TW201620553A - Anti-cd98 antibody-drug conjugate - Google Patents

Abstract

Provided is an anti-CD98 antibody-drug conjugate comprising an anti-CD98 antibody, a linker, and a drug, or a pharmacologically acceptable salt of the anti-CD98 antibody-drug congugate, as an anti-tumor drug having exceptional anti-tumor effects and safety as well as exceptional therapeutic effects, wherein: the linker is selected from the group consisting of -(succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2-O-CH2-C(=O)-, -(succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2-O-CH2-C(=O)-, and -(succinimid-3-yl-N)-CH2CH2-C(=O)-NH-CH2CH2-O-CH2CH2-O-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-; the drug is a compound represented by the formula; a nitrogen atom of a position-1 amino group of the drug bonds to a carbonyl moiety of the linker; and the anti-CD98 antibody bonds to a succinimide moiety of the linker.

Description

anti-CD98 antibody-drug conjugate

The present invention relates to an antibody-drug conjugate that binds an anti-tumor activity anti-CD98 antibody to an anti-tumor drug-intervening linker moiety.

The cytotoxic drug is bound to an antibody-drug conjugate (ADC) of an antibody that binds to an antigen present on the surface of the cancer cell and can be internalized into the cell, since the drug can be selectively delivered to Cancer cells can be expected to accumulate drugs in cancer cells and destroy cancer cells (see Non-Patent Documents 1 to 3). As the ADC, for example, Mylotarg (registered trademark; gemtuzumab ozogamicin) which binds anti-CD33 antibody to calicheamicin is recognized as a therapeutic drug for acute myeloid leukemia. Furthermore, Adcetris (registered trademark; brentuximab vedotin), which binds to anti-CD30 antibody and auristatin E, has been approved as Hodgkin's lymphoma and A therapeutic drug for undifferentiated large cell lymphoma (see Non-Patent Document 4). The drug contained in the ADC which has been approved so far is based on DNA or tubulin.

As a low molecular compound which is antitumor, a camptothecin derivative which inhibits topoisomerase I and exhibits an antitumor effect is known. Where

Antitumor compound shown (exateca n, chemical name: (1S, 9S)-1-amino-9-ethyl-5-fluoro-2,3-dihydro-9-hydroxyl -4-methyl-1H,12H-benzo[de]pyrano[3',4':6,7]吲 And [1,2-b]quinoline-10,13(9H,15H)-dione ((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)) A base derivative (Patent Documents 1 and 2). This compound is different from the current clinical use of irinotecan, and the anti-tumor effect does not require activation by enzymes. In addition, SN-38, which is the active body of Alenuo Dikeken, has stronger topoisomerase I inhibitory activity than topotecan which is also used clinically, and has various cancer cells in vitro. Stronger cell killing activity. In particular, by the expression of P-glycoprotein, it also exhibits an effect on cancer cells which are resistant to SN-38 and the like. Further, the mouse human tumor has a strong antitumor effect in the subcutaneous transplantation mode, and although it has been clinically tested, it has not yet been marketed (see Non-Patent Documents 5 to 10). It is unclear whether ezetidine is effective as an ADC.

DE-310 system allows ecitacol to separate GGFG peptide interval The substance is bonded to a complex of a biodegradable carboxymethyldextran polyol polymer (Patent Document 3). By pre-pharmaceutically stimulating the polymer, the retention of high blood retention is maintained, and the permeability of the tumor neovascularization and the retention of the tumor tissue are further utilized to passively improve the directionality to the tumor site. DE-310 is continuously liberated by the cleavage of the peptide spacer caused by the enzyme, and the active form of exenotecan and the glycine-conjugated ezetidine. As a result, the pharmacokinetics was improved, and the evaluation mode of various tumors in non-clinical trials, even the total amount of ezetidine contained in DE-310 was reduced compared with the dose of exemitic single agent. It also presents a higher effectiveness than the administration of a single dose of citrate. Regarding DE-310, effective cases have also been confirmed by clinical trials, and it has been confirmed that the active body is more concentrated in tumors than normal tissues. On the other hand, there is no significant difference between the accumulation of DE-310 and the active body to the tumor and the accumulation to the normal tissue, and there is no report of passive targeting in humans (see Non-Patent Documents 11 to 14). As a result, DE-310 has not yet reached the market, and it is not clear whether or not it is effective to use the drug as a target.

As a related compound of DE-310, it is also known to insert a moiety represented by -NH-(CH ₂ ) ₄ -C(=O)- into a -GGFG- spacer and ezetidine, and -GGFG _{-NH- (CH 2) 4 -C (} = O) - as the complex of the spacer structure (Patent Document 4), but the anti-tumor effect of the same composite is completely unknown.

The CD98 system contains approximately 40kDa of multi-pass light A heterodimer of a type II single-pass transmembrane heavy chain of about 80 to 85 kDa having a chain and a disulfide bond (Non-Patent Document 15). The CD98 heavy chain (known as CD98hc, 4F2 or FRP-1) is encoded in the mouse by the Slc3a2 gene and encoded in the human SLC3A2 gene. CD98hc is a type II transmembrane protein with extracellular regions, transmembrane regions, and cytoplasmic tails. The CD98 system forms a heterodimer by a disulfide bond between at least one of the six existing CD98 light chains (amino acid transport protein, LAT-1, LAT-2, etc.) and the extracellular region of CD98hc. It is known that the anti-CD98 antibody having this CD98 as a target has antitumor activity and immunosuppressive activity (Patent Documents 5 to 11).

Prior technical literature Patent literature

Patent Document 1 Japanese Patent Publication No. 5-59061

Patent Document 2 Japanese Patent Publication No. 8-337584

Patent Document 3 International Publication WO1997/46260 Booklet

Patent Document 4 International Publication WO2000/25825 Booklet

Patent Document 5 International Publication WO2007/114496 Brochure

Patent Document 6 International Publication WO2008/017828 Booklet

Patent Document 7 International Publication WO2009/043922 Booklet

Patent Document 8 International Publication WO2009/090553 Booklet

Patent Document 9 JP-A-2012-092068

Patent Document 10 International Publication WO2011/118804 Booklet

Patent Document 11 International Publication WO2013/078377 Booklet

Non-patent literature

Non-Patent Document 1 Ducry, L., et al., Bioconjugate Chemw (2010) 21, 5-13.

Non-Patent Document 2 Alley, S. C., et al., Current Opinion in Chemical Biology (2010) 14, 529-537.

Non-Patent Document 3 Damle N.K., Expert Opin. Biol. Ther. (2004) 4, 1445-1452.

Non-Patent Document 4 Senter P. D., et al., Nature Biotechnology (2012) 30, 631-637.

Non-Patent Document 5 Kumazawa, E., Tohgo, A., Exp. Opin. Invest. Drugs (1998) 7, 625-632.

Non-Patent Document 6 Mitsui, I., Kumazawa, E., Hirota, Y., et al., Jpn J. Cancer Res. (1995) 86, 776-786.

Non-Patent Document 7 Takiguchi, S., Tohgo, A., et al., Jpn J. Cancer Res. (1997) 88, 760-769.

Non-Patent Document 8 Joto, N. et al., Int J Cancer (1997) 72, 680-686.

Non-Patent Document 9 Kumazawa, E. et al., Cancer Chemother. Pharmacol. (1998) 42, 210-220.

Non-Patent Document 10 De Jager, R., et al., Ann N Y Acad Sci (2000) 922, 260-273.

Non-Patent Document 11 Inoue, K. et al., Polymer Drugs in the Clinical Stage, Edited by Maeda et al. (2003), 145-153.

Non-Patent Document 12 Kumazawa, E. et al., Cancer Sci (2004) 95, 168-175.

Non-Patent Document 13 Soepenberg, O. et al., Clinical Cancer Research, (2005) 11, 703-711.

Non-Patent Document 14 Wente M. N. et al., Investigational New Drugs (2005) 23, 339-347.

Non-Patent Document 15 Joseph M. Cantor et al., Jornal of Cell Science (2012) 125, 1373-1382.

[Summary of the Invention]

In the case of tumor treatment by an antibody, it has been observed that even if an antibody recognizes an antigen and binds to a tumor cell, the antitumor effect is insufficient, and there is a case where an antitumor antibody having a higher effect is required. Further, in the anti-tumor low-molecular compound, even if it is excellent in antitumor effect, there are many problems such as side effects or toxic surfaces, and it is also a problem to improve safety and obtain a more excellent therapeutic effect. That is, the subject of the present invention is to obtain and provide an antitumor agent having an excellent antitumor effect and an excellent anti-tumor effect and an excellent therapeutic effect.

Anti-CD98 anti-system can be the target of tumor cells The body, that is, by having an antibody capable of recognizing the characteristics of a tumor cell, a property capable of binding to a tumor cell, or a property which can be internalized into a tumor cell, by converting the antitumor compound ixeticon to The antibody-drug conjugate that binds to the same antibody and mediates the same antibody, the present inventors believe that the following effects may be achieved: (1) The ethicitol derivative can be transported to tumor cells to make ezetidine The anti-tumor effect of Kang derivatives is specifically exerted in tumor cells, (2) the anti-tumor effect can be exerted at the same time, and the dose of ezetike derivatives is reduced more than when administered alone. (3) A higher safety can be achieved because the effect of the ezetimicon derivative on normal cells can be alleviated.

Therefore, the inventors have created a connection of a specific structure, The anti-CD98 antibody-drug conjugate in which the anti-CD98 antibody was bound to exenotecan was successfully obtained, and the compound was found to exert an excellent antitumor effect, and the present invention was completed.

That is, the present invention relates to: (1) an anti-CD98 antibody-drug conjugate comprising an anti-CD98 antibody, a linker and a drug anti-CD98 antibody-drug conjugate or a pharmacologically acceptable salt thereof, wherein The linker is selected from the group consisting of a linker of the formula: -(amber succinimide-3-yl-N)-CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ -C(=O)-GGFG-NH- CH ₂ -O-CH ₂ -C(=O)-;-(succinimide-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 -(succinimide-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)-; The drug is a compound represented by the following formula:

The nitrogen atom of the amine group at the 1-position of the drug binds to the carbonyl moiety of the linker, and the anti-CD98 anti-system binds to the amber quinone imine moiety of the linker; (2) the antibody-drug conjugate as described in the above (1) Or a pharmacologically acceptable salt thereof, wherein the average drug binding amount per antibody is in the range of 2 to 8; (3) the antibody-drug conjugate as described in the above (1) or pharmacologically acceptable thereof a salt having an average number of bindings per one antibody of from 3 to 6; (4) an antibody-drug conjugate according to the above (2) or (3) or a pharmacologically acceptable salt thereof, wherein each (1) The antibody-drug conjugate according to the above (1), or a pharmacologically acceptable salt thereof, wherein each antibody is one antibody per one antibody, which is determined by a reverse phase chromatography (RPC) method; The antibody-drug conjugate according to any one of the above items (1) to (5), or a pharmacologically acceptable salt thereof, wherein the conjugate is of the formula :-( 3-yl succinate (PEI) _{-N) -CH 2 CH 2 CH 2} CH 2 CH 2 -C (= O) -GGFG-NH-CH 2 -O-CH 2 -C (= O)-; (7) The antibody-drug conjugate according to any one of the above (1) to (5) Their pharmacologically tolerable salt thereof, wherein the linker of the formula :-( 3-yl succinate (PEI) _{-N) -CH 2 CH 2 CH 2} CH 2 CH 2 -C (= O) -GGFG-NH _{-CH 2 CH 2 -O-CH 2} -C (= O) -; (8) according to (1) to (5) according to any one of the antibodies - the binding drugs thereof or a pharmacologically acceptable salt Wherein the linker is of the formula: -(amber succinimide-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)-; (9) The antibody-drug binding according to any one of the above (1) to (8) Or a pharmacologically acceptable salt thereof, wherein the anti-CD98 anti-system is combined with a moiety consisting of amino acid residues 460-541 of SEQ ID NO: 38; (10) as described above (1) to (9) The antibody-drug conjugate according to any one of the above, wherein the anti-CD98 anti-system comprises: an amino acid sequence represented by SEQ ID NO: 19 or 1 or 1 in the amino acid sequence CDRH1 consisting of several amino acid residues added, deleted or substituted amino acid sequence; amino acid sequence represented by SEQ ID NO: 20 or having one or several amino acid residues in the amino acid sequence Base is added, a CDRH2 consisting of a substituted or substituted amino acid sequence; an amino acid sequence represented by SEQ ID NO: 21 or an amine group having 1 or several amino acid residues added, deleted or substituted in the amino acid sequence CDRH3 consisting of an acid sequence; an amino acid sequence represented by SEQ ID NO: 22 or a CDRL1 consisting of an amino acid sequence in which one or several amino acid residues of the amino acid sequence are added, deleted or substituted ; an amino acid sequence represented by SEQ ID NO: 23 or a CDR2 comprising an amino acid sequence in which one or more amino acid residues are added, deleted or substituted; and SEQ ID NO: 24 The represented amino acid sequence or the 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; and binds to the CD98 heavy chain; (11) The antibody-drug conjugate according to any one of the above (1), wherein the anti-CD98 antibody comprises: the amino acid sequence represented by SEQ ID NO: 19; CDRH1; CDRH2 consisting of the amino acid sequence represented by SEQ ID NO: 20; SEQ ID NO: 21 CDRH3 consisting of the amino acid sequence shown; CDRL1 consisting of the amino acid sequence represented by SEQ ID NO: 22; CDRL2 consisting of the amino acid sequence represented by SEQ ID NO: 23; and SEQ ID NO: 24 The antibody-drug conjugate according to any one of the above (1) to (11), or a pharmacologically acceptable salt thereof, is the same as the above-mentioned (1) to (11). Wherein the anti-CD98 anti-system comprises a heavy chain variable region and/or a light chain variable region, the heavy chain variable region being associated with an amino group of amino acid residues 20 to 135 of SEQ ID NO: 8. An acid sequence having at least 90% identity, the light chain variable region having 90% identity with respect to an amino acid sequence consisting of amino acid residues 21 to 135 of SEQ ID NO: 10; The antibody-drug conjugate according to any one of the above-mentioned items, wherein the anti-CD98 anti-system comprises: a heavy chain selected from the group consisting of the following groups, or a pharmacologically acceptable salt thereof The heavy chain of the variable region: (1) an amino acid sequence consisting of amino acid residues 20 to 135 of SEQ ID NO: 12 or 14; (2) amino acid relative to (1) a sequence of amino acid having at least 95% identity; and (3) an amino acid sequence having one or more amino acids deleted, substituted or added to the amino acid sequence of (1); a light chain comprising a light chain variable region selected from the group consisting of: (4) an amino acid sequence consisting of amino acid residues 21 to 135 of SEQ ID NO: 16 or 18; (5) relative to (4) an amino acid sequence having an amino acid sequence of at least 95% identical; and (6) an amino acid sequence of (4), wherein one or more amino acids are deleted, substituted or The antibody-drug conjugate according to any one of the above items (1) to (11), wherein the anti-CD98 antibody comprises: a sequence identification number, or a pharmacologically acceptable salt thereof The heavy chain variable region heavy chain composed of the amino acid sequence consisting of the amino acid residues 20 to 135 of 12 and the amino acid residues 21 to 135 of SEQ ID NO: 16 (15) The antibody-drug conjugate according to any one of the above (1) to (11), or a pharmacologically acceptable salt thereof, in the light chain of the light chain variable region; , wherein the anti-CD98 antibody comprises a heavy chain of a heavy chain variable region comprising an amino acid sequence consisting of amino acid residues 20 to 135 of SEQ ID NO: 12 and an amino group comprising SEQ ID NO: 18 of Nos. 21 to 135 (16) The antibody-drug conjugate according to any one of the above (1) to (11), or a pharmacologically active substance thereof, wherein the light chain of the light chain variable region is composed of the amino acid sequence of the acid residue; An acceptable salt, wherein the anti-CD98 antibody comprises: a heavy chain variable region comprising an amino acid sequence consisting of amino acid residues 20 to 135 of SEQ ID NO: 14 and comprising a sequence recognition a light chain of a light chain variable region composed of an amino acid sequence consisting of amino acid residues No. 21 to 135 of No. 18; (17) as described in any one of (1) to (11) above An antibody-drug conjugate or a pharmacologically acceptable salt thereof, wherein the anti-CD98 antibody comprises: a heavy amino acid sequence consisting of amino acid residues 20 to 465 of SEQ ID NO: 12 a light chain comprising an amino acid sequence consisting of a chain and an amino acid residue of Nos. 21 to 240 of SEQ ID NO: 1; (18) as described in any one of the above items (1) to (11) Antibody-drug a conjugate or a pharmacologically acceptable salt thereof, wherein the anti-CD98 antibody comprises: a heavy chain comprising the amino acid sequence consisting of amino acid residues 20 to 465 of SEQ ID NO: 12, and a sequence comprising the same a light chain comprising an amino acid sequence consisting of amino acid residues 21 to 240 of the identification number 18; (19) an antibody-drug binding according to any one of the above (1) to (11) Or a pharmacologically acceptable salt thereof, wherein the anti-CD98 antibody comprises: a heavy chain comprising an amino acid sequence consisting of amino acid residues 20 to 465 of SEQ ID NO: 14 and comprising sequence recognition a light chain composed of an amino acid sequence consisting of amino acid residues 21 to 240; (20) an anti-CD98 antibody or an antigen-binding fragment thereof, and the sequence identification number 38 (21) The anti-CD98 antibody or the antigen-binding fragment of the antibody according to the above (20), which comprises: the amino acid represented by SEQ ID NO: 19. a sequence or an amino acid sequence in which one or more amino acid residues are added, deleted or substituted in the amino acid sequence CDRH1; an amino acid sequence represented by SEQ ID NO: 20 or a CDRH2 consisting of an amino acid sequence in which one or several amino acid residues are added, deleted or substituted in the amino acid sequence; SEQ ID NO: 21 The amino acid sequence represented or the amino acid sequence 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 amino acid represented by SEQ ID NO: 22 a sequence or a CDRL1 consisting of an amino acid sequence in which one or several amino acid residues are added, deleted or substituted in the amino acid sequence; or an amino acid sequence represented by SEQ ID NO: 23 or The acid sequence has a CDRL2 consisting of an amino acid sequence in which one or several amino acid residues are added, deleted or substituted; and the amino acid sequence represented by SEQ ID NO: 24 or 1 in the amino acid sequence A CDRL3 consisting of several amino acid residues added, deleted or substituted with an amino acid sequence

(22) The anti-CD98 antibody or the antigen-binding fragment of the antibody according to the above (20) or (21), which comprises: CDRH1 consisting of the amino acid sequence represented by SEQ ID NO: 19; The CDRH2 composed of the amino acid sequence; CDRH3 composed of the amino acid sequence represented by SEQ ID NO: 21; CDRL1 composed of the amino acid sequence represented by SEQ ID NO: 22; CDRL2 composed of the amino acid sequence represented by SEQ ID NO: 23; The anti-CD98 antibody according to any one of the above (20) to (22), or the antigen-binding fragment of the antibody, which comprises the sequence identification An amino acid sequence consisting of amino acid residues No. 20 to 135 of No. 8, having a heavy chain variable region of at least 90% identity and/or No. 21 to 135 of sequence identification number 410 The amino acid sequence consisting of the amino acid residue, the light chain variable region having at least 90% identity; (24) the anti-CD98 antibody according to any one of the above (20) to (22) or An antigen-binding fragment of the antibody comprising a heavy chain selected from the group consisting of heavy chain variable regions of the following composition: (1) amino acid residues 20 to 135 of SEQ ID NO: 12 or 14 An amino acid sequence; (2) an amino acid sequence having at least 95% identity with respect to the amino acid sequence of (1); and (3) an amino acid of (1) a sequence, an amino acid sequence having one or more amino acids deleted, substituted or added; and a light chain comprising a light chain variable region selected from the group consisting of: (4) an amino acid sequence consisting of amino acid residues 21 to 135 of SEQ ID NO: 16 or 18; (5) having at least 95% or more with respect to the amino acid sequence of (4) And (6) an amino acid sequence of (4), wherein one or more amino acids are deleted, substituted or added an amino acid sequence; (25) as described above (20) The anti-CD98 antibody according to any one of (22), wherein the antigen-binding fragment of the antibody comprises an amino acid sequence consisting of amino acid residues 20 to 135 of SEQ ID NO: 12; a heavy chain of a heavy chain variable region and a light chain of a light chain variable region comprising an amino acid sequence consisting of amino acid residues 21 to 135 of SEQ ID NO: 16; (26) The anti-CD98 antibody according to any one of the above (20), wherein the antigen-binding fragment of the antibody comprises an amino group comprising amino acid residues 20 to 135 of SEQ ID NO: 12. a light chain of a light chain variable region composed of a heavy chain variable region consisting of an acid sequence and an amino acid sequence comprising amino acid residues 21 to 135 of SEQ ID NO: 18; (27) As mentioned above (20)~(22) An anti-CD98 antibody according to any one of the above, which comprises an amino acid sequence comprising an amino acid sequence consisting of amino acid residues 20 to 135 of SEQ ID NO: 14 and having a variable heavy chain a light chain of a light chain variable region comprising a heavy chain of the region and an amino acid sequence comprising amino acid residues 21 to 135 of SEQ ID NO: 18; The anti-CD98 antibody according to any one of the above-mentioned items (20) to (22), wherein the amino acid-containing fragment of the antibody comprising the amino acid group No. 20 to 465 of SEQ ID NO: 12 is contained. a light chain variable region consisting of a heavy chain variable region composed of an amino acid sequence and an amino acid sequence comprising amino acid residues 21 to 240 of SEQ ID NO: 16 (29) The anti-CD98 antibody according to any one of the above (20) to (22), or an antigen-binding fragment of the antibody, comprising: an amino group of SEQ ID NO: 12 to No. 20 to 465 The light chain of the heavy chain variable region consisting of the amino acid sequence consisting of acid residues and the amino acid sequence consisting of amino acid residues 21 to 240 of SEQ ID NO: 18 (30) The anti-CD98 antibody according to any one of the above (20) to (22), or the antigen-binding fragment of the antibody, comprising: 20 to 465 containing the sequence identification number 14 Amino acid sequence consisting of the heavy chain variable region consisting of the amino acid sequence consisting of amino acid residues and the amino acid sequence consisting of amino acid residues 21 to 240 of SEQ ID NO: 18. (31) An antibody-drug conjugate or a pharmacologically acceptable salt thereof, which comprises the anti-CD98 antibody according to any one of the above (20) to (30) or An antigen-binding fragment of the antibody; (32) A pharmaceutical composition comprising the antibody-drug conjugate according to any one of the above (1) to (19) and (31) or a pharmacologically acceptable salt thereof Or an anti-CD98 antibody according to any one of the above (20) to (30) or an antigen-binding fragment of the antibody as an active ingredient; (33) The pharmaceutical composition according to the above (32), which is used for anti-tumor or anti-cancer; (34) The pharmaceutical composition according to (33) above, wherein the tumor or cancer is lung cancer, kidney cancer, urothelial carcinoma , colorectal 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, lymph An antibody-drug conjugate according to any one of the above (1) to (19) and (31), wherein the tumor, the acute myeloid leukemia, the acute lymphocytic leukemia, the chronic myelogenous leukemia, or the multiple myeloma; Or a pharmaceutically acceptable salt thereof, or an anti-CD98 antibody according to any one of the above (20) to (30), or an antigen-binding fragment of the antibody, for use in the manufacture of a pharmaceutical composition; (36) The antibody-drug conjugate according to any one of the above items (1) to (19), or a pharmaceutically acceptable salt thereof, or the antibody according to any one of the above (20) to (30) A CD98 antibody or antigen-binding fragment thereof, for use in the treatment of tumors and/or cancer.

