DNA sequence encoding the variable region of the human antibody that binds to the antigen can be determined.

  If the DNA sequence of scFv that binds to an antigen is clarified, a human antibody can be obtained by preparing an expression vector having the sequence and introducing it into an appropriate host for expression (WO92 / 01047, WO92 / 20791, WO93 / 06213, WO93 / 11236, WO93 / 19172, WO95 / 01438, WO95 / 15388, Annu. Rev. Immunol (1994) 12, p.433-455, Nature Biotechnology (2005) 23 (9), p. 1105-1116).

  If a newly produced human antibody binds to the partial peptide or partial conformation to which the M23 antibody binds, it can be determined that the human antibody binds to the same epitope as the M23 antibody. In addition, by confirming that the human antibody competes for binding of M23 antibody to CD98 (that is, the human antibody prevents binding of M23 antibody to CD98), the sequence or structure of a specific epitope can be determined. Even if not determined, it can be determined that the human antibody binds to the same epitope as the M23 antibody. When it is confirmed that the epitope is the same, it is strongly expected that the human antibody has a biological activity equivalent to that of the M23 antibody.

  The chimeric antibody, humanized antibody, or human antibody obtained by the above method can be evaluated for binding to an antigen by a known method or the like, and a suitable antibody can be selected.

  An example of another index for comparing antibody properties is antibody stability. Differential scanning calorimetry (DSC) is a device that can quickly and accurately measure the thermal denaturation midpoint (Tm), which is a good indicator of the relative structural stability of proteins. By measuring the Tm value using DSC and comparing the values, the difference in thermal stability can be compared. It is known that the storage stability of antibodies shows a certain degree of correlation with the thermal stability of antibodies (Lori Burton, et.al., Pharmaceutical Development and Technology (2007) 12, p.265-273). A suitable antibody can be selected using stability as an index. Other indicators for selecting antibodies include high yields in appropriate host cells and low aggregation in aqueous solutions. For example, since the antibody with the highest yield does not necessarily exhibit the highest thermal stability, it is necessary to select the most suitable antibody for human administration based on a comprehensive judgment based on the above-mentioned indicators. .

  Antibodies used in the present invention include antibody modifications. The modified body means that the antibody is chemically or biologically modified. Chemical modifications include chemical modifications to the amino acid backbone, chemical modifications of N-linked or O-linked carbohydrate chains, and the like. For biological modifications, post-translational modifications (eg N- or O-glycosylation, N- or C-terminal processing, deamidation, aspartic acid isomerization, methionine oxidation) And those having a methionine residue added to the N-terminus by expression using a prokaryotic host cell. In addition, those modified to allow detection or isolation of the antibody or antigen used in the present invention, for example, an enzyme label, a fluorescent label, and an affinity label are also included in the meaning of such a modified product. Such a modified antibody used in the present invention is useful for improving the stability and blood retention of the original antibody, reducing antigenicity, and detecting or isolating such antibody or antigen.

  In addition, it is possible to enhance antibody-dependent cytotoxic activity by regulating the modification of the sugar chain bound to the antibody (glycosylation, defucosylation, etc.). WO99 / 54342, WO00 / 61739, WO02 / 31140, and the like are known as techniques for regulating antibody sugar chain modification, but are not limited thereto. The antibody used in the present invention includes an antibody in which the sugar chain modification is regulated.

  The anti-CD98 antibody used in the present invention can also be obtained by once isolating the antibody gene and then introducing it into a suitable host. Specific examples of the antibody gene include a combination of a gene encoding the heavy chain sequence and a gene encoding the light chain sequence of the antibody described herein. When transforming a host cell, the heavy chain sequence gene and the light chain sequence gene can be inserted into the same expression vector, or can be inserted into separate expression vectors. is there.

  When eukaryotic cells are used as hosts, animal cells, plant cells, and eukaryotic microorganisms can be used. In particular, as animal cells, mammalian cells such as COS cells (Gluzman, Y. Cell (1981) 23, p.175-182, ATCC CRL-1650) which are monkey cells, mouse fibroblast NIH3T3 (ATCC No. CRL-1658) and Chinese hamster ovary cells (CHO cells, ATCC CCL-61) dihydrofolate reductase-deficient strain (Urlaub, G. and Chasin, LAProc. Natl. Acad. Sci. USA (1980) 77, p .4126-4220).

  When prokaryotic cells are used, for example, Escherichia coli and Bacillus subtilis can be mentioned.

  An antibody can be obtained by introducing a desired antibody gene into these cells by transformation and culturing the transformed cells in vitro. In the culture, the yield may vary depending on the sequence of the antibody, and it is possible to select an antibody having an equivalent binding activity that can be easily produced as a drug using the yield as an index. Therefore, the antibody used in the present invention includes a step of culturing the transformed host cell and a step of collecting the target antibody or a functional fragment of the antibody from the culture obtained in the step. The antibody obtained by the method for producing the antibody is also included.

  It is known that the lysine residue at the carboxyl terminus of the heavy chain of an antibody produced in cultured mammalian cells is deleted (Journal of Chromatography A, 705: 129-134 (1995)). It is known that two amino acid residues of glycine and lysine at the chain carboxyl terminus are deleted and a proline residue located at the carboxyl terminus is newly amidated (Analytical Biochemistry, 360: 75-83 (2007) ). However, deletion and modification of these heavy chain sequences do not affect the antigen-binding ability and effector functions (such as complement activation and antibody-dependent cytotoxicity) of the antibody. Accordingly, the present invention also includes the modified antibody and a functional fragment of the antibody, a deletion in which one or two amino acids are deleted from the heavy chain carboxyl terminus, and the amidated deletion. (For example, a heavy chain in which a proline residue at the carboxyl terminal site is amidated). However, as long as the antigen binding ability and the effector function are maintained, the deletion of the carboxyl terminus of the heavy chain of the antibody used in the present invention is not limited to the above type. The two heavy chains constituting the antibody used in the present invention may be any one of full length and a heavy chain selected from the group consisting of the above-mentioned deletion forms, or a combination of any two of them. It may be. The amount ratio of each deletion can be affected by the type and culture conditions of cultured mammalian cells that produce the antibody used in the present invention, but the main component of the antibody used in the present invention is both two heavy chains. And the case where one amino acid residue at the carboxyl terminus is deleted.

  Examples of the isotype of the antibody used in the present invention include IgG (IgG1, IgG2, IgG3, IgG4), and preferably IgG1 or IgG2.

  The antibody used in the present invention may be a functional fragment of an antibody having an antigen-binding portion of the antibody or a modified product thereof. A fragment of the antibody can be obtained by treating the antibody with a proteolytic enzyme such as papain or pepsin or by modifying the antibody gene by a genetic engineering technique and expressing it in an appropriate cultured cell. Among such antibody fragments, a fragment that retains all or part of the functions of the full-length antibody molecule can be referred to as an antibody functional fragment.

  The biological activity of the anti-CD98 antibody used in the present invention includes antigen-binding activity, activity that is internalized in cells that express the antigen by binding to the antigen, activity that neutralizes the activity of the antigen, and activity of the antigen is enhanced. Functions, antibody-dependent cytotoxicity (ADCC) activity, complement-dependent cytotoxicity (CDC) activity and antibody-dependent cell-mediated phagocytosis (ADCP). Is a binding activity to the CD98 heavy chain, preferably an activity that is internalized in a CD98-expressing cell by binding to the CD98 heavy chain. Furthermore, the antibody used in the present invention may have ADCC activity, CDC activity and / or ADCP activity in addition to cell internalization activity.

  The antigen-binding activity of the antibody can be confirmed using flow cytometry.

  The activity internalized in cells is (1) an assay that visualizes antibodies taken into cells using a secondary antibody (fluorescent label) that binds to the therapeutic antibody (Cell Death and Differentiation (2008) 15, 751-761), (2) Assays that measure the amount of fluorescence incorporated into cells using a secondary antibody (fluorescent label) that binds to a therapeutic antibody (Molecular Biology of the Cell Vol. 15, 5268-5282, December 2004) or (3) Mab-ZAP assay, where immunotoxin binding to therapeutic antibodies is used to release toxins and suppress cell proliferation when taken up into cells (BioTechniques 28: 162-165, January 2000) Can be confirmed. As the immunotoxin, a diphtheria toxin catalytic domain and a protein G recombinant complex protein can also be used.

  The obtained antibody can be purified to homogeneity. For separation and purification of antibodies, separation and purification methods used for ordinary proteins may be used. For example, antibodies can be separated and purified by appropriately selecting and combining column chromatography, filter filtration, ultrafiltration, salting out, dialysis, preparative polyacrylamide gel electrophoresis, isoelectric focusing, etc. (Strategies for Protein Purification and Characterization: A Laboratory Course Manual, Daniel R. Marshak et al. eds., Cold Spring Harbor Laboratory Press (1996); Antibodies: A Laboratory Manual. Ed Harlow and David Lane, Cold Spring Harbor Laboratory (1988)) However, it is not limited to these.

  Examples of the chromatography include affinity chromatography, ion exchange chromatography, hydrophobic chromatography, gel filtration chromatography, reverse phase chromatography, adsorption chromatography and the like.

  These chromatography can be performed using liquid chromatography such as HPLC or FPLC.

  Examples of the column used for affinity chromatography include a protein A column and a protein G column. For example, as a column using a protein A column, Hyper D, POROS, Sepharose F.F. (Pharmacia) and the like can be mentioned.

  It is also possible to purify an antibody using a carrier on which an antigen is immobilized, utilizing the binding property to the antigen.

The anti-CD98 antibody used in the present invention is not particularly limited as long as it can bind to the CD98 heavy chain, but is preferably the following antibody:
(1) an antibody having the property of being internalized in a CD98-expressing cell by binding to a CD98 heavy chain;
(2) The antibody or the antibody according to (1) above, wherein CD98 is human CD98;
(3) CDRs in the heavy chain of the antibody, CDRH1 consisting of the amino acid sequence set forth in SEQ ID NO: 19, CDRH2 consisting of the amino acid sequence set forth in SEQ ID NO: 20, and CDRH3 consisting of the amino acid sequence set forth in SEQ ID NO: 21, and The CDR in the light chain of the antibody has CDRL1 consisting of the amino acid sequence shown in SEQ ID NO: 22, CDRL2 consisting of the amino acid sequence shown in SEQ ID NO: 23, and CDRL3 consisting of the amino acid sequence shown in SEQ ID NO: 24 ( The antibody according to 1) or (2);
(4) The antibody according to any one of (1) to (3) above, wherein the constant region is a human-derived constant region;
(5) The antibody according to any one of (1) to (4), which is humanized;
(6) The variable region of the heavy chain of the antibody
(A) the amino acid sequence of amino acid numbers 20 to 135 in SEQ ID NO: 12,
(B) the amino acid sequence of amino acid numbers 20 to 135 in SEQ ID NO: 14,
(C) an amino acid sequence having at least 95% homology to the sequence of (a) or (b), and
(D) an amino acid sequence selected from the group consisting of amino acid sequences in which one or several amino acids are deleted, substituted or added in the sequence of (a) or (b),
Consisting of an amino acid sequence selected from the group consisting of
The variable region of the light chain of the antibody
(E) the amino acid sequence of amino acid numbers 21 to 135 in SEQ ID NO: 16,
(F) the amino acid sequence of amino acid numbers 21 to 135 in SEQ ID NO: 18,
(G) an amino acid sequence having at least 95% homology to the sequence of (e) or (f), and
(H) an amino acid sequence in which one or several amino acids are deleted, substituted or added in the sequence of (e) or (f),
The antibody according to (5) above, comprising an amino acid sequence selected from the group consisting of:
(7) the combination of the variable region of the heavy chain of the antibody and the variable region of the light chain of the antibody,
A combination of the variable region of the heavy chain consisting of the amino acid sequence set forth in amino acid numbers 20 to 135 in SEQ ID NO: 12 and the variable region of the light chain consisting of the amino acid sequence set forth in amino acid numbers 21 to 135 in SEQ ID NO: 16;
A combination of a variable region of a heavy chain consisting of the amino acid sequence set forth in amino acid numbers 20 to 135 in SEQ ID NO: 12 and a variable region of a light chain consisting of the amino acid sequence set forth in amino acid numbers 21 to 135 in SEQ ID NO: 18;
A combination of a variable region of a heavy chain consisting of the amino acid sequence set forth in amino acid numbers 20 to 135 in SEQ ID NO: 14 and a variable region of a light chain consisting of the amino acid sequence set forth in amino acid numbers 21 to 135 in SEQ ID NO: 16; A combination of a variable region of a heavy chain consisting of the amino acid sequence set forth in amino acid numbers 20 to 135 and a light chain variable region consisting of the amino acid sequence set forth in amino acid numbers 21 to 135 of SEQ ID NO: 18;
The antibody according to (6) above, which is a combination selected from the group consisting of:
(8) The combination of the variable region of the antibody heavy chain and the variable region of the antibody light chain is:
A combination of the variable region of the heavy chain consisting of the amino acid sequence set forth in amino acid numbers 20 to 135 in SEQ ID NO: 12 and the variable region of the light chain consisting of the amino acid sequence set forth in amino acid numbers 21 to 135 in SEQ ID NO: 16;
A combination of a variable region of a heavy chain consisting of the amino acid sequence set forth in amino acid numbers 20 to 135 in SEQ ID NO: 12 and a variable region of a light chain consisting of the amino acid sequence set forth in amino acid numbers 21 to 135 in SEQ ID NO: 18, or
A combination of a variable region of a heavy chain consisting of the amino acid sequence set forth in amino acid numbers 20 to 135 in SEQ ID NO: 14 and a variable region of a light chain consisting of the amino acid sequence set forth in amino acid numbers 21 to 135 in SEQ ID NO: 18;
The antibody according to (6) above,
(9) The combination of heavy chain and light chain is
A combination of a heavy chain consisting of the amino acid sequence set forth in amino acid numbers 20 to 465 in SEQ ID NO: 12 and a light chain consisting of the amino acid sequence set forth in amino acid numbers 21 to 240 in SEQ ID NO: 16;
A combination of a heavy chain consisting of the amino acid sequence set forth in amino acid numbers 20 to 465 in SEQ ID NO: 12 and a light chain consisting of the amino acid sequence set forth in amino acid numbers 21 to 240 in SEQ ID NO: 18;
A combination of a heavy chain consisting of the amino acid sequence set forth in amino acid numbers 20 to 465 in SEQ ID NO: 14 and a light chain consisting of the amino acid sequence set forth in amino acid numbers 21 to 240 in SEQ ID NO: 16; and
A combination of a heavy chain consisting of the amino acid sequence set forth in amino acid numbers 20 to 465 in SEQ ID NO: 14 and a light chain consisting of the amino acid sequence set forth in amino acid numbers 21 to 240 in SEQ ID NO: 18;
The antibody according to (6) above, which is a combination selected from the group consisting of:
(10) The combination of heavy chain and light chain is
A combination of a heavy chain consisting of the amino acid sequence set forth in SEQ ID NO: 12 and a light chain consisting of the amino acid sequence set forth in SEQ ID NO: 16,
A combination of a heavy chain consisting of the amino acid sequence set forth in SEQ ID NO: 12 and a light chain consisting of the amino acid sequence set forth in SEQ ID NO: 18, or
A combination of a heavy chain consisting of the amino acid sequence set forth in SEQ ID NO: 14 and a light chain consisting of the amino acid sequence set forth in SEQ ID NO: 18.
Or the antibody according to (6) above, or
(11) A step of culturing a host cell transformed with an expression vector containing a polynucleotide encoding the antibody according to any one of (1) to (10) above, and a culture obtained in the step An antibody obtained by a method for producing the antibody, comprising the step of collecting the antibody of interest from the method.

(Drug)
The drug used in the present invention has the following formula:

Exatecan ((1S, 9S) -1-amino-9-ethyl-5-fluoro-2,3-dihydro-9-hydroxy-4-methyl-1H, 12H-benzo [de], a camptothecin derivative represented by Pyrano [3 ', 4': 6,7] Indolizino [1,2-b] quinoline-10,13 (9H, 15H) -dione, although exatecan has excellent antitumor activity, Exatecan can be easily obtained by a known method, and the amino group at position 1 can be preferably used as a binding site to the linker structure. In some cases, it is released into tumor cells in a bound state, but it is an excellent compound that exhibits an excellent antitumor effect even in such a state.

  Exatecan has a camptothecin structure, so in an acidic aqueous medium (for example, about pH 3), the equilibrium is biased to a structure in which a lactone ring is formed (ring-closed), whereas in a basic aqueous medium (for example, about pH 10), the lactone ring is It is known that the equilibrium is biased to the ring-opened structure (ring-opened body). Even drug conjugates into which exatecan residues corresponding to such a ring-closed structure and ring-opened structure are introduced are expected to have an equivalent antitumor effect, and any of them are included in the scope of the present invention. Nor.

  In an antibody-drug conjugate, the number of drugs bound to one antibody molecule is an important factor affecting the effectiveness and safety. Antibody-drug conjugates are manufactured by specifying reaction conditions such as the amount of raw materials and reagents to be reacted so that the number of drug bonds is constant. Unlike, it is usually obtained as a mixture of different numbers of drugs combined. The number of drugs bound to one antibody molecule is specified and expressed as an average value, that is, the average number of drug bonds. In the present invention, unless otherwise indicated, the number of drug bindings is, unless it indicates an antibody-drug conjugate having a specific drug binding number contained in an antibody-drug conjugate mixture having a different drug binding number. Mean value. The number of Exatecan binding to the antibody molecule is controllable, and about 1 to 10 Exatecan can be bound as the average number of drugs per antibody, preferably 2 to 8, more preferably There are 3 to 8 pieces. A person skilled in the art can design a reaction that binds the required number of drugs to the antibody from the description in Examples below, and can obtain an antibody-drug conjugate in which the number of bindings of exatecan is controlled. .

(Linker)
The linker used in the present invention is
-(Succinimid-3-yl-N) -CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ -C (= O) -GGFG-NH-CH ₂ -O-CH ₂ -C (= O)-,
-(Succinimid-3-yl-N) -CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ -C (= O) -GGFG-NH-CH ₂ CH ₂ -O-CH ₂ -C (= O)-, or
-(Succinimid-3-yl-N) -CH ₂ CH ₂ -C (= O) -NH-CH ₂ CH ₂ O-CH ₂ CH ₂ O-CH ₂ CH ₂ -C (= O) -GGFG-NH -CH ₂ CH ₂ CH ₂ -C (= O)-
It is a linker shown by the structure of “-(Succinimid-3-yl-N)-” has the following formula:

It has the structure represented by these. The 3rd position in this partial structure is a binding site to the anti-CD98 antibody. This binding to the antibody at the 3-position is characterized by binding by forming a thioether. The nitrogen atom at position 1 of this structural moiety is bound to the methylene carbon atom present in the linker containing the structure. That is, the binding part between the antibody and the linker is represented by the following formula:

(Wherein “antibody-S-” is derived from an antibody and n ¹ is 2 or 5).

  “GGFG” is a tetrapeptide consisting of glycine-glycine-phenylalanine-glycine.

  The linker used for this invention can be prepared according to the method as described in the Example mentioned later, for example.

(Antibodies having sulfide groups)
An antibody having a sulfide group, that is, “antibody-S-” can be obtained by a method well known to those skilled in the art (Hermanson, GT, Bioconjugate Techniques, pp. 56-136, pp. 456-493, Academic Press (1996). )). For example, a method in which Traut's reagent is allowed to act on the amino group of an antibody, a method in which N-succinimidyl S-acetylthioalkanoate is allowed to act on the amino group of an antibody, and then a hydroxylamine is allowed to act, N-succinimidyl 3- (pyridyldithio) ) Method of reacting propionate and then reducing agent, hinge on intra-antibody by reducing agent such as dithiothreitol, 2-mercaptoethanol, tris (2-carboxyethyl) phosphine hydrochloride (TCEP) Examples of the method include, but are not limited to, a method of generating a sulfhydryl group by reducing a part of disulfide bonds.

  Specifically, TCEP as a reducing agent is used in an amount of 0.3 to 3 molar equivalents per hinge part disulfide in the antibody, and reacted with the antibody in a buffer containing a chelating agent, whereby the hinge part disulfide in the antibody Can be obtained in which the antibody is partially or completely reduced.

  Examples of chelating agents include ethylenediaminetetraacetic acid (EDTA) and diethylenetriaminepentaacetic acid (DTPA). These may be used at a concentration of 1 to 20 mM.

  As the buffer solution, sodium phosphate, sodium borate, sodium acetate solution and the like can be used. Specifically, an antibody having a partially or completely reduced sulfhydryl group can be obtained by reacting with TCEP at 4 to 37 ° C. for 1 to 4 hours.

(Antibody-drug conjugate)
The bond between the drug and the linker, and the bond between the antibody having a sulfide bond and the drug linker can be performed, for example, according to the method described in Examples described later. That is, the antibody-drug conjugate of the present invention can be prepared, for example, according to the method described in Examples described later.

The antibody-drug conjugate of the present invention has NH ₂ -CH ₂ -O-CH ₂ -C (= O)-(NH-DX), NH _2- CH ₂ CH ₂ —O—CH ₂ —C (═O) — (NH—DX) or NH ₂ —CH ₂ CH ₂ CH ₂ —C (═O) — (NH—DX). Where “-(NH-DX)” is:

The nitrogen atom of the amino group at position 1 is bonded to the carbonyl group of the linker.

In the case of NH ₂ -CH ₂ -O-CH ₂ -C (= O)-(NH-DX), since the aminal structure in the same molecule is unstable, it further self-decomposes to HO-CH ₂ -C It was confirmed that (= O)-(NH-DX) was released. These compounds can also be suitably used as an intermediate for producing the antibody-drug conjugate of the present invention.

  The antibody-drug conjugate of the present invention may absorb moisture, become adsorbed water, or become hydrated by being left in the air or recrystallized, and contains such water. Compounds and salts are also encompassed by the present invention.

The present invention can also include antibody-drug conjugates in which one or more of the atoms making up the antibody-drug conjugate is replaced with an isotope of that atom. There are two types of isotopes: radioactive isotopes and stable isotopes. Examples of isotopes include hydrogen isotopes ( ² H and ³ H), carbon isotopes ( ¹¹ C, ¹³ C and ¹⁴ C), nitrogen isotopes ( ¹³ N and ¹⁵ N), oxygen isotopes ( ¹⁵ O, ¹⁷ O and ¹⁸ O), fluorine isotopes ( ¹⁸ F) and the like. Compositions comprising isotope-labeled antibody-drug conjugates are useful, for example, as therapeutic agents, prophylactic agents, research reagents, assay reagents, diagnostic agents, in vivo diagnostic imaging agents, and the like. Isotope-labeled antibody-drug conjugates and any ratio mixtures of isotope-labeled antibody-drug conjugates are all encompassed by the present invention. An isotope-labeled antibody-drug conjugate is produced by a method known in the art, for example, by using an isotope-labeled raw material instead of the raw material in the production method of the present invention described later. Can do.

  Since the antibody-drug conjugate of the present invention exhibits cytotoxic activity against cancer cells, it can be used as an active ingredient of a pharmaceutical composition for treatment and / or prevention against cancer.

  The types of cancer are lung cancer, renal cancer, urothelial cancer, colon cancer, prostate cancer, glioblastoma multiforme, ovarian cancer, pancreatic cancer, breast cancer, melanoma, liver cancer, bladder cancer, stomach cancer, cervical cancer, head and neck Cancer, esophageal cancer, lymphoma, acute myelogenous leukemia, acute lymphocytic leukemia, chronic myelogenous leukemia, multiple myeloma, etc. These are cancers derived from cancer cells expressing CD98. It is not limited to.

  The CD98 antibody used in the present invention or the antibody-drug conjugate of the present invention is used as an active ingredient of a pharmaceutical composition for treating an autoimmune disease or a pharmaceutical composition for suppressing rejection to transplantation. be able to.

  The pharmaceutical composition containing the antibody-drug conjugate of the present invention is preferably systemically or locally when administered to a mammal (eg, human, horse, cow, pig, etc., preferably human). Can be administered parenterally.

  Parenteral routes of administration include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous and subcutaneous routes. Examples of the administration method include infusion and bolus injection, and infusion is preferred.

  The pharmaceutical composition of the present invention can be prepared by selecting an appropriate form according to the administration method and preparing various preparations that are usually used.

  For example, antibody-drug conjugates of the present invention and sterile liquids (eg, water and oils (eg oils, animals, plants, or oils of synthetic origin (eg, oils such as oil, animals, plants or synthetic sources) such as those described in “Remington's Pharmaceutical Sciences” by EW Martin). , Peanut oil, soybean oil, mineral oil, sesame oil, etc.))), saline solution, dextrose solution, glycerol solution, etc., wetting agent, emulsifier, pH buffering agent, etc. A pharmaceutical composition can be prepared.

  The pharmaceutical composition of the present invention may also contain a solubilizer, a local anesthetic (eg, lignocaine) for relieving pain at the injection site, and the like. The pharmaceutical composition of the present invention may be supplied in a form in which the active ingredient and the solvent are contained in separate containers. Also, when the pharmaceutical composition of the invention is administered by infusion, it may be administered, for example, in an infusion bottle containing the active ingredient and sterile pharmaceutical grade water or saline. When the pharmaceutical composition of the present invention is administered by injection, the active ingredient may be mixed with sterile water for injection or saline before administration.

  The pharmaceutical composition of the present invention may comprise an anti-CD98 antibody-drug conjugate and at least one other cancer therapeutic agent. The antibody-drug conjugate of the present invention can also be administered together with other cancer therapeutic agents, thereby enhancing the anticancer effect. Other anticancer agents used for such purposes may be administered to an individual simultaneously or separately with the antibody-drug conjugate, or may be administered at different intervals. Examples of such cancer therapeutic agents include abraxane, carboplatin, cisplatin, gemcitabine, irinotecan (CPT-11), paclitaxel, pemetrexed, sorafenib, vinblastin, drugs described in International Publication No. WO2003 / 038043, and LH-RH analogs (Leuprorelin, goserelin, etc.), estramustine phosphate, estrogen antagonists (tamoxifen, raloxifene, etc.), aromatase inhibitors (anastrozole, letrozole, exemestane, etc.), etc. The drug is not limited as long as it has an activity.

  The pharmaceutical composition of the present invention may be provided as a lyophilized formulation or a liquid formulation. When provided as a lyophilized formulation, it may be a formulation containing appropriate formulation additives used in this field. Similarly, liquid preparations may be preparations containing appropriate preparation additives used in this field.

  Although the composition of the pharmaceutical composition of the present invention and the concentration of the active ingredient vary depending on the administration method, the anti-CD98 antibody-drug conjugate contained in the pharmaceutical composition of the present invention is compatible with the antigen of the antibody-drug conjugate. That is, in terms of the dissociation constant (Kd value) for the antigen, the higher the affinity (the lower the Kd value), the more effective the drug effect can be achieved. Therefore, in determining the dose of the antibody-drug conjugate, the dose can be set based on the affinity state between the antibody-drug conjugate and the antigen. When the antibody-drug conjugate of the present invention is administered to humans, for example, about 0.001 to 100 mg / kg may be administered once or multiple times at intervals of 1 to 180 days.

  The present invention will be specifically described with reference to the following examples, but the present invention is not limited thereto. Moreover, these are not limitedly interpreted in any sense. In addition, in this specification, reagents, solvents and starting materials not particularly described are readily available from commercially available sources.

(Example 1) Monoclonal antibody production and antibody screening (1-1) Immunization
4-6 week old BALB / cAnNCrlCrlj mice (Charles River Japan) were used. On the 0th, 7th, 15th and 24th days, 5 × 10 ⁶ MCF7 cells (ATCC HTB-22) detached with Bersen (Life Technology) were suspended in PBS and administered subcutaneously to the back. . On day 31, 5 × 10 ⁶ cells were intravenously administered, and the spleen was collected on the same day and used for hybridoma production.

(1-2) Preparation of hybridoma Spleen cells and mouse myeloma P3X63Ag8U.1 cells (ATCC CRL-1597) were fused using PEG4000 (IBL) to prepare hybridomas. As a result, 1760 clone hybridomas were established from mice immunized with MCF7 cells. The resulting hybridoma culture supernatant was used to screen antibody-producing hybridomas by CDC assay.

(1-3) Hybridoma screening (CDC assay)
MCF7 cells were diluted with 10% fetal bovine serum (FBS) -containing RPMI medium (Life Technologies) so as to obtain 5000 cells / 80 μL, added to a 96-well plate at 80 μL / well, and cultured overnight. The hybridoma culture supernatant was added to a plate plated with cells at 20 μL / well and allowed to stand at 4 ° C. for 1 hour. 1 mL of sterile water per vial was added on ice to diluted lyophilized rabbit complement (Cedarlane). The mixture was allowed to stand for 1 minute, mixed, and then mixed with 19 mL of 0.1% BSA / RPMI1640 medium (BSA Sigma). 20 μL / well of the complement dilution was added to the plate to which the hybridoma culture supernatant was added, and the reaction was performed at 37 ° C. for 1 hour.

  The plate was left at room temperature for 30 minutes and returned to room temperature. 120 μL of CellTiter-Glo reagent (Promega) was added to each well and reacted at room temperature for 10 minutes. The amount of luminescence was measured with a plate reader (ARVO HTS Prekin Elmer). It was judged that wells with low light emission induced complement-dependent cell death. Hybridomas producing culture supernatants that induced such complement-dependent cell death were selected. As a result, 15 screening positive hybridoma clones were obtained.

(Example 2) Preparation of antibody from hybridoma 8-10 week-old mice or nude mice (BALB) that were intraperitoneally administered with 0.5 ml of pristane (2,6,10,14-tetramethylpentadecane) and raised in advance for 2 weeks / c nude mouse, female, CLEA Japan) were suspended in PBS with the monoclonal antibody-producing hybridoma 1-2 × 10 ⁶ cells obtained in Example 1, and 0.5 mL was injected intraperitoneally. After 10 to 21 days, ascites was collected after the hybridoma was changed to ascites tumor. The resulting ascites was centrifuged to remove solids, and after addition of PBS twice or more, purification was performed using a MabSelect Sure HiTrap 5 ml column (GE Healthcare Life Sciences), and IgG bound to the column in the neutral region. Fractions were collected and used as purified monoclonal antibodies.