(37) A method for treating a tumor and/or a cancer, which comprises the antibody-drug conjugate according to any one of the above (1) to (19) and (31) or a pharmacologically acceptable salt thereof or The therapeutically effective amount of the anti-CD98 antibody according to any one of the above (20) to (30) or the antigen-binding fragment of the antibody is administered to a mammal; (38) a polynucleotide encoding the same as (20) above (30) an antibody according to any one of the items; (39) A polynucleotide according to the above (38), which comprises a polynucleotide selected from the group consisting of: (1) Nos. 58 to 405 of SEQ ID NO: 11 or 13. a nucleotide sequence consisting of nucleotides; (2) a nucleotide sequence having at least 95% identity with respect to the nucleotide sequence of (1); (3) and a nucleoside of (1) a nucleotide sequence consisting of a nucleotide sequence complementary to a nucleotide sequence, a nucleotide sequence retained by a polynucleotide which hybridizes under stringent conditions; and (4) a nucleotide sequence of (1), having one or several nucleotide sequences a nucleotide sequence in which a nucleotide is deleted, substituted or added; and comprises a nucleotide selected from the group consisting of: (5) nucleotides 61 to 405 of SEQ ID NO: 15 or 17. a nucleotide sequence; (6) a nucleotide sequence having at least 95% identity with respect to the nucleotide sequence of (5); (7) a nucleoside complementary to the nucleotide sequence of (5) a nucleotide sequence consisting of a polynucleotide consisting of an acid sequence which is hybridized under stringent conditions; and (8) a nucleotide sequence of (5), wherein one or several nucleotides are deleted or substituted Or added core (40) The polynucleotide according to the above (39), which comprises the polynucleotide consisting of the nucleotide sequence of nucleotides 58 to 405 of SEQ ID NO: 11 and sequence recognition a polynucleotide consisting of a nucleotide sequence consisting of nucleotides 61 to 405 of No. 15; (41) The polynucleotide according to the above (39), which comprises the polynucleotide consisting of the nucleotide sequence of nucleotides 58 to 405 of SEQ ID NO: 11 and the sequence identification number 17 A polynucleotide comprising the nucleotide sequence of nucleotides 61 to 405; (42) The polynucleotide according to the above (39), which comprises: SEQ ID NO: 13 No. 58 to No. 405 a polynucleotide consisting of a nucleotide sequence consisting of a nucleotide and a nucleotide sequence consisting of nucleotides 61 to 405 of SEQ ID NO: 17; (43) a polynucleotide The vector comprising the polynucleotide according to any one of the above (38) to (42); (44) a transgenic host cell comprising the multinuclear according to any one of the above (38) to (42) And (45) a method for producing an antibody according to any one of the above (20) to (30), comprising: The step of purifying the antibody from the culture product by culturing the host cell as described in the above (44) or (45).

An anti-tumor effect and safety can be achieved by binding an anti-CD98 antibody-drug conjugate of exenote to an anti-CD98 antibody by mediating a linker of a specific structure.

Figure 1 shows the nucleotide sequence (SEQ ID NO: 7) and amino acid sequence (SEQ ID NO: 8) of the heavy chain of the chimeric M23 antibody.

Figure 2 shows the nucleotide sequence (SEQ ID NO: 9) and the amino acid sequence (SEQ ID NO: 10) of the chimeric M23 antibody light chain.

Figure 3 shows the nucleotide sequence of the hM23-H1 heavy chain (SEQ ID NO: 11) and the amino acid sequence (SEQ ID NO: 12).

Figure 4 shows the nucleotide sequence of the hM23-H2 heavy chain (SEQ ID NO: 13) and the amino acid sequence (SEQ ID NO: 14).

Figure 5 shows the nucleotide sequence (SEQ ID NO: 15) and amino acid sequence (SEQ ID NO: 16) of the hM23-L1 type light chain.

Figure 6 shows the nucleotide sequence (SEQ ID NO: 17) and amino acid sequence (SEQ ID NO: 18) of the hM23-L2 type light chain.

Figure 7 shows the amino acid sequence of CDRH1 of M23 antibody (SEQ ID NO: 19), amino acid sequence of CDRH2 (SEQ ID NO: 20), amino acid sequence of CDRH3 (SEQ ID NO: 21), amine of CDRL1 The acid sequence (SEQ ID NO: 22), the amino acid sequence of CDRL2 (SEQ ID NO: 23) and the amino acid sequence of CDRL3 (SEQ ID NO: 24).

Figure 8 shows the cell internalization ability of the M23 antibody.

Figure 9 is a graph showing the anti-tumor effect of hM23-H1L1-drug conjugates on human Burkitt's lymphoma transplanted mice.

Figure 10 is a graph showing the anti-tumor effect of hM23-H1L2-drug conjugates on human Burkitt's lymphoma transplanted mice.

Figure 11 is a graph showing the anti-tumor effect of hM23-H2L2-drug conjugates on human Burkitt's lymphoma transplanted mice.

Figure 12 is a graph showing the anti-tumor effect of hM23-H1L1-drug conjugates on human Burkitt's lymphoma transplanted mice.

Fig. 13 is a view showing the results of analyzing the epitope of the M23 antibody by expressing cells expressing a fusion protein of human CD98 and FLAG in which a part of the amino acid sequence was substituted into a mouse sequence.

Fig. 14 is a graph showing the results of expression of the fusion protein on the cell membrane of a cell expressed by a fusion protein of human CD98 and FLAG which was substituted with a part of the amino acid sequence into a mouse sequence.

[Formation for implementing the invention]

In this specification, "cancer" and "tumor" are used in the same sense.

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

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

As used herein, "polypeptide" and "protein" are used in the same sense.

In the present specification, "cell" includes cells and cultured cells in an individual animal.

In the present specification, "CD98" is used in the same sense as the CD98 protein. Since CD98 contains a heavy chain and a light chain, the "CD98 heavy chain" and the "CD98 light chain" are each used in the same sense as the CD98 heavy chain protein and the CD98 light chain protein. In addition, in this specification, "CD98" can be interchanged with any of "CD98 heavy chain" and "CD98 light chain", or "CD98 heavy chain" or "CD98 light chain" unless otherwise specified. use.

As used in this specification, "anti-CD98 antibody" means CD98 Heavy chain bound antibodies.

As used in this specification, "cell injury" means in any form The pathological changes to cells are not limited to direct trauma, but also refer to all cells such as DNA cleavage, formation of base dimers, cleavage of chromosomes, damage of cell division devices, and reduction of various enzyme activities. Damage to construction or function.

As used herein, "cell injury activity" means causing Said the cell injury.

In this specification, "antibody-dependent cellular injury activity" It means "antibody dependent cellular cytotoxicity (ADCC) activity", and NK cells damage the action activity of target cells such as tumor cells by antibodies.

In this specification, "complement dependent cell injury activity "Sex" means "complement dependent cytotoxicity (CDC) activity", and complement acts to damage the activity of a target cell such as a tumor cell by an antibody.

In this specification, "antigen-binding fragments of antibodies" are also known The "functional fragment of an antibody" means a partial fragment of an antibody having an activity of binding to an antigen, and comprises Fab, F(ab')2, scFv and the like. Further, Fab' of a fragment of one of the variable regions of the antibody treated with F(ab')2 under reducing conditions is also contained in the antigen-binding fragment of the antibody. However, as long as it has the ability to bind to an antigen, it is not limited to such a molecule. Further, such antigen-binding fragments include not only a full-length molecule of the antibody protein, but also a protein produced by a suitable host cell using a genetically engineered antibody gene.

In this specification, "Fab'" is as described above, and F(ab')2 A fragment of one of the variable regions of the antibody treated under reducing conditions. However, Fab' produced using a genetically engineered antibody gene is also included in the Fab' of the present invention.

As used herein, "antigenic epitope" means a partial peptide or partial steric structure of CD98 to which a particular anti-CD98 antibody binds. The epitope of a part of the peptide of CD98 can be determined by a method well known to those skilled in the art, such as immunoassay, for example, the following method. First, various partial structures of the antigen are produced. For the preparation of partial structures, well-known oligopeptide synthesis techniques can be used. For example, a series of polypeptides which are sequentially shortened from the C-terminus or the N-terminus of CD98, and which are sequentially shortened by an appropriate length, are prepared by using a genetic recombination technique well known to those skilled in the art to review the reactivity of antibodies against them. After determining the approximate identification site, the short peptide is synthesized and the epitope is determined by reviewing the reactivity with the peptide. Further, the epitope of the partial steric structure of the binding antigen of the specific antibody can be determined by X-ray structure, and can be determined by the amino acid residue of the antigen adjacent to the antibody.

As used herein, "an antibody that binds to the same epitope" means a heterologous antibody that binds to a common epitope. If a partial peptide or a partial steric structure of the binding of the first antibody has a second antibody binding, it can be determined that the first antibody and the second antibody system bind to the same epitope. Further, by confirming that the second antibody competes for binding of the first antibody to the antigen (ie, the second antibody prevents the binding of the first antibody to the antigen), even if the sequence or structure of the specific epitope is not determined, It is determined that the first antibody and the second antibody system bind to the same epitope. Further, 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 can be expected to have the same activity. Accordingly, if a partial peptide of the binding of the first anti-CD98 antibody binds to the second anti-CD98 antibody, it can be determined that the first antibody binds to the same epitope of CD98 as the second antibody. Further, by confirming binding to CD98 with respect to the first anti-CD98 antibody, the second anti-CD98 antibody competes, and it is possible to determine that the first antibody and the second anti-system bind to the same epitope of CD98.

For the purposes of this specification, "CDR" means the complementarity determining region (CDR: Complemetarity detemining region). It is known that the heavy and light chains of an antibody molecule each have three CDRs. The CDR is also called a hypervariable domain. In the variable region of the heavy and light chains of the antibody, the variability of the primary structure is an extremely high site, and the polypeptide chain is one of the heavy chain and the light chain. They are separated from 3 locations. In the present specification, regarding the CDRs of the antibody, the CDRs of the heavy chain are labeled as CDRH1, CDRH2, CDRH3 from the N-terminal side of the heavy chain amino acid sequence, and the CDRs of the light chain are labeled from the N-terminal side of the light chain amino acid sequence as CDRL1, CDRL2, CDRL3. These sites are close to each other in steric structure and determine the specificity for the bound antigen.

In this manual, "a few" means 2~10, 2~9, 2~8, 2~7, 2~6, 2~5, 2~4, 2~3 Or two, preferably two.

In the present specification, the "average drug binding number" is also referred to as Drug-to-Antibody Ratio (DAR), and means the average number of drugs bound to the antibody 1 molecule. In this specification, "identity" and "homology" are used in the same meaning.

(CD98)

CD98 is a heterodimer composed of a multi-pass light chain of approximately 40 kDa and a double-sulfur-bonded type II single-pass transmembrane heavy chain of approximately 80-85 kDa (non-patented) Document 15). The CD98 heavy chain (known as CD98hc, 4F2, or FRP-1) is encoded in the mouse by the Slc3a2 gene and in humans by the SLC3A2 gene. CD98hc is a type II transmembrane protein with extracellular regions, transmembrane regions, and cytoplasmic tails. The CD98 system has at least one of the six CD98 light chains (LAT-1, LAT-2, etc.) and the CD98hc extracellular region to form a heterodimer by a disulfide bond. The CD98 heavy chain is involved in the message of integrin, and the CD98 light chain is involved in the transport of amino acids.

The nucleotide sequence of the CD98 heavy chain and the amino acid sequence are disclosed in a public database. For example, in the case of a nucleotide sequence, reference may be made to NM_001012662, NM_001013251 (GenBank), and in the case of an amino acid sequence, Refer to NP_001012680, NP_001013269 (GenBank), and the like. It has also been disclosed in the present specification that the nucleotide sequence NM_001012662 is the sequence identifier 37 and the amino acid sequence NP_001012680 is the sequence identifier number 38.

The CD98 system can be directly purified from CD98 expression cells of human, non-human mammals (rats, mice, etc.), or can be used to prepare a cell membrane fraction of the cells, and CD98 can be synthesized in vitro. Or can be obtained by genetic manipulation to produce host cells. For genetic manipulation, specifically, to incorporate CD98 cDNA The vector of expression can be obtained by synthesizing in a solution containing enzymes, matrices and energy substances necessary for transcription and translation, or by transforming host cells of other prokaryotes or eukaryotes by expressing CD98. The protein. Further, CD98 expression cells by the above-described gene manipulation or cell strains expressing CD98 may be used as CD98.

Further, in the amino acid sequence of the above CD98 heavy chain, 1 or A protein in which a plurality of amino acids are substituted, deleted and/or added with an amino acid sequence and which has the same biological activity as the protein is also contained in the CD98 heavy chain.

(anti-CD98 antibody)

The anti-CD98 antibody used in the present invention is not particularly limited as long as it binds to the CD98 heavy chain. The anti-system has the property of recognizing the characteristics of the tumor cells expressing CD98, the characteristics of binding to the tumor cells, and the intrinsic properties incorporated into the tumor cells.

The anti-CD98 anti-system used in the present invention can be purified by immunizing an animal with any polypeptide selected from the amino acid sequence of the CD98 heavy chain or the CD98 heavy chain by using a method generally practiced in the art, and purifying the antibody produced in vivo. And get. The biological species of CD98 which is an antigen is not limited to humans, and CD98 of animals other than humans such as mice and rats can also be immunized against animals. In this case, by testing the cross-linking of the antibody which binds to the obtained heterologous CD98 to human CD98, an antibody suitable for human diseases can be selected.

Further, according to well-known methods (for example, Kohler and Milstein, Nature (1975) 256, p. 495-497; Kennet, R. ed., Monoclonal Antibodies, p. 365-367, Plenum Press, N.Y. (1980)), a monoclonal antibody can also be obtained by fusion of antibody-producing cells producing anti-CD98 antibodies to myeloma cells to establish a fusion tumor.

Further, the CD98 heavy chain system which is an antigen can be obtained by causing a gene of the CD98 heavy chain to be produced in a host cell by genetic manipulation.

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

(1) Modulation of antigen

The antigen for producing an anti-CD98 antibody may, for example, be a polypeptide consisting of a CD98 heavy chain or at least 6 consecutive partial amino acid sequences thereof, or an arbitrary amino acid sequence or a derivative of an additional carrier, Cells that express the heavy chain of CD98, etc.

The CD98 system can be directly purified from human tumor tissues or tumor cells, and can be obtained by synthesizing CD98 in vitro or by genetic manipulation in a host cell.

For genetic manipulation, in particular, the cDNA of CD98 is incorporated into a renderable vector, synthesized in a solution containing the enzymes, matrix and energy substances necessary for transcription and translation, or by making other prokaryotes, or The host cell of the nuclear organism is transformed, and by expressing CD98, an antigen can be obtained.

Further, it is also possible to obtain an antigen secreting protein by expressing a fusion protein in which the extracellular region of CD98 which is a membrane protein is linked to the constant region of the antibody in a suitable host or vector system.

The cDNA of CD98 can, for example, behave as CD98 The cDNA library of cDNA serves as a template for polymerase chain reaction (hereinafter referred to as "PCR") by using a primer that specifically amplifies the cDNA of CD98 (refer to Saiki, RK, et al., Science (1988) 239, p. Obtained by the so-called PCR method of 487-489).

For the in vitro synthesis of the polypeptide, for example, Roche Diagnostics' Rapid Translation System (RTS), but is not limited to this.

For the host of prokaryotic cells, for example, the large intestine can be cited Escherichia coli or Bacillus subtilis. The host gene is transformed into a host cell such as the target cell, and the host cell is transformed into a plastid vector containing a replicator (i.e., an origin of replication, and a regulatory sequence) which is suitable as a host species. Further, in the case of a vector, it is preferred that the transforming cell has a sequence which can confer selectivity to a phenotype (phenotype).

Host cells of eukaryotic cells contain vertebrates, insects, For cells such as yeast, for vertebrate cells, for example, COS cells of monkey cells are often used (Gluzman, Y. Cell (1981) 23, p. 175-182, ATCC CRL-1650; ATCC: American Type Culture Collection ), mouse fibroblast NIH3T3 (ATCC No. CRL-1658) or Chinese hamster ovary cells (CHO cells, ATCC CCL-61) dihydrofolate reductase deficiency strain (Urlaub, G. and Chasin, LA Proc.Natl .Acad. Sci. USA (1980) 77, p. 4126-4220), etc., but is not limited thereto.

The morph obtained as described above can be generally implemented in accordance with this field. The method is cultured, and the polypeptide of interest is produced intracellularly or extracellularly by the culture.

For the medium used for the culture, it can be used accordingly. The host cell is appropriately selected from various conventional media, and if it is Escherichia coli, for example, an antibiotic such as ampicillin or IPMG may be added to the LB medium as necessary.

By the above culture, the intracellular or extracellular body of the transformed body The produced recombinant protein can be isolated and purified by various well-known separation methods using physical properties or chemical properties of the protein.

In terms of the method, specifically, for example, A typical liquid precipitant treatment, ultrafiltration, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, affinity chromatography, and the like, various liquid chromatography, dialysis methods, combinations thereof, and the like.

Also, by linking 6 residues to the expressed recombinant protein The histidine acid tag formed by the base can be efficiently purified by the nickel affinity column. Alternatively, the protein A column can be efficiently purified by linking the Fc region of the IgG to the expressed recombinant protein.

By combining the above methods, the polypeptide can be easily produced in large quantities for the purpose of high yield and high purity.

(2) Manufacture of anti-CD98 monoclonal antibody

Examples of the antibody that specifically binds to CD98 include a monoclonal antibody that specifically binds to CD98, but the method for obtaining the same is as follows.

In the manufacture of monoclonal antibodies, in general, the following procedures are necessary. That is, (a) a step of modulating an antigen, (b) a step of vaccinating the antigen by injecting the antigen into the animal, examining the antibody valence by taking blood, determining a period of spleen sputum extraction, and modulating the antibody-producing cell, (c) a step of modulating myeloma, (d) a step of fusing antibody-producing cells with myeloma cells, (e) selecting a step of producing a fusion tumor population of an antibody of interest, and (f) dividing into a single cell-selecting strain (selection) Step, (g) a step of culturing a fusion tumor to produce a monoclonal antibody, or a step of producing a monoclonal antibody by culturing a fusion tumor, and (h) reviewing the physiological activity of the thus produced monoclonal antibody, and Binding specificity, or checking as a step of identifying the characteristics of the reagent.

Hereinafter, the method for producing the monoclonal antibody will be described in detail according to the above procedure. However, the method for producing the antibody is not limited thereto, and for example, antibody-producing cells other than spleen cells and myeloma may be used.

(a) Steps of modulating the antigen

In the case of the antigen, CD98 or a part thereof prepared by the above method, a membrane fraction prepared by expressing a recombinant cell by CD98, a CD98 expressing recombinant cell, a CD98 expressing cell strain, and the technical field to which the present invention pertains can be used. A part of the peptide of CD98 chemically synthesized by a method known to those skilled in the art.

(b) Steps of modulating antibody-producing cells

The antigen obtained in the step (a) is mixed with a Freund's complete or incomplete adjuvant, or an adjuvant such as potash alum, and the experimental animal is immunized as an immunogen. Or the CD98 expressing cells are immunized to the experimental animals as immunogens. The experimental animal system can use the animal used in the well-known fusion tumor preparation method unimpeded. Specifically, for example, mice, rats, goats, sheep, cows, horses, and the like can be used. However, it is preferable to use a mouse or a rat as an animal to be immunized from the viewpoint of easiness of obtaining myeloma cells in which the cells are fused with the extracted antibody.

Further, the strains of mice and rats actually used are not particularly limited, 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., in the case of rats, for example, Wistar, Low, Lewis, Sprague, Dawley, ACI can be used. , BN, Fischer, etc.

Such mouse and rat lines can be obtained, for example, from experimental animal breeding and sales companies such as CLEA Corporation of Japan, Charles River Corporation of Japan, and the like.

Among them, in the case of immunized animals, the mouse BALB/c strain is preferred, and the rat Wistar and Low strains are preferred in consideration of fusion affinity with the myeloma cells described later.

Further, in consideration of the homology between the human and the mouse of the antigen, it is preferable to use a mouse which has reduced the biological mechanism by removing the autologous antibody, that is, using an autoimmune disease mouse.

Further, the age of the mice in the immunization of these mice or rats is preferably 3 to 12 weeks old, more preferably 4 to 6 weeks old.

For the method of immunizing animals, for example, Weir, DM, Handbook of Experimental Immunology Vol. I. II. III., Blackwell Scientific Publications, Oxford (1987), Kabat, EA and Mayer, MM, Experimental Immunochemistry, A well-known method described by Charles C Thomas Publisher Springfield, Illinois (1964).

In such an immunoassay, a method of administering an antigen to the skin of an animal is preferred. In order to improve the immune efficiency, the first half is administered intradermally, and only the second half or the final intravenous administration is more preferable.

The timing of administration of the antigen varies depending on the type of the immunized animal, individual differences, etc., but in general, the number of antigen administrations is 3 to 6 times, and the interval of administration is preferably 1 to 3 weeks, and the number of administrations is 4~ It is better to have 5 times and the interval of administration is 1 to 2 weeks.

Further, the dose of the antigen varies depending on the type of the animal, the individual difference, and the like, but is generally 0.05 to 5 mg, preferably about 0.1 to 0.5 mg.

The immunization system is administered after 1 to 6 weeks, preferably after 1 to 4 weeks, more preferably 1 to 3 weeks. In the case where the immunogen is a cell, 1 × 10 ⁶ to 1 × 10 ⁷ cells are used.

In addition, the amount of the antigen to be administered in the case of the additional immunization varies depending on the type and size of the animal, but in general, for example, the case of the mouse is 0.05 to 5 mg, preferably 0.1 to 0.5 mg, more preferably 0.1~0.2mg or so. In the case where the immunogen is a cell, 1 × 10 ⁶ to 1 × 10 ⁷ cells are used.

1 to 10 days after the above immunization, preferably 2 to 5 days More preferably, after 2 to 3 days, the spleen cells or lymphocytes containing the antibody-producing cells are aseptically taken out from the immunized animal. At this time, the antibody valence is measured, and if an animal having a sufficiently high antibody valence is used as a supply source of the antibody-producing cells, the efficiency of subsequent operations can be improved.

The method for measuring the antibody valence used herein may, for example, be an RIA method or an ELISA method, but is not limited thereto. The case of the ELISA method can be carried out in the order as described below.

First, the purified or partially purified antigen is adsorbed on the solid phase surface of a 96-well plate or the like for ELISA, and the unadsorbed solid phase surface of the antigen is covered with a protein not associated with the antigen, for example, by bovine serum albumin (in This specification, sometimes referred to as BSA), covers the surface, and then contacts the serially diluted sample (for example, mouse serum) as a first antibody to bind the antigen to the antibody in the sample.

Further, as a second antibody, an antibody against an antibody recognized by an enzyme is added to a mouse antibody, and after washing, a substrate of the enzyme is added, and a change in absorbance due to matrix decomposition is measured, thereby calculating Antibody price.

The isolation of cells produced by antibodies to spleen cells or lymphocytes of immunized animals can be carried out according to well-known methods (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, the spleen can be finely cut and the cells are filtered through a stainless steel mesh to be freed from the Eagle's minimum essential medium. (Eagle's minimal essential medium, MEM) is a general method for separating antibody-producing cells.

(c) Steps to modulate myeloma

The myeloma cells used for cell fusion are not particularly limited, and a well-known cell strain can be appropriately selected and used. However, in consideration of the convenience of selecting a fusion tumor from a fusion cell, it is preferable to use a HGPRT (hypoxanthine-guanine phosphoribosyl transferase) defective strain whose 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/) from mice. 0), MPC11-45.6TG1.7 (45.6TG), FO, S149/5XXO, BU.1, etc.; 210.RSY3.Ag.1.2.3 (Y3) from rats; U266AR from humans (SKO- 007), GM1500. GTG-A12 (GM1500), UC729-6, LICR-LOW-HMy2 (HMy2), 8226AR/NIP4-1 (NP41), and the like. Such HGPRT-deficient strains are, for example, available from ATCC and the like.

Such cell lines are in a suitable medium, such as 8-azaguanine medium (bromoic acid, 2-mercaptoethanol, gentamycin, and fetal bovine serum are added to RPMI-1640 medium). Instructions, sometimes referred to as "FBS"), medium containing 8-azaguanine, Iscove's Modified Dulbecco's Medium (IMDM), or Dulbecco's Subculture was performed in Dulbecco's Modified Eagle Medium (DMEM), but subculture was performed in a normal medium (for example, ASF104 medium (manufactured by Ajinomoto Co., Ltd.) containing 10% FBS) 3 to 4 days before cell fusion. On the day of integration, ensure the number of cells above 2 × 10 ⁷ .

(d) Steps for cell fusion

Fusion of antibody-producing cells to myeloma cells is based on well-known methods (Weir, DM, Handbook of Experimental Immunology Vol. I. II. III., Blackwell Scientific Publications, Oxford (1987); Kabat, EA and Mayer, MM, Experimental Immunochemistry , Charles C Thomas Publisher Springfield, Illinois (1964), etc., can be suitably carried out under the condition that the cell survival rate is not extremely lowered.

In such a method, for example, a chemical method in which antibody-producing cells are mixed with myeloma cells, a physical method using electrical stimulation, and the like can be used in a high-concentration polymer solution such as polyethylene glycol. Here, specific examples of the above chemical methods are as follows.

That is, in the case of using polyethylene glycol as a high-concentration polymer solution, in a polyethylene glycol solution having a molecular weight of 1,500 to 6,000, preferably 2,000 to 4,000, at 30 to 40 ° C, preferably at 35 to 38 ° C. At the temperature, the antibody-producing cells are mixed with the myeloma cells for 1 to 10 minutes, preferably 5 to 8 minutes.