(Example 3) Identification of an antigen to which an antibody produced by a hybridoma binds
An SLC3A2 gene expression vector encoding CD98 was prepared using a commercially available SLC3A2 gene clone (IOH-4673, Life Technologies) and a Gateway expression vector pDEST40 (Life Technologies).

NIH-3T3 cells (ATCC CRL-1658) were adjusted to 1 x 10 ⁵ cells / ml in RPMI medium (Life Technologies) containing 10% fetal bovine serum (FBS), and pcDNA-DEST40-SLC3A2 was NIH -3T3 cells were transfected with FUGENE6 (Roche Applied Science), and further cultured at 37 ° C. under 5% CO ₂ for 2 nights. Similarly, cells treated with FUGEN6 without plasmid were used as a CD98 negative control. The transfected NIH-3T3 cells were trypsinized, washed with RPMI containing 10% FBS, and then suspended in PBS containing 5% FBS. The resulting cell suspension was used for flow cytometry analysis.

  After centrifugation of the prepared cell suspension and removal of the supernatant, the hybridoma culture supernatant or purified antibody is added to and suspended in NIH-3T3 cells transfected with each vector, and the suspension is allowed to stand at 4 ° C. for 0.5 to 1 hour. I put it. After washing twice with 5% FBS-containing PBS, Fluorescein-conjugated goat IgG fraction to mouse IgG (Whole Molecule) (ICN Pharmaceuticals, # 55493) diluted 400-fold with 5% FBS-containing PBS was added and suspended. The mixture was allowed to stand at 4 ° C for 0.5 to 1 hour. After washing twice with 5% FBS-containing PBS, resuspended in 5% FBS-containing PBS containing 2 μg / ml 7-aminoactinomycin D (Invitrogen (Molecular Probes)) and detected with a flow cytometer (FC500: BeckmanCoulter) Went. Data analysis was performed with Flowjo (TreeStar). After excluding 7-aminoactinomycin D-positive dead cells at the gate, a histogram of FITC fluorescence intensity of live cells was created. A sample (hybridoma culture supernatant or purified antibody) in which the histogram of CDH-expressing NIH-3T3 cells is shifted to the strong fluorescence intensity side of the fluorescence intensity histogram of NIH-3T3 cells not transfected with the negative control plasmid. The resulting hybridoma was obtained as an anti-CD98 antibody-producing hybridoma. This hybridoma is referred to as CD98 hybridoma M23, and the antibody produced by CD98 hybridoma M23 is referred to as M23 antibody or M23.

(Example 4) Determination of nucleotide sequence of cDNA encoding variable region of M23 antibody gene and production of chimeric M23 antibody

(4-1) Determination of nucleotide sequence of cDNA encoding variable region of M23 antibody gene (4-1-1) Preparation of mRNA from CD98 hybridoma M23
In order to amplify cDNA containing the variable region of M23 antibody, mRNA was prepared from CD98 hybridoma M23 using mRNA Isolation kit (Roche applied science).

(4-1-2) Synthesis of cDNA (5'-RACE-Ready cDNA)
cDNA (5′-RACE-Ready cDNA) was synthesized using 100 ng of mRNA prepared in 4-1-1 and SMARTer RACE cDNA Amplification Kit (CLONTECH).

(4-1-3) Amplification and sequencing of cDNA containing heavy chain variable region of M23 antibody by 5'-RACE PCR As a primer for PCR amplification of heavy chain variable region cDNA, UPM (Universal Primer A Mix: attached to SMARTer RACE cDNA Amplification Kit) and an oligonucleotide having a sequence of 5′-GGCATCCTAGAGTCACCGAGGAGCCAGTTG-3 ′ (SEQ ID NO: 25: primer mG2aVR1) were used. The UPM attached to the SMARTer RACE cDNA Amplification Kit (CLONTECH) was used, and mG2aVR1 was designed from the sequence of the constant region of the mouse heavy chain (IgG2a) in the database.

  The cDNA containing the variable region of the heavy chain of the M23 antibody was amplified by 5'-RACE PCR using this primer combination and the cDNA synthesized in 4-1-2 (5'-RACE-Ready cDNA) as a template. PCR was performed using a touchdown PCR program according to the manual of SMARTer RACE cDNA Amplification Kit (CLONTECH) using KOD-Plus- (TOYOBO) as Polymerase.

  The cDNA containing the variable region of the heavy chain amplified by 5'-RACE PCR is purified using the MinElute PCR Purification Kit (QIAGEN), then cloned using the Zero Blunt TOPO PCR Cloning Kit (Invitrogen), and the cloned heavy chain Sequence analysis of the nucleotide sequence of the cDNA containing the variable region of the strand was performed. As a sequence primer, an oligonucleotide having the sequence of 5′-GGCATCCTAGAGTCACCGAGGAGCCAGTTG-3 ′ (SEQ ID NO: 25: primer mG2aVR1) designed from the sequence of the constant region of the mouse heavy chain in the database, and NUP (Nested Universal Primer A: SMARTer RACE (Attached to cDNA Amplification Kit) was used.

  Sequence analysis was performed using a gene sequence analyzer (ABI PRISM 3700 DNA Analyzer; Applied Biosystems, or Applied Biosystems 3730xl Analyzer; Applied Biosystems), and GeneAmp 9700 (Applied Biosystems) was used for the sequence reaction.

  The nucleotide sequence of the cDNA encoding the determined variable region of the heavy chain of the M23 antibody is shown in SEQ ID NO: 1, and the amino acid sequence is shown in SEQ ID NO: 2.

(4-1-4) Amplification and sequencing of cDNA containing the light chain variable region of M23 antibody by 5'-RACE PCR
As primers for PCR amplification of the variable region cDNA of the light chain gene of M23 antibody, UPM (Universal Primer A Mix: included with SMARTer RACE cDNA Amplification Kit) and 5'-AGTCCAACTGTTCAGGACGCCATTTTGTCG-3 '(SEQ ID NO: 26: Primer An oligonucleotide having the sequence mKVR2) was used. The UPM attached to the SMARTer RACE cDNA Amplification Kit (CLONTECH) was used, and mKVR2 was designed from the sequence of the mouse light chain constant region in the database.

  The cDNA containing the variable region of the light chain of the M23 antibody was amplified by 5′-RACE PCR using this primer combination and the cDNA synthesized in 4-1-2 (5′-RACE-Ready cDNA) as a template.

  PCR was performed using a touchdown PCR program according to the manual of SMARTer RACE cDNA Amplification Kit (CLONTECH) using KOD-Plus- (TOYOBO) as Polymerase.

  The cDNA containing the variable region of the light chain amplified by 5'-RACE PCR was purified using the MinElute PCR Purification Kit (QIAGEN), then cloned using the Zero Blunt TOPO PCR Cloning Kit (Invitrogen) and cloned. Sequence analysis of the nucleotide sequence of the cDNA containing the variable region of the strand was performed. As a sequence primer, an oligonucleotide having the sequence of 5′-AGTCCAACTGTTCAGGACGCCATTTTGTCG-3 ′ (SEQ ID NO: 26: primer mKVR2) designed from the sequence of the constant region of the mouse light chain in the database, and NUP (Nested Universal Primer A: SMARTer RACE (Attached to cDNA Amplification Kit) was used.

  Sequence analysis was performed using a gene sequence analyzer (ABI PRISM 3700 DNA Analyzer; Applied Biosystems, or Applied Biosystems 3730xl Analyzer; Applied Biosystems), and GeneAmp 9700 (Applied Biosystems) was used for the sequence reaction.

  The determined nucleotide sequence of the cDNA encoding the light chain variable region of the M23 antibody is shown in SEQ ID NO: 3, and the amino acid sequence thereof is shown in SEQ ID NO: 4.

(4-2) Preparation of chimeric M23 antibody (4-2-1) Construction of chimeric and humanized antibody light chain expression vector pCMA-LK Plasmid pcDNA3.3-TOPO / LacZ (Invitrogen) was constructed with restriction enzymes XbaI and PmeI. About 5.4 kb fragment obtained by digestion and DNA fragment containing nucleotide sequence encoding human κ chain secretion signal and human κ chain constant region shown in SEQ ID NO: 5 were obtained from In-Fusion Advantage PCR Cloning Kit (CLONTECH) And bound to produce pcDNA3.3 / LK.

Using pcDNA3.3 / LK as a template, PCR was performed with the following primer set, and the resulting 3.8 kb fragment was phosphorylated and self-ligated to downstream the signal sequence, cloning site, and human κ chain constant. A chimeric and humanized antibody light chain expression vector pCMA-LK with regions was constructed.
Primer set
5'-TATACCGTCGACCTCTAGCTAGAGCTTGGC-3 '(SEQ ID NO: 27: Primer 3.3-F1)
5'-GCTATGGCAGGGCCTGCCGCCCCGACGTTG-3 '(SEQ ID NO: 28: Primer 3.3-R1)

(4-2-2) Construction of chimeric and humanized antibody IgG1-type heavy chain expression vector pCMA-G1
A DNA fragment obtained by digesting pCMA-LK with XbaI and PmeI to remove the kappa chain secretion signal and the human kappa chain constant region, and the nucleotide sequence encoding the human heavy chain secretion signal sequence shown in SEQ ID NO: 6 and the human IgG1 constant region Combined DNA fragments using In-Fusion Advantage PCR Cloning Kit (CLONTECH), chimeric and humanized antibody IgG1 type heavy chain with signal sequence, cloning site and human IgG1 heavy chain constant region downstream of CMV promoter An expression vector pCMA-G1 was constructed.

(4-2-3) Construction of chimeric M23 antibody heavy chain expression vector
Using the cDNA containing the heavy chain variable region of M23 antibody obtained in 4-1-3 as a template, DNA containing cDNA encoding the heavy chain variable region with KOD-Plus- (TOYOBO) and the following primer set The fragment was amplified and inserted into the chimeric and humanized IgG1-type heavy chain expression vector pCMA-G1 with the restriction enzyme BlpI, using the In-Fusion HD PCR cloning kit (CLONTECH), to obtain the chimeric M23 antibody heavy A chain expression vector was constructed. The obtained expression vector was designated as “pCMA-G1 / cM23”. The nucleotide sequence of the chimeric M23 antibody heavy chain is shown in SEQ ID NO: 7, and the amino acid sequence is shown in SEQ ID NO: 8. The nucleotide sequence of SEQ ID NO: 7 and the amino acid sequence of SEQ ID NO: 8 are also shown in FIG.

Primer set for chimeric M23 antibody heavy chain
5'-CCAGATGGGTGCTGAGCCAGGTCCAGCTGCAGCAGTCTGGAGCTGAG-3 '(SEQ ID NO: 29: primer M23H-F)
5'-GGGCCCTTGGTGGAGGCTGCAGAGACAGTGACCAGAGTCCCTTGGCC-3 '(SEQ ID NO: 30: primer M23H-R)

(4-2-4) Construction of chimeric M23 antibody light chain expression vector
Using the cDNA containing the light chain variable region of M23 antibody obtained in 4-1-4 as a template, KAN-Plus- (TOYOBO) and DAN containing the cDNA encoding the light chain variable region with the following primer set Amplifying the fragment and inserting the chimeric and humanized antibody light chain expression general-purpose vector pCMA-LK with the restriction enzyme BsiWI and using the In-Fusion HD PCR cloning kit (CLONTECH) to insert the chimeric M23 antibody. A light chain expression vector was constructed. The obtained expression vector was designated as “pCMA-LK / cM23”. The nucleotide sequence of the light chain of the chimeric M23 antibody is shown in SEQ ID NO: 9, and the amino acid sequence is shown in SEQ ID NO: 10. The nucleotide sequence of SEQ ID NO: 9 and the amino acid sequence of SEQ ID NO: 10 are also shown in FIG.

Primer set for chimeric M23 antibody light chain
5'-ATCTCCGGCGCGTACGGCGACATTGTGATGTCACAGTCTCCATCCTCC-3 '(SEQ ID NO: 31: Primer M23L-F)
5'-GGAGGGGGCGGCCACAGCCCGTTTGATTTCCAGCTTGGTGCCTCC-3 '(SEQ ID NO: 32: primer M23L-R)

(4-2-5) Production of chimeric M23 antibody FreeStyle 293F cells (Invitrogen) were subcultured according to the manual. Logarithmically growing 1.2 × 10 ⁹ FreeStyle 293F cells (Invitrogen) seeded in 3L Fernbach Erlenmeyer Flask (CORNING), diluted with FreeStyle293 expression medium (Invitrogen) to prepare 1.0 × 10 ⁶ cells / ml After that, the cells were cultured with shaking at 90 rpm for 1 hour in an incubator at 37 ° C. and 8% CO ₂ . Polyethyleneimine (Polyscience, # 24765) 3.6mg was dissolved in Opti-Pro SFM (Invitrogen) 20ml, then light chain expression vector (0.8mg) and heavy chain prepared using PureLink HiPure Plasmid kit (Invitrogen) The expression vector (0.4 mg) was added to 20 ml of Opti-Pro SFM (Invitrogen). 20 ml of the expression vector / Opti-Pro SFM mixture was added to 20 ml of the Polyethyleneimine / Opti-Pro SFM mixture, gently stirred, allowed to stand for another 5 minutes, and then added to FreeStyle 293F cells. The culture supernatant obtained by shaking culture at 90 rpm for 7 days in a 37 ° C., 8% CO ₂ incubator was filtered with a Disposable Capsule Filter (ADVANTEC # CCS-045-E1H). The chimeric M23 antibody obtained by the combination of pCMA-G1 / cM23 and pCMA-LK / cM23 was named “cM23 antibody”.

(4-2-6) Purification of cM23 antibody
The antibody was purified from the culture supernatant obtained in 4-2-5 in two steps using rProtein A affinity chromatography (under 4-6 ° C) and ceramic hydroxyapatite (under room temperature). The buffer replacement step after rProtein A affinity chromatography purification and ceramic hydroxyapatite purification was performed at 4-6 ° C. First, the culture supernatant was applied to MabSelectSuRe (GE Healthcare Bioscience, HiTrap column) equilibrated with PBS. After all of the culture supernatant entered the column, the column was washed with PBS having a column volume of 2 times or more. Next, elution was performed with a 2M arginine hydrochloride solution (pH 4.0), and fractions containing the antibody were collected. After the fraction was replaced with PBS by dialysis (Thermo Scientific, Slide-A-Lyzer Dialysis Cassette), an antibody solution diluted 5-fold with a buffer of 5 mM sodium phosphate / 50 mM MES / pH 7.0 was diluted with 5 mM NaPi / This was applied to a ceramic hydroxyapatite column (Nippon Bio-Rad, Bio-Scale CHT Type-I Hydroxyapatite Column) equilibrated with 50 mM MES / 30 mM NaCl / pH 7.0 buffer. Linear gradient elution with sodium chloride was performed and the fractions containing antibody were collected. The fraction was subjected to liquid replacement with HBSor (25 mM histidine / 5% sorbitol, pH 6.0) by dialysis (Thermo Scientific, Slide-A-Lyzer Dialysis Cassette). Finally, it was concentrated with Centrifugal UF Filter Device VIVASPIN20 (fractionated molecular weight UF10K, Sartorius, 4 ° C.), and the IgG concentration was adjusted to 10 mg / ml or more to obtain a purified sample.

(Example 5) Design of humanized antibody of mouse anti-CD98 monoclonal antibody (5-1) Design of humanized version of M23 antibody (5-1-1) Molecular modeling of variable region of M23 antibody
Molecular modeling of the variable region of M23 was performed by a method known as homology modeling (Methods in Enzymology, 203, 121-153, (1991)). Primary sequence of variable region of human immunoglobulin registered in Protein Data Bank (Nuc. Acid Res. 35, D301-D303 (2007)) (Three-dimensional structure derived from X-ray crystal structure is available) Was compared with the variable region of M23 determined in Example 4. As a result, 1MJU was selected as having the highest sequence homology among antibodies that are similarly defective in the framework with respect to the variable region of the heavy chain of the M23 antibody. 3MBX was also selected as having the highest sequence homology to the light chain variable region of the M23 antibody. The three-dimensional structure of the framework region was created by combining the 1MJU and 3MBX coordinates corresponding to the heavy and light chains of the M23 antibody to obtain a “framework model”. A representative conformation for each CDR was then incorporated into the framework model.

  Finally, in order to obtain a possible molecular model of the variable region of the M23 antibody in terms of energy, an energy calculation was performed to eliminate adverse interatomic contacts. The above procedure was performed using a commercially available protein tertiary structure analysis program Discovery Studio (Accelrys).

(5-1-2) Design of amino acid sequence for humanized M23 antibody The construction of humanized M23 antibody is known as CDR grafting (Proc. Natl. Acad. Sci. USA 86, 1000029-10033 (1989)). Went by. Acceptor antibodies were selected based on amino acid homology within the framework regions. The sequence of the M23 antibody framework region was compared with all human frameworks in the Kabat database of antibody amino acid sequences (Nuc. Acid Res. 29, 205-206 (2001)). As a result, the HuMc3 antibody Was selected as an acceptor due to its 80% sequence homology. The amino acid residues of the framework region for HuMc3 were aligned with the amino acid residues for the M23 antibody to identify the positions where different amino acids were used. The positions of these residues are analyzed using the three-dimensional model of the M23 antibody constructed above, and the donor residues to be graphed on the acceptor are Queen et al. (Proc. Natl. Acad Sci. USA 86,10029-10033 (1989)). The sequence of the humanized M23 antibody was constructed as described in the examples below by transferring several selected donor residues into the acceptor antibody.

(5-2) Humanization of M23 antibody heavy chain (5-2-1) hM23-H1 type heavy chain The amino acid number 24 (glutamine) of the cM23 antibody heavy chain shown in SEQ ID NO: 8 is valine, amino acid number 30 ( Leucine) to valine, amino acid number 31 (valine) to lysine, amino acid number 32 (arginine) to lysine, amino acid number 35 (threonine) to alanine, amino acid number 57 (lysine) to arginine, amino acid number 59 ( Arginine) to alanine, amino acid number 67 (isoleucine) to methionine, amino acid number 86 (lysine) to arginine, amino acid number 87 (alanine) to valine, amino acid number 89 (leucine) to isoleucine, amino acid number 93 ( Lysine) to threonine, amino acid number 95 (serine) to threonine, amino acid number 101 (glutamine) to glutamic acid, amino acid number 106 (threonine) ) To arginine, amino acid number 108 (aspartic acid) to glutamic acid, amino acid number 110 (serine) to threonine, amino acid number 111 (serine) to alanine, amino acid number 114 (phenylalanine) to tyrosine, amino acid number 135 ( The humanized M23 antibody heavy chain designed to replace alanine with serine was named “hM23-H1 type heavy chain”.

  The amino acid sequence of the hM23-H1 type heavy chain is set forth in SEQ ID NO: 12. The sequence consisting of amino acid residues 1 to 19 of the amino acid sequence of SEQ ID NO: 12, the sequence consisting of amino acid residues 20 to 135, the sequence consisting of amino acid residues 136 to 465 are a signal sequence and a heavy chain, respectively. It corresponds to the variable region and heavy chain constant region. The nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 12 is set forth in SEQ ID NO: 11. The sequence consisting of the 1st to 57th nucleotides, the sequence consisting of the 58th to 405th nucleotides, and the sequence consisting of the 406th to 1395th nucleotides of the nucleotide sequence of SEQ ID NO: 11 are respectively a signal sequence, a heavy chain variable region sequence, and a heavy chain constant. The normal region sequence is encoded. The nucleotide sequence of SEQ ID NO: 11 and the amino acid sequence of SEQ ID NO: 12 are also shown in FIG.

(5-2-2) hM23-H2 type heavy chain The amino acid number 30 (leucine) of the cM23 heavy chain shown in SEQ ID NO: 8 is valine, amino acid number 31 (valine) is lysine, and amino acid number 32 (arginine) is Amino acid number 35 (threonine) to alanine, amino acid number 57 (lysine) to arginine, amino acid number 59 (arginine) to alanine, amino acid number 67 (isoleucine) to methionine, amino acid number 89 (leucine) to lysine Isoleucine, amino acid number 95 (serine) to threonine, amino acid number 101 (glutamine) to glutamic acid, amino acid number 108 (aspartic acid) to glutamic acid, amino acid number 110 (serine) to threonine, amino acid number 111 (serine) To alanine, amino acid number 114 (phenylalanine) to tyrosine, amino acid number 135 (alanine) to sericin In, it was named "hM23-H2-type heavy chain" a designed humanized M23 antibody heavy chain involves replacing.

  The amino acid sequence of the hM23-H2 type heavy chain is set forth in SEQ ID NO: 14. The sequence consisting of amino acid residues 1 to 19 of the amino acid sequence of SEQ ID NO: 14, the sequence consisting of amino acid residues 20 to 135, the sequence consisting of amino acid residues 136 to 465 are a signal sequence and a heavy chain, respectively. It corresponds to the variable region and heavy chain constant region. The nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 14 is set forth in SEQ ID NO: 13. The sequence consisting of the 1st to 57th nucleotides, the sequence consisting of the 58th to 405th nucleotides, and the sequence consisting of the 406th to 1395th nucleotides of the nucleotide sequence of SEQ ID NO: 13 are respectively a signal sequence, a heavy chain variable region sequence, and a heavy chain constant. The normal region sequence is encoded. The nucleotide sequence of SEQ ID NO: 13 and the amino acid sequence of SEQ ID NO: 14 are also shown in FIG.

(5-3) Humanization of M23 antibody light chain (5-3-1) hM23-L1 type light chain Amino acid number 25 (serine) of the cM23 antibody light chain represented by SEQ ID NO: 10 is converted into threonine, amino acid number 29 ( Serine) to aspartic acid, amino acid number 35 (valine) to leucine, amino acid number 38 (lysine) to arginine, amino acid number 39 (valine) to alanine, amino acid number 41 (methionine) to isoleucine, amino acid number 42 (Threonine) to asparagine, amino acid number 69 (serine) to proline, amino acid number 89 (threonine) to serine, amino acid number 103 (asparagine) to serine, amino acid number 104 (valine) to leucine, amino acid number 109 (Leucine) to valine, amino acid number 126 (glycine) to glutamine, amino acid number 130 (leucine) to valine, amino acid number 135 (alanine) The humanized M23 antibody light chain designed to be replaced with threonine was named “hM23-L1 type light chain”.

  The amino acid sequence of the hM23-L1 type light chain is set forth in SEQ ID NO: 16. The sequence consisting of amino acid residues 1 to 20 of the amino acid sequence of SEQ ID NO: 16, the sequence consisting of amino acid residues 21 to 135, the sequence consisting of amino acid residues 136 to 240 are respectively a signal sequence and a light chain It corresponds to the variable region and the light chain constant region. The nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 16 is set forth in SEQ ID NO: 15. The sequence consisting of the 1st to 60th nucleotides of the nucleotide sequence of SEQ ID NO: 15, the sequence consisting of the 61st to 405th nucleotides, and the sequence consisting of the 406th to 720th nucleotides are respectively a signal sequence, a light chain variable region sequence and a light chain constant. The normal region sequence is encoded. The nucleotide sequence of SEQ ID NO: 15 and the amino acid sequence of SEQ ID NO: 16 are also shown in FIG.

(5-3-2) hM23-L2 type light chain The amino acid number 25 (serine) of the cM23 antibody light chain shown in SEQ ID NO: 10 of the sequence listing is threonine, amino acid number 29 (serine) is aspartic acid, amino acid number 35 (valine) to leucine, amino acid number 38 (lysine) to arginine, amino acid number 39 (valine) to alanine, amino acid number 41 (methionine) to isoleucine, amino acid number 42 (threonine) to asparagine, amino acid number 69 (serine) to proline, amino acid number 89 (threonine) to serine, amino acid number 103 (asparagine) to serine, amino acid number 104 (valine) to leucine, amino acid number 109 (leucine) to valine, amino acid number Humanized M23 antibody designed to replace 130 (leucine) with valine and amino acid number 135 (alanine) with threonine The light chain was named “hM23-L2 type light chain”.

  The amino acid sequence of the hM23-L2 type light chain is set forth in SEQ ID NO: 18 in the sequence listing. The sequence consisting of amino acid residues 1 to 20 in the amino acid sequence of SEQ ID NO: 18, the sequence consisting of amino acid residues 21 to 135, the sequence consisting of amino acid residues 136 to 240 are respectively a signal sequence and a light chain It corresponds to the variable region and the light chain constant region. The nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 18 is described in SEQ ID NO: 17 of the sequence listing. The sequence consisting of the 1st to 60th nucleotides of the nucleotide sequence of SEQ ID NO: 17, the sequence consisting of the 61st to 405th nucleotides, and the sequence consisting of the 406th to 720th nucleotides are respectively a signal sequence, a light chain variable region sequence and a light chain constant. The normal region sequence is encoded. The nucleotide sequence of SEQ ID NO: 17 and the amino acid sequence of SEQ ID NO: 18 are also shown in FIG.

(Example 6) Construction of humanized M23 antibody expression vector and production of antibody (6-1) Construction of humanized M23 antibody heavy chain expression vector (6-1-1) Construction of hM23-H1 type heavy chain expression vector Synthesis of a DNA fragment (nucleotide number 36 to 422) containing a DNA sequence encoding the variable region of the hM23-H1 type heavy chain shown in nucleotide numbers 58 to 405 of the nucleotide sequence of the hM23-H1 type heavy chain shown in SEQ ID NO: 11. (GENEART artificial gene synthesis service). Using the synthesized DNA fragment as a template, KOD-Plus- (TOYOBO) and the following primer set are used to amplify a DNA fragment containing the DNA sequence encoding the variable region of the hM23-H1 type heavy chain. The hM23-H1 type heavy chain expression vector was constructed by inserting the type heavy chain expression vector pCMA-G1 into a position cleaved with the restriction enzyme BlpI using an In-Fusion HD PCR cloning kit (CLONTECH). The obtained expression vector was designated as “pCMA-G1 / hM23-H1”.
Primer set
5'-AGCTCCCAGATGGGTGCTGAGC-3 '(SEQ ID NO: 33: Primer EG-Inf-F)
5'-GGGCCCTTGGTGGAGGCTGAGC-3 '(SEQ ID NO: 34: primer EG1-Inf-R)

(6-1-2) Construction of hM23-H2 type heavy chain expression vector The variable region of hM23-H2 type heavy chain shown in nucleotide numbers 58 to 405 of the nucleotide sequence of hM23-H2 type heavy chain shown in SEQ ID NO: 13 A DNA fragment (nucleotide numbers 36 to 422) containing the DNA sequence to be encoded was synthesized (GENEART Artificial Gene Synthesis Service), and an hM23-H2 type heavy chain expression vector was constructed in the same manner as in 6-1-1. The obtained expression vector was designated as “pCMA-G1 / hM23-H2”.

(6-2) Construction of humanized M23 antibody light chain expression vector (6-2-1) Construction of hM23-L1 type light chain expression vector Nucleotide number of nucleotide sequence of hM23-L1 type light chain shown in SEQ ID NO: 15 A DNA fragment (nucleotide numbers 38 to 420) containing a DNA sequence encoding the variable region of the hM23-L1 type light chain shown in 61 to 405 was synthesized (GENEART Artificial Gene Synthesis Service). Using the synthesized DNA fragment as a template, KOD-Plus- (TOYOBO) and the following primer set are used to amplify a DNA fragment containing the DNA sequence encoding the variable region of the hM23-L1 type light chain. The hM23-L1-type light chain expression vector was constructed by inserting the chain expression vector pCMA-LK into the site cleaved with the restriction enzyme BsiWI using an In-Fusion HD PCR cloning kit (CLONTECH). The obtained expression vector was designated as “pCMA-LK / hM23-L1”.
Primer set
5'-CTGTGGATCTCCGGCGCGTACGGC-3 '(SEQ ID NO: 35: Primer CM-LKF)
5'-GGAGGGGGCGGCCACCGTACG-3 '(SEQ ID NO: 36: primer KCL-Inf-R)

(6-2-2) Construction of hM23-L2 type light chain expression vector The variable region of the hM23-L2 type light chain represented by nucleotide numbers 61 to 405 of the nucleotide sequence of the hM23-L2 type light chain represented by SEQ ID NO: 17 A DNA fragment (nucleotide numbers 38 to 420) containing the DNA sequence to be encoded was synthesized (GENEART Artificial Gene Synthesis Service), and an hM23-L2 type light chain expression vector was constructed in the same manner as in 6-2-1. The obtained expression vector was designated as “pCMA-LK / hM23-L2”.

(6-3) Production and purification of humanized M23 antibody (6-3-1) Production of humanized M23 antibody Humanized M23 antibody was produced in the same manner as in 4-2-5. The humanized M23 antibody obtained by combining pCMA-G1 / hM23-H1 and pCMA-LK / hM23-L1 is called `` hM23-H1L1 '', pCMA-G1 / hM23-H1 and pCMA-LK / hM23-L2 The humanized M23 antibody obtained by the combination as `` hM23-H1L2 '' and the humanized M23 antibody obtained by the combination of pCMA-G1 / hM23-H2 and pCMA-LK / hM23-L2 as `` hM23-H2L2 '' Named.

(6-3-2) Purification of humanized M23 antibody
The antibody was purified from the culture supernatant obtained in 6-3-1 by the same method as in 4-2-6.

(Production of ADC)
Common operation A: Concentration of antibody or antibody-drug conjugate aqueous solution
An antibody or antibody-drug conjugate solution is placed in a container of Amicon Ultra (50,000 MWCO, Millipore Corporation) and centrifuged using a centrifuge (Allegra X-15R, Beckman Coulter, Inc.) (2000G-3800G, 5 Concentrate the antibody or antibody-drug conjugate solution by centrifugation for ~ 20 minutes.

Common operation B: Antibody concentration measurement
The antibody concentration is measured using a UV measuring instrument (Nanodrop 1000, Thermo Fisher Scientific Inc.) according to the method specified by the manufacturer. At that time, a different 280 nm extinction coefficient (1.3 mLmg ⁻¹ cm ^{−1 to} 1.8 mLmg ⁻¹ cm ⁻¹ ) is used for each antibody.