(e) Steps for selecting a fusion tumor group

The selection method of the fusion tumor obtained by the above cell fusion is not particularly limited, but a HAT (hypoxanthine. Aminoguanidine thymidine) selection method is usually used (Kohler et al., Nature (1975) 256, p. 495; Milstein et al., Nature (1977) 266, p. 550).

This method is not effective for use in amine hydrazine It is effective to obtain a fusion tumor by the myeloma cells of the HGPRT-deficient strain.

That is, by using unfused cells and fusion tumors as HAT Nutrient culture can selectively retain only the fusion tumor that is resistant to aminoguanidine and can proliferate.

(f) Steps of dividing into single cell colonies (selection)

For the method of colonization of fusion tumors, for example, a well-known method such as methylcellulose method, soft agarose method, critical dilution method, or the like can be used (for example, refer to Barbara, BMand Stanley, MS: Selected Methods in Cellular Immunology, WH). Freeman and Company, San Francisco (1980)). Among these methods, a three-dimensional culture method such as a methyl cellulose method or the like is preferred. For example, a fusion tumor group formed by cell fusion is suspended in a methylcellulose medium such as ClonaCell-HY Selection Medium D (#03804, manufactured by StemCell Technologies), and the resulting fusion tumor colony is recovered by recycling. The acquisition of a single fusion tumor is possible. Each of the recovered fusion tumor selection strains is cultured, and the recombinant antibody culture supernatant in the obtained fusion tumor culture supernatant is selected to be a CD98 monoclonal antibody to produce a fusion tumor strain.

As an example of the fusion tumor strain thus established, CD98 fusion tumor M23 can be cited. Further, in the present specification, the antibody produced by the CD98 fusion tumor M23 is described as "M23 antibody" or only "M23".

The heavy chain variable region of the M23 antibody has the amino acid sequence shown in SEQ ID NO: 2 of the Sequence Listing. Further, the light chain variable region of the M23 antibody has an amino acid sequence represented by SEQ ID NO: 4 in the Sequence Listing.

(g) Steps for producing monoclonal antibodies

The fusion tumor thus selected can be efficiently obtained by culturing the monoclonal antibody, but before the culture, the fusion tumor system producing the monoclonal antibody of interest is selected.

This screening can be carried out by a method known per se.

The measurement of the antibody valence can be carried out according to, for example, the ELISA method described in the item (b) above.

The fusion tumor obtained by the above method can be stored in a frozen state in liquid nitrogen or in a freezer below -80 °C.

The completed tumor line was cultured by changing the medium from HAT medium to normal medium.

A large number of cultures are carried out by spin culture using a large culture flask or spinner culture. The supernatant in the large-scale culture from this can be purified by a method known to a person skilled in the art using Protein A column purification, etc., to obtain a monoclonal antibody which specifically binds to the protein of the present invention. .

Further, by injecting a fusion tumor into a mouse of the same strain (for example, BALB/c described above) or the peritoneal cavity of a Nu/Nu mouse, the fusion tumor is proliferated, and a large amount of the monoclonal antibody of the present invention can be obtained. ascites.

In the case of intraperitoneal administration, 2,6,10,14-tetramethylpentadecane (2,6,10,14-tetramethylpentadecane; pristane) was administered beforehand (before 3 to 7 days). When the mineral oil is equal to, a larger amount of ascites can be obtained.

For example, in the hybridoma intraperitoneally with strains of mice previously immunized inhibitors, does not activate the T cells, in 20 days the 10 ⁶ to 10 ⁷ of hybridoma. The selected cells were administered to the peritoneal cavity freely (0.5 ml) in serum-free medium, usually in the abdomen, and the ascites was accumulated, and ascites was taken from the mice. By this method, a monoclonal antibody having a concentration of about 100 times or more is obtained as compared with the culture medium.

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

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

(h) Verification of monoclonal antibodies

The determination of the isotype and subtype of the monoclonal antibodies thus obtained can be carried out as follows.

First, an Ouchterlony method, an ELISA method, or an RIA method can be cited as an identification method.

The Euclidean two-way diffusion method is simple, but in the case of a low concentration of a single antibody, concentration operation is necessary.

On the other hand, in the case of using the ELISA method or the RIA method, the culture supernatant is directly reacted with the antigen-adsorbing solid phase, and further, by using antibodies corresponding to various immunoglobulin isotypes and subtypes as the second antibody, the single antibody can be identified. The isotype and subtype of the strain antibody.

Moreover, as a simpler method, a commercially available kit for identification (for example, Mouse typer kit: manufactured by Bio-Rad Co., Ltd.) or the like can be used.

Furthermore, the protein may be quantified based Folin Lowry method and the absorbance at 280nm (1.4 (OD ₂₈₀₎ = Immunoglobulin 1mg / ml) determined by the method is carried out.

Furthermore, the steps (a) to (h) of (2) are performed again and In the case of obtaining a monoclonal antibody independently, it is also possible to obtain an antibody having the same cytotoxic activity as the M23 antibody. An example of such an antibody is an antibody that binds to the same epitope as the M23 antibody. When a newly produced monoclonal antibody binds to a partial peptide or a partial steric structure of a binding to the M23 antibody, it can be determined that the monoclonal antibody binds to the same epitope as the M23 antibody. Furthermore, by confirming binding to CD98 relative to the M23 antibody, the monoclonal antibody competes with the system (i.e., the monoclonal antibody system prevents the binding of the M23 antibody to CD98), even if the sequence or structure of the specific epitope is not determined. Alternatively, the monoclonal antibody can be determined to bind to the same epitope as the CD98 antibody. The epitope is confirmed to be the same, and the monoclonal antibody is strongly expected to have antigen binding ability or biological activity equivalent to that of the M23 antibody.

(3) Other antibodies

The anti-system for use in the present invention includes, in addition to the above-mentioned monoclonal antibody against CD98, a recombinant antibody which artificially changes for the purpose of reducing the heterologous antigenicity to humans, for example, a chimeric antibody. Humanized antibodies, human antibodies, and the like. These antibodies can be produced using known methods.

In the case of a chimeric antibody, an antibody in which the variable region and the constant region of the antibody are heterologous to each other, for example, a chimeric antibody in which a variable region of an antibody derived from mouse or rat is ligated to a constant region derived from human is exemplified. (See Proc. Natl. Acad. Sci. USA, 81, 6851-6855 (1984)).

Chimeric antibody from mouse anti-human CD98 antibody M23 An antibody comprising a heavy chain comprising a heavy chain variable region consisting of 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, There may be any constant region from humans.

As an example of such a chimeric antibody, an inclusion can be cited. The heavy chain of the amino acid sequence consisting of amino acid residues 20 to 465 of SEQ ID NO: 8 and the amino group consisting of amino acid residues 21 to 240 of SEQ ID NO: 10 An antibody to the light chain of an acid sequence. Further, in the heavy chain sequence represented by SEQ ID NO: 8, the amino acid sequence consisting of amino acid residues 1 to 19 is a message sequence, and amino acid residues 20 to 135 are used. The amino acid sequence formed is a variable region, and the amino acid sequence consisting of the amino acid residues 136 to 465 is a constant region. Further, in the light chain sequence represented by SEQ ID NO: 10 in the sequence listing, the amino acid sequence consisting of amino acid residues 1 to 20 is a message sequence, and the amino group of Nos. 21 to 135 is used. The amino acid sequence composed of the acid residue is a variable region, and the amino acid sequence composed of the amino acid residues 136 to 240 is a constant region.

Sequence of heavy chain amino acid sequence represented by SEQ ID NO: 8 The nucleotide sequence shown in column identification number 7 is encoded. The nucleotide sequence consisting of nucleotides 1 to 57 of the nucleotide sequence represented by SEQ ID NO: 7 is a heavy chain message sequence encoding an antibody, and is composed of nucleotides 58 to 405. The nucleotide sequence encodes a heavy chain variable region of an antibody, and the nucleotide sequence consisting of nucleotides Nos. 406 to 1395 encodes a heavy chain constant region of an antibody.

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

The sequence of sequence identification numbers 7 and 8 is described in the first The sequence of sequence identification numbers 9 and 10 is shown in Fig. 2.

In the case of humanized antibodies, it can be cited that only the CDRs are incorporated. An antibody against a human antibody (refer to Nature (1986) 321, p. 522-525), in addition to the CDR sequence by CDR grafting, a part of the framework of the amino acid residue is also grafted to the antibody of the human antibody ( International Open Booklet WO90/07861).

For humanized antibodies from the M23 antibody (in this case) In the case of the term "humanized M23 antibody", "humanized M23", "hM23 antibody" or "hM23", as long as the six CDR sequences of the M23 antibody are retained, it is not limited to a specific human. Antibody. Further, the heavy chain variable region of the M23 antibody retains CDRH1 (NYLIE) composed of the amino acid sequence represented by SEQ ID NO: 19 and CDRH2 (VINPGSGVTNYNEKFKG) composed of the amino acid sequence represented by SEQ ID NO: 20. And CDRH3 (AEAWFAY) composed of the amino acid sequence shown in SEQ ID NO: 21. Further, the light chain variable region of the M23 antibody retains the amino acid sequence represented by SEQ ID NO: 22 in the sequence listing. CDRL1 (QRSGYPWT) composed of CDRL1 (KSSQSLLYSSNQKNYLA), amino acid sequence represented by SEQ ID NO: 23, and CDRL2 (QRYYGYPWT) composed of amino acid sequence represented by SEQ ID NO: 24.

As an example of a humanized antibody of mouse antibody M23, any combination of the following heavy and light chains may be mentioned: a heavy chain selected from a heavy chain variable region comprising the following group: (1) SEQ ID NO: 12 Or an amino acid sequence composed of an amino acid residue of No. 20 to No. 135 of (14), and an amino acid sequence having at least 95% or more identity with respect to the amino acid sequence of the above (1) And (3) an amino acid sequence in which one or more amino acids of the amino acid sequence of the above (1) are deleted, substituted or added; and a light chain variable region selected from the group consisting of the following Light chain: (4) an amino acid sequence consisting of amino acid residues 21 to 135 of SEQ ID NO: 16 or 18, and (5) having at least an amino acid sequence of the above (4) More than 95% of the amino acid sequence of the same identity, and (6) an amino acid sequence in which one or more of the amino acids of the above (4) amino acid sequence are deleted, substituted or added.

Further, in the case of the substitution of an amino acid, a preserved amino acid substitution is preferred. The preserved amino acid substitution refers to the substitution produced within the amino acid group associated with the amino acid side chain. Preferred amino acid groups are as follows: Acidic group = aspartic acid and glutamic acid; basic group = lysine, arginine and histidine; non-polar group = alanine, valine, leucine, isoleucine , proline, phenylalanine, methionine and tryptophan; and non-electrode family = glycine, aspartame, glutamic acid, cysteine, serine, threonine And tyrosine.

Other suitable amino acid groups are as follows: aliphatic hydroxy group = serine and threonine; guanamine containing group = aspartame and glutamic acid; aliphatic group = alanine, Proline, leucine and isoleucine; and aromatic groups = phenylalanine, tryptophan and tyrosine.

The amino acid substitution is preferably carried out in a range in which the properties of the substance having the original amino acid sequence are not lowered.

The antibody of a preferred combination of the above-mentioned heavy chain and light chain may be exemplified by an antibody comprising an amino acid sequence consisting of amino acid residues 20 to 135 having SEQ ID NO: 12. An antibody having a light chain of a light chain variable region comprising a heavy chain variable region heavy chain and an amino acid sequence consisting of amino acid residues 21 to 135 of SEQ ID NO: 16 a heavy chain variable region heavy chain composed of an amino acid sequence consisting of amino acid residues 20 to 135 of SEQ ID NO: 12 and amino acid Nos. 21 to 135 having SEQ ID NO: 18. An antibody to a light chain of a light chain variable region composed of an amino acid sequence composed of a residue, a heavy chain comprising 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 an amine having SEQ ID NO: 16 of Nos. 21 to 135 An antibody to a light chain of a light chain variable region composed of an amino acid sequence composed of a base acid residue, and a heavy chain comprising 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 an amine having SEQ ID NO: 18 of Nos. 21 to 135 An antibody to a light chain of a light chain variable region composed of an amino acid sequence composed of a base acid residue.

For a better combination of antibodies, the following antibodies can be cited: a heavy chain comprising a heavy chain variable region consisting of an amino acid sequence consisting of amino acid residues 20 to 135 of SEQ ID NO: 12 and an amine having SEQ ID NO: 16 of Nos. 21 to 135 An antibody to a light chain of a light chain variable region composed of an amino acid sequence composed of a base acid residue, a heavy chain comprising a heavy chain variable region consisting of an amino acid sequence consisting of amino acid residues 20 to 135 of SEQ ID NO: 12 and an amine having SEQ ID NO: 18 of Nos. 21 to 135 An antibody to a light chain of a light chain variable region composed of an amino acid sequence composed of a base acid residue, and a heavy chain comprising 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 an amine having SEQ ID NO: 18 of Nos. 21 to 135 An antibody to a light chain of a light chain variable region composed of an amino acid sequence composed of a base acid residue.

Further, as for other suitable combinations of antibodies, the following antibodies: heavy chain and sequence identification number consisting of amino acid sequences consisting of amino acid residues 20 to 465 of SEQ ID NO: 12 can be mentioned. 16th An antibody to a light chain composed of an amino acid sequence composed of an amino acid residue of 21 to 240, An amine comprising an amino acid sequence consisting of amino acid residues 20 to 465 of SEQ ID NO: 12 and an amino acid residue of amino acid residues 21 to 240 of SEQ ID NO: 18. An antibody to a light chain composed of a base acid sequence, A heavy chain comprising an amino acid sequence consisting of amino acid residues 20 to 465 of SEQ ID NO: 14 and an amino acid residue of amino acid residues 21 to 240 of SEQ ID NO: 16 An antibody to a light chain composed of a base acid sequence, and A heavy chain comprising an amino acid sequence consisting of amino acid residues 20 to 465 of SEQ ID NO: 14 and an amino acid residue of amino acid residues 21 to 240 of SEQ ID NO: 18 An antibody to a light chain composed of a base acid sequence.

In the case of a more suitable combination of antibodies, the following antibodies may be mentioned: a heavy chain comprising the amino acid sequence consisting of amino acid residues 20 to 465 of SEQ ID NO: 12 and a sequence identifier of 16 An amino acid sequence consisting of an amino acid sequence consisting of amino acid residues of amino acids 21 to 240, and an amino acid sequence comprising amino acid residues 20 to 465 of SEQ ID NO: 12 The light chain of the heavy chain and the amino acid sequence consisting of amino acid residues 21 to 240 of SEQ ID NO: 18, and A heavy chain comprising an amino acid sequence consisting of amino acid residues 20 to 465 of SEQ ID NO: 14 and an amino acid residue of amino acid residues 21 to 240 of SEQ ID NO: 18 An antibody to a light chain composed of a base acid sequence.

By combining sequences having high identity with the above-described heavy chain amino acid sequence and light chain amino acid sequence, it is possible to select an anti-system having the same biological activity as each of the above antibodies. Such identity is generally 80% or more of the identity, preferably 90% or more of the identity, more preferably 95% or more of the identity, and most preferably 99% or more of the identity. Further, the amino acid sequence in which one or more amino acid residues of the amino acid sequence combined with the heavy chain or the light chain are substituted, deleted or added may also be selected to have the same biological activity as each of the above-mentioned antibodies. Active antibody.

The identity between the two types of amino acid sequences can be achieved by using 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) is determined by the default parameters of the system. The Blast algorithm can also be used by, for example, accessing the Internet at www.ncbi.nlm.nih.gov/blast.

Further, in the heavy chain amino acid sequence represented by SEQ ID NO: 12 or 14, the amino acid sequence message sequence consisting of the amino acid residues 1 to 19, the amino acid of the 20th to the 135th The amino acid sequence of the residue is a variable region, and the amino group consisting of amino acid residues 136 to 465 The acid sequence is a constant region. The heavy chain amino acid sequence shown in SEQ ID NO: 12 or 14 is each encoded by the nucleotide sequence shown in SEQ ID NO: 11 or 13. The nucleotide sequence consisting of nucleotides 1 to 57 of each nucleotide sequence is a heavy chain message sequence encoding an antibody, and the nucleotide sequence consisting of nucleotides 58 to 405 encodes an antibody. The heavy chain variable region, the nucleotide sequence consisting of nucleotides 406 to 1395, encodes a heavy chain constant region of an antibody. The sequence of sequence identification numbers 11 and 12 is shown in Fig. 3, and the sequence of sequence identification numbers 13 and 14 is shown in Fig. 4.

Also, the sequence identification number 16 or 18 of the sequence table is light In the chain amino acid sequence, the amino acid sequence message sequence consisting of the amino acid residues 1 to 20, and the amino acid sequence of the amino acid residues 21 to 135 are variable. In the region, the amino acid sequence 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 each encoded by the nucleotide sequence shown in SEQ ID NO: 15 or 17. The nucleotide sequence consisting of nucleotides 1 to 60 of each nucleotide sequence is a light chain message sequence encoding an antibody, and the nucleotide sequence consisting of nucleotides 61 to 405 encodes an antibody. The light chain variable region, the nucleotide sequence consisting of nucleotides 406-720, encodes the light chain constant region of the antibody. The sequence of sequence identification numbers 15 and 16 is shown in Fig. 5, and the sequence of sequence identification numbers 17 and 18 is shown in Fig. 6.

The identity between these nucleotide sequences and the nucleotide sequences of other antibodies can be determined by the Blast algorithm.

Further, examples of the antibody of the present invention include human antibodies which bind to the same epitope of the M23 antibody. anti-CD98 human resistance system Means a human antibody having only the gene sequence of an antibody derived from a human chromosome. The anti-CD98 human anti-system can produce a mouse by using a human antibody having a human chromosome fragment containing a gene of a heavy chain and a light chain of a human antibody (see 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. P.69-73 (Kitagawa, Y., Matsuda, T. and Iijima, S., eds.), Kluwer Academic Publishers, 1999.; Tomizuka, K. et al., Proc. Natl. Acad. Sci. USA ( Obtained in 2000) 97, p. 722-727, etc.).

Such human antibody-producing mice, in particular, can be The locus of the intrinsic immunoglobulin heavy chain and the light chain is disrupted, and is introduced into the locus of the human immunoglobulin heavy chain and the light chain by a yeast artificial chromosome (YAC) vector or the like. Recombinant animals, making the production of genetically knockout animals and genetically modified animals, and making these animals mated with each other.

Also, by genetic recombination techniques, each code is so The cDNA of the heavy chain and the light chain of the human antibody is preferably obtained by transforming the eukaryotic cell by the vector containing the cDNA, and culturing the transformed cell which produces the recombinant human monoclonal antibody, thereby obtaining the culture supernatant. This antibody.

Among them, as a host, for example, eukaryotic cells can be used, Preferred are mammalian cells such as CHO cells, lymphocytes or myeloma.

Also, it is known that it is obtained from the human antibody library. Method for displaying human antibodies by bacteria (cf. Wormstone, IM et 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.).

For example, a variable region of a human antibody can be used as a single strand The antibody (scFv) is expressed on the surface of the phage, and the phage display method of the antigen-binding phage is selected (Nature Biotechnology (2005), 23, (9), p. 1105-1116).

The basis of the phage selected by analysis of binding to the antigen Thus, the DNA sequence encoding the variable region of the human antibody that binds to the antigen can be determined.

If the DNA sequence of the scFv that binds to the antigen is clear A human antibody can be obtained by producing a expression vector having the sequence, introducing it into a suitable host, and performing the expression (WO92/01047, WO92/20791, WO93/06213, WO93/11236, WO93/19172, WO95/01438 WO 95/15388, Annu. Rev. Immunol (1994) 12, p. 433-455, Nature Biotechnology (2005) 23 (9), p. 1105-1116).

Newly produced human antibodies that bind to the M23 antibody When a partial peptide or a partial steric structure is combined, it can be determined that the human antibody binds to the same epitope as the M23 antibody. Furthermore, by confirming binding to CD98 relative to the M23 antibody, the human antibody is competitive (ie, the human antibody interferes with the binding of the M23 antibody to CD98), even if specific The sequence or structure of the epitope is not determined, and it is also determined that the human antibody binds to the same epitope as the M23 antibody. In the case where the epitope is confirmed to be the same, the human antibody is strongly expected to have the same biological activity as the M23 antibody.

Chimeric antibody obtained by the above method, humanized anti-antibody The human or human anti-system can evaluate the binding to an antigen by a known method or the like, and a suitable antibody can be selected.

In the case of comparing other indicators of the nature of the antibody In other words, the stability of the antibody can be cited. A differential scanning thermal analyzer (DSC) is a device that quickly and correctly measures the thermal denaturation midpoint (Tm) that is a good indicator of the relative structural stability of proteins. The Tm value is measured using DSC, and the difference in thermal stability can be compared by comparing the values. It is known that the preservation stability of antibodies is somewhat related to the thermal stability of antibodies (Lori Burton, et al., Pharmaceutical Development and Technology (2007) 12, p. 265-273), with thermal stability as an indicator. A suitable antibody can be selected. Other indicators for selecting antibodies include high yield in a suitable host cell and low agglutination in an aqueous solution. For example, the highest antibody that is not limited to yield exhibits the highest thermal stability, so it is necessary to comprehensively judge based on the above-mentioned indicators to select the antibody most suitable for human administration.

The antibodies used in the present invention also comprise modifications of the antibodies. The modified system means a chemical or biological modification of the antibody. The chemical modification includes a bond to a chemical moiety of an amino acid backbone, a N-bond or a chemical modification of an O-bonded carbohydrate chain, and the like. Biological modifications include post-translational modifications (eg, for N-bonding or O-bonding) The addition of a sugar chain, processing at the N-terminus or C-terminus, deamination, isomerization of aspartic acid, oxidation of methionine, by expression of a prokaryotic host cell, and at the N-terminus Additional methionine residues, etc. Further, in order to be able to detect the antibody or antigen used in the present invention or to be isolated, for example, an enzyme marker, a fluorescent marker, and an affinity marker are also included in the meaning of the modifier. Thus, the modified antibody used in the present invention is useful for improving the stability and blood retention of the original antibody, reducing the antigenicity, detecting or detaching the antibody or antigen.

Also, by modulating the sugar chain modification (glycoside) bound to the antibody Glycosylation, fucose, etc., can enhance antibody-dependent cellular damage activity. WO99/54342, WO00/61739, WO02/31140 and the like are known, but are not limited thereto, in terms of a technique for adjusting the sugar chain modification of an antibody. The antibodies used in the present invention also comprise the glycoconjugate-modified antibody.

The anti-CD98 anti-system used in the present invention can also be used once After isolation of the antibody gene, it is obtained by introducing it into an appropriate host. Specific examples of the antibody gene include a gene encoding a heavy chain sequence of the antibody described in the present specification and a gene encoding a light chain sequence. When the host cell is transformed, the heavy chain sequence gene and the light chain sequence gene line may be inserted into the same expression vector, or may be inserted into different expression vectors.

When eukaryotic cells are used as a host, they can be used. Biological cells, plant cells, eukaryotic microorganisms. Particularly, in terms of animal cells, mammalian cells, for example, COS cells of monkey cells (Gluzman, Y. Cell (1981) 23, p. 175-182, ATCC CRL-1650), mouse fiber can be cited. Virgin cell NIH3T3 (ATCC No. CRL-1658) or Chinese hamster ovary cells (CHO cells, ATCC CCL-61) dihydrofolate reductase-deficient strain (Urlaub, G. and Chasin, LA Proc. Natl. Acad. Sci .USA (1980) 77, p. 4126-4220).

In the case of using prokaryotic cells, for example, Escherichia coli or Bacillus subtilis can be cited.

The target antibody gene is introduced into these cells by transformation, and the antibody can be obtained by culturing the transformed cells in vitro. In the case of the culture, depending on the sequence of the antibody, the yield may be different from among the antibodies having the same binding activity, and the yield may be selected as an indicator for the pharmaceutical producer. Accordingly, the antibody used in the present invention also comprises an antibody obtained by the method for producing the antibody, which comprises the steps of culturing the above-described transformed host cell, and the culture obtained from the step. The step of taking an antibody of interest or an antigen-binding fragment of the antibody.