Common operation C: Buffer exchange of antibody
A NAP-25 column (Cat. No. 17-0852-02, GE Healthcare Japan Corporation) using Sephadex G-25 carrier, sodium chloride (137 mM) and ethylenediaminetetraacetic acid (EDTA, 5 mM) according to the method specified by the manufacturer In a phosphate buffer solution (10 mM, pH 6.0; sometimes referred to as “PBS6.0 / EDTA” in this specification). After applying 2.5 mL of the antibody aqueous solution to each NAP-25 column, fraction (3.5 mL) eluted with 3.5 mL of PBS6.0 / EDTA is collected. This fraction is concentrated by common operation A, the antibody concentration is measured using common operation B, and then the antibody concentration is adjusted to 10 mg / mL using PBS 6.0 / EDTA.

Common operation D: Purification of antibody-drug conjugate
Equilibrate the NAP-25 column with acetate buffer (10 mM, pH 5.5; sometimes referred to herein as “ABS”) containing Sorbitol (5%). An antibody-drug conjugate aqueous solution (2.5 mL) was placed on the NAP-25 column and eluted with a buffer defined by the manufacturer to fractionate the antibody fraction. Repeat this gel filtration purification procedure to place the preparative fraction on the NAP-25 column and elute with a buffer solution a total of 2 to 3 times, so that unbound drug linkers and low molecular weight compounds (Tris (2-carboxyethyl) Antibody-drug conjugates are obtained without phosphine hydrochloride (TCEP), N-acetyl-L-cysteine (NAC) and dimethyl sulfoxide).

Common operation E: Measurement of antibody concentration in antibody-drug conjugate and average number of drugs bound per molecule of antibody The concentration of bound drug in antibody-drug conjugate is determined by UV at two wavelengths of 280 nm and 370 nm of antibody-drug conjugate aqueous solution. It can be calculated by performing the following calculation after measuring the absorbance.

The total absorbance at a certain wavelength is equal to the sum of the absorbances of all absorbing species present in the system [addition of absorbance], so the molar extinction coefficient of the antibody and drug changes before and after conjugation of the antibody and drug. Assuming that there is no antibody, the antibody concentration and the drug concentration in the antibody-drug conjugate are represented by the following relational expression.
A ₂₈₀ = A _{D, 280} + A _{A, 280} = ε _{D, 280} C _D + ε _{A, 280} C _A Formula (1)
A ₃₇₀ = A _{D, 370} + A _{A, 370} = ε _{D, 370} C _D + ε _{A, 370} C _A Formula (2)

Here, A ₂₈₀ represents the absorbance of the antibody-drug conjugate aqueous solution at ₂₈₀ nm, A ₃₇₀ represents the absorbance of the antibody-drug conjugate aqueous solution at ₃₇₀ nm, A _{A, 280} represents the absorbance of the antibody at ₂₈₀ nm, and A _{A , 370} represents the absorbance of the antibody at ₃₇₀ nm, _{AD, 280} represents the absorbance of the conjugate precursor at ₂₈₀ nm, _{AD, 370} represents the absorbance of the conjugate precursor at ₃₇₀ nm, and ε _{A, 280} represents the absorbance _{at 280} nm. Indicates the molar extinction coefficient of the antibody, ε _{A, 370} indicates the molar extinction coefficient of the antibody at ₃₇₀ nm, ε _{D, 280} indicates the molar extinction coefficient of the conjugate precursor at ₂₈₀ nm, and ε _{D, 370} indicates the conjugate at ₃₇₀ nm. shows the molar extinction coefficient of the precursor, C _a antibody - indicates antibody concentration in drug conjugates, C _D antibody - shows the drug concentration in the drug conjugate.

Here, for ε _{A, 280} , ε _{A, 370} , ε _{D, 280} and ε _{D, 370} , values prepared in advance (calculated estimation values or actual values obtained from UV measurement of compounds) are used. For example, ε _{A, 280} can be estimated from the amino acid sequence of an antibody by a known calculation method (Protein Science, 1995, vol. 4, 2411-2423). ε _{A, 370} is usually zero. ε _{D, 280} and ε _{D, 370} are measured by measuring the absorbance of a solution in which the conjugate precursor to be used is dissolved at a certain molar concentration, and the Lambert-Beer law (absorbance = molar concentration × molar extinction coefficient × cell optical path). Long). By measuring A ₂₈₀ and A ₃₇₀ of the antibody-drug conjugate aqueous solution and substituting these values into the equations (1) and (2), CA _A and C _D can be obtained. Furthermore C _D can be drug average binding per antibody determined by the dividing in C _A.

(Example 7) Production of hM23-H1L1 ADC (1)

Step 1: tert-butyl (4-{[(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] quinolin-1-yl] amino} -4-oxobutyl) carbamate
4- (tert-Butoxycarbonylamino) butanoic acid (0.237 g, 1.13 mmoL) was dissolved in dichloromethane (10 mL), N-hydroxysuccinimide (0.130 g, 1.13 mmoL) and 1-ethyl-3- (3-dimethylamino) Propyl) carbodiimide hydrochloride (0.216 g, 1.13 mmol) was added and stirred for 1 hour. The resulting mixture was added dropwise to N, N-dimethylformamide solution (10 mL) containing exatecan mesylate (0.500 g, 0.94 mmoL) and triethylamine (0.157 mL, 1.13 mmoL) and stirred at room temperature for one day. . The solvent was distilled off under reduced pressure, and the obtained residue was purified by silica gel column chromatography [chloroform to chloroform: methanol = 8: 2 (v / v)] to obtain the title compound (0.595 g, quantitative). .
¹ H-NMR (400 MHz, DMSO-d ₆ ) δ: 0.87 (3H, t, J = 7.2 Hz), 1.31 (9H, s), 1.58 (1H, t, J = 7.2 Hz), 1.66 (2H, t , J = 7.2Hz), 1.89-1.82 (2H, m), 2.12-2.21 (3H, m), 2.39 (3H, s), 2.92 (2H, t, J = 6.5Hz), 3.17 (2H, s) , 5.16 (1H, d, J = 19.2Hz), 5.24 (1H, d, J = 18.8Hz), 5.42 (2H, s), 5.59-5.55 (1H, m), 6.53 (1H, s), 6.78 ( 1H, t, J = 6.3Hz), 7.30 (1H, s), 7.79 (1H, d, J = 11.0Hz), 8.40 (1H, d, J = 8.6Hz).
MS (APCI) m / z: 621 (M + H) ⁺ .

Step 2: 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] indolidino [1,2-b] quinolin-1-yl] butanamide trifluoroacetate compound obtained in step 1 above ( 0.388 g, 0.61 mmoL) was dissolved in dichloromethane (9 mL). Trifluoroacetic acid (9 mL) was added and stirred for 4 hours. The solvent was distilled off under reduced pressure, and the obtained residue was purified by silica gel column chromatography [chloroform to chloroform: methanol: water = partition organic layer of 7: 3: 1 (v / v / v)] to give the title compound (0.343 g, quantitative) was obtained.
¹ H-NMR (400 MHz, DMSO-d ₆ ) δ: 0.87 (3H, t, J = 9.4 Hz), 1.90-1.80 (4H, m), 2.15-2.13 (2H, m), 2.27 (2H, t, J = 7.0Hz), 2.41 (3H, s), 2.87-2.82 (2H, m), 3.18 (2H, dd, J = 8.0, 3.7Hz) 5.15 (1H, d, J = 19.2Hz), 5.26 (1H , d, J = 18.8Hz), 5.43 (2H, s), 5.61-5.56 (1H, m), 6.57 (1H, s), 7.32 (1H, s), 7.72 (3H, brs), 7.82 (1H, d, J = 10.9Hz), 8.55 (1H, d, J = 8.6Hz).
MS (APCI) m / z: 521 (M + H) ⁺ .

Step 3: N- (tert-butoxycarbonyl) glycylglycyl-L-phenylalanyl-N- (4-{[(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] indolidino [1,2-b] quinoline-1- Yl] amino} -4-oxobutyl) glycinamide
N- (tert-butoxycarbonyl) glycylglycyl-L-phenylalanylglycine (0.081 g, 0.19 mmoL) was dissolved in dichloromethane (3 mL) and N-hydroxysuccinimide (0.021 g, 0.19 moL) and 1-ethyl-3- (3-Dimethylaminopropyl) carbodiimide hydrochloride (0.036 g, 0.19 mmol) was added and stirred for 3.5 hours. The obtained mixture was added dropwise to an N, N-dimethylformamide solution (1.5 mL) to which the compound obtained in the above Step 2 (0.080 g, 0.15 mmol) was added, and stirred at room temperature for 4 hours. The solvent was distilled off under reduced pressure, and the resulting residue was purified by silica gel column chromatography [chloroform to chloroform: methanol = 8: 2 (v / v)] to obtain the title compound (0.106 g, 73%). .
¹ H-NMR (400 MHz, DMSO-d ₆ ) δ: 0.87 (3H, t, J = 7.4 Hz), 1.36 (9H, s), 1.71 (2H, m), 1.86 (2H, t, J = 7.8 Hz) ), 2.15-2.19 (4H, m), 2.40 (3H, s), 2.77 (1H, dd, J = 12.7, 8.8Hz), 3.02 (1H, dd, J = 14.1, 4.7Hz), 3.08-3.11 ( 2H, m), 3.16-3.19 (2H, m), 3.54 (2H, d, J = 5.9Hz), 3.57-3.77 (4H, m), 4.46-4.48 (1H, m), 5.16 (1H, d, J = 19.2Hz), 5.25 (1H, d, J = 18.8Hz), 5.42 (2H, s), 5.55-5.60 (1H, m), 6.53 (1H, s), 7.00 (1H, t, J = 6.3 Hz), 7.17-7.26 (5H, m), 7.31 (1H, s), 7.71 (1H, t, J = 5.7Hz), 7.80 (1H, d, J = 11.0Hz), 7.92 (1H, t, J = 5.7Hz), 8.15 (1H, d, J = 8.2Hz), 8.27 (1H, t, J = 5.5Hz), 8.46 (1H, d, J = 8.2Hz).
MS (APCI) m / z: 939 (M + H) ⁺ .

Step 4: Glycylglycyl-L-phenylalanyl-N- (4-{[(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] indolidino [1,2-b] quinolin-1-yl] amino} -4-oxobutyl ) Glycinamide trifluoroacetate The compound obtained in Step 3 above (1.97 g, 2.10 mmol) was dissolved in dichloromethane (7 mL). Trifluoroacetic acid (7 mL) was added to the resulting solution and stirred for 1 hour. The solvent was distilled off under reduced pressure, toluene was added and azeotroped, and the resulting residue was subjected to silica gel column chromatography [chloroform to chloroform: methanol: water = 7: 3: 1 (v / v / v) partition organic layer The title compound (1.97 g, 99%) was obtained.
¹ H-NMR (400 MHz, DMSO-d ₆ ) δ: 0.87 (3H, t, J = 7.4 Hz), 1.71-1.73 (2H, m), 1.82-1.90 (2H, m), 2.12-2.20 (4H, m), 2.40 (3H, s), 2.75 (1H, dd, J = 13.7, 9.4Hz), 3.03-3.09 (3H, m), 3.18-3.19 (2H, m), 3.58-3.60 (2H, m) , 3.64 (1H, d, J = 5.9Hz), 3.69 (1H, d, J = 5.9Hz), 3.72 (1H, d, J = 5.5Hz), 3.87 (1H, dd, J = 16.8, 5.9Hz) , 4.50-4.56 (1H, m), 5.16 (1H, d, J = 19.2Hz), 5.25 (1H, d, J = 18.8Hz), 5.42 (2H, s), 5.55-5.60 (1H, m), 7.17-7.27 (5H, m), 7.32 (1H, s), 7.78-7.81 (2H, m), 7.95-7.97 (3H, m), 8.33-8.35 (2H, m), 8.48-8.51 (2H, m ).
MS (APCI) m / z: 839 (M + H) ⁺ .

Step 5: N- {3- [2- (2-{[3- (2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl) propanoyl] amino} ethoxy) ethoxy] propanoyl} glycylglycyl -L-phenylalanyl-N- (4-{[(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] indolidino [1,2-b] quinolin-1-yl] amino} -4-oxobutyl) glycinamide Above To a solution of the compound obtained in step 4 (100 mg, 0.119 mmoL) in N, N-dimethylformamide (1.20 mL), diisopropylethylamine (20.8 μL, 0.119 mmoL) and 3- (2- (2- (3-maleimidopropane) N-succinimidyl (amido) ethoxy) ethoxy) propanoate (50.7 mg, 0.119 mmol) was added and stirred at room temperature for 1 hour. The solvent was distilled off under reduced pressure, and the resulting residue was purified by silica gel column chromatography [chloroform to chloroform: methanol = 5: 1 (v / v)] to obtain the title compound (66.5 mg, 48%). .
¹ H-NMR (400 MHz, DMSO-d ₆ ) δ: 0.85 (3H, t, J = 7.4 Hz), 1.65-1.74 (2H, m), 1.77-1.90 (2H, m), 2.07-2.19 (4H, m), 2.30 (2H, t, J = 7.2Hz), 2.33-2.36 (2H, m), 2.38 (3H, s), 2.76 (1H, dd, J = 13.7, 9.8Hz), 2.96-3.18 (9H , m), 3.42-3.44 (4H, m), 3.53-3.76 (10H, m), 4.43 (1H, td, J = 8.6,4.7Hz), 5.14 (1H, d, J = 18.8Hz), 5.23 ( 1H, d, J = 18.8Hz), 5.38 (1H, d, J = 17.2Hz), 5.42 (1H, d, J = 17.2Hz), 5.52-5.58 (1H, m), 6.52 (1H, s), 6.98 (2H, s), 7.12-7.17 (1H, m), 7.18-7.25 (4H, m), 7.29 (1H, s), 7.69 (1H, t, J = 5.5Hz), 7.78 (1H, d, J = 11.3Hz), 7.98-8.03 (2H, m), 8.11 (1H, d, J = 7.8Hz), 8.16 (1H, t, J = 5.7Hz), 8.23 (1H, t, J = 5.9Hz) , 8.44 (1H, d, J = 9.0Hz).
MS (APCI) m / z: 1149 (M + H) ⁺ .

Process 6: hM23-H1L1 ADC (1)

(i) Reduction of antibody hM23-H1L1 prepared in Example 6 was prepared to 10 mg / mL in PBS 6.0 / EDTA using common operation B (using 1.65 as the 280 nm extinction coefficient) and C. This solution (1.00 mL) is collected in a 2 mL polypropylene tube, and 10 mM TCEP (Tokyo Chemical Industry Co., Ltd.) aqueous solution (0.0315 mL; 4.6 equivalents per antibody molecule) and 1 M dipotassium hydrogen phosphate aqueous solution (Nacalai Tesque, Inc .; 0.015 mL). After confirming that the pH of this solution was within 7.0 ± 0.1, the disulfide bond at the hinge part in the antibody was reduced by incubating at 37 ° C. for 2 hours.