Further, it is known that the amino acid terminal of the heavy chain of the antibody produced by the mammalian cultured cells is deficient in the amino acid residue (Journal of Chromatography A, 705: 129-134 (1995)), and the same heavy chain carboxy terminal is known. The two amino acid residues of glycine and lysine are deleted, and the proline residue at the new carboxy terminus is amidated (Analytical Biochemistry, 360: 75-83 (2007)). However, deletions and modifications of such heavy chain sequences have no effect on the antigen binding ability of the antibody and the effector function (activation of complement or antibody-dependent cellular damage, etc.). Accordingly, the present invention also encompasses an antibody that receives the modification and an antigen-binding fragment of the antibody, which may be exemplified by 1 or 2 at the carboxy terminus of the heavy chain. The deletion of the amino acid is removed, and the deletion is obtained by guanidination (for example, a heavy chain which is amidated by a proline residue of a carboxy terminal moiety). However, the deletion of the carboxy terminus of the heavy chain of the antibody used in the present invention is not limited to the above species as long as the antigen binding ability and the effector function are retained. The two-strand heavy chain constituting the antibody used in the present invention may be any one selected from the group consisting of a full length and a heavy chain of the above-described deletion body, or may be combined in any two. The amount of each of the deletion bodies is affected by the type and culture conditions of the mammalian cultured cells which produce the antibody used in the present invention, but the main component of the antibody used in the present invention is exemplified by both of the two heavy chains. A case where the amino acid residue of one of the carboxyl termini is deleted.

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

Further, the anti-system used in the present invention may be an antigen-binding fragment of an antibody having an antigen-binding portion of an antibody or a modification 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 genetic engineering and appropriately culturing the cells. Among such antibody fragments, a fragment which retains all or a part of the function of the full-length molecule of the antibody may be referred to as an antigen-binding fragment of the antibody. For example, in the case of a fragment of an antibody, Fab, F(ab') 2, Fv, or Fv of a heavy chain and a light chain may be exemplified by a linker of a single-chain Fv (scFv) or a bispecific antibody (diabodies). ), linear antibodies, and multispecific antibodies formed by antibody fragments. Further, Fab' of one of the valency fragments of the variable region of the antibody treated with F(ab')2 under reducing conditions is also contained in a fragment of the antibody.

The anti-system used in the present invention may be an antibody-drug conjugate that binds an anti-neoplastic compound. The antitumor compound is not particularly limited as long as it has a substituent or a partial structure which can bind to the structure of the linker. Part or all of the antitumor compound-based linker is cleaved in the tumor cells and the anti-tumor compound is partially free to exhibit an anti-tumor effect. If the linker and the drug-binding moiety are cleaved, the anti-tumor compound is released in its original structure, and its original anti-tumor effect is exerted.

As the antitumor compound, for example, doxorubicin, daunorubicin, mitomycin C, bleomycin, cyclocytidine may be mentioned. ), vincristine, vinblastine, methotrexate, platinum anti-tumor agent (cisplatin or its derivatives), Taxol or its derivatives The camptothecin or a derivative thereof (the antitumor agent described in JP-A-6-87746) is preferably exenote.

The biological activity of the anti-CD98 antibody used in the present invention includes an antigen-binding activity, an activity of internalizing to a cell expressing the antigen by binding to an antigen, an activity of neutralizing an antigen activity, and an activity of enhancing antigen activity. Antibody-dependent cellular damage (ADCC) activity, complement-dependent cellular damage (CDC) activity, and antibody-dependent cellular mediator phagocytosis (ADCP), but the antibodies used in the present invention have the function of binding to the CD98 heavy chain. Activity, preferably internalized to the CD98 expressing cells by binding to the CD98 heavy chain. Furthermore, the anti-system used in the present invention may have ADCC activity, CDC activity and/or ADCP activity in addition to the cell internalization activity.

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

The activity system internalized to the cells can be confirmed by the following methods: (1) A test method for visualizing an antibody incorporated into a cell by a fluorescence microscope using a secondary antibody (fluorescent marker) bound to a therapeutic antibody (Cell) Death and Differentiation (2008) 15, 751-761), (2) A test method for measuring the amount of fluorescence incorporated into cells using a secondary antibody (fluorescent marker) that binds to a therapeutic antibody (Molecular Biology of the Cell Vol. 15, 5268-5282, December 2004), or (3) using an immunotoxin that binds to a therapeutic antibody, once incorporated into a cell, the toxin is released and the cell proliferation is inhibited by the Mab-ZAP assay (BioTechniques 28: 162-165, January 2000). In the case of an immunotoxin, a recombinant complex protein of a diphtheria toxin catalytic region and Protein G can also be used.

The obtained antibody can be purified to homogeneity. For the separation and purification of the antibody, a separation and purification method used for a usual protein can be used. For example, if properly selected, combined column chromatography, filter filtration, ultrafiltration, salting out, dialysis, preparation of polypropylene guanamine colloidal electrophoresis, isoelectric point electrophoresis, etc., the antibody can be isolated and purified (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 this.

Examples of the color layer analysis include affinity chromatography, ion exchange chromatography, hydrophobic chromatography, colloidal filtration chromatography, inverse phase chromatography, and adsorption chromatography.

Such chromatographic analysis can be carried out using liquid chromatography analysis of HPLC, FPLC, and the like.

For the column used for the affinity chromatography, a Protein A column and a Protein G column can be cited. For example, examples of the column using the Protein A column include Hyper D, POROS, and Sepharose F.F. (GE Healthcare).

Further, it is also possible to use a carrier for immobilizing an antigen and to purify the antibody by binding to an antigen.

The anti-CD98 anti-system 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) has internalization into CD98-expressing cells by binding to a CD98 heavy chain. (2) CD98 is an antibody or the antibody according to the above (1) of human CD98; (3) CDRH1 having the amino acid sequence of SEQ ID NO: 19, and an amine of SEQ ID NO: 20. The CDRH2 composed of the carboxylic acid sequence and the CDRH3 composed of the amino acid sequence described in SEQ ID NO: 21 are the CDRs in the heavy chain of the antibody, and the CDRL1 sequence consisting of the amino acid sequence described in SEQ ID NO: 22 The antibody according to the above (1) or (2), wherein the CDRL2 composed of the amino acid sequence of the identification number 23 and the amino acid sequence of the amino acid sequence of SEQ ID NO: 24 are used as the CDR in the light chain of the antibody (4) The constant region is an antibody according to any one of the above (1) to (3) from the human constant region; (5) The antibody according to any one of the above (1) to (4), wherein the antibody according to the above (5), the variable region of the heavy chain of the antibody is selected from the group consisting of the following Group of amino acid sequences

(a) the amino acid sequence described in amino acid numbers 20 to 135 in SEQ ID NO: 12, (b) the amino acid sequence described in amino acid numbers 20 to 135 in SEQ ID NO: 14, and (c) relative a sequence of (a) or (b), an amino acid sequence having at least 95% identity, and (d) an amino acid selected from the group consisting of (a) or (b) having one or more amino acids. a group of amino acid sequences consisting of deleted, substituted or added amino acid sequences; the variable region of the light chain of the antibody consisting of an amino acid sequence selected from the group consisting of

(e) the amino acid sequence described in amino acid numbers 21 to 135 in SEQ ID NO: 16 and (f) the amino acid sequence described in amino acid numbers 21 to 135 in SEQ ID NO: 18, and (g) relative a sequence of (e) or (f), an amino acid sequence having at least 95% identity, and (h) a sequence of (e) or (f) having 1 or more amino acids (A) The antibody according to the above (6), wherein the variable region of the heavy chain of the antibody and the variable region of the light chain of the antibody are selected from the group consisting of the following: The variable region of the heavy chain composed of the amino acid sequence described in amino acid numbers 20 to 135 of SEQ ID NO: 12 and the amino acid sequence of amino acid numbers 21 to 135 of SEQ ID NO: 16 The variable region of the light chain, the variable region of the heavy chain composed of the amino acid sequence of amino acid numbers 20 to 135 in SEQ ID NO: 12, and the amino acid number 21 of SEQ ID NO: 18. The combination of the variable regions of the light chain composed of the amino acid sequence described in ~135, and the variable region and sequence of the heavy chain composed of the amino acid sequence described in amino acid numbers 20 to 135 of SEQ ID NO: 14 The combination of the variable regions of the light chain composed of the amino acid sequence described in the amino acid numbers 21 to 135 of the identification number 16 and the amino acid sequence of the amino acid number 20 to 135 of the sequence identification number 14 a combination of a variable region of the heavy chain and a variable region of a light chain composed of an amino acid sequence described in amino acid numbers 21 to 135 in SEQ ID NO: 18; (8) as described in the above (6) The combination of the antibody, the variable region of the heavy chain of the antibody, and the variable region of the light chain of the antibody is the combination of the following: amino acid of SEQ ID NO: 12. The variable region of the heavy chain composed of the amino acid sequence of No. 20 to 135 and the combination of the variable regions of the light chain of the amino acid sequence of amino acid numbers 21 to 135 of SEQ ID NO: 16 The variable region of the heavy chain composed of the amino acid sequence described in the amino acid numbers 20 to 135 of the sequence identification number 12 and the amino acid sequence of the amino acid number 21 to 135 of the sequence identification number 18 a combination of variable regions of the constructed light chain, or The variable region of the heavy chain composed of the amino acid sequence described in amino acid numbers 20 to 135 of SEQ ID NO: 14 and the amino acid sequence of amino acid numbers 21 to 135 of SEQ ID NO: 18 The combination of the variable regions of the light chain; (9) The antibody according to the above (6), wherein the combination of the heavy chain and the light chain is selected from the group consisting of the following: amino acid number 20 to 465 in SEQ ID NO: 12. The heavy chain composed of the amino acid sequence described, the combination of the light chain composed of the amino acid sequence described in amino acid numbers 21 to 240 in SEQ ID NO: 16 and the amino acid number 20 in SEQ ID NO: 12 The heavy chain composed of the amino acid sequence described in ~465 and the light chain composed of the amino acid sequence described in amino acid numbers 21 to 240 in SEQ ID NO: 18, and the amino acid of SEQ ID NO: 14. The heavy chain composed of the amino acid sequence of Nos. 20 to 465 and the light chain of the amino acid sequence described in the amino acid numbers 21 to 240 of SEQ ID NO: 16 and the sequence identification number 14 The heavy chain composed of the amino acid sequence described in amino acid numbers 20 to 465 and the amino acid number 21 to 24 in the sequence identification number 18. (10) The antibody according to the above (6), wherein the combination of the heavy chain and the light chain is the combination of the following: amino acid number 20 in SEQ ID NO: 12. The heavy chain composed of the amino acid sequence described in ~465 and the light chain composed of the amino acid sequence described in amino acid numbers 21 to 240 in SEQ ID NO: 16 and the amino acid of SEQ ID NO: 12 a combination of the heavy chain composed of the amino acid sequence of Nos. 20 to 465 and the light chain of the amino acid sequence of amino acid numbers 21 to 240 of SEQ ID NO: 18 or The heavy chain composed of the amino acid sequence described in amino acid numbers 20 to 465 in SEQ ID NO: 14 and the light chain composed of the amino acid sequence described in amino acid numbers 21 to 240 in SEQ ID NO: 18 Or (11) an antibody obtained by the method for producing an antibody comprising the expression vector comprising the polynucleotide encoding the antibody according to any one of the above (1) to (10) The step of transforming the host cell and the step of taking the antibody of interest from the culture obtained in this step.

(drug)

The drug used in the present invention is a camptothecin derivative represented by the following formula: (1S, 9S)-1-amino-9-ethyl-5-fluoro-2,3-dihydro- 9-hydroxy-4-methyl-1H,12H-benzo[de]pyrano[3',4':6,7]吲 And [1,2-b]quinoline-10,13(9H,15H)-dione):

Although ezetidine has excellent antitumor activity, it has not yet It is commercially available as an anti-tumor drug. The exemitix can be easily obtained by a known method, and the amine group at the 1-position can be preferably used as a site of the structure of the binding linker. Further, exenatide is also liberated in tumor cells in a state in which one part of the conjugate is bound, but even in such a state, it is an excellent compound which exhibits an excellent antitumor effect.

It is known that ezetidine has a camptothecin structure, In an acidic aqueous medium (for example, about pH 3), the equilibrium is biased toward a structure formed by a lactone ring (closed loop), and on the other hand, in an alkaline aqueous medium (for example, about pH 10), the equilibrium lactone ring is an open-loop structure. (open body). The introduction of a drug conjugate corresponding to the ectic remains of such a closed-loop structure and an open-loop structure is also expected to have an equivalent anti-tumor effect, and it is needless to say that the state of either one is also included in the scope of the present invention.

For antibody-drug conjugates, drugs for antibody 1 molecule The combination number is an important factor affecting its effectiveness and safety. The production of antibody-drug conjugates is the number of combinations of drugs in a certain number of ways, specifying the raw materials for the reaction. The reaction conditions such as the amount of the reagent to be used are carried out, but unlike the chemical reaction of the low molecular compound, it is usually obtained by using a mixture of drugs in a different number. The mean value of the drug binding number of the antibody 1 molecule, that is, the average drug binding number is specified. In the present invention, in combination with the principle of non-violation, that is, in the case of an antibody-drug conjugate having a specific drug-binding number contained in an antibody-drug conjugate mixture having different drug binding numbers, the combination of drugs The number system means the average. The binding number of the antibody to the antibody molecule can be controlled, and the average number of bindings per antibody can be 1~10. The combination of hexahydrate or the like is preferably 2 to 8, more preferably 3 to 8. Further, if it is a person having ordinary knowledge in the technical field to which the present invention pertains, an antibody-drug that controls the binding amount of exenotecan can be obtained by designing a reaction in which a necessary number of drugs are bound to an antibody as described in Examples to be described later. Conjugate.

(connector)

The linker used in the present invention is -(succinimide-3-yl-N)-CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ -C(=O)-GGFG-NH-CH ₂ -O-CH ₂ -C(=O)-,-(amber 醯imino-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 -(succinimide-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)-

The linker shown in the structure may be a linker selected from any of these. Among these, -(succinimide-3-yl-N)-CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ -C(=O)-GGFG-NH-CH ₂ -O-CH ₂ -C(= O)-

It is especially good. Further, "-(amber ylidene-3-yl-N)-" has a structure represented by the following formula:

The 3 position in this part of the structure is a binding site against the CD98 antibody. The binding to the antibody at the 3 position is characterized by the formation of a thioether and bonding. The nitrogen atom of the 1-position of this structural moiety is bonded to the carbon atom of the methylene group present in the linker contained in this structure. That is, the binding moiety of the antibody to the linker is as follows:

(In the formula, "antibody-S-" is derived from an antibody, and n ¹ is 2 or 5).

"GGFG" is a tetrapeptide composed of glycine-glycine-phenylalanine-glycine.

The linker used in the present invention can be prepared according to, for example, the method described in the examples below.

(antibody-drug conjugate)

The antibody-drug conjugate of the present invention can be obtained at the end by the method described in the examples by converting an anti-CD98 antibody to a disulfide bond of a hinge region to a thiol group. It is produced by reacting any of the following drug-linker intermediate compounds having a maleimide group.

An anti-CD98 anti-system having a thiol group can be obtained by a method well known to those skilled in the art to which the present invention pertains (Hermanson, GT, Bioconjugate Techniques, pp. 56-136, pp. 456-493, Academic Press (1996). ). For example, a method of allowing Traut's reagent to act on an amine group of an antibody; a method of allowing a hydroxylamine to act after an N-amber succinimide S-ethyl sulfonate is applied to an amine group of an antibody; a method of causing a reducing agent after the action of amber quinone imino 3-(pyridyldithio)propionate; making dithiothreitol, 2-mercaptoethanol, ginseng (2-carboxyethyl) A method in which a reducing agent such as a phosphonate hydrochloride (TCEP) acts on an antibody to reduce a disulfide bond in the hinge region of the antibody to form a hydrogenthio group, but is not limited thereto.

Specifically, TCEP is used as a reducing agent to counteract the disulfide bond in the hinge region of the body by using 0.3 to 3 molar equivalents per cell, and reacting with the antibody in a buffer containing a chelating agent to obtain an inner hinge region of the antibody. An antibody that is partially or completely reduced by a sulfur bond.

Examples of the chelating agent include ethylenediaminetetraacetic acid (EDTA) or diethylenetriamine 5acetic acid (DTPA). These are preferably used at a concentration of 1 to 20 mM.

As the buffer, sodium phosphate or sodium borate, sodium acetate solution or the like can be used. Specifically, the anti-system reacts with TCEP at 4 to 37 ° C for 1 to 4 hours to obtain an antibody having a partially or completely reduced hydrogenthio group.

The antibody having such a thiol group can be used to produce 2 to 20 drug-bound antibody-drug conjugates per antibody per 2 to 20 molar equivalents of the drug-linker intermediate compound. Specifically, it is preferred to add a solution in which the drug-linker intermediate compound is dissolved in a buffer containing an antibody having a thiol group. Among them, in the case of a buffer, a sodium acetate solution, sodium phosphate or sodium borate is preferably used. The pH at the time of the reaction is 5 to 9, more preferably about pH 7. As the solvent for dissolving the drug-linker intermediate compound, dimethyl sulfoxide (DMSO), dimethylformamide (DMF), dimethylacetamide (DMA), N-methyl group can be used. An organic solvent such as 2-pyridone (NMP).

The organic solvent solution in which the drug-linker intermediate compound is dissolved is added to a buffer containing an antibody having a thiol group in an amount of 1 to 20% v/v to facilitate the reaction. The reaction temperature is 0 to 37 ° C, preferably 10 to 25 ° C, and the reaction time is 0.5 to 2 hours. The reaction system can terminate the reactivity of the unreacted drug-linker intermediate compound by inactivation of the thiol-containing reagent. The thiol-containing reagent is, for example, cysteine or N-ethinyl-L-cysteine (NAC). More specifically, 1 to 2 molar equivalents of NAC are added with respect to the drug-linker intermediate compound used, and the reaction can be terminated by culturing for 10 to 30 minutes at room temperature.

The antibody-drug conjugates produced are identified by concentration, buffer exchange, purification, antibody concentration, and the average number of drug bindings per molecule of the antibody by the following common procedures, and the antibody-drug conjugate can be identified.

1. Concentration of antibody or antibody-drug conjugate solution

The antibody or antibody-drug conjugate solution was placed in a container of Amicon Ultra (50,000 MWCO, Millipore Corporation), and centrifuged using a centrifuge (Allegra X-15R, Beckman Coulter, Inc.) (centrifugation at 2000G to 3800G 5~) 20 minutes), the antibody or antibody-drug conjugate solution was concentrated.

2. Determination of antibody concentration

The antibody concentration was measured using a UV analyzer (Nanodrop 1000, Thermo Fisher Scientific Inc.) according to the method specified by the manufacturer. At this time, each antibody used a different 280 nm absorbance coefficient (1.3 mL mg ^-1 cm ^-1 ~ 1.8 mL mg ^-1 cm ^-1 ).

3. Buffer exchange of antibodies

A NAP-25 column (Cat. No. 17-0852-02, GE Healthcare Japan Corporation) of Sephadex G-25 support will be used, containing sodium chloride (137 mM) and ethylenediamine according to the method specified by the manufacturer. Tetraacetic acid (EDTA, 5 mM) in phosphate buffer (10 mM, pH 6.0; in the present specification, referred to as "PBS6.0/EDTA") was equilibrated. To each of the NAP-25 column, 2.5 mL of an aqueous antibody solution was placed, and then fractionated (3.5 mL) was eluted with PBS6.0/EDTA 3.5 mL. This division was concentrated by the common operation A, and the antibody concentration was measured using the common operation B, and then the antibody concentration was adjusted to 10 mg/mL using PBS6.0/EDTA.

4. Purification of antibody-drug conjugates

The NAP-25 column was equilibrated with a sorbitol (5%) acetate buffer (10 mM, pH 5.5; referred to herein as "ABS"). On this NAP-25 column, an antibody-drug conjugate reaction aqueous solution (2.5 mL) was placed, and eluted by a manufacturer's prescribed amount of buffer to separate the antibody fractions. The fractionation portion is again placed in the NAP-25 column, and the eluted colloid filtration purification operation is repeated in the buffer for a total of 2 to 3 times to obtain the unbound drug linker and the low molecular compound (see (2- An antibody-drug conjugate of carboxyethyl)phosphine hydrochloride (TCEP), N-acetyl-L-cysteine (NAC), and dimethylhydrazine.

5. Determination of antibody concentration in antibody-drug conjugates and average drug binding number per antibody of antibody (UV method)

The concentration of the binding drug in the antibody-drug conjugate can be calculated by measuring the UV absorbance at two wavelengths of 280 nm and 370 nm of the aqueous antibody-drug conjugate solution, and then performing the following calculation.

Since the total absorbance in a certain wavelength is equal to the sum of the absorbances of all the absorbed chemical species present in the system (addition of absorbance), it is assumed that the Mohr absorption coefficient of the antibody and the drug is not before and after the combination of the antibody and the drug. When changing, the antibody concentration and drug concentration in the antibody-drug conjugate are as shown in the following relationship.

A ₂₈₀ =A _D,280 +A _A,280 =ε _D,280 C _D +ε _A,280 C _A (1)

A ₃₇₀ =A _D,370 +A _A,370 =ε _D,370 C _D +ε _A,370 C _A (2)

Wherein A ₂₈₀ represents the absorbance of the antibody-drug conjugate aqueous solution in 280 nm, A ₃₇₀ represents the absorbance of the antibody-drug conjugate aqueous solution in ₃₇₀ nm, A _{A, 280} represents the absorbance of the antibody in ₂₈₀ nm, and A _{A, 370} represents 370 nm. The absorbance of the antibody, A _{D, 280} represents the absorbance of the conjugate precursor in 280 nm, A _{D, 370} represents the absorbance of the conjugate precursor in ₃₇₀ nm, and ε _{A, 280} represents the molar absorptivity of the antibody in ₂₈₀ nm , ε _{A, 370} represents the Mohr absorption coefficient of the antibody in ₃₇₀ nm, ε _{D, 280} represents the Mohr absorption coefficient of the conjugate precursor in ₂₈₀ nm, and ε _{D, 370} represents the Mohr absorption of the conjugate precursor in ₃₇₀ nm coefficient, C _a indicates an antibody - an antibody drug conjugate in a concentration, C _D represents the antibody - concentration of the drug in the drug conjugate.

Among them, ε _{A, 280} , ε _{A, 370} , ε _{D, 280} , ε _{D, 370} are values prepared in advance (calculated estimated value or measured value obtained by UV measurement of the compound). For example, ε _{A, 280} is an amino acid sequence derived from an antibody and can be estimated by a known calculation method (Protein Science, 1995, vol. 4, 2411-2423). The ε _{A, 370} system is usually zero. ε _{D, 280} and ε _{D, 370} can be measured by the absorbance of a solution of the conjugate precursor used in a molar concentration, by Lambert-Beer law (absorbance = molar concentration × Obtained by Mohr absorption coefficient × cell light path length). A ₂₈₀ and A ₃₇₀ of the aqueous solution of the antibody-drug conjugate are measured, and the values of these values are substituted into the formulas (I) and (II) to solve the simultaneous equation, and C _A and C _D can be obtained. Furthermore, by dividing C _D by C _A , the average number of drugs per antibody per antibody can be determined.

6. Determination of the average number of bindings of each molecule of the antibody in the antibody-drug conjugate (RPC method)

The average number of drug bindings per antibody in the antibody-drug conjugate can be analyzed by the following high-speed liquid chromatography using reversed phase chromatography (RPC) method in addition to the UV method described above ( HPLC) was determined by analysis.

[6-1. Preparation of sample for HPLC analysis (reduction of antibody-drug conjugate)]

The antibody-drug conjugate solution (about 1 mg/mL, 60 μL) was mixed with a dithiothreitol (DTT) aqueous solution (100 mM, 15 μL). The mixture was incubated at 37 ° C for 30 minutes, and a sample of the double-sulfur bond between the L chain and the H chain of the antibody-drug conjugate was cleaved for HPLC analysis.

[6-2. HPLC analysis]

HPLC analysis was carried out under the following measurement conditions.

HPLC system: Agilent 1290 HPLC system (Agilent Technologies)

Detector: UV absorbance meter (measuring wavelength: 280 nm)

Column: PLRP-S (2.1×50mm, 8μm, 1000Å; Agilent Technologies, P/NPL1912-1802)

Column temperature: 80 ° C

Mobile phase A: 0.04% aqueous solution of trifluoroacetic acid (TFA)

Mobile phase B: acetonitrile solution containing 0.04% TFA

Gradient program: 29% - 36% (0 minutes - 12.5 minutes), 36% - 42% (12.5 - 15 minutes), 42% - 29% (15 minutes - 15.1 minutes), 29% - 29% (15.1 minutes - 25 minutes)

Sample injection amount: 15 μL

[6-3. Data Analysis]

[6-3-1] L chain (L ₀ ) and H chain (H ₀ ) of the antibody to the unbound drug, the drug-bound L chain (the drug has a combined L chain: L ₁ ) and the H chain ( The drug has a combined H chain: H ₁ , the drug has two bound H chains: H ₂ , and the drug has three bound H chains: H ₃ ). The hydrophobicity increases in proportion to the number of drugs bound and remains time-dependent. large, so to _{L 0, L 1, H 0} , H 1, H 2, H ₃ of the sequence to be eluted. By comparing the hold times of L ₀ and H ₀ , the detected peaks can be assigned to any of L ₀ , L ₁ , H ₀ , H ₁ , H ₂ , and H ₃ .