(ii) Conjugation of antibody and drug linker After incubating the above solution in a room temperature water bath for 10 minutes, a 10 mM dimethyl sulfoxide solution (0.0579 mL; 9.2 equivalents per antibody molecule) of the compound obtained in Step 5 above was added, The drug linker was bound to the antibody by stirring at room temperature for 60 minutes using a tube rotator (MTR-103, ASONE Corporation). Next, a 100 mM NAC (Sigma-Aldrich Co. LLC) aqueous solution (0.0126 mL; 18.4 equivalents per molecule of antibody) was added, and the mixture was further stirred at room temperature for 20 minutes to stop the reaction of the drug linker.

(iii) Purification The above solution was purified by common operation D to obtain 6.0 mL of a solution containing the title antibody-drug conjugate.

(iv) Characteristic evaluation Using the common operation E, the following characteristic values were obtained.

Antibody concentration: 1.50 mg / mL, antibody yield: 9.00 mg (90%), average number of drugs bound per antibody molecule (n): 6.0.

(Example 8) Production of hM23-H1L2 ADC (1)

Process 1: hM23-H1L2 ADC (1)
Using the hM23-H1L2 prepared in Example 6 and the compound obtained in Step 7 of Example 7, the title antibody-drug conjugate was obtained in the same manner as in Step 7 of Example 7.

Antibody concentration: 1.54 mg / mL, antibody yield: 9.24 mg (92%), average drug binding number per antibody molecule (n): 6.0.

(Example 9) Production of hM23-H2L2 ADC (1)

Process 1: hM23-H2L2 ADC (1)
The title antibody-drug conjugate was obtained in the same manner as in Example 7, Step 6, using hM23-H2L2 prepared in Example 6 and the compound obtained in Step 7 of Example 7.

Antibody concentration: 1.45 mg / mL, antibody yield: 8.70 mg (87%), average number of drugs bound per antibody molecule (n): 6.0.

(Example 10) Production of hM23-H1L1 ADC (2)

Step 1: ({N-[(9H-Fluoren-9-ylmethoxy) carbonyl] glycyl} amino) methyl acetate
N-9-fluorenylmethoxycarbonylglycylglycine (4.33 g, 12.2 mmol), tetrahydrofuran (120 ml) and toluene (40.0 ml) were mixed with pyridine (1.16 ml, 14.7 mmol) and lead tetraacetate (6.84 g). 14.7 mmol) was added and the mixture was refluxed for 5 hours. The reaction mixture was cooled to room temperature, insoluble material was removed by celite filtration, and the mixture was concentrated under reduced pressure. The obtained residue was dissolved in ethyl acetate, washed with water and saturated brine, and the organic layer was dried over anhydrous magnesium sulfate. After the solvent was distilled off under reduced pressure, the obtained residue was purified by silica gel column chromatography [hexane: ethyl acetate = 9: 1 (v / v) to ethyl acetate] to give the title compound (3.00 g, 67 %).
¹ H-NMR (400 MHz, CDCl ₃ ) δ: 2.07 (3H, s), 3.90 (2H, d, J = 5.1 Hz), 4.23 (1H, t, J = 7.0 Hz), 4.46 (2H, d, J = 6.6Hz), 5.26 (2H, d, J = 7.0Hz), 5.32 (1H, brs), 6.96 (1H, brs), 7.32 (2H, t, J = 7.3Hz), 7.41 (2H, t, J = 7.3Hz), 7.59 (2H, d, J = 7.3Hz), 7.77 (2H, d, J = 7.3Hz).

Step 2: Benzyl [({{N-[(9H-fluoren-9-ylmethoxy) carbonyl] glycyl} amino) methoxy] acetate The compound obtained in Step 1 above (3.68 g, 10.0 mmoL) and benzyl glycolate (4.99 g, To a solution of 30.0 mmoL) in tetrahydrofuran (40.0 mL) was added potassium tert-butoxide (2.24 g, 20.0 mmoL) at 0 ° C., and the mixture was stirred at room temperature for 15 minutes. Ethyl acetate and water were added to the reaction solution at 0 ° C., and the mixture was extracted with ethyl acetate and chloroform. The obtained organic layer was dried over sodium sulfate and filtered. The solvent was distilled off under reduced pressure, and the resulting residue was dissolved in dioxane (40.0 mL) and water (10.0 mL). Sodium bicarbonate (1.01 g, 12.0 mmoL), 9-fluorenylmethyl chloroformate (2.59 g) , 10.0 mmoL), and stirred at room temperature for 2 hours. Water was added to the reaction solution, extraction was performed with ethyl acetate, and the resulting organic layer was dried over sodium sulfate and filtered. The solvent was distilled off under reduced pressure, and the obtained residue was purified by silica gel column chromatography [hexane: ethyl acetate = 100: 0 (v / v) to 0: 100] to give the title compound (1.88 g, 40%) Got.
¹ H-NMR (400 MHz, CDCl ₃ ) δ: 3.84 (2H, d, J = 5.5 Hz), 4.24 (3H, t, J = 6.5 Hz), 4.49 (2H, d, J = 6.7 Hz), 4.88 ( 2H, d, J = 6.7Hz), 5.15-5.27 (1H, m), 5.19 (2H, s), 6.74 (1H, brs), 7.31-7.39 (7H, m), 7.43 (2H, t, J = 7.4Hz), 7.61 (2H, d, J = 7.4Hz), 7.79 (2H, d, J = 7.4Hz).

Step 3: [({{N-[(9H-Fluoren-9-ylmethoxy) carbonyl] glycyl} amino) methoxy] acetic acid The compound obtained in Step 2 above (1.88 g, 3.96 mmoL) was added to ethanol (40.0 mL), ethyl acetate Dissolved in (20.0 ml). Palladium carbon catalyst (376 mg) was added, and the mixture was stirred at room temperature for 2 hours under a hydrogen atmosphere. The insoluble material was removed by Celite filtration, and the solvent was distilled off under reduced pressure to obtain the title compound (1.52 g, quantitative).
¹ H-NMR (400 MHz, DMSO-d ₆ ) δ: 3.62 (2H, d, J = 6.3 Hz), 3.97 (2H, s), 4.18-4.32 (3H, m), 4.60 (2H, d, J = 6.7Hz), 7.29-7.46 (4H, m), 7.58 (1H, t, J = 5.9Hz), 7.72 (2H, d, J = 7.4Hz), 7.90 (2H, d, J = 7.4Hz), 8.71 (1H, t, J = 6.5Hz).

Step 4: 9H-fluoren-9-ylmethyl (2-{[(2-{[(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] quinolin-1-yl] amino} -2 -Oxoethoxy) methyl] amino} -2-oxoethyl) carbamate obtained in ice-cooled exatecan mesylate (0.283 g, 0.533 mmoL), N-hydroxysuccinimide (61.4 mg, 0.533 mmoL), and step 3 above N, N-Diisopropylethylamine (92.9μL, 0.533mmoL) and N, N'-dicyclohexylcarbodiimide (0.143g, 0.693mmoL) in a solution of the compound (0.205g, 0.533mmoL) in N, N-dimethylformamide (10.0mL) And stirred at room temperature for 3 days. The solvent was distilled off under reduced pressure, and the obtained residue was purified by silica gel column chromatography [chloroform to chloroform: methanol: water = partition organic layer of 7: 3: 1 (v / v / v)] to give the title compound (0.352 g, 82%) was obtained.
¹ H-NMR (400 MHz, DMSO-d ₆ ) δ: 0.81 (3H, t, J = 7.4 Hz), 1.73-1.87 (2H, m), 2.06-2.20 (2H, m), 2.34 (3H, s) , 3.01-3.23 (2H, m), 3.58 (2H, d, J = 6.7Hz), 3.98 (2H, s), 4.13-4.25 (3H, m), 4.60 (2H, d, J = 6.7Hz), 5.09-5.22 (2H, m), 5.32-5.42 (2H, m), 5.50-5.59 (1H, m), 6.49 (1H, s), 7.24-7.30 (3H, m), 7.36 (2H, t, J = 7.4Hz), 7.53 (1H, t, J = 6.3Hz), 7.66 (2H, d, J = 7.4Hz), 7.75 (1H, d, J = 11.0Hz), 7.84 (2H, d, J = 7.4 Hz), 8.47 (1H, d, J = 8.6Hz), 8.77 (1H, t, J = 6.7Hz).
MS (ESI) m / z: 802 (M + H) ⁺ .

Step 5: N-[(2-{[(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] indolidino [1,2-b] quinolin-1-yl] amino} -2-oxoethoxy) methyl] glycinamide Piperidine (1.1 mL) was added to a solution of the compound obtained in Step 4 (0.881 g, 1.10 mmol) in N, N-dimethylformamide (11.0 mL), and the mixture was stirred at room temperature for 2 hours. The solvent was distilled off under reduced pressure to obtain a mixture containing the title compound. This mixture was used in the next reaction without further purification.

Step 6: N-[(9H-Fluoren-9-ylmethoxy) carbonyl] glycylglycyl-L-phenylalanyl-N-[(2-{[(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] indolidino [1,2 -b] quinolin-1-yl] amino} -2-oxoethoxy) methyl] glycinamide Under ice-cooling, the mixture obtained in step 5 above (0.439 mmoL), N-hydroxysuccinimide (0.101 g, 0.878 mmoL) and N -[(9H-Fluoren-9-ylmethoxy) carbonyl] glycylglycyl-L-phenylalanine (compound described in JP 2002-60351 A) (0.440 g, 0.878 mmoL) N, N-dimethylformamide (50.0 mL) N, N′-dicyclohexylcarbodiimide (0.181 g, 0.878 mmol) was added to the solution, and the mixture was stirred at room temperature for 4 days. The solvent was distilled off under reduced pressure, and the resulting residue was purified by silica gel column chromatography [chloroform to chloroform: methanol = 9: 1 (v / v)] to obtain the title compound (0.269 g, 58%). .
MS (ESI) m / z: 1063 (M + H) ⁺ .

Step 7: Glycylglycyl-L-phenylalanyl-N-[(2-{[(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] quinolin-1-yl] amino} -2- Oxoethoxy) methyl] glycinamide To a solution of the compound obtained in Step 6 above (0.269 g, 0.253 mmoL) in N, N-dimethylformamide (4.00 mL), piperidine (0.251 mL, 2.53 mmoL) was added, and then at room temperature for 2 hours. Stir. The solvent was distilled off under reduced pressure to obtain a mixture containing the title compound. This mixture was used in the next reaction without further purification.

Step 8: N- [6- (2,5-Dioxo-2,5-dihydro-1H-pyrrol-1-yl) hexanoyl] glycylglycyl-L-phenylalanyl-N-[(2-{[(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] quinolin-1-yl] amino} -2-oxoethoxy) methyl] glycinamide N, N of the compound obtained in Step 7 above (0.253 mmoL) -To a dimethylformamide (10.0 mL) solution, 6-maleimidohexanoic acid N-succinimidyl (0.156 g, 0.506 mmoL) was added and stirred at room temperature for 3 days. The solvent was distilled off under reduced pressure, and the resulting residue was purified by silica gel column chromatography [chloroform to chloroform: methanol = 9: 1 (v / v)] to obtain the title compound (0.100 g, 38%). .
¹ H-NMR (400 MHz, DMSO-d ₆ ) δ: 0.83 (3H, t, J = 7.2 Hz), 1.09-1.21 (2H, m), 1.33-1.47 (4H, m), 1.75-1.90 (2H, m), 2.00-2.23 (4H, m), 2.36 (3H, s), 2.69-2.81 (1H, m), 2.94-3.03 (1H, m), 3.06-3.22 (2H, m), 3.23-3.74 ( 6H, m), 3.98 (2H, s), 4.39-4.50 (1H, m), 4.60 (2H, d, J = 6.7Hz), 5.17 (2H, s), 5.39 (2H, s), 5.53-5.61 (1H, m), 6.50 (1H, s), 6.96 (2H, s), 7.11-7.24 (5H, m), 7.28 (1H, s), 7.75 (1H, d, J = 11.0Hz), 7.97 ( 1H, t, J = 5.7Hz), 8.03 (1H, t, J = 5.9Hz), 8.09 (1H, d, J = 7.8Hz), 8.27 (1H, t, J = 6.5Hz), 8.48 (1H, d, J = 9.0Hz), 8.60 (1H, t, J = 6.5Hz).
MS (ESI) m / z: 1034 (M + H) ⁺ .

Process 9: hM23-H1L1 ADC (2)
(i) Reduction of antibody hM23-H1L1 prepared in Example 6 was prepared to 10 mg / mL in PBS 6.0 / EDTA using common operation B (using 1.65 as the 280 nm extinction coefficient) and C. This solution (1.00 mL) is collected in a 2 mL polypropylene tube, and 10 mM TCEP (Tokyo Chemical Industry Co., Ltd.) aqueous solution (0.0158 mL; 2.3 equivalents per antibody molecule) and 1 M dipotassium hydrogen phosphate aqueous solution (Nacalai Tesque, Inc .; 0.015 mL). After confirming that the pH of this solution was within 7.0 ± 0.1, the disulfide bond at the hinge part in the antibody was reduced by incubating at 37 ° C. for 2 hours.

(ii) Conjugation of antibody and drug linker The above solution was incubated in a room temperature water bath for 10 minutes, and then dimethyl sulfoxide (0.0228 mL) was added. Next, a 10 mM dimethyl sulfoxide solution (0.0268 mL; 4.6 equivalents per antibody molecule) of the compound obtained in the above step 8 was added, and the mixture was stirred at room temperature for 60 minutes using a tube rotator (MTR-103, ASONE CORPORATION) A drug linker was attached to the antibody. Next, a 100 mM NAC (Sigma-Aldrich Co. LLC) aqueous solution (0.0063 mL; 9.2 equivalents per antibody molecule) was added, and the mixture was further stirred at room temperature for 20 minutes to stop the reaction of the drug linker.

(iii) Purification The above solution was purified by common operation D to obtain 6.0 mL of a solution containing the title antibody-drug conjugate.

(iv) Characteristic evaluation Using the common operation E, the following characteristic values were obtained.

Antibody concentration: 1.53 mg / mL, antibody yield: 9.18 mg (92%), average number of drugs bound per antibody molecule (n): 2.9

Example 11 Production of hM23-H1L1 ADC (3)

Process 1: hM23-H1L1 ADC (3)
Using the hM23-H1L1 prepared in Example 6 and the compound obtained in Step 8 of Example 10, the title antibody-drug conjugate was obtained in the same manner as in Step 6 of Example 7.

Antibody concentration: 1.59 mg / mL, antibody yield: 9.54 mg (95%), average number of drugs bound per antibody molecule (n): 5.4.

(Example 12) Production of hM23-H1L2 ADC (2)

Process 1: hM23-H1L2 ADC (2)
Using the hM23-H1L2 prepared in Example 6 and the compound obtained in Step 10 of Example 10, the title antibody-drug conjugate was obtained in the same manner as in Step 9 of Example 10.

Antibody concentration: 1.52 mg / mL, antibody yield: 9.12 mg (91%), average number of drugs bound per antibody molecule (n): 3.0.

(Example 13) Production of hM23-H1L2 ADC (3)

Process 1: hM23-H1L2 ADC (3)
Using the hM23-H1L2 prepared in Example 6 and the compound obtained in Step 8 of Example 10, the title antibody-drug conjugate was obtained in the same manner as in Step 6 of Example 7.

Antibody concentration: 1.57 mg / mL, antibody yield: 9.42 mg (94%), average number of drugs bound per antibody molecule (n): 5.7.