[6-3-2] Since the drug linker has UV absorption, the peak area value of the L chain, the H chain and the drug linker is corrected according to the following formula in accordance with the number of binding of the drug linker.

Here, the molar absorption coefficient (280 nm) of the L chain and the H chain in each antibody can be determined by a known calculation method (Protein Science, 1995, vol. 4, 2411-2423) using the L chain and H from each antibody. The putative value of the amino acid sequence of the chain. Moreover, the molar absorption coefficient (280 nm) of the drug linker can be used to convert each drug linker with mercaptoethanol or N -ethylmercaptocysteine to convert the maleimide group to amber. The measured molar absorption coefficient (280 nm) of the amine thioether compound.

[6-3-3] Calculate the ratio (%) of each chain peak area in total with respect to the peak area correction value according to the following formula.

A _Li , A _Hi : L _i , H _i peak area correction value

[6-3-4] The average number of drug bindings per molecule of the antibody in the antibody-drug conjugate was calculated according to the following formula.

Average drug binding number = (L ₀ peak area ratio × 0 + L ₀ peak area ratio × 1 + H ₀ peak area ratio × 0 + H ₁ peak area ratio × 1 + H ₂ peak area ratio × 2 + H ₃ peak area Ratio × 3) / 100 × 2

After the antibody-drug conjugate of the present invention is moved within the tumor cell, the linker moiety is cleaved, 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) is released in tumor cells. Among them, "-(NH-DX)" is expressed by the following formula:

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

Further, in the case of NH ₂ -CH ₂ -O-CH ₂ -C(=O)-(NH-DX), it has been confirmed that the structure of the amine acetal having the same molecule is unstable, and further autolysis is carried out to have HO- CH ₂ -C(=O)-(NH-DX) is free. These compounds are also preferably used as intermediates for the production of the antibody-drug conjugates of the present invention.

The antibody-drug conjugate of the present invention may be a hydrate formed by being placed in the atmosphere, or subjected to recrystallization or purification, and absorbing water or adsorbed water, thereby hydrating the compound or pharmacology. Upper tolerable salts are also included in the present invention.

In the case where the antibody-drug conjugate of the present invention has a basic group such as an amine group, a pharmacologically acceptable acid addition salt can be formed as desired. Examples of such acid addition salts include hydrogen halides such as hydrofluoric acid salts, hydrochloride salts, hydrobromide salts, and hydroiodides; nitrates, perchlorates, sulfates, and phosphates. Oral acid salt; lower alkane sulfonate such as methanesulfonate, trifluoromethanesulfonate or ethanesulfonate; arylsulfonate such as besylate or p-toluenesulfonate Organic acid salts of formate, acetate, trifluoroacetate, malate, fumarate, succinate, citrate, tartrate, oxalate, maleate, etc. Or an amino acid salt of ornidamine, glutamate, aspartate or the like.

The antibody-drug conjugate of the present invention has an acidic group such as a carboxyl group, and a pharmacologically acceptable base addition salt can be formed as desired. Examples of such a base addition salt include an alkali metal salt such as a sodium salt, a potassium salt or a lithium salt; an alkaline earth metal salt such as a calcium salt or a magnesium salt; an inorganic salt such as an ammonium salt; and a dibenzylamine. salt, a porphyrin salt, an alkyl phenylglycine salt, an ethyl diamine salt, an N-methyl glucosamine salt, a diethylamine salt, a triethylamine salt, a cyclohexylamine salt, Dicyclohexylamine salt, N,N'-dibenzylethylidene diamine salt, diethanolamine salt, N-benzyl-N-(2-phenylethoxy)amine salt, piperazine An organic amine salt such as a salt, a tetramethylammonium salt or a hydroxymethylaminomethane salt.

The present invention may further comprise an antibody-drug conjugate in which one or more atoms constituting the antibody-drug conjugate are substituted with an isotope of its atom. There are two types of radioisotopes and stable isotopes in the isotope system. Examples of isotopes include hydrogen isotopes ( ² H and ³ H), carbon isotopes ( ¹¹ C, ¹³ C and ¹⁴ C), and nitrogen. Isotopes ( ¹³ N and ¹⁵ N), isotopes of oxygen ( ¹⁵ O, ¹⁷ O and ¹⁸ O), isotopes of fluorine ( ¹⁸ F), etc. The composition containing the isotope-labeled antibody-drug conjugate is used as, for example, a therapeutic agent, a prophylactic agent, a research reagent, an analytical reagent, a diagnostic agent, an in vivo imaging diagnostic agent, and the like. Mixtures of isotopically labeled antibody-drug conjugates and isotopically labeled antibody-drug conjugates are also all encompassed by the present invention. The isotope-labeled antibody-drug conjugate system can be produced by a method known in the art, for example, by using an isotope-labeled starting material in place of the raw material in the production method of the present invention to be described later.

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

That is, the anti-CD98 antibody-drug conjugate of the present invention can be used as a chemotherapeutic agent which is the main treatment method for cancer treatment, and as a result, growth of cancer cells can be delayed, proliferation can be inhibited, and cancer can be destroyed. cell. By this, in cancer patients, it is possible to achieve the treatment effect of maintaining the life of a cancer patient by eliminating the symptoms caused by cancer and improving QOL. Even if the destruction of cancer cells is not achieved, the cancer patients can achieve higher QOL and achieve longer-term survival by inhibiting or controlling the proliferation of cancer cells.

In addition to the use of the drug alone in such drug therapy, the adjuvant therapy can also be used as a combination with other therapies, and can be combined with surgery, radiation therapy, hormone therapy, and the like. Furthermore, it can also be used as a medicament for drug therapy in neoadjuvant therapy.

In addition to the above therapeutic use, the proliferation of the finely metastatic cancer cells is suppressed, and the effect of destruction can be expected. In particular, when the expression of CD98 is confirmed in a primary cancer cell, the anti-CD98 antibody-drug conjugate of the present invention can be expected to have an effect of inhibiting and preventing cancer metastasis. For example, an effect of suppressing or destroying cancer cells in a body fluid during transfer, and an effect of suppressing or destroying fine cancer cells immediately after implantation into any tissue can be expected. Accordingly, in particular, it is expected to have an effect of suppressing and preventing cancer metastasis after surgical cancer removal.

The anti-CD98 antibody-drug conjugate of the present invention can be administered to a cancer tissue in addition to being administered as a systemic therapy, and a therapeutic effect is expected.

As for the type of cancer, lung cancer, kidney cancer, and urethra can be cited. Epithelial cancer, colorectal 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 myeloid leukemia, acute lymphocytic leukemia, chronic myelogenous leukemia, multiple myeloma, etc. However, it is not limited to this as long as it is cancer derived from cancer cells expressing CD98.

CD98 antibody or antibody of the present invention used in the present invention The drug conjugate system can be used as an active ingredient of a pharmaceutical composition for treating an autoimmune disease or a pharmaceutical composition for inhibiting rejection of a transplant.

Medicinal composition containing the antibody-drug conjugate of the present invention The system may be administered systemically or locally, preferably parenterally, if administered to a mammal (e.g., human, horse, cow, pig, etc., preferably human).

Examples of the parenteral administration route include intradermal, intramuscular, intraperitoneal, intravenous, and subcutaneous routes, but are not limited thereto. Examples of the method of administration include injection, bolus injection, and the like, but injection is preferred.

The pharmaceutical composition of the present invention can be prepared in a suitable form depending on the method of administration, and can be prepared by a preparation method of various preparations which are usually used.

For example, the antibody-drug conjugate of the present invention, the sterilized liquid described in "Remington's Pharmaceutical Sciences" by EW Martin, and the like (for example, water and oil (including petroleum, animal, plant, or synthetic origin oil) may be mixed (for example). , peanut oil, soybean oil, mineral oil, sesame oil, etc.))), a salt solution, a dextrose aqueous solution, an aqueous solution of glycerin or the like, a wetting agent, an emulsifier, an additive such as a pH buffering agent, etc., to prepare the pharmaceutical composition of the present invention. Things.

The pharmaceutical composition of the present invention may further contain a solubilizing agent, a local paralysis agent for relieving pain in the injection site (for example, lignocaine), and the like. The pharmaceutical composition of the present invention may be supplied in such a manner that an active ingredient and a solvent are placed in separate containers. Further, the case of administration by injecting the pharmaceutical composition of the present invention can be administered, for example, by injecting a bottle containing an active ingredient and sterilized pharmaceutical grade water or saline. When the pharmaceutical composition of the present invention is administered by injection, the active ingredient may be administered by mixing with the sterilized water for injection or saline before administration.

The pharmaceutical composition of the present invention may contain 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, according to which Can enhance the anti-cancer effect. Other anticancer agents used for such purposes may be administered to the individual simultaneously, separately, or sequentially with the antibody-drug conjugate, or may be administered by changing the respective administration intervals. Examples of such a cancer therapeutic agent include carboplatin, cisplatin, gemcitabine, irinotecan (CPT-11), paclitaxel, and pemetrex. Pemetrexed, sorafenib, vinblastine, Pharmacopoeia of International Publication No. WO2003/038043, and LH-RH analogues (leuprorelin, Gosher) Goserelin, etc., estramustine phosphate, estrogen antagonist (tamoxifen, raloxifene, etc.), aromatic ring transformase ( Aromatase) an inhibitor (anastrozole, letrozole, exemestane, etc.), etc., but is not limited thereto as long as it is an agent having antitumor activity.

The pharmaceutical composition of the present invention may also be a freeze-dried preparation or A liquid preparation is provided. Where a lyophilized formulation is provided, it may be a formulation containing suitable formulation additives for use in the art. Also in the liquid preparation, it may be a preparation containing an appropriate preparation additive used in the field.

The composition of the pharmaceutical composition of the present invention and the concentration of the active ingredient The degree also varies depending on the administration method, but the anti-CD98 antibody-drug conjugate contained in the pharmaceutical composition of the present invention, the affinity for the antigen in the antibody-drug conjugate, that is, the dissociation constant for the antigen (Kd) Point), the higher the affinity (the lower the Kd value), even if a small amount of the dose can make the effect Wave. Accordingly, when the dose of the antibody-drug conjugate is determined, the dose can be set based on the affinity of the antibody-drug conjugate to 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 at intervals of 1 to 180 days.

The present invention will be specifically described by the following examples, but The invention is not limited to these examples. Again, these are not to be construed in a limiting sense. Moreover, the reagents, solvents, and starting materials which are not specifically described in the present specification can be easily obtained from a commercially available supply source.

[Examples] (Example 1) Preparation of individual antibodies and screening of antibodies (1-1) Immunization

BALB/cAnNCrlCrlj mice (Charles River, Japan) of 4-6 weeks old were used. On day 0, day 7, day 15, and day 24, 5 x 106 MCF7 cells (ATCCHTB-22) stripped with Versene (Life Technologies) were suspended in PBS and administered to the back of the skin. On the 31st day, the same cells were administered in 5 × 106 veins, and the spleen was taken on the same day to be used for fusion tumor production.

(1-2) Production of fusion tumor

The spleen cells were combined with mouse myeloma P3X63Ag8U.1 cells (ATCC CRL-1597), and PEG4000 (IBL) was used to prepare a cell-fused fusion tumor. As a result, a fusion tumor of 1760 strains was established from mice immunized with MCF7 cells. Screening by CDC analysis of antibody-producing fusion tumors was performed using the obtained fusion tumor culture supernatant.

(1-3) Screening of fusion tumors (CDC analysis)

MCF7 cells were diluted to 5000 cells/80 μL using RPMI medium (Life Technologies) containing 10% fetal bovine serum (FBS), added to a 96-well plate at 80 μL/well, and cultured overnight. The fusion tumor culture supernatant was added to a plate inoculated with cells at 20 μL/well, and allowed to stand at 4 ° C for 1 hour. On ice, 1 mL of sterilized water was added to each bottle of the diluted frozen product (Cedarlane) of rabbit complement. After standing for 1 minute, mixing, and mixing with 19 mL of 0.1% BSA/RPMI 1640 medium (BSA Sigma). The same complement dilution was added to a disk containing the mixed tumor culture supernatant at 20 μL/well, and the reaction was carried out at 37 ° C for 1 hour.

The plate was placed 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 flat disk reader (ARVO HTS Prekin Elmer). A well with a small amount of luminescence was judged to induce complement-dependent cell death. A fusion tumor that produces a culture supernatant that induces complement-dependent cell death is selected. As a result, screening positive fusion tumor colonies of 15 selected strains were obtained.

(Example 2) Antibody modulation from self-fused tumor

8 to 10 weeks old mice or nude mice (BALB/c) administered intraperitoneally with decane (Pristane; 2,6,10,14-tetramethylpentadecane; 0.5 ml) for 2 weeks. Nude mice, females, CLEA, Japan, intraperitoneal injection of the monoclonal antibodies obtained in Example 1 to produce 1~2×10 6 cells of the fusion tumor were suspended in PBS, and 0.5 mL was intraperitoneally injected. After 10 to 21 days, ascites was taken after the ascites of the fusion tumor was cancerated. Centrifugal fraction of ascites obtained After removing the solid component, the PBS was added twice or more, and then purified by a Mab Select Sure HiTrap 5 ml column (GE Healthcare LifeSciences), and the IgG fraction bound to the column in the neutral region was recovered as a purification. Individual antibodies.

(Example 3) Identification of antigen bound by antibody produced by fusion tumor (3-1) Binding specificity (3-1-1) Adjustment of antigen gene expression cells

The SLC3A2 gene expression vector encoding CD98 was produced using a commercially available SLC3A2 gene selection strain (IOH-4673, Life Technologies) and the Gateway expression vector pDEST40 (Life Technologies).

NIH-3T3 cells (ATCC CRL-1658) were adjusted to 1 × 10 ⁵ cells/ml in RPMI medium (Life Technologies) containing 10% fetal bovine serum (FBS), using FUGENE6 (Roche Applied Science) pcDNA-DEST40-SLC3A2 was transfected into NIH-3T3 cells and further cultured for 2 nights at 37 ° C under 5% CO ₂ . Cells treated with the same plastid-free FUGEN6 were used as a CD98 negative control group. The transfected NIH-3T3 cells were trypsinized, washed with RPMI containing 10% FBS, and suspended in PBS containing 5% FBS. The obtained cell suspension was used for flow cytometry analysis.

(3-1-2) Flow cytometry analysis

The cell suspension prepared by 3-1-1 was centrifuged, and after removing the supernatant, the fusion culture supernatant or purified antibody was added to the NIH-3T3 cells transfected with each vector and suspended, and allowed to stand at 4 ° C. ~1 hour. Take After washing twice with 5% FBS in PBS, it was suspended by adding 40-fold diluted Fluorescein-conjugated goat IgG fraction to mouse IgG (integrin; ICN Pharmaceuticals, #55493) in PBS containing 5% FBS. °C is allowed to stand for 0.5~1 hour. After washing twice with 5% FBS in PBS, it was resuspended in PBS containing 5% FBS containing 2 μg/ml 7-Amino Actinomycin D (Invitrogen (Molecular Probes)) to flow cytometer (FC500: Beckman Coulter) for testing. Data analysis was performed by Flowjo (TreeStar). After the 7-aminoactinomycin D-positive dead cells were removed at the gate, a histogram of the FITC fluorescence intensity of the living cells was made. The histogram of the NIH-3T3 cells shifted to the strong fluorescence intensity side (the fusion tumor culture supernatant) was generated from the histogram of the fluorescence intensity of the NIH-3T3 cells which were not introduced into the plastid in the negative control group. The fusion tumor of the antibody or the purified antibody was obtained as an anti-CD98 antibody to produce a fusion tumor. This fusion tumor is referred to as CD98 fusion tumor M23, and the antibody produced by CD98 fusion tumor M23 is referred to as M23 antibody or M23.

(3-2) Determination of epitopes (3-2-1) Modulation of antigen gene expression cells

The cell line 293α cells stably transfected with integrin αv and integrin β3 in HEK293 cells were seeded in a collagen-coated 100 mm dish at 2.6×10 6 cells/dish (manufactured by AGC TEC HNO GLASS) ), cultured in DMEM medium containing 10% FBS at 37 ° C under 5% CO 2 for one night. On the next day, a part of the sequence was replaced with mouse pcDNA3.1-FLAG-hCD98-mouse206-271, pcDNA3.1-FLAG-hCD98-mouse262-331, pcD NA3.1-FLAG-hCD98-mouse322-401, pcDNA3.1-FLAG-hCD98-mouse392-471, pcDNA3.1-FLAG-hCD98-mouse462-541, pcDNA3.1-FLAG-hCD98-mouse 532-631, The mouse CD98 expression vector pGEM-T-mouse CD98 was introduced into 293α cells using Lipo fectamine 2000 transfection reagent, and further cultured overnight at 37 ° C under 5% CO 2 . On the next day, the expression vector was introduced into cells and treated with TrypLE Express (manufactured by Life Technologies). The cells were washed with PBS containing 3% FBS, and then suspended in PBS containing 3% FBS. The obtained cell suspension was used for flow cytometry analysis.

(3-2-2) Flow cytometry analysis

The cell suspension prepared by 3-2-1 was centrifuged, and after removing the supernatant, a expression vector of 2 × 10 ⁵ cells was introduced into the cells, and each of the M23 antibodies and the individual ANTI produced in the mouse as a positive control group were added. The -FLAG M2 antibody and the mouse IgG2A as a negative control group were suspended at 10 μg/mL, and allowed to stand at 4 ° C for 15 minutes. After washing once with PBS containing 3% FBS, an anti-mouse IgG FITC conjugate diluted 500-fold in PBS containing 3% FBS was added and suspended, and allowed to stand at 4 ° C for 20 minutes. After washing with PBS containing 3% FBS, it was resuspended in PBS containing 3% FBS, and detected by a flow cytometer (BD FACSC ant II: BD Biosciences). Data analysis is performed in Flowjo. A histogram of the fluorescence intensity of FITC was prepared, and the histogram of the fluorescence intensity of the mouse IgG2A in the negative control group was not displaced relative to the unstained person, and the histogram of the M23 antibody was introduced into the cell with respect to the expression vector. In the case where the middle displacement is on the side of the strong fluorescence intensity, it is judged to be combined. As a result, the M23 anti-system was pcDNA3.1-FLAG-hCD98-mouse206-271, pcDNA3.1-FLAG-hCD98-mouse262-331, pcDNA3.1-FLAG-hCD98-mouse322-401, pcDNA3.1-FLAG-hCD98- Mouse392-471 or pcDNA3.1-FLAG-hCD98-mouse532-631 was introduced into the cell for binding, and the cells introduced into pcDNA3.1-FLAG-hCD98-mouse462-541 were unbound. Accordingly, the M23 anti-system revealed a sequence different from mouse CD98 in the amino acid sequence of human CD98 shown in SEQ ID NO: 38 of the Sequence Listing (Fig. 13). Further, each antigen was expressed on the cell membrane using an anti-FLAG antibody of the positive control group (Fig. 14), and M23 was also confirmed not to bind to mouse CD98 (Fig. 13).

(Example 4) Determination of nucleotide sequence of cDNA encoding variable region of M23 antibody gene and preparation of chimeric M23 antibody (4-1) Determination of the nucleotide sequence of the cDNA encoding the variable region of the M23 antibody gene (4-1-1) Modulation of mRNA from CD98 fusion tumor M23

To amplify the cDNA containing the variable region of the M23 antibody, mRNA was prepared from the CD98 fusion tumor M23 using an mRNA single isolation kit (Roche applied science).

Synthesis of (4-1-2) cDNA (5'-RACE-Ready cDNA)

The synthesis of cDNA (5'-RACE-Ready cDNA) was carried out using 100 ng of mRNA prepared by 4-1-1 and a SMARTer RACE cDNA amplification kit (CLONTECH).

(4-1-3) Amplification and Sequence Determination of cDNA Containing Heavy Chain Variable Region of M23 Antibody by 5'-RACE PCR

For the primer for PCR amplification of the cDNA of the variable region of the heavy chain gene, UPM (Universal Primer A Mix: attached to the SMARTer RACE cDNA amplification kit) and 5'-GGCATCCTAGAGTCACCGAGGAGCCAGTTG-3' (sequence recognition) were used. No. 25: Oligonucleotide of the sequence of the primer mG2aVR1). The UPM system was used in the SMARTer RACE cDNA Amplification Kit (CLONTECH), and the mG2aVR1 was designed from the sequence of the constant region of the mouse heavy chain (IgG2a) of the database.

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

The cDNA containing the heavy chain variable region amplified by 5'-RACE PCR was purified using a MinElute PCR purification kit (QIAGEN), and then selected using the Zero Blunt TOPO PCR selection kit (Invitrogen). Sequence analysis of the nucleotide sequence of the cDNA containing the variable region of the selected heavy chain was carried out. As a sequence primer, an oligonucleotide designed using 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 of the library, and NUP (Nested Universal) Primer A: attached to the SMARTer RACE cDNA amplification kit).

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

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

(4-1-4) Amplification and sequence determination of cDNA containing the light chain variable region of M23 antibody by 5'-RACE PCR

As a primer for amplifying a variable region of the light chain gene of the M23 antibody, UPM (Universal Primer A Mix: attached to the SMARTer RACE cDNA amplification kit) and 5'-AGTCCAACTGTTCAGGACGCCATTTTGTCG-3' (sequence recognition) No. 26: Oligonucleotide of the sequence of the primer mKVR2). The UPM system was used in the SMARTer RACE cDNA Amplification Kit (CLONTECH), and the mKVR2 was designed from the sequence of the constant region of the mouse light chain of the library.

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

The PCR system was carried out using a KOD-Plus- (TOYOBO) as a polymerase according to the manual of the SMARTer RACE cDNA Amplification Kit (CLONTECH), using a touchdown PCR program.

Variable light chain containing amplification amplified by 5'-RACE PCR The cDNA of the region was purified using a MinElute PCR purification kit (QIAGEN), and then subjected to colonization using a Zero Blunt TOPO PCR selection kit (Invitrogen) to carry out cDNA containing the variable region of the selected light chain. Sequence analysis of nucleotide sequences. As a sequence primer, an oligonucleotide designed using 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 of the library, and NUP (Nested Universal) Primer A: attached to the SMARTer RACE cDNA amplification kit).

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

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

(4-2) Production of chimeric M23 antibody (4-2-1) Construction of chimeric and humanized antibody light chain expression vector pCMA-LK

A fragment of about 5.4 kb obtained by digestion of the plastid pcDNA3.3-TOPO/LacZ (Invitrogen) with the restriction enzymes XbaI and PmeI, ie, the sequence containing the human kappa chain secretion and the human kappa chain constant region shown in SEQ ID NO: 5. DNA fragment of the nucleotide sequence, used In-Fusion Advantage PCR selection kit (CLONTECH) was combined to make pcDNA3.3/LK.

Using pcDNA3.3/LK as a template, the following primer set PCR is performed, and the obtained 3.8 kb fragment is phosphorylated and then constructed by self-ligation to construct a chimeric and human sequence having a message sequence, a selection site, and a human kappa chain constant region downstream of the CMV promoter. The antibody light chain expression vector pCMA-LK.

Primer group

5'-TATACCGTCGACCTCTAGCTAGAGCTTGGC-3' (sequence identification number 27: primer 3.3-F1)

5'-GCTATGGCAGGGCCTGCCGCCCCGACGTTG-3' (SEQ ID NO: 28: Introduction 3.3-R1)

(4-2-2) Construction of chimeric and humanized antibody IgG1 heavy chain expression vector pCMA-G1

Using the In-Fusion Advantage PCR Kit (CLONTECH) to digest pCMA-LK with XbaI and PmeI to remove the kappa chain secretion message and the DNA fragment of the human kappa chain constant region, and the human with the coding sequence number 6 The DNA fragment of the chain secretion message sequence and the nucleotide sequence of the human IgG1 constant region is ligated, and the chimeric and humanized antibody IgG1 type having the message sequence, the selection site and the human IgG1 heavy chain constant region are constructed downstream of the CMV promoter. The strand expresses the vector pCMA-G1.