(Example 14) Production of hM23-H2L2 ADC (2)

Process 1: hM23-H2L2 ADC (2)
Using the hM23-H2L2 prepared in Example 6 and the compound obtained in Step 10 of Example 10, the title antibody-drug conjugate was obtained in the same manner as in Step 9 of Example 10.

Antibody concentration: 1.50 mg / mL, antibody yield: 9.00 mg (90%), average number of drugs bound per antibody molecule (n): 3.0.

(Example 15) Production of hM23-H2L2 ADC (3)

Process 1: hM23-H2L2 ADC (3)
Using the hM23-H2L2 produced in Example 6 and the compound obtained in Step 10 of Example 10, the title antibody-drug conjugate was obtained in the same manner as in Step 6 of Example 7.

Antibody concentration: 1.51 mg / mL, antibody yield: 9.06 mg (91%), average number of drugs bound per antibody molecule (n): 5.8.

(Example 16) Production of hM23-H1L1 ADC (4)

Step 1: tert-butyl [2- (2-{[(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] indolidino [1,2-b] quinolin-1-yl] amino} -2-oxoethoxy) ethyl] Carbamate
Instead of 4- (tert-butoxycarbonylamino) butanoic acid, {2-[(tert-butoxycarbonyl) amino] ethoxy} acetic acid (J. Med. Chem., 1992, 35, 2928) (1.55 g, The title compound (2.56 g, 73%) was obtained in the same manner as in Example 7, Step 1.
¹ H-NMR (400 MHz, DMSO-d ₆ ) δ: 0.87 (3H, t, J = 7.3 Hz), 1.26 (9H, s), 1.81-1.91 (2H, m), 2.13-2.22 (2H, m) , 2.40 (3H, s), 3.08-3.26 (4H, m), 3.43-3.53 (2H, m), 4.00 (1H, d, J = 15.1Hz), 4.05 (1H, d, J = 15.1Hz), 5.14 (1H, d, J = 18.7Hz), 5.22 (1H, d, J = 18.7Hz), 5.40 (1H, d, J = 16.6Hz), 5.44 (1H, d, J = 16.6Hz), 5.59- 5.66 (1H, m), 6.53 (1H, s), 6.86 (1H, t, J = 5.4Hz), 7.31 (1H, s), 7.79 (1H, d, J = 10.9Hz), 8.49 (1H, d , J = 9.1Hz).
MS (APCI) m / z: 637 (M + H) ⁺ .

Step 2: 2- (2-Aminoethoxy) -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] indolidino [1,2-b] quinolin-1-yl] acetamide trifluoroacetate salt in step 1 above The obtained compound (1.50 g, 2.36 mol) was reacted in the same manner as in Step 7 of Example 7 to obtain the title compound (1.50 g, quantitative).
¹ H-NMR (400 MHz, DMSO-d ₆ ) δ: 0.87 (3H, t, J = 7.5 Hz), 1.81-1.92 (2H, m), 2.15-2.23 (2H, m), 2.41 (3H, s) , 3.05 (2H, t, J = 5.1Hz), 3.15-3.23 (2H, m), 3.71 (2H, t, J = 5.1Hz), 4.10 (2H, s), 5.19 (1H, d, J = 18.7 Hz), 5.24 (1H, d, J = 18.7Hz), 5.43 (2H, s), 5.58-5.66 (1H, m), 6.55 (1H, s), 7.33 (1H, s), 7.73-7.84 (4H , m), 8.55 (1H, d, J = 9.1Hz).
MS (APCI) m / z: 537 (M + H) ⁺ .

Step 3: N- (tert-butoxycarbonyl) glycylglycyl-L-phenylalanyl-N- [2- (2-{[(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] indolidino [1,2-b] quinoline 1-yl] amino} -2-oxoethoxy) ethyl] glycinamide The compound obtained in Step 2 above (554 mg, 0.85 mmol) was reacted in the same manner as in Step 7 of Example 7 to give the title compound (775 mg, 95% )
¹ H-NMR (400 MHz, DMSO-d ₆ ) δ: 0.85 (3H, t, J = 7.3 Hz), 1.36 (9H, s), 1.78-1.89 (2H, m), 2.13-2.22 (2H, m) , 2.39 (3H, s), 2.71 (1H, dd, J = 13.4,9.8Hz), 2.95 (1H, dd, J = 13.4,4.3Hz), 3.09-3.23 (1H, m), 3.23-3.32 (2H , m), 3.40-3.62 (8H, m), 3.73 (1H, dd, J = 16.5,5.5Hz), 4.03 (2H, s), 4.39-4.47 (1H, m), 5.17 (1H, d, J = 18.9Hz), 5.25 (1H, d, J = 18.9Hz), 5.41 (1H, d, J = 16.8Hz), 5.45 (1H, d, J = 16.8Hz), 5.57-5.64 (1H, m), 6.54 (1H, s), 6.99 (1H, t, J = 5.8Hz), 7.13-7.26 (5H, m), 7.31 (1H, s), 7.76-7.82 (2H, m), 7.90 (1H, t, J = 5.2Hz), 8.13 (1H, d, J = 7.9Hz), 8.27 (1H, t, J = 5.8Hz), 8.49 (1H, d, J = 8.5Hz).
MS (APCI) m / z: 955 (M + H) ⁺ .

Step 4: Glycylglycyl-L-phenylalanyl-N- [2- (2-{[(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] quinolin-1-yl] amino}- 2-Oxoethoxy) ethyl] glycinamide trifluoroacetate The compound obtained in Step 3 above (630 mg, 0.659 mmol) was reacted in the same manner as in Step 7 of Example 7 to obtain the title compound (588 mg, 92%). .
¹ H-NMR (400 MHz, DMSO-d ₆ ) δ: 0.86 (3H, t, J = 7.3 Hz), 1.79-1.90 (2H, m), 2.13-2.22 (2H, m), 2.39 (3H, s) , 2.71 (1H, dd, J = 13.4,10.1Hz), 2.99 (1H, dd, J = 13.4,4.3Hz), 3.09-3.23 (1H, m), 3.24-3.32 (3H, m), 3.41-3.71 (7H, m), 3.86 (1H, dd, J = 16.8,5.8Hz), 4.04 (2H, s), 4.52 (1H, td, J = 9.0,4.1Hz), 5.17 (1H, d, J = 18.9 Hz), 5.25 (1H, d, J = 18.9Hz), 5.41 (1H, d, J = 16.5Hz), 5.45 (1H, d, J = 16.5Hz), 5.56-5.65 (1H, m), 6.55 ( 1H, s), 7.13-7.26 (5H, m), 7.32 (1H, s), 7.80 (1H, d, J = 11.0Hz), 7.87-8.01 (4H, m), 8.29-8.36 (2H, m) , 8.46-8.55 (2H, m).
MS (APCI) m / z: 855 (M + H) ⁺ .

Step 5: N- [6- (2,5-Dioxo-2,5-dihydro-1H-pyrrol-1-yl) hexanoyl] glycylglycyl-L-phenylalanyl-N- [2- (2-{[( 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] quinolin-1-yl] amino} -2-oxoethoxy) ethyl] glycinamide instead of diisopropylethylamine, triethylamine (31.4 μL, 0.22 mmoL) N-succinimidyl 6-maleimidohexanoate (95.3 mg, 0.31 mmoL) instead of N-succinimidyl 3- (2- (2- (3-maleimidopropanamide) ethoxy) ethoxy) propanoate Example 7 The title compound (162 mg, 62%) was obtained in the same manner as in Step 5.
¹ H-NMR (400 MHz, DMSO-d ₆ ) δ: 0.86 (3H, t, J = 7.6 Hz), 1.13-1.22 (2H, m), 1.40-1.51 (4H, m), 1.78-1.90 (2H, m), 2.09 (2H, t, J = 7.6Hz), 2.14-2.21 (2H, m), 2.39 (3H, s), 2.74 (1H, dd, J = 13.6,9.7Hz), 2.96 (1H, dd , J = 13.6,4.5Hz), 3.08-3.24 (1H, m), 3.24-3.30 (1H, m), 3.33-3.40 (4H, m), 3.47-3.68 (7H, m), 3.72 (1H, dd , J = 16.6,5.7Hz), 4.03 (2H, s), 4.42 (1H, td, J = 8.6,4.2Hz), 5.17 (1H, d, J = 18.7Hz), 5.25 (1H, d, J = 18.7Hz), 5.40 (1H, d, J = 17.2Hz), 5.44 (1H, d, J = 17.2Hz), 5.57-5.64 (1H, m), 6.52 (1H, s), 6.99 (2H, s) , 7.13-7.25 (5H, m), 7.31 (1H, s), 7.74-7.81 (2H, m), 7.99 (1H, t, J = 5.7Hz), 8.03-8.11 (2H, m), 8.22 (1H , t, J = 5.7Hz), 8.47 (1H, d, J = 9.1Hz).
MS (APCI) m / z: 1048 (M + H) ⁺ .

Process 6: hM23-H1L1 ADC (4)
Using the hM23-H1L1 prepared in Example 6 and the compound obtained in Step 5 above, the title antibody-drug conjugate was obtained in the same manner as in Example 10, Step 9.

Antibody concentration: 1.47 mg / mL, antibody yield: 8.82 mg (88%), average number of drugs bound per antibody molecule (n): 2.8.

(Example 17) Production of hM23-H1L1 ADC (5)

Process 1: hM23-H1L1 ADC (5)
Using the hM23-H1L1 prepared in Example 6 and the compound obtained in Step 5 of Example 16, the title antibody-drug conjugate was obtained in the same manner as in Step 6 of Example 7.

Antibody concentration: 1.47 mg / mL, antibody yield: 8.82 mg (88%), average number of drugs bound per antibody molecule (n): 5.3.

(Example 18) Production of hM23-H1L2 ADC (4)

Process 1: hM23-H1L2 ADC (4)
Using the hM23-H1L2 prepared in Example 6 and the compound obtained in Step 5 of Example 16, the title antibody-drug conjugate was obtained in the same manner as in Step 10 of Example 10.

Antibody concentration: 1.49 mg / mL, antibody yield: 8.94 mg (89%), average number of drugs bound per antibody molecule (n): 3.0.

Example 19 Production of hM23-H1L2 ADC (5)

Process 1: hM23-H1L2 ADC (5)
Using the hM23-H1L2 prepared in Example 6 and the compound obtained in Step 5 of Example 16, the title antibody-drug conjugate was obtained in the same manner as in Step 6 of Example 7.

Antibody concentration: 1.57 mg / mL, antibody yield: 9.42 mg (94%), average number of drugs bound per antibody molecule (n): 5.8.

(Example 20) Production of hM23-H2L2 ADC (4)

Process 1: hM23-H2L2 ADC (4)
Using the hM23-H1L1 prepared in Example 6 and the compound obtained in Step 5 of Example 16 above, the title antibody-drug conjugate was obtained in the same manner as in Step 10 of Example 10.

Antibody concentration: 1.48 mg / mL, antibody yield: 8.88 mg (89%), average drug binding number per antibody molecule (n): 3.1.

(Example 21) Production of hM23-H2L2 ADC (5)

Process 1: hM23-H2L2 ADC (5)
Using the hM23-H1L1 prepared in Example 6 and the compound obtained in Step 5 of Example 16 above, the title antibody-drug conjugate was obtained in the same manner as in Step 6 of Example 7.

Antibody concentration: 1.56 mg / mL, antibody yield: 9.36 mg (94%), average number of drugs bound per antibody molecule (n): 5.9.

(Test Example 1) Internalization activity of cM23 antibody
The internalization activity of the cM23 antibody was examined according to Mol Biol Cell. 2004; 15: 5268-5282.

cM23, anti-transferrin receptor antibody (positive control) and control hIgG1 (negative control) were labeled with Alexa488. Each Alexa488-labeled antibody was adjusted to 2 μg / mL with ice-cold RPMI 1640 supplemented with 10% FBS, and 50 μL was dispensed into a 96-well U-bottom plate. Semi-confluent NCI-H322 cells were collected, suspended in 4 × 10 ⁶ cells / mL with RPMI 1640 supplemented with ice-cold 10% FBS, and 50 μL of cells were dispensed onto the antibody-dispensed plate. After stirring with a plate mixer, the mixture was incubated at 4 ° C. for 1 hour, 150 μL of ice-cold wash buffer (PBS containing 3% FBS) was added, and the supernatant was removed by centrifugation. Further, 200 μL of ice-cold wash buffer was added to remove the supernatant twice.
(1) Sample with 4 ° C incubation only: Add ice-cold wash buffer (1000-fold diluted LIVE / DEAD Fixable Near-IR Dead Cell Stain Kit (dead cell dye, Invitrogen # L10119)) or Anti- 75 μL of Alexa-488 solution (25 μg / mL Anti-Alexa-488 antibody, 1000-fold diluted dead cell dye added) was added, transferred to a new plate, and incubated on ice for 30 minutes. Add ice-cold fixing solution (PBS containing 2% PFA and 3% FBS) at 150 μL / well, dispense 150 μL each of the 2: 1 solution of the fixing solution and wash buffer into the empty well, and add 100 μL of the sample. Fluorescence was measured with FACS Canto (Becton Dickinson).
(2) 37 ° C. incubation sample: Add 100 μL of RPMI 1640 supplemented with 10% FBS warmed to 37 ° C. to the washed cells, suspend in a plate mixer, transfer to a 96-well V bottom plate, 1 hour or 3 hours 37 Incubated at 5 ° C. under 5% CO ₂ . 100 μL of ice-cold Wash Buffer was added, and the entire amount was transferred to a 96-well round bottom plate and centrifuged. After removing the supernatant, add 75 μL of ice-cold wash buffer (1000-fold diluted dead cell dye added) or Anti-Alexa-488 solution (25 μg / mL, 1000-fold diluted dead cell dye added), transfer to a new plate and place on ice Incubated for 30 minutes. An ice-cold fixing solution was added at 150 μL / well, 150 μL each of a 2: 1 solution of the fixing solution and the wash buffer was dispensed into each vacant well, 100 μL of the sample was added, and fluorescence was measured with a FACS Canto.

  As shown in FIG. 8, about 16% of the internalization activity of cM23 was recognized by the evaluation using Alexa488-labeled cM23 on NCI-H322 cells. In FIG. 8, TfR Ab is an abbreviation for anti-transferrin receptor antibody labeled with Alexa488.

(Test Example 2) Measurement of antigen binding activity of hM23 antibody (2-1) Measurement of antigen binding activity using antibody (culture supernatant) Antibody and antigen (Recombinant Human SLC3A2 / MDU1 (Sino Biological Inc.)) The dissociation constant is measured using a capture method that uses Biacore T200 (GE Healthcare Bio-Sciences) to capture (capture) the immobilized anti-human IgG (Fc) antibody as a ligand and measure the antigen as an analyte. went. Anti-human IgG (Fc) antibody (Human anibody capture kit, GE Healthcare Bio-Sciences) was covalently bound to sensor chip CM5 (GE Healthcare Bio-Sciences) by about 1000 RU by an amine coupling method. The reference cell was similarly fixed. As a running buffer, HBS-EP + (10 mM HEPES pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.05% Surfactant P20, GE Healthcare Bio-Sciences) was used. Culture supernatant containing antibody whose concentration is adjusted with HBS-EP + so that 20-30RU capture can be obtained by adding 60 μs at a flow rate of 10 μL / min to a measurement side cell immobilized with anti-human IgG (Fc) antibody ( The culture supernatant obtained in 6-3-1) was added. Antigen dilution series solutions (0.077-100 nM and 0 nM) were added to the reference cell and the measurement side cell at a flow rate of 30 μL / min for 300 seconds, and then the dissociation phase for 600 seconds was monitored. As a regeneration solution, 3M MgCl ₂ was added twice at a flow rate of 10 μL / min for 60 seconds. For analysis of data, the binding rate constant kon, dissociation rate constant koff and dissociation constant (KD; KD = koff / kon) were calculated using the 1: 1 Binding model of analysis software (Biacore T200 Evaluation Software, version 1.0). did.