(4-2-3) Construction of chimeric M23 antibody heavy chain expression vector

By using the cDNA of the variable region of the heavy chain of the M23 antibody obtained in 4-1-3 as a template, KOD-Plus-(TOYOBO) And the primer set described below amplifies the DNA fragment containing the cDNA encoding the variable region of the heavy chain, and uses the In-Fusion HD PCR selection kit (CLONTECH) to insert the chimeric and humanized IgG1 type. The strand expression vector pCMA-G1 was constructed by restriction enzyme BlpI cleavage to construct a chimeric M23 antibody heavy chain expression vector. The obtained performance vector was named "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 described in Figure 1.

Chimeric M23 antibody heavy chain primer set

5'-CCAGATGGGTGCTGAGCCAGGTCCAGCTGCAGCAGTCTGGAGCTGAG-3' (SEQ ID NO: 29: Introduction M23H-F)

5'-GGGCCCTTGGTGGAGGCTGCAGAGACAGTGACCAGAGTCCCTTGGCC-3' (SEQ ID NO: 30: Introduction M23H-R)

(4-2-4) Construction of chimeric M23 antibody light chain expression vector

By using cDNA containing the variable region of the light chain of the M23 antibody obtained in 4-1-4 as a template, KOD-Plus- (TOYOBO Co., Ltd.) and the following primer set will contain a variable region encoding a light chain. The DAN fragment of the cDNA was amplified, and the In-Fusion HD PCR selection kit (CLONTECH) was inserted into the chimeric and humanized IgG1 type light chain to express the universal vector pCMA-LK to limit the enzyme BsiWI. A light chain expression vector of a chimeric M23 antibody. The obtained performance vector was named "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 described in Figure 2.

Chimeric M23 antibody light chain introduction group

5'-ATCTCCGGCGCGTACGGCGACATTGTGATGTCACAGTCTCCATCCTCC-3' (sequence identification number 31: introduction M23L-F)

5'-GGAGGGGGCGGCCACAGCCCGTTTGATTTCCAGCTTGGTGCCTCC-3' (SEQ ID NO: 32: Introduction M23L-R)

(4-2-5) Production of chimeric M23 antibody

FreeStyle 293F cells (Invitrogen) were subcultured according to the manual. FreeStyle 293F cells (Invitrogen) having a logarithmic growth period of 1.2 × 10 ⁹ were inoculated into 3 L Fernbach Erlenmeyer Flask (CORNING), diluted with FreeStyle 293 expression medium (Invitrogen) to prepare 1.0 × 10 ⁶ cells/ml. The culture was shaken at 90 rpm for one hour at 37 ° C in an 8% CO ₂ incubator. 3.6 mg of polyethylenimine (Polyscience, #24765) was dissolved in 20 ml of Opti-Pro SFM (Invitrogen), followed by a light chain expression vector (0.8 mg) prepared using PureLink HiPure Plasmid kit (Invitrogen). The heavy chain expression vector (0.4 mg) was added to 20 ml of Opti-Pro SFM (Invitrogen). To 20 ml of the polyethylenimine/Opti-Pro SFM mixture, 20 ml of the expression carrier/Opti-Pro SFM mixture was added and stirred slowly, and then left for 5 minutes, and then added to FreeStyle 293F cells. The culture supernatant obtained by shaking and culturing for 7 days at 90 rpm in an incubator at 37 ° C in an 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".

Purification of (4-2-6) cM23 antibody

From the culture supernatant obtained at 4-2-5, the antibody was purified in two stages using rProtein A affinity chromatography (at 4-6 ° C) and ceramic hydroxyapatite (at room temperature). The buffer substitution step after purification of rProtein A affinity chromatography and ceramic hydroxyapatite was carried out at 4-6 °C. Initially, the culture supernatant was used for MabSelect SuRe (GE Healthcare Bioscience, HiTrap column) equilibrated with PBS. After the culture supernatant was placed in the column, the column was washed with PBS having a column volume of 2 times or more. Next, it was eluted with a 2 M arginine hydrochloride solution (pH 4.0), and fractions containing the antibody were collected. The fraction was replaced by PBS by dialysis (Thermo Scientific, Slide-A-Lyzer Dialysis Cassette), and then the 5-fold diluted antibody solution was applied to the buffer in 5 mM sodium phosphate/50 mM MES/pH 7.0 buffer. A buffered equilibrated ceramic hydroxyapatite column (Japan BioRad, Bio-Scale CHT Type-I Hydroxyapatite Column) of 5 mM NaPi/50 mM MES/30 mM NaCl/pH 7.0. The linear concentration gradient elution by sodium chloride was carried out, and the fraction containing the antibody was collected. This was divided into a solution of HBSor (25 mM histidine/5% sorbitol, pH 6.0) by dialysis (Thermo Scientific, Slide-A-Lyzer Dialysis Cassette). Finally, it was concentrated by Centrifugal UF Filter Device VIVASPIN20 (divided molecular weight UF10K, Sartorius, 4 ° C) to prepare a purified sample having an IgG concentration of 10 mg/ml or more.

(Example 5) Design of humanized antibody against mouse anti-CD98 monoclonal antibody (5-1) Design of humanized variant of M23 antibody (5-1-1) Molecular modeling of the variable region of the M23 antibody

Molecular modeling of the variable region of M23 by homology A well-known method of homology modeling (Methods in Enzymology, 203, 121-153 (1991)) is carried out. The first sequence of the variable region of human immunoglobulin registered in the Protein Data Bank (Nuc. Acid Res., 35, D301-D303 (2007)) (three-dimensional structure derived from X-ray crystal structure) It is possible to compare with the variable region of M23 determined in Example 4. As a result, the 1MJU line was selected from the variable region of the heavy chain of the M23 antibody, and the antibody having the defect in the same frame was selected to have the highest sequence homology. Further, 3MBX was selected with the highest sequence homology with respect to the variable region of the light chain of the M23 antibody. The three-dimensional structure of the framework region can be produced by combining the coordinates of 1 MJU and 3 MBX corresponding to the heavy and light chains of the M23 antibody to obtain a "frame work model". Second, the configuration represented by the respective CDRs is incorporated into the framework mode.

Finally, in terms of energy, in order to obtain M23 resistance A molecular model of the likelihood of a variable region of a body, performing an energy calculation to remove contact between unfavorable atoms. The above procedure was carried out using a commercially available protein three-dimensional structure analysis program Discovery Studio (Accelrys).

(5-1-2) Design of the amino acid sequence of the humanized M23 antibody

The construction of the humanized M23 antibody is carried out by a known method of CDR grafting (Proc. Natl. Acad. Sci. U.S.A. 86, 10029-10033 (1989)). The acceptor antibody (acceptor antiboby) is selected based on the amino acid homology in the framework region. The sequence of the framework region of the M23 antibody, and the Kabat database of the amino acid sequence of the antibody (Nuc. Acid Res., 29, 205-206 (2001)) A comparison of all human frameworks revealed that the HuMc3 antibody was selected as an acceptor due to 80% sequence homology of the framework region. The amino acid residue in the framework region of HuMc3 was juxtaposed with the amino acid residue of the M23 antibody to identify the position at which the isomeric amino acid was used. The positions of these residues are based on the use of the three-dimensional model of the M23 antibody constructed above, and the donor residues that should be grafted at the receptor are by Queen et al. (Proc. Natl. Acad. Sci .USA, 86, 10029-10033 (1989)) was selected based on the benchmark given. The sequence of the humanized M23 antibody was constructed as described in the following examples by transferring the selected donor residues into the acceptor antibody.

(5-2) Humanization of M23 antibody heavy chain (5-2-1) hM23-H1 heavy chain

The amino acid number 24 (glutamic acid) of the heavy chain of the cM23 antibody shown in SEQ ID NO: 8 is substituted with lysine, and the amino acid number 30 (leucine) is substituted with lysine or amino acid. No. 31 (proline) substituted with lysine, amino acid number 32 (arginine) substituted with lysine, amino acid number 35 (threonine) substituted with alanine, amino acid number 57 ( Substituted by arginine for arginine, amino acid number 59 (arginine) substituted with alanine, amino acid number 67 (isoleucine) substituted with methionine, amino acid number 86 (from Amine acid) is substituted with arginine, amino acid number 87 (alanine) is substituted for lysine, amino acid number 89 (leucine) is substituted for isoleucine, amino acid number 93 (ionic acid) Substituted for threonine, amino acid number 95 (serine acid) substituted with threonine, amino acid number 101 (glutamic acid) substituted with glutamic acid, amino acid number 106 (threonine) Substituted for arginine, amino acid number 108 (aspartic acid) substituted with glutamic acid, amino acid number 110 (serine) Substituted for threonine, amino acid number 111 (serine acid) substituted with alanine, amino acid number 114 (phenylalanine) substituted with tyrosine, amino acid number 135 (alanine) substituted with serine The designed humanized M23 antibody heavy chain was named "hM23-H1 heavy chain".

The amino acid sequence of the hM23-H1 heavy chain is described in the sequence Identification number 12. The sequence consisting of the amino acid residues 1 to 19 of the amino acid sequence of SEQ ID NO: 12, the sequence of amino acid residues 20 to 135, and the amino acid of No. 136 to 465 The sequence consisting of residues corresponds to a message sequence, a heavy chain variable region, and a heavy chain constant region. The nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 12 is described in SEQ ID NO: 11. a sequence consisting of nucleotides 1 to 57 of the nucleotide sequence of SEQ ID NO: 11, a sequence consisting of nucleotides 58 to 405, and nucleotides 406 to 1395 The sequence lines are each a coding message sequence, a heavy chain variable region sequence, and a heavy chain constant region sequence. The nucleotide sequence of SEQ ID NO: 11 and the amino acid sequence of SEQ ID NO: 12 are also described in Figure 3.

(5-2-2) hM23-H2 heavy chain

Substituting the amino acid number 30 (leucine) of the cM23 heavy chain shown in SEQ ID NO: 8 with lysine, amino acid number 31 (proline) with lysine, amino acid number 32 (arginine) substituted with lysine, amino acid number 35 (threonine) substituted with alanine, amino acid number 57 (isoamine) substituted with arginine, amino acid number 59 (spermine The acid is substituted with alanine, amino acid number 67 (isoleucine) is substituted for methionine, amino acid number 89 (leucine) is substituted with isoleucine, amino acid number 95 (silamine) Acid) substituted with threonine, amino acid number 101 (glutamic acid) Substituting glutamic acid, amino acid number 108 (aspartic acid) for glutamic acid, amino acid number 110 (serine acid) for substitution with threonine, amino acid number 111 (serine) for The designed humanized M23 antibody heavy chain substituted with alanine, amino acid number 114 (phenylalanine) substituted with tyrosine, amino acid number 135 (alanine) and substituted with serine was named "hM23-H2 heavy chain".

The amino acid sequence of the hM23-H2 heavy chain is described in the sequence Identification number 14. a sequence consisting of amino acid residues 1 to 19 of the amino acid sequence of SEQ ID NO: 14, a sequence consisting of amino acid residues 20 to 135, and amino acids 136 to 465 The sequence consisting of residues corresponds to a message sequence, a heavy chain variable region, and a heavy chain constant region. The nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 14 is described in SEQ ID NO: 13. a sequence consisting of nucleotides 1 to 57 of the nucleotide sequence of SEQ ID NO: 13, a sequence consisting of nucleotides 58 to 405, and nucleotides 406 to 1395 The sequences each encode a message sequence, a heavy chain variable region sequence, and a heavy chain constant region sequence. The nucleotide sequence of SEQ ID NO: 13 and the amino acid sequence of SEQ ID NO: 14 are also described in Figure 4.

(5-3) Humanization of M23 antibody light chain (5-3-1) hM23-L1 light chain

Substituting the amino acid number 25 (serine acid) of the cM23 antibody light chain shown in SEQ ID NO: 10 with threonine, amino acid number 29 (serine acid) for aspartic acid, amino acid No. 35 (proline) substituted with leucine, amino acid number 38 (isoamine) substituted with arginine, amino acid number 39 (proline) substituted with alanine, amino acid number 41 ( Methyl sulfide Amine acid) is substituted with isoleucine, amino acid number 42 (threonine) is substituted for aspartame, amino acid number 69 (serine) is substituted for valine, amino acid number 89 (su Amine acid) is substituted with serine, amino acid number 103 (asparagine) is substituted for serine, amino acid number 104 (proline) is substituted with leucine, amino acid number 109 (alkaline amine) The acid) is substituted with lysine, the amino acid number 126 (glycine) is substituted with glutamic acid, the amino acid number 130 (leucine) is substituted with lysine, and the amino acid number 135 (alanine) is substituted. The designed humanized M23 antibody light chain substituted with threonine was named "hM23-L1 light chain".

The amino acid sequence of hM23-L1 light chain is described in sequence No. 16. a sequence consisting of amino acid residues 1 to 20 of the amino acid sequence of SEQ ID NO: 16, a sequence consisting of amino acid residues 21 to 135, and amino acids 136 to 240 The sequence consisting of residues corresponds to a message sequence, a light chain variable region, and a light chain constant region. The nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 16 is described in SEQ ID NO: 15. a sequence consisting of nucleotides 1 to 60 of the nucleotide sequence of SEQ ID NO: 15, a sequence consisting of nucleotides 61 to 405, and nucleotides 406 to 720 The sequences each encode a message sequence, a light chain variable region sequence, and a light chain constant region sequence. The nucleotide sequence of SEQ ID NO: 15 and the amino acid sequence of SEQ ID NO: 16 are also described in Figure 5.

(5-3-2) hM23-L2 light chain

Substituting the amino acid number 25 (serine acid) of the cM23 antibody light chain shown in SEQ ID NO: 10 in the sequence listing with threonine, amino acid number 29 (serine acid), and aspartic acid, Amino acid number 35 (guanamine) Acid substituted with leucine, amino acid number 38 (isoamine) substituted with arginine, amino acid number 39 (proline) substituted with alanine, amino acid number 41 (methionine) Substituted as isoleucine, amino acid number 42 (threonine) substituted with aspartame, amino acid number 69 (serine acid) substituted with lysine, amino acid number 89 (threonine) Substituted for serine acid, amino acid number 103 (asparagine) substituted with serine, amino acid number 104 (proline) substituted with leucine, amino acid number 109 (leucine) Designed humanized M23 antibody light chain substituted with lysine, amino acid number 130 (leucine) substituted with lysine, amino acid number 135 (alanine) substituted with threonine, named "hM23 -L2 type light chain".

The amino acid sequence of the hM23-L2 light chain is described in the sequence The serial number of the table is 18. a sequence consisting of amino acid residues 1 to 20 of the amino acid sequence of SEQ ID NO: 18, a sequence consisting of amino acid residues 21 to 135, and amino acids 136 to 240 The sequence consisting of residues corresponds to a message sequence, a light chain variable region, and a 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. a sequence consisting of nucleotides 1 to 60 of the nucleotide sequence of SEQ ID NO: 17, a sequence consisting of nucleotides 61 to 405, and nucleotides 406 to 720 The sequences each encode a message sequence, a light chain variable region sequence, and a light chain constant region sequence. The nucleotide sequence of SEQ ID NO: 17 and the amino acid sequence of SEQ ID NO: 18 are also described in Figure 6.

(Example 6) Construction of humanized M23 antibody expression vector and production of antibody (6-1) Construction of heavy chain expression vector of humanized M23 antibody (6-1-1) Construction of hM23-H1 heavy chain expression vector

A DNA fragment (nucleotide) encoding a DNA sequence encoding the variable region of the hM23-H1 heavy chain represented by nucleotide numbers 58 to 405 of the nucleotide sequence of the hM23-H1 heavy chain represented by SEQ ID NO: 11 was synthesized. Acid No. 36~422) (GENEART, Inc., Artificial Gene Synthesis Service). Using the synthesized DNA fragment as a template, the DNA fragment containing the DNA sequence encoding the variable region of the hM23-H1 heavy chain was amplified with KOD-Plus- (TOYOBO) and the following primer set, using In-Fusion The HD PCR selection kit (CLONTECH) was inserted into the chimeric and humanized antibody IgG1 heavy chain expression vector pCMA-G1 to limit the break of the enzyme BlpI, and the hM23-H1 heavy chain expression vector was constructed. The obtained expression vector was named "pCMA-G1/hM23-H1".

Primer group

5'-AGCTCCCAGATGGGTGCTGAGC-3' (sequence identification number 33: primer EG-Inf-F)

5'-GGGCCCTTGGTGGAGGCTGAGC-3' (SEQ ID NO: 34: primer EG1-Inf-R)

(6-1-2) Construction of hM23-H2 heavy chain expression vector

A DNA fragment (nucleotide) encoding a DNA sequence encoding the variable region of the hM23-H2 heavy chain represented by nucleotide numbers 58 to 405 of the nucleotide sequence of the hM23-H2 heavy chain represented by SEQ ID NO: 13 Acid No. 36 to 422) (GENEART, Inc., artificial gene synthesis service), hM23-H2 heavy chain expression vector was constructed in the same manner as in 6-1-1. The obtained expression vector was named "pCMA-G1/hM23-H2".

(6-2) Construction of a light chain expression vector for humanized M23 antibody (6-2-1) Construction of hM23-L1 light chain expression vector

A DNA fragment encoding a DNA sequence encoding the variable region of the hM23-L1 type light chain represented by nucleotide numbers 61 to 405 of the nucleotide sequence of the hM23-L1 type light chain represented by SEQ ID NO: 15 (nucleotide) Acid number 38~420) (GENEART, artificial gene synthesis service). Using the synthesized DNA fragment as a template, the DNA fragment containing the DNA sequence encoding the variable region of the hM23-L1 type light chain was amplified with KOD-Plus- (TOYOBO) and the following primer set, by using In-Fusion An HD PCR selection kit (CLONTECH) was inserted into the chimeric and humanized antibody light chain expression vector pCMA-LK to restriction the enzyme BsiWI, and the hM23-L1 type light chain expression vector was constructed. The obtained performance vector was named "pCMA-LK/hM23-L1".

Primer group

5'-CTGTGGATCTCCGGCGCGTACGGC-3' (sequence identification number 35: primer CM-LKF)

5'-GGAGGGGGCGGCCACCGTACG-3' (SEQ ID NO: 36: Introduction KCL-Inf-R)

(6-2-2) Construction of hM23-L2 light chain expression vector

A DNA fragment (nucleus) encoding the DNA sequence of 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 Glycoside No. 38-420) (GENEART, Inc., Artificial Gene Synthesis Service), hM23-L2 type light chain expression vector was constructed in the same manner as 6-2-1. The obtained performance vector was named "pCMA-LK/hM23-L2".

(6-3) Production and purification of humanized M23 antibody (6-3-1) Production of humanized M23 antibody

The humanized M23 antibody was produced in the same manner as 4-2-5. The humanized M23 antibody obtained by the combination of pCMA-G1/hM23-H1 and pCMA-LK/hM23-L1 was named "hM23-H1L1" and will be pCMA-G1/hM23-H1 and pCMA-LK/ The humanized M23 antibody obtained by the combination of hM23-L2 was named "hM23-H1L2", and the humanized M23 antibody obtained by the combination of pCMA-G1/hM23-H2 and pCMA-LK/hM23-L2 was named as "hM23-H2L2".

(6-3-2) Purification of humanized M23 antibody

From the culture supernatant obtained in 6-3-1, the antibody was purified in the same manner as in 4-2-6.

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

Step 1: (4-{[(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-di- oxy-2,3,9,10,13 ,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]吲 And [1,2-b]quinolin-1-yl]amino}-4-oxobutylbutyl)carbamic acid tert-butyl butyl ester

4-(tertiary butoxycarbonylamino)butyric acid (0.237 g, 1.13 mmol) was dissolved in dichloromethane (10 mL), and N-hydroxysuccinimide (0.130 g, 1.13 mmol) and 1-B were added. Base-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (0.216 g, 1.13 mmol) was stirred for 1 hour. The obtained mixture was added dropwise to an N,N-dimethylformamide solution (10 mL) to which mesylate (0.500 g, 0.94 mmoL) and triethylamine (0.157 mL, 1.13 mmoL) were added. Stir at room temperature for one day. The solvent was evaporated under reduced pressure, and the residue was purified mjjjjjlilililililililililililililili

^{1 H-NMR (400MHz, DMSO} -d 6) δ: 0.87 (3H, t, J = 7.2Hz), 1.31 (9H, s), 1.58 (1H, t, J = 7.2Hz), 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 = 19.2 Hz), 5.24 (1H, d, J = 18.8 Hz), 5.42 (2H, s), 5.59-5.55 (1H, m), 6.53 (1H, s), 6.78 ( 1H, t, J = 6.3 Hz), 7.30 (1H, s), 7.79 (1H, d, J = 11.0 Hz), 8.40 (1H, d, J = 8.6 Hz).

MS (APCI) m/z: 621 (M+H) ⁺ .

Step 2: 4-Amino-N-[(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-di- oxy-2,3,9, 10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]吲 And [1,2-b]quinolin-1-yl]butanamine trifluoroacetate

The compound obtained in the above Step 1 (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 analyzed by chromatography. [Organic layer of chloroform to chloroform:methanol:water = 7:3:1 (v/v/v)) was purified to give the title compound (0.343 g, quantitative).

^{1 H-NMR (400MHz, DMSO} -d 6) δ: 0.87 (3H, t, J = 9.4Hz), 1.90-1.80 (4H, m), 2.15-2.13 (2H, m), 2.27 (2H, t, J=7.0 Hz), 2.41 (3H, s), 2.87-2.82 (2H, m), 3.18 (2H, dd, J=8.0, 3.7 Hz) 5.15 (1H, d, J = 19.2 Hz), 5.26 (1H) , d, J = 18.8 Hz), 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 = 10.9 Hz), 8.55 (1H, d, J = 8.6 Hz).

MS (APCI) m/z: 521 (M+H) ⁺ .

Step 3: N-(tertiary butoxycarbonyl)glycine decylglycine fluorenyl-L-phenylpropylamine fluorenyl-N-(4-{[(1S,9S)-9-ethyl-5- Fluorin-9-hydroxy-4-methyl-10,13-di-oxy-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3' , 4':6,7]吲 And [1,2-b]quinolin-1-yl]amino}-4-oxobutyl)glycine

N-(tertiary butoxycarbonyl)glycine mercaptoglycine-L-phenylpropylamine mercaptoglycine (0.081 g, 0.19 mmol) was dissolved in dichloromethane (3 mL), and N-hydroxy group was added. Amber succinimide (0.021 g, 0.19 mol) and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (0.036 g, 0.19 mmol) were stirred for 3.5 hours. The obtained mixture was added dropwise to a solution of N,N-dimethylformamide (1.5 mL), which was obtained from the compound obtained in the above step 2 (0.080 g, 0.15 mmol), and stirred at room temperature for 4 hours. The solvent was evaporated under reduced pressure, and the residue was purified mjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjj

^{1 H-NMR (400MHz, DMSO} -d 6) δ: 0.87 (3H, t, J = 7.4Hz), 1.36 (9H, s), 1.71 (2H, m), 1.86 (2H, t, J = 7.8Hz ), 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 = 19.2 Hz), 5.25 (1H, d, J = 18.8 Hz), 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 = 5.7 Hz), 7.80 (1H, d, J = 11.0 Hz), 7.92 (1H, t, J =5.7 Hz), 8.15 (1H, d, J = 8.2 Hz), 8.27 (1H, t, J = 5.5 Hz), 8.46 (1H, d, J = 8.2 Hz).

MS (APCI) m/z: 939 (M+H) ⁺ .

Step 4: Glycidylglycine indenyl-L-phenylpropylamine fluorenyl-N-(4-{[(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl -10,13-di-oxy-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]吲 And [1,2-b]quinolin-1-yl]amino}-4-yloxybutyl)glycine guanamine trifluoroacetate

The compound obtained in the above Step 3 (1.97 g, 2.10 mmol) was dissolved in dichloromethane (7 mL). Trifluoroacetic acid (7 mL) was added to the obtained solution and stirred for 1 hour. The solvent was distilled off under reduced pressure, and toluene was added to be azeotroped, and the obtained residue was purified by chromatography on silica gel column chromatography [chloroform-chloroform:methanol:water=7:3:1 (v/v/v). 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, 9.4 Hz), 3.0 3-3.09 (3H, m), 3.18-3.19 (2H, m), 3.58-3.60 (2H, m ), 3.64 (1H, d, J = 5.9 Hz), 3.69 (1H, d, J = 5.9 Hz), 3.72 (1H, d, J = 5.5 Hz), 3.87 (1H, dd, J = 16.8, 5.9 Hz) ), 4.50-4.56 (1H, m), 5.16 (1H, d, J = 19.2 Hz), 5.25 (1H, d, J = 18.8 Hz), 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-di- oxo-2,5-dihydro-1H-pyrrol-1-yl)propanyl]amine} Ethoxy)ethoxy]propanyl}glycine mercaptoamine-L-phenylpropylamine fluorenyl-N-(4-{[(1S,9S)-9-ethyl-5-fluoro -9-hydroxy-4-methyl-10,13-di- oxy-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3', 4':6,7]吲 And [1,2-b]quinolin-1-yl]amino}-4-oxobutyl)glycine

To a solution of the compound (100 mg, 0.119 mmol) obtained in the above step 4, N,N-dimethylformamide (1.20 mL), diisopropylethylamine (20.8 μL, 0.119 mmoL) and 3- (2-(2-(3-butene diimide acrylamide) ethoxy) ethoxy) propionic acid N-succinimide (50.7 mg, 0.119 mmoL), and stirred at room temperature 1 hour. The solvent was evaporated under reduced pressure, and the residue was purified mjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjj

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 = 7.2 Hz), 2.33 - 2.36 (2H, m), 2.38 (3H, s), 2.76 (1H, dd, J = 13.7, 9.8 Hz), 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, 4.7 Hz), 5.14 (1H, d, J = 18.8 Hz), 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 = 5.5 Hz), 7.78 (1H, d, J = 11.3 Hz), 7.98-8.03 (2H, m), 8.11 (1H, d, J = 7.8 Hz), 8.16 (1H, t, J = 5.7 Hz), 8.23 (1H, t, J = 5.9) Hz), 8.44 (1H, d, J = 9.0 Hz).

MS (APCI) m/z: 1149 (M+H) ⁺ .

Step 6: hM23-H1L1 ADC(1)

(i) Buffer exchange and concentration adjustment of antibodies

The hM23-H1L1 produced in Example 6 was subjected to a NAP-25 column (Cat. No. 17-0852-02, GE Healthcare Japan Corporation) of a Sephadex G-25 support in accordance with the method specified by the manufacturer to contain chlorine. Sodium phosphate (137 mM) and ethyldiaminetetraacetic acid (EDTA, 5 mM) phosphate buffer (10 mM, pH 6.0; in the present specification, referred to as "PBS6.0/EDTA") were equilibrated. To each of the NAP-25 column, 2.5 mL of an aqueous antibody solution was placed, and then fractionated (3.5 mL) was eluted with PBS6.0/EDTA 3.5 mL. The fraction was placed in a container of Amicon Ultra (50,000 MWCO, Millipore Corporation) into an antibody or antibody-drug conjugate solution for centrifugation using a centrifuge (Allegra X-15R, Beckman Coulter, Inc.) (2000G) The antibody or antibody-drug conjugate solution was concentrated by centrifugation at ~3800G for 5-20 minutes. The antibody concentration was measured using a UV analyzer (Nanodrop 1000, Thermo Fisher Scientific Inc.) according to the method specified by the manufacturer. At this time, use the 280 nm absorption coefficient After measuring the antibody concentration (1.65 mL mg-1 cm-1), the antibody concentration was adjusted to 10 mg/mL using PBS6.0/EDTA.

(ii) Reduction of antibodies

This solution (1.00 mL) was taken in a 2 mL polypropylene tube, and 10 mM TCEP (Tokyo Chemical Industry Co., Ltd.) aqueous solution (0.0315 mL; 4.6 equivalents relative to the antibody molecule) and 1 M potassium hydrogen phosphate aqueous solution (Nacalai) were added. Tesque, Inc.; 0.015 mL). After confirming that the pH of the solution was within 7.0 ± 0.1, the disulfide bond in the inner hinge region of the antibody was reduced by incubation at 37 ° C for 2 hours.

(iii) Combination of antibody and drug linker

After the solution was incubated in a normal temperature water bath for 10 minutes, a 10 mM dimethyl sulfoxide solution (0.0579 mL; 9.2 equivalents relative to the antibody molecule) of the compound obtained in the above step 5 was added, and a tube mixer (Tube) was used. Rotator) (MTR-103, ASONE Co., Ltd.) was stirred at room temperature for 60 minutes to bind the drug linker to the antibody. Next, a 100 mM NAC (Sigma-Aldrich Co. LLC) aqueous solution (0.0126 mL; 18.4 equivalents relative to the antibody molecule) was added, and the mixture was stirred at room temperature for 20 minutes to stop the reaction of the drug conjugate.

(iv) purification

The NAP-25 column was equilibrated with a sorbitol (5%) acetate buffer (10 mM, pH 5.5; referred to herein as "ABS"). On this NAP-25 column, the antibody-drug conjugate reaction aqueous solution (2.5 mL) was loaded, and the amount of the buffer specified by the manufacturer was eluted, and the antibody fraction was fractionated. The fractionation was again loaded on the NAP-25 column, and the colloid filtration purification operation in the buffer solution was repeated for 2~3. Secondly, the removal of unbound drug linkers or low molecular compounds (t-(2-carboxyethyl)phosphine hydrochloride (TCEP), N-acetyl-L-cysteine (NAC) and dimethyl groups) A solution of the antibody-drug conjugate of Aachen) was 6.0 mL.

(v) Characteristic evaluation

Common operation A: Determination of the antibody concentration in the antibody-drug conjugate and the average number of bindings of the drug per molecule (1)

The concentration of the binding drug in the antibody-drug conjugate was determined by measuring the UV absorbance at two wavelengths of 280 nm and 370 nm of the antibody-drug conjugate aqueous solution, and the following calculation was performed.

Since the total absorbance in a certain wavelength is equal to the sum of the absorbances of all the absorbed chemical species present in the system (additiveness of absorbance), it is assumed that the Mohr absorption coefficient of the antibody and the drug does not change before and after the combination of the antibody and the drug. The antibody concentration and drug concentration in the antibody-drug conjugate are shown in the following relationship.

A280=AD, 280+AA, 280=εD, 280CD+εA, 280CA (1)

A370=AD,370+AA,370=εD,370CD+εA,370CA (2)

A280 represents the absorbance of the antibody-drug conjugate aqueous solution at 280 nm, A370 represents the absorbance of the antibody-drug conjugate aqueous solution at 370 nm, AA, 280 represents the absorbance of the antibody at 280 nm, and AA, 370 represents the absorbance of the antibody at 370 nm, AD, 280 represents the absorbance of the conjugate precursor in 280 nm, AD, 370 represents the absorbance of the conjugate precursor in 370 nm, εA, 280 represents the Mohr absorbance coefficient of the antibody in 280 nm, and εA, 370 represents the antibody in 370 nm Moer The absorbance coefficient, εD, 280 represents the Mohr absorbance coefficient of the conjugate precursor in 280 nm, εD, 370 represents the Mohr absorbance coefficient of the conjugate precursor in 370 nm, CA represents the antibody concentration in the antibody-drug conjugate, CD Represents the concentration of the drug in the antibody-drug conjugate.

Among them, εA, 280, εA, 370, εD, 280, εD, 370 are used for values prepared in advance (calculation of estimated values or measured values obtained by UV measurement of compounds). ε _{A, 280} is an amino acid sequence derived from an antibody and can be estimated by a known calculation method (Protein Science, 1995, vol. 4, 2411-2423). In the case of hM23-H1L1, hM23-H1L2, and hM23-H2L2, 240400 was used as an estimated value depending on the amino acid sequence. ε _{A, 370} as zero. The molar absorption coefficient (280 nm, 370 nm) of the drug linker can be measured by the reaction of the drug linker with mercaptoethanol and the conversion of the maleimide group to the amber sulfimine sulfide. Coefficient (280 nm, 370 nm) (in the present example, ε _{D, 280} = 4964, ε _{D, 370} = 18982) was used. A ₂₈₀ and A ₃₇₀ of the antibody-drug conjugate aqueous solution were measured, and C _A and C _D were obtained by substituting the equivalent values into the formulas (1) and (2). Further dividing C _D C _A binding and determine the average number of drugs per antibody.

Common Operation B: Determination of the average number of bindings of each molecule of the antibody in the antibody-drug conjugate (2)

The average number of drug bindings per antibody of the antibody-drug conjugate is determined in addition to the aforementioned common operation A, by high speed liquid chromatography (HPLC) analysis using the following method.

[B-1. Modulation of sample for HPLC analysis (reduction of antibody-drug conjugate)]

The antibody-drug conjugate solution (about 1 mg/mL, 60 μL) was mixed with a dithiothreitol (DTT) aqueous solution (100 mM, 15 μL). The mixture was incubated at 37 ° C for 30 minutes, and a sample of the double-sulfur bond between the L chain and the H chain of the antibody-drug conjugate was cut for HPLC analysis.

[B-2. HPLC analysis]

HPLC analysis was carried out under the following measurement conditions.

HPLC system: Agilent 1290 HPLC system (Agilent Technologies)

Detector: UV absorbance meter (measuring wavelength: 280 nm)

Column: PLRP-S (2.1×50mm, 8μm, 1000□; Agilent Technologies, P/N PL1912-1802)

Column temperature: 80 ° C

Mobile phase A: 0.04% aqueous solution of trifluoroacetic acid (TFA)

Mobile phase B: acetonitrile solution containing 0.04% TFA

Gradient program: 29% - 36% (0 minutes - 12.5 minutes), 36% - 42% (12.5 - 15 minutes), 42% - 29% (15 minutes - 15.1 minutes), 29% - 29% (15.1 minutes - 25 minutes)

Sample injection amount: 15 μL

[B-3. Data Analysis]

[B-3-1] L chain (L ₀ ) and H chain (H ₀ ) of the antibody to which the drug is not bound, the drug-bound L chain (the drug is a combined L chain: L ₁ ) and the H chain ( The drug is a bound H chain: H ₁ , the drug is two bound H chain: H ₂ , the drug is three bound H chain: H ₃ ) the system is proportional to the number of bound drugs and the hydrophobicity is increased while maintaining Since the time becomes large, it is eluted in the order of L ₀ , L ₁ , H ₀ , H ₁ , H ₂ , and H ₃ . By comparing the hold times of L ₀ and H ₀ , the detected peaks can be assigned to any of L ₀ , L ₁ , H ₀ , H ₁ , H ₂ , and H ₃ .

[B-3-2] Since the drug linker has UV absorption, the peak area value is corrected according to the following formula using the molar absorption coefficient of the L chain, the H chain and the drug linker in accordance with the number of binding of the drug linker.

Here, the molar absorption coefficient (280 nm) of the L chain and the H chain in each antibody can be determined by a known calculation method (Protein Science, 1995, vol. 4, 2411-2423) using the L chain and H from each antibody. The putative value of the amino acid sequence of the chain. In the case of hM23-H1L1, hM23-H1L2, and hM23-H2L2, according to the amino acid sequence, 41370 is used as the Mohr absorption coefficient of the L chain, and 77810 is used as the estimated value of the Mohr absorption coefficient of the H chain. Further, the molar absorption coefficient (280 nm) of the drug linker was the measured molar absorption coefficient (280 nm) obtained by the above-mentioned common operation A.

[B-3-3] The ratio (%) of each chain peak area in total with respect to the peak area correction value was calculated according to the following formula.

_{A Li, A Hi: L i} , H i each peak area correction value

[B-3-4] The average number of drug bindings per molecule of the antibody in the antibody-drug conjugate was calculated according to the following formula.

Average drug binding number = (L ₀ peak area ratio × 0 + L ₀ peak area ratio × 1 + H ₀ peak area ratio × 0 + H ₁ peak area ratio × 1 + H ₂ peak area ratio × 2 + H ₃ peak area Ratio × 3) / 100 × 2

The following characteristic values were obtained using the common operations A and B. Antibody concentration: 1.50 mg/mL, antibody yield: 9.00 mg (90%), average drug binding number per antibody (n): 6.0 as determined by the common operation A; each molecule of the antibody determined by the common operation B Mean drug binding number (n): 7.1.

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

Step 1: hM23-H1L2 ADC(1)

Using the hM23-H1L2 produced in Example 6 and the compound obtained in Step 5 of Example 7, the title antibody-drug conjugate was obtained in the same manner as in Step 6 of Example 7.

Antibody concentration: 1.54 mg/mL, antibody yield: 9.24 mg (92%), average drug binding number per antibody (n): 6.0 as determined by Common Procedure A; antibody per molecule as determined by Common Procedure B Mean drug binding number (n): 7.1.

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

Step 1: hM23-H2L2 ADC(1)

Using the hM23-H2L2 produced in Example 6 and the compound obtained in Step 5 of Example 7, the title antibody-drug conjugate was obtained by the same procedure as in Step 6 of Example 7.

Antibody concentration: 1.45 mg/mL, antibody yield: 8.70 mg (87%), average drug binding number per antibody (n): 6.0 as determined by the common operation A; each molecule of the antibody determined by the common operation B Mean drug binding number (n): 7.1.

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

Step 1: ({N-[(9H-茀-9-ylmethoxy)carbonyl]glycidyl}amino)methyl acetate

Pyridine (1.16 ml, 14.7 mmol) was added to a mixture containing N-9-fluorenylmethoxycarbonylglycineglycine (4.33 g, 12.2 mmol), tetrahydrofuran (120 ml) and toluene (40.0 ml). Lead tetraacetate (6.84 g, 14.7 mmol) was heated under reflux for 5 hours. After cooling the reaction mixture to room temperature, the insoluble material was removed by filtration over Celite, and concentrated under reduced pressure. The obtained residue was dissolved in ethyl acetate, washed with water and brine, and then evaporated. After the solvent was evaporated under reduced pressure,yield crystals crystals crystals crystals crystals %).

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 = 6.6 Hz), 5.26 (2H, d, J = 7.0 Hz), 5.32 (1H, brs), 6.96 (1H, brs), 7.32 (2H, t, J = 7.3 Hz), 7.41 (2H, t, J =7.3 Hz), 7.59 (2H, d, J = 7.3 Hz), 7.77 (2H, d, J = 7.3 Hz).

Step 2: [({N-[(9H-茀-9-ylmethoxy)carbonyl]]glycidyl}amino)methoxy]benzyl acetate

In a solution of the compound obtained in the above step 1 (3.68 g, 10.0 mmol) and benzyl glycolate (4.99 g, 30.0 mmol) in tetrahydrofuran (40.0 mL), potassium tert-butoxide (2.24 g, 20.0) was added at 0 °C. MmoL) and stirred at room temperature for 15 minutes. Ethyl acetate and water were added to the reaction mixture, and the mixture was evaporated to ethyl acetate. The solvent was distilled off under reduced pressure, and the obtained residue was dissolved in two Acetone (40.0 mL) and water (10.0 mL) were added, and sodium hydrogencarbonate (1.01 g, 12.0 mmoL) and 9-decylmethyl chloroformate (2.59 g, 10.0 mmol) were stirred at room temperature for 2 hours. Water was added to the reaction solution, and the mixture was extracted with ethyl acetate. The solvent was evaporated under reduced pressure.yield eluted eluted elut elut elut elut elut elut elut elut .

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= 7.4 Hz), 7.61 (2H, d, J = 7.4 Hz), 7.79 (2H, d, J = 7.4 Hz).

Step 3: [({N-[(9H-茀-9-ylmethoxy)carbonyl]]glycidyl}amino)methoxy]acetic acid

The compound obtained in the above step 2 (1.88 g, 3.96 mmol) was dissolved in ethanol (40.0 mL) and ethyl acetate (20.0 ml). A palladium carbon catalyst (376 mg) was added and stirred at room temperature for 2 hours under a hydrogen atmosphere. The insoluble material was removed by filtration of celite, and the solvent was evaporated under reduced pressure to give the title compound (1.52 g, quantitative).

^{1 H-NMR (400MHz, DMSO} -d 6) δ: 3.62 (2H, d, J = 6.3Hz), 3.97 (2H, s), 4.18-4.32 (3H, m), 4.60 (2H, d, J = 6.7 Hz), 7.29-7.46 (4H, m), 7.58 (1H, t, J = 5.9 Hz), 7.72 (2H, d, J = 7.4 Hz), 7.90 (2H, d, J = 7.4 Hz), 8.71 (1H, t, J = 6.5Hz).

Step 4: 9H-Indol-9-ylmethyl (2-{[(2-{[(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13- Bilateral oxy-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]吲 And [1,2-b]quinolin-1-yl]amino}-2-oxoethoxyethoxy)methyl]amino}-2-oxoethyl)amine formate

Under ice cooling, the mesylate salt (0.283 g, 0.533 mmol), N-hydroxysuccinimide (61.4 mg, 0.533 mmol), and the compound obtained in the above step 3 (0.205 g, 0.533 mmoL) N,N-Diisopropylethylamine (92.9 μL, 0.533 mmol) and N,N'-dicyclohexylcarbodiimide were added to a solution of N,N-dimethylformamide (10.0 mL). (0.143 g, 0.693 mmoL), and stirred at room temperature for 3 days. The solvent was evaporated under reduced pressure, and the residue was purified mjjjjjjjjjjjj g, 82%).

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 =7.4 Hz), 7.53 (1H, t, J = 6.3 Hz), 7.66 (2H, d, J = 7.4 Hz), 7.75 (1H, d, J = 11.0 Hz), 7.84 (2H, d, J = 7.4) Hz), 8.47 (1H, d, J = 8.6 Hz), 8.77 (1H, t, J = 6.7 Hz).

MS (ESI) m/z: 802 (M+H) ⁺ .

Step 5: N-[(2-{[(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-di- oxy-2,3,9, 10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]吲 And [1,2-b]quinolin-1-yl]amino}-2-oxoethoxyethoxy)methyl]glycine decylamine

Piperazine (1.1 mL) was added to a solution of the compound (0.881 g, 1.10 mmol) of N,N-dimethylformamide (11.0 mL), and the mixture was stirred at room temperature for 2 hours. The solvent was evaporated under reduced pressure to give a mixture. This mixture was used in the following reaction without further purification.

Step 6: N-[(9H-fluoren-9-ylmethoxy)carbonyl]glycine-glycine-indenyl-L-phenylpropylamine-yl-N-[(2-{[(1S,9S)) -9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-di- oxy-2,3,9,10,13,15-hexahydro-1H,12H-benzo[ De]pyrano[3',4':6,7]吲 And [1,2-b]quinolin-1-yl]amino}-2-oxoethoxyethoxy)methyl]glycine decylamine

The mixture obtained in the above step 5 (0.439 mmoL), N-hydroxysuccinimide (0.101 g, 0.878 mmol) and N-[(9H-fluoren-9-ylmethoxy)carbonyl]glycol under ice cooling N, N-dimethylformamide (50.0 mL) was added to a solution of decylglycine decyl-L-phenylalanine (a compound described in JP-A-2002-60351; 0.440 g, 0.878 mmol); N'-Dicyclohexylcarbodiimide (0.181 g, 0.878 mmol) was stirred at room temperature for 4 days. The solvent was evaporated under reduced pressure, and the residue was evaporated, mjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjj

MS (ESI) m / z: 1063 (M + H) +.

Step 7: Glycidylglycine indenyl-L-phenylpropylamine-yl-N-[(2-{[(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl) -10,13-di-oxy-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]吲 And [1,2-b]quinolin-1-yl]amino}-2-oxoethoxyethoxy)methyl]glycine decylamine

Piperazine (0.251 mL, 2.53 mmoL) was added to a solution of the compound (0.269 g, 0.253 mmol) of N,N-dimethylformamide (4.00 mL) obtained in the above step 6 and stirred at room temperature 2 hour. The solvent was evaporated under reduced pressure to give a mixture. This mixture was used in the following reaction without further purification.

Step 8: N-[6-(2,5-di- oxo-2,5-dihydro-1H-pyrrol-1-yl)hexyl]glycine decylglycine fluorenyl-L-phenyl Alaninyl-N-[(2-{[(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-di- oxy-2,3,9 ,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]吲 And [1,2-b]quinolin-1-yl]amino}-2-oxoethoxyethoxy)methyl]glycine decylamine

To a solution of the compound (0.253 mmoL) obtained in the above step 7 in N,N-dimethylformamide (10.0 mL), 6-m-butyleneimine hexane acid N-succinimide group The ester (0.156 g, 0.506 mmol) was stirred at room temperature for 3 days. The solvent was evaporated under reduced pressure, and the residue was purified mjjjjlililililililililililililililili

^{1 H-NMR (400MHz, DMSO} -d 6) δ: 0.83 (3H, t, J = 7.2Hz), 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 = 6.7 Hz), 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 = 11.0 Hz), 7.97 ( 1H, t, J = 5.7 Hz), 8.03 (1H, t, J = 5.9 Hz), 8.09 (1H, d, J = 7.8 Hz), 8.27 (1H, t, J = 6.5 Hz), 8.48 (1H, d, J = 9.0 Hz), 8.60 (1H, t, J = 6.5 Hz).

MS (ESI) m / z: 1034 (M + H) ⁺ .

Step 9: hM23-H1L1 ADC(2)

(i) Buffer exchange and concentration adjustment of antibodies

The hM23-H1L1 produced in Example 6 was subjected to a chlorine-containing method using a NAP-25 column (Cat. No. 17-0852-02, GE Healthcare Japan Corporation) of Sephadex G-25 support according to the method specified by the manufacturer. Sodium (137 mM) and ethyldiamine Tetraacetic acid (EDTA, 5 mM) phosphate buffer (10 mM, pH 6.0; in the case of "PBS6.0/EDTA" in this specification) was equilibrated. To each of the NAP-25 column, 2.5 mL of an aqueous antibody solution was placed, and then fractionated (3.5 mL) was eluted with PBS6.0/EDTA 3.5 mL. This fraction was placed in a container of Amicon Ultra (50,000 MWCO, Millipore Corporation) into an antibody or antibody-drug conjugate solution for centrifugation using a centrifuge (Allegra X-15R, Beckman Coulter, Inc.) (2000G~ 3800G, centrifugation for 5-20 minutes), concentrate the antibody or antibody-drug conjugate solution. The antibody concentration was measured using a UV analyzer (Nanodrop 1000, Thermo Fisher Scientific Inc.) according to the method specified by the manufacturer. At this time, the antibody concentration was measured using an absorbance coefficient of 280 nm (1.65 mL mg-1 cm-1), and then the antibody concentration was adjusted to 10 mg/mL using PBS6.0/EDTA.

(ii) Reduction of antibodies

This solution (1.00 mL) was taken up to a 2 mL polypropylene tube, and 10 mM TCEP (Tokyo Chemical Industry Co., Ltd.) aqueous solution (0.0158 mL; 2.3 equivalents relative to the antibody molecule) and 1 M potassium hydrogen phosphate aqueous solution (Nacalai) were added. Tesque, Inc.; 0.015 mL). After confirming that the pH of the solution was within 7.0 ± 0.1, the disulfide bond in the inner hinge region of the antibody was reduced by incubation at 37 ° C for 2 hours.

(iii) Combination of antibody and drug linker

After the solution was incubated in a room temperature water bath for 10 minutes, dimethyl hydrazine (0.0228 mL) was added. Next, a 10 mM dimethyl hydrazine solution (0.0268 mL; 4.6 equivalents relative to the antibody molecule) of the compound obtained in the above step 8 was added, and a tube mixer (Tube) was used. Rotator) (MTR-103, ASONE Co., Ltd.) was stirred at room temperature for 60 minutes to bind the drug linker to the antibody. Next, a 100 mM NAC (Sigma-Aldrich Co. LLC) aqueous solution (0.0063 mL; 9.2 equivalents per molecule of the antibody) was added, and the mixture was stirred at room temperature for 20 minutes to stop the reaction of the drug conjugate.

(v) purification

The NAP-25 column was equilibrated with sorbitol (5%) in acetate buffer (10 mM, pH 5.5). On this NAP-25 column, the antibody-drug conjugate reaction aqueous solution (2.5 mL) was loaded, and the amount of the buffer specified by the manufacturer was eluted, and the antibody fraction was fractionated. The fractionation fraction was again loaded on the NAP-25 column, and the colloid filtration purification operation in the buffer solution was repeated 2 to 3 times to obtain the unbound drug linker or the low molecular compound (see (2-carboxyl group). 6.0 mL of a solution containing an antibody-drug conjugate of ethyl)phosphine hydrochloride (TCEP), N-ethinyl-L-cysteine (NAC) and dimethylhydrazine.

(v) Characteristic evaluation

Using the common operations A and B (using εD, 280 = 5178 (measured value), εD, 370 = 20217 (actual measured value)), the following characteristic values were obtained.

Antibody concentration: 1.53 mg/mL, antibody yield: 9.18 mg (92%), average drug binding number per antibody (n) of 2.9 as determined by common procedure A; antibody per molecule as determined by common procedure B Mean drug binding number (n): 3.6.

(Example 11) Production of hM23-H1L1 ADC (3)

Step 1: hM23-H1L1 ADC(3)

Using the hM23-H1L1 produced 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. Using a common procedure A and B (use ε _{D, 280} = 5178 _{(found), ε D, 370 = 20217} ( Found)), the following characteristic values is obtained.