(2-2) Measurement of antigen binding activity using purified antibodies
Using the antibody obtained in 6-3-2, binding rate constant, dissociation rate constant and dissociation constant were calculated in the same manner as described above.

(Test Example 3) Cytotoxic activity of ADC Human plasmacytoma NCI-H929, human Burkitt lymphoma Ramos, and human melanoma MeWo were purchased from the American Type Culture Collection. NCI-H929 cells and Ramos cells were seeded on a 96-well plate at 2 × 10 ³ cells / well and MeWo cells at 1 × 10 ³ cells / well. At the same time, each antibody and ADC serially diluted in medium were added (final). Concentration: 0.1, 1, 10, 100, 1000 ng / mL, each n = 2). The cells were cultured in an incubator set at 37 ° C. and 5% CO ₂ for 6 days, and the amount of ATP was measured using Cell Titer Glo (Promega KK). The cell survival rate was calculated by the following formula, and the 50% survival inhibitory concentration IC ₅₀ (ng / mL) was calculated using a regression line. The results are shown in the table.
Cell viability (%) = 100 × (TB) / (CB)
T: Amount of luminescence in ADC-treated cell well
C: Average luminescence of untreated cell wells (n = 2)
B: Average light emission of blank well (n = 2)

  hM23H1L1, hM23H1L2 and hM23H2L2 did not show in vitro cytotoxic activity, but their ADCs showed cytotoxic activity.

(Test Example 4) ADC antitumor effect
Five-week-old female SCID mice (Charles River Japan) were acclimatized for 7 days under SPF conditions before using the experiment. Mice were fed a chow (FR-2, Funabashi Farms Co., Ltd) and given water with 1-5 ppm of chlorine added. Ramos cells (8 × 10 ⁷ cells / mL) suspended in physiological saline were implanted subcutaneously at 100 μL / mouse into the right axilla of SCID mice (Day 0). On Day 10, the major axis and minor axis of the tumor were measured with a digital caliper (CD-15CX, Mitutoyo Corp.), and grouping was performed based on the tumor volume calculated according to the following formula (n = 6).
Tumor volume (mm ³ ) = 1/2 × major axis (mm) × [minor axis (mm)] ²

  On the day of grouping, the antibody or ADC was diluted to 0.3 mg / mL with ABS and administered into the tail vein at 10 mL / kg (3 mg / kg). Thereafter, the tumor diameter was measured twice a week. Changes in tumor volume are shown in FIGS.

  hM23H1L1, hM23H1L2 and hM23H2L2 showed tumor growth inhibitory activity, but their ADCs showed stronger antitumor activity than antibody alone.

SEQ ID NO: 1: Nucleotide sequence of cDNA encoding heavy chain variable region of M23 antibody SEQ ID NO: 2: Amino acid sequence of heavy chain variable region of M23 antibody SEQ ID NO: 3: cDNA encoding light chain variable region of M23 antibody Nucleotide sequence SEQ ID NO: 4: amino acid sequence of the light chain variable region of M23 antibody SEQ ID NO: 5: nucleotide sequence encoding human κ chain secretion signal and human κ chain constant region SEQ ID NO: 6: human heavy chain secretion signal and human IgG1 Nucleotide sequence SEQ ID NO: 7 encoding the constant region SEQ ID NO: 7 Chimeric M23 antibody heavy chain nucleotide sequence SEQ ID NO: 8 Chimeric M23 antibody heavy chain amino acid sequence SEQ ID NO: 9 Chimeric M23 antibody light chain nucleotide sequence SEQ ID NO: 10 Chimeric M23 antibody Light chain amino acid sequence SEQ ID NO: 11: hM23-H1-type heavy chain nucleotide sequence SEQ ID NO: 12: hM23-H1-type heavy chain amino acid sequence SEQ ID NO: 13: hM23-H2-type heavy chain NUC Otide sequence SEQ ID NO: 14: amino acid sequence of hM23-H2 type light chain SEQ ID NO: 15: nucleotide sequence of hM23-L1 type light chain SEQ ID NO: 16: amino acid sequence of hM23-L1 type light chain SEQ ID NO: 17: hM23-L2 type light chain SEQ ID NO: 18: amino acid sequence of hM23-L2 type light chain SEQ ID NO: 19: amino acid sequence of CDRH1 of M23 antibody SEQ ID NO: 20: amino acid sequence of CDRH2 of M23 antibody SEQ ID NO: 21: amino acid sequence of CDRH3 of M23 antibody SEQ ID NO: 22: Amino acid sequence of CDRL1 of M23 antibody SEQ ID NO: 23: Amino acid sequence of CDRL2 of M23 antibody SEQ ID NO: 24: Amino acid sequence of CDRL3 of M23 antibody SEQ ID NO: 25: Nucleotide sequence of primer mG2aVR1 SEQ ID NO: 26: Nucleotide sequence of primer mKVR2 SEQ ID NO: 27: nucleotide sequence of primer 3.3-F1 SEQ ID NO: 28: nucleotide sequence of primer 3.3-R1 SEQ ID NO: 29: nucleotide sequence of primer M23H-F SEQ ID NO: 30 Primer M23H-R nucleotide sequence SEQ ID NO: 31: Primer M23L-F nucleotide sequence SEQ ID NO: 32: Primer M23L-R nucleotide sequence SEQ ID NO: 33: Primer EG-Inf-F nucleotide sequence SEQ ID NO: 34: Primer EG1-Inf -R nucleotide sequence SEQ ID NO: 35: Primer CM-LKF nucleotide sequence SEQ ID NO: 36: Primer KCL-Inf-R nucleotide sequence

Claims (25)

An anti-CD98 antibody-drug conjugate comprising an anti-CD98 antibody, a linker and a drug, or a salt thereof,
Where the linker is:
- (Succinimid-3-yl- N) -CH 2 CH 2 CH 2 CH 2 CH 2 -C (= O) -GGFG-NH-CH 2 -O-CH 2 -C (= O) -;
- (Succinimid-3-yl- N) -CH 2 CH 2 CH 2 CH 2 CH 2 -C (= O) -GGFG-NH-CH 2 CH 2 -O-CH 2 -C (= O) -; and
-(Succinimid-3-yl-N) -CH ₂ CH ₂ -C (= O) -NH-CH ₂ CH ₂ O-CH ₂ CH ₂ O-CH ₂ CH ₂ -C (= O) -GGFG-NH -CH ₂ CH ₂ CH ₂ -C (= O)-;
A linker selected from the group consisting of
The drug has the following formula:
A compound represented by
The nitrogen atom of the amino group at position 1 of the drug binds to the carbonyl moiety of the linker,
An anti-CD98 antibody binds to the succinimide part of the linker,
Anti-CD98 antibody-drug conjugate.   2. The antibody-drug conjugate or salt thereof according to claim 1, wherein the average number of drugs bound per antibody is in the range of 1 to 10.   2. The antibody-drug conjugate or a salt thereof according to claim 1, wherein the average number of drugs bound per antibody is in the range of 2 to 8.   2. The antibody-drug conjugate or a salt thereof according to claim 1, wherein the average number of drugs bound per antibody ranges from 3 to 8. Anti-CD98 antibody
CDRH1 consisting of the amino acid sequence represented by SEQ ID NO: 19 or an amino acid sequence in which one or several amino acid residues are added, deleted or substituted in the amino acid sequence;
CDRH2 consisting of the amino acid sequence represented by SEQ ID NO: 20 or an amino acid sequence in which one or several amino acid residues are added, deleted or substituted in the amino acid sequence;
CDRH3 consisting of the amino acid sequence represented by SEQ ID NO: 21 or an amino acid sequence in which one or several amino acid residues are added, deleted or substituted in the amino acid sequence;
CDRL1 consisting of the amino acid sequence represented by SEQ ID NO: 22 or an amino acid sequence in which one or several amino acid residues are added, deleted or substituted in the amino acid sequence;
CDRL2 consisting of the amino acid sequence represented by SEQ ID NO: 23 or an amino acid sequence in which one or several amino acid residues are added, deleted or substituted in the amino acid sequence; and the amino acid sequence represented by SEQ ID NO: 24, CDRL3 consisting of an amino acid sequence in which one or several amino acid residues are added, deleted or substituted in the amino acid sequence;
The antibody-drug conjugate or salt thereof according to any one of claims 1 to 4, which has at least one CDR selected from the group consisting of and binds to a CD98 heavy chain.   The anti-CD98 antibody has a heavy chain variable region having at least 80% sequence identity to the amino acid sequence represented by SEQ ID NO: 2 and / or at least 80% sequence to the amino acid sequence represented by SEQ ID NO: 4 The antibody-drug conjugate or salt thereof according to any one of claims 1 to 4, comprising a light chain variable region having identity.   The anti-CD98 antibody has a heavy chain having at least 80% sequence identity to the amino acid sequence represented by SEQ ID NO: 12 or 14, and / or at least 80% to the amino acid sequence represented by SEQ ID NO: 16 or 18 The antibody-drug conjugate or salt thereof according to any one of claims 1 to 4, comprising a light chain having the following sequence identity.   The anti-CD98 antibody consists of a heavy chain consisting of the amino acid sequence represented by SEQ ID NO: 12 or 14 and a light chain consisting of the amino acid sequence represented by SEQ ID NO: 16 or 18. Antibody-drug conjugates or salts thereof.   The antibody-drug according to any one of claims 1 to 4, wherein the anti-CD98 antibody comprises a heavy chain consisting of the amino acid sequence represented by SEQ ID NO: 12 and a light chain consisting of the amino acid sequence represented by SEQ ID NO: 16. Conjugate or salt thereof.   The antibody-drug according to any one of claims 1 to 4, wherein the anti-CD98 antibody consists of a heavy chain consisting of the amino acid sequence represented by SEQ ID NO: 12 and a light chain consisting of the amino acid sequence represented by SEQ ID NO: 18. Conjugate or salt thereof.   The antibody-drug according to any one of claims 1 to 4, wherein the anti-CD98 antibody consists of a heavy chain consisting of the amino acid sequence represented by SEQ ID NO: 14 and a light chain consisting of the amino acid sequence represented by SEQ ID NO: 16. Conjugate or salt thereof.   The antibody-drug according to any one of claims 1 to 4, wherein the anti-CD98 antibody consists of a heavy chain consisting of the amino acid sequence represented by SEQ ID NO: 14 and a light chain consisting of the amino acid sequence represented by SEQ ID NO: 18. Conjugate or salt thereof. CDRH1 consisting of the amino acid sequence represented by SEQ ID NO: 19 or an amino acid sequence in which one or several amino acid residues are added, deleted or substituted in the amino acid sequence;
CDRH2 consisting of the amino acid sequence represented by SEQ ID NO: 20 or an amino acid sequence in which one or several amino acid residues are added, deleted or substituted in the amino acid sequence;
CDRH3 consisting of the amino acid sequence represented by SEQ ID NO: 21 or an amino acid sequence in which one or several amino acid residues are added, deleted or substituted in the amino acid sequence;
CDRL1 consisting of the amino acid sequence represented by SEQ ID NO: 22 or an amino acid sequence in which one or several amino acid residues are added, deleted or substituted in the amino acid sequence;
CDRL2 consisting of the amino acid sequence represented by SEQ ID NO: 23 or an amino acid sequence in which one or several amino acid residues are added, deleted or substituted in the amino acid sequence; and the amino acid sequence represented by SEQ ID NO: 24, CDRL3 consisting of an amino acid sequence in which one or several amino acid residues are added, deleted or substituted in the amino acid sequence;
An anti-CD98 antibody having at least one CDR selected from the group consisting of and binding to a CD98 heavy chain.   A heavy chain variable region having at least 80% sequence identity to the amino acid sequence represented by SEQ ID NO: 2 and / or a light chain having at least 80% sequence identity to the amino acid sequence represented by SEQ ID NO: 4 14. The anti-CD98 antibody of claim 13, comprising a chain variable region.   A heavy chain having at least 80% sequence identity to the amino acid sequence represented by SEQ ID NO: 12 or 14, and / or at least 80% sequence identity to the amino acid sequence represented by SEQ ID NO: 16 or 18. 14. The anti-CD98 antibody of claim 13, comprising a light chain possessed by it.   14. The anti-CD98 antibody according to claim 13, comprising a heavy chain consisting of the amino acid sequence represented by SEQ ID NO: 12 or 14, and a light chain consisting of the amino acid sequence represented by SEQ ID NO: 16 or 18.   14. The anti-CD98 antibody according to claim 13, comprising a heavy chain consisting of the amino acid sequence represented by SEQ ID NO: 12 and a light chain consisting of the amino acid sequence represented by SEQ ID NO: 16.   14. The anti-CD98 antibody according to claim 13, comprising a heavy chain consisting of the amino acid sequence represented by SEQ ID NO: 12 and a light chain consisting of the amino acid sequence represented by SEQ ID NO: 18.   14. The anti-CD98 antibody according to claim 13, comprising a heavy chain consisting of the amino acid sequence represented by SEQ ID NO: 14 and a light chain consisting of the amino acid sequence represented by SEQ ID NO: 16.   14. The anti-CD98 antibody according to claim 13, comprising a heavy chain consisting of the amino acid sequence represented by SEQ ID NO: 14 and a light chain consisting of the amino acid sequence represented by SEQ ID NO: 18.   A pharmaceutical composition comprising the antibody-drug conjugate according to any one of claims 1 to 12 or a salt thereof or the anti-CD98 antibody according to any one of claims 13 to 20 as an active ingredient.   22. A pharmaceutical composition according to claim 21 for antitumor or anticancer.   Tumor or cancer is lung cancer, renal cancer, urothelial cancer, colon cancer, prostate cancer, glioblastoma multiforme, ovarian cancer, pancreatic cancer, breast cancer, melanoma, liver cancer, bladder cancer, stomach cancer, cervical cancer, head and neck 23. The pharmaceutical composition according to claim 22, which is cancer, esophageal cancer, lymphoma, acute myelogenous leukemia, acute lymphocytic leukemia, chronic myelogenous leukemia or multiple myeloma.   An antibody-drug conjugate according to any one of claims 1 to 12, or a pharmaceutically acceptable salt thereof, or an anti-CD98 antibody according to claims 13 to 20 for producing a pharmaceutical composition. use.   A tumor comprising administering to a mammal a therapeutically effective amount of the antibody-drug conjugate or salt thereof according to any one of claims 1 to 12 or the anti-CD98 antibody according to any one of claims 13 to 20. And / or a method of treating cancer.