Antibody concentration: 1.59 mg/mL, antibody yield: 9.54 mg (95%), average number of drug bindings per molecule of antibody determined by the common operation A (n): 5.4; each molecule of the antibody determined by the common operation B Mean drug binding number (n): 6.7.

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

Step 1: hM23-H1L2 ADC(2)

Using the hM23-H1L2 produced in Example 6 and the compound obtained in Step 8 of Example 10, the title antibody-drug conjugate was obtained by the same procedure as in Step 10 of Example 10.

Antibody concentration: 1.52 mg/mL, antibody yield: 9.12 mg (91%), average number of drug bindings per molecule of antibody determined by the common operation A (n): 3.0; each molecule of the antibody determined by the common operation B Mean drug binding number (n): 3.7.

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

Step 1: hM23-H1L2 ADC(3)

Using the hM23-H1L2 produced in Example 6 and the compound obtained in Step 8 of Example 10, the title antibody-drug conjugate was obtained by the same procedure as in Step 6 of Example 7. Using the common operations A and B (using ε _{D, 280} = 5178 (measured value), ε _{D, 370} = 20217 (measured value)), the following characteristic values were obtained.

Antibody concentration: 1.57 mg/mL, antibody yield: 9.42 mg (94%), the average number of bindings of the drug per molecule of the antibody determined by the common operation A (n): 5.7; the average number of bindings of the drug per molecule of the antibody determined by the common operation B (n): 7.1.

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

Step 1: hM23-H2L2 ADC(2)

Using the hM23-H2L2 produced in Example 6 and the compound obtained in Step 8 of Example 10, the title antibody-drug conjugate was obtained by the same procedure as in Step 10 of Example 10.

Antibody concentration: 1.50 mg/mL, antibody yield: 9.00 mg (90%), the average number of drug bindings per molecule of the antibody determined by the common operation A (n): 3.0; the average number of drug bindings per molecule of the antibody measured by the common operation B (n): 3.8.

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

Step 1: hM23-H2L2 ADC(3)

Using the hM23-H2L2 produced in Example 6 and the compound obtained in Step 8 of Example 10, the title antibody-drug conjugate was obtained by the same procedure as in Step 6 of Example 7. Using the common operations A and B (using ε _{D, 280} = 5178 (measured value), ε _{D, 370} = 20217 (measured value)), the following characteristic values were obtained.

Antibody concentration: 1.51 mg/mL, antibody yield: 9.06 mg (91%), the average number of bindings of the drug per molecule of the antibody determined by the common operation A (n): 5.8; the average number of bindings of the drug per molecule of the antibody measured by the common operation B (n): 7.1.

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

Step 1: [2-(2-{[(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-di- oxy-2,3,9, 10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]吲 And [1,2-b]quinolin-1-yl]amino}-2-oxoethoxyethoxy)ethyl]aminecarboxylic acid tert-butyl butyl ester

Using {2-[(tris-butoxycarbonyl)amino]ethoxy}acetic acid (J. Med. Chem., 1992, Vol. 35, p. 2928; 1.55 g, 6.01 mmol) The title compound (2.56 g, 73%) was obtained.

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 = 15.1 Hz), 4.05 (1H, d, J = 15.1 Hz), 5.14 (1H, d, J = 18.7 Hz), 5.22 (1H, d, J = 18.7 Hz), 5.40 (1H, d, J = 16.6 Hz), 5.44 (1H, d, J = 16.6 Hz), 5.59- 5.66 (1H, m), 6.53 (1H, s), 6.86 (1H, t, J = 5.4 Hz), 7.31 (1H, s), 7.79 (1H, d, J = 10.9 Hz), 8.49 (1H, d , J = 9.1 Hz).

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-di-oxy -2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]吲 And [1,2-b]quinolin-1-yl]acetamide trifluoroacetate

The compound obtained in the above step 1 (1.50 g, 2.36 mol) was reacted in the same manner as in Step 2 of Example 7 to give 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 = 5.1 Hz), 3.15-3.23 (2H, m), 3.71 (2H, t, J = 5.1 Hz), 4.10 (2H, s), 5.19 (1H, d, J = 18.7) Hz), 5.24 (1H, d, J = 18.7 Hz), 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 = 9.1 Hz).

MS (APCI) m/z: 537 (M+H) ⁺ .

Step 3: N-(tertiary butoxycarbonyl)glycine mercaptoglycine fluorenyl-L-phenylpropylamine fluorenyl-N-[2-(2-{[(1S,9S)-9-ethyl) -5-fluoro-9-hydroxy-4-methyl-10,13-di-oxy-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyran [3',4':6,7]吲 And [1,2-b]quinolin-1-yl]amino}-2-oxoethoxyethoxy)ethyl]glycine decylamine

The compound obtained in the above step 2 (554 mg, 0.85 mmol) was obtained to give the title compound (775 mg, 95%).

^{1 H-NMR (400MHz, DMSO} -d 6) δ: 0.85 (3H, t, J = 7.3Hz), 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, 9.8 Hz), 2.95 (1H, dd, J = 13.4, 4.3 Hz), 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, 5.5 Hz), 4.03 (2H, s), 4.39-4.47 (1H, m), 5.17 (1H, d, J =18.9 Hz), 5.25 (1H, d, J = 18.9 Hz), 5.41 (1H, d, J = 16.8 Hz), 5.45 (1H, d, J = 16.8 Hz), 5.57-5.64 (1H, m), 6.54 (1H, s), 6.99 (1H, t, J = 5.8 Hz), 7.13 - 7.26 (5H, m), 7.31 (1H, s), 7.76-7.82 (2H, m), 7.90 (1H, t, J = 5.2 Hz), 8.13 (1H, d, J = 7.9 Hz), 8.27 (1H, t, J = 5.8 Hz), 8.49 (1H, d, J = 8.5 Hz).

MS (APCI) m/z: 955 (M+H) ⁺ .

Step 4: Glycidylglycine decyl-L-phenylpropylamine fluorenyl-N-[2-(2-{[(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4) -methyl-10,13-di- oxy-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7 ]吲 And [1,2-b]quinolin-1-yl]amino}-2-oxoethoxyethoxy)ethyl]glycine decyl trifluoroacetate

The compound obtained in the above step 3 (630 mg, 0.659 mmol) was obtained.

^{1 H-NMR (400MHz, DMSO} -d 6) δ: 0.86 (3H, t, J = 7.3Hz), 1.79-1.90 (2H, m), 2.13-2.22 (2H, m), 2.39 (3H, s) , 2.71 (1H, dd, J = 13.4, 10.1 Hz), 2.99 (1H, dd, J = 13.4, 4.3 Hz), 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, 5.8 Hz), 4.04 (2H, s), 4.52 (1H, td, J = 9.0, 4.1 Hz), 5.17 (1H, d, J = 18.9) Hz), 5.25 (1H, d, J = 18.9 Hz), 5.41 (1H, d, J = 16.5 Hz), 5.45 (1H, d, J = 16.5 Hz), 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 = 11.0 Hz), 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-Di-Ethyloxy-2,5-dihydro-1H-pyrrol-1-yl)hexyl]glycine-glycine-indenyl-L-phenyl Alaninyl-N-[2-(2-{[(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-di- oxo-2,3 ,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]吲 And [1,2-b]quinolin-1-yl]amino}-2-oxoethoxyethoxy)ethyl]glycine decylamine

Triethylamine (31.4 μL, 0.22 mmoL) was used instead of diisopropylethylamine, and N-succinimide (65.3 mg, 0.31 mmoL) was used instead of 6-maleimide. 3-(2-(2-(3-butene diimide acrylamide) ethoxy) ethoxy) propionic acid N-succinimide, in the same manner as in Step 5 of Example 7, The title compound was obtained (162 mg, 62%).

^{1 H-NMR (400MHz, DMSO} -d 6) δ: 0.86 (3H, t, J = 7.6Hz), 1.13-1.22 (2H, m), 1.40-1.51 (4H, m), 1.78-1.90 (2H, m), 2.09 (2H, t, J = 7.6 Hz), 2.14 - 2.21 (2H, m), 2.39 (3H, s), 2.74 (1H, dd, J = 13.6, 9.7 Hz), 2.96 (1H, dd , J=13.6, 4.5 Hz), 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, 5.7 Hz), 4.03 (2H, s), 4.42 (1H, td, J = 8.6, 4.2 Hz), 5.17 (1H, d, J = 18.7 Hz), 5.25 (1H, d, J = 18.7 Hz), 5.40 (1H, d, J = 17.2 Hz), 5.44 (1H, d, J = 17.2 Hz), 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 = 5.7 Hz), 8.03 - 8.11 (2H, m), 8.22 (1H) , t, J = 5.7 Hz), 8.47 (1H, d, J = 9.1 Hz).

MS (APCI) m/z: 1048 (M+H) ⁺ .

Step 6: hM23-H1L1 ADC(4)

Using the hM23-H1L1 produced in Example 6 and the compound obtained in the above Step 5, the title antibody-drug conjugate was obtained by the same procedure as in Step 10 of Example 10. Using the common operations A and B (using ε _{D, 280} = 5193 (measured value), ε _{D, 370} = 20347 (actual measured value)), the following characteristic values were obtained.

Antibody concentration: 1.47 mg/mL, antibody yield: 8.82 mg (88%), average drug binding number per molecule of antibody determined by the common operation A (n): 2.8; antibody per molecule measured by the common operation B Mean drug binding number (n): 3.6.

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

Step 1: hM23-H1L1 ADC(5)

The title antibody-drug conjugate was obtained in the same manner as in the step 6 of Example 7 using the compound obtained in the mH23-H1L1 of Example 6 and the compound obtained in the step 5 of Example 16. Using the common operations A and B (using ε _{D, 280} = 5193 (measured value), ε _{D, 370} = 20347 (actual measured value)), the following characteristic values were obtained.

Antibody concentration: 1.47 mg/mL, antibody yield: 8.82 mg (88%), the average number of bindings of the drug per molecule of the antibody determined by the common operation A (n): 5.3; the average number of bindings of the drug per molecule of the antibody measured by the common operation B (n): 6.7.

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

Step 1: hM23-H1L2 ADC(4)

The title antibody-drug conjugate was obtained in the same manner as in the step 9 of Example 10, using the compound obtained in the m. Using the common operations A and B (using ε _{D, 280} = 5193 (measured value), ε _{D, 370} = 20347 (actual measured value)), the following characteristic values were obtained.

Antibody concentration: 1.49 mg/mL, antibody yield: 8.94 mg (89%), the average number of bindings of the drug per molecule of the antibody measured by the common operation A (n): 3.0; the average number of bindings of the drug per molecule of the antibody measured by the common operation B (n): 3.4.

(Example 19) Production of hM23-H1L2 ADC (5)

Step 1: hM23-H1L2 ADC(5)

Using the hM23-H1L2 produced in Example 6 and the compound obtained in Step 5 of Example 16, the title antibody-drug conjugate was obtained by the same procedure as in Step 6 of Example 7. Using the common operations A and B (using ε _{D, 280} = 5193 (measured value), ε _{D, 370} = 20347 (actual measured value)), the following characteristic values were obtained.

Antibody concentration: 1.57 mg/mL, antibody yield: 9.42 mg (94%), average number of drug bindings per molecule of antibody determined by the common operation A (n): 5.8; each molecule of the antibody determined by the common operation B Mean drug binding number (n): 6.9.

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

Step 1: hM23-H2L2 ADC(4)

Using the hM23-H1L1 produced in Example 6 and the compound obtained in Step 5 of Example 16 above, the title antibody-drug conjugate was obtained by the same procedure as in Step 10 of Example 10. Using the common operations A and B (using ε _{D, 280} = 5193 (measured value), ε _{D, 370} = 20347 (actual measured value)), the following characteristic values were obtained.

Antibody concentration: 1.48 mg/mL, antibody yield: 8.88 mg (89%), the average number of bindings of the drug per molecule of the antibody determined by the common operation A (n): 3.1; the average number of bindings of the drug per molecule of the antibody measured by the common operation B (n): 3.9.

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

Step 1: hM23-H2L2 ADC(5)

Using the hM23-H1L1 produced in Example 6 and the compound obtained in the above Step 16 of Example 16, the title antibody-drug conjugate was obtained by the same procedure as in Step 6 of Example 7. Using the common operations A and B (using ε _{D, 280} = 5193 (measured value), ε _{D, 370} = 20347 (actual measured value)), the following characteristic values were obtained.

Antibody concentration: 1.56 mg/mL, antibody yield: 9.36 mg (94%), the average number of bindings of the drug per molecule of the antibody determined by the common operation A (n): 5.9; the average number of bindings of the drug per molecule of the antibody determined by the common operation B (n): 7.1.

(Example 22) Production of hM23-H1L1 ADC (6)

Step 1: hM23-H1L1 ADC(6)

(i) Buffer exchange and concentration adjustment of antibodies

The hM23-H1L1 produced in Example 6 was subjected to a NAP-25 column (Cat. No. 17-0852-02, GE Healthcare Japan Corporation) of a Sephadex G-25 support in accordance with the method specified by the manufacturer to contain chlorine. Sodium phosphate (137 mM) and ethyldiaminetetraacetic acid (EDTA, 5 mM) phosphate buffer (10 mM, pH 6.0; in the present specification, referred to as "PBS6.0/EDTA") were equilibrated. To each of the NAP-25 column, 2.5 mL of an aqueous antibody solution was placed, and then fractionated (3.5 mL) was eluted with PBS6.0/EDTA 3.5 mL. The fraction was placed in a container of Amicon Ultra (50,000 MWCO, Millipore Corporation) into an antibody or antibody-drug conjugate solution for centrifugation using a centrifuge (Allegra X-15R, Beckman Coulter, Inc.) (2000G) The antibody or antibody-drug conjugate solution was concentrated by centrifugation at ~3800G for 5-20 minutes. The antibody concentration was measured using a UV analyzer (Nanodrop 1000, Thermo Fisher Scientific Inc.) according to the method specified by the manufacturer. At this time, the antibody concentration was measured using an absorbance coefficient of 280 nm (1.65 mL mg ^-1 cm ^-1 ), and then the antibody concentration was adjusted to 10 mg/mL using PBS6.0/EDTA.

(ii) Reduction of antibodies

This solution (10 mL) was taken in a 50 mL polypropylene tube, and a 10 mM aqueous solution of TCEP (0.377 mL; 5.5 equivalents per molecule of the antibody) and 1 M aqueous potassium dihydrogen phosphate solution (0.287 mL) were added. After confirming that the pH of the solution was within 7.2 ± 0.1, the disulfide bond in the hinge region of the antibody was reduced by incubation at 37 ° C for 2 hours.

(iii) Combination of antibody and drug linker

After the solution was incubated in a water bath at 15 ° C for 5 minutes, a 10 mM dimethyl sulfoxide solution (0.617 mL; 9.0 equivalents per molecule of the antibody) of the compound obtained in the step 8 of Example 10 was added, and at 15 ° C. The mixture was stirred for 30 minutes in a water bath to allow the drug linker to bind to the antibody. Next, a 100 mM aqueous solution of NAC (0.088 mL; 12.9 equivalents per molecule of the antibody) was added, and the mixture was stirred at room temperature for 20 minutes to stop the reactivity of the drug linker.

(iv) purification

The NAP-25 column was equilibrated with sorbitol (5%) in acetate buffer (10 mM, pH 5.5). On this NAP-25 column, the antibody-drug conjugate reaction aqueous solution (2.5 mL) was loaded, and the amount of the buffer specified by the manufacturer was eluted, and the antibody fraction was fractionated. The fraction was again loaded on the NAP-25 column and filtered by colloidal dissolution. The purification operation is repeated 2~3 times in total to obtain the removal of unbound drug linker or low molecular compound (t-(2-carboxyethyl)phosphine hydrochloride (TCEP), N-acetyl-L-cysteine (NAC) and dimethyl hydrazine) 35 mL of a solution containing an antibody-drug conjugate.

(v) Characteristic evaluation

Using the common operations A and B (using ε _{D, 280} = 5178 (measured value), ε _{D, 370} = 20217 (measured value)), the following characteristic values were obtained.

Antibody concentration: 2.59 mg/mL, antibody yield: 90.7 mg (91%), average number of drug bindings per molecule of antibody determined by the common operation A (n): 6.4; each molecule of the antibody determined by the common operation B Mean drug binding number (n): 7.8.

(Example 23) Production of hM23-H1L1 ADC (7)

Step 1: hM23-H1L1 ADC(7)

(i) Buffer exchange and concentration adjustment of antibodies

The hM23-H1L1 produced in Example 6 was subjected to a NAP-25 column (Cat. No. 17-0852-02, GE Healthcare Japan Corporation) of a Sephadex G-25 support in accordance with the method specified by the manufacturer to contain chlorine. Sodium phosphate (137 mM) and ethyldiaminetetraacetic acid (EDTA, 5 mM) phosphate buffer (10 mM, pH 6.0; in the present specification, referred to as "PBS6.0/EDTA") were equilibrated. To each of the NAP-25 column, 2.5 mL of an aqueous antibody solution was placed, and then fractionated (3.5 mL) was eluted with PBS6.0/EDTA 3.5 mL. The fraction was placed in a container of Amicon Ultra (50,000 MWCO, Millipore Corporation) into an antibody or antibody-drug conjugate solution for centrifugation using a centrifuge (Allegra X-15R, Beckman Coulter, Inc.) (2000G) The antibody or antibody-drug conjugate solution was concentrated by centrifugation at ~3800G for 5-20 minutes. The antibody concentration was measured using a UV analyzer (Nanodrop 1000, Thermo Fisher Scientific Inc.) according to the method specified by the manufacturer. At this time, the antibody concentration was measured using an absorbance coefficient of 280 nm (1.65 mL mg ^-1 cm ^-1 ), and then the antibody concentration was adjusted to 10 mg/mL using PBS6.0/EDTA.

(ii) Reduction of antibodies

This solution (10 mL) was taken in a 50 mL polypropylene tube, and a 10 mM aqueous solution of TCEP (0.178 mL; 2.6 equivalents per molecule of the antibody) and 1 M potassium hydrogen phosphate aqueous solution (0.287 mL) were added. After confirming that the pH of the solution was within 7.2 ± 0.1, the disulfide bond in the inner hinge region of the antibody was reduced by incubation at 37 ° C for 1 hour.

(iii) Combination of antibody and drug linker

After the solution was incubated in a water bath at 15 ° C for 5 minutes, a 10 mM dimethyl sulfoxide solution (0.308 mL; 4.5 equivalents per molecule of the antibody) of the compound obtained in the step 8 of Example 10 was added, and at 15 ° C. The mixture was stirred for 1 hour in a water bath to bind the drug linker to the antibody. Next, a 100 mM aqueous solution of NAC (0.088 mL; 12.9 equivalents per molecule of the antibody) was added, and the mixture was stirred at room temperature for 20 minutes to stop the reactivity of the drug linker.

(iv) purification

The NAP-25 column was equilibrated with sorbitol (5%) in acetate buffer (10 mM, pH 5.5). On this NAP-25 column, the antibody-drug conjugate reaction aqueous solution (2.5 mL) was loaded, and the amount of the buffer specified by the manufacturer was eluted, and the antibody fraction was fractionated. The fractionation fraction was again loaded on the NAP-25 column, and the colloid filtration purification operation in the buffer solution was repeated 2 to 3 times to obtain the unbound drug linker or the low molecular compound (see (2-carboxyl group). 35 mL of a solution containing an antibody-drug conjugate of ethyl)phosphine hydrochloride (TCEP), N-acetyl-L-cysteine (NAC) and dimethyl hydrazine.

(v) Characteristic evaluation

Using the common operations A and B (using εD, 280 = 5178 (measured value), εD, 370 = 20217 (actual measured value)), the following characteristic values were obtained.

Antibody concentration: 2.63 mg/mL, antibody yield: 92.1 mg (92%), average drug binding number per molecule of antibody determined by the common operation A (n): 3.3; antibody per molecule measured by the common operation B Mean drug binding number (n): 3.9.

(Test Example 1) Internalization activity of cM23 antibody

The internalization activity of the cM23 antibody was investigated according to Mol Biol Cell. 2004; 15: 5268-5282.

cM23, a transferrin receptor antibody (positive control group) and a control group hIgG1 (negative control group) were identified by Alexa488. Each Alexa488-labeled antibody was adjusted to 2 μg/mL with ice-cold RPMI 1640 supplemented with 10% FBS, and 50 μL of each column in a 96-well U chassis. The semi-fused NCI-H322 cells were recovered, suspended in ice-cold RPMI 1640 supplemented with 10% FBS to 4 × 10 ⁶ cells/mL, and 50 μL of each of the cells was dispensed into the well-dispensed disk. After stirring in a flat-plate mixer, the mixture was incubated at 4 ° C for 1 hour, and 150 μL of ice-cold washing buffer (PBS supplemented with 3% FBS) was added, and the supernatant was removed by centrifugation. Further, 200 μL of ice-cold washing buffer was added, and the supernatant was removed twice.

(1) Samples cultured only at 4 ° C: Add ice-cold wash buffer to the cells that have been washed as above (add 1000-fold diluted LIVE/DEAD Fixable Near-IR Dead Cell Stain Kit (Invitrogen #L10119) Or 75 μL of Anti-Alexa-488 solution (25 μg/mL anti-Alexa-488 antibody, 1000-fold diluted dead cell dye), transferred to a fresh plate and incubated on ice for 30 minutes. 150 μL/well was added with ice-cold fixative solution (PBS supplemented with 2% PFA and 3% FBS), 150 μL of fixative solution and 2:1 solution of wash buffer were dispensed into the empty wells, 100 μL of sample was added and FACS Canto (FACS Canto) Becton Dickinson) measures fluorescence.

(2) Culture samples at 37 ° C: Add 100 μL of RPMI 1640 with 10% FBS warmed to 37 ° C in the above-mentioned washed cells, suspend in a flat-plate mixer, and transfer to a 96-well V-bottom plate. Incubate at 3 hours or 3 hours and at 37 ° C, 5% CO ₂ . 100 μL of ice-cold wash buffer was added and the whole amount was transferred to a 96-well round bottom plate and centrifuged. After removing the supernatant, add 75 μL of ice-cold wash buffer (add 1000-fold diluted dead cell dye) or Anti-Alexa-488 solution (25 μg/mL, add 1000-fold diluted dead cell dye), and transfer to a new plate and ice. Incubate for 30 minutes. An ice-cold fixing solution was added at 150 μL/well, and 150 μL of a 2:1 solution of the fixing solution and the washing buffer was dispensed into each of the empty wells, 100 μL of the sample was added, and fluorescence was measured by FACS Canto.

As shown in Figure 8, for NCI-H322 cells, by Using the Alexa 488 marker cM23 evaluation, an internalization activity of about 16% of cM23 was observed. Further, the TfR Ab in Fig. 8 is an abbreviation of the transferrin receptor antibody identified by Alexa488.

(Test Example 2) Determination of antigen binding activity of hM23 antibody (2-1) Determination of antigen binding activity using antibody (culture supernatant)

The dissociation constant of the antibody and antigen (Recombinant Human SLC3A2/MDU1 (Sino Biological Inc.)) was determined by using Biacore T200 (GE Healthcare Bio-Sciences) as an antibody to immobilized anti-human IgG (Fc) antibody. The capture method is performed by capturing the antigen and measuring the antigen as an analyte. An anti-human IgG (Fc) antibody (Human anibody capture kit, GE Healthcare Bio-Sciences) was covalently bound to about 1000 RU by an amine coupling method to Sensorchip CM5 (GE Healthcare Bio-Sciences). The reference cells were also immobilized in the same manner. 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 as a running buffer. The measurement-side cells in which the anti-human IgG (Fc) antibody was immobilized were added at a flow rate of 10 μL/min for 60 seconds to obtain a 20-30 RU capture method, and the culture containing the antibody adjusted by the HBS-EP+ concentration was added. The supernatant (culture supernatant obtained in 6-3-1). The dilution series solution (0.077-100 nM and 0 nM) of the antigen was added to the reference cells and the assay side cells at a flow rate of 30 μL/min for 300 seconds, followed by monitoring the dissociation phase for 600 seconds. As the regeneration solution, at a flow rate of 10μL / min was added 3M MgCl ₂ 60 seconds, add 2 times. The analysis of the data was performed using the 1:1 Binding mode of the analysis software (Biacore T200 Evaluation Software, version 1.0), and the binding rate constant kon, the dissociation rate constant koff, and the dissociation constant (KD; KD=koff/kon) were calculated.

(2-2) Determination of antigen binding activity using purified antibodies

The binding rate constant, the dissociation rate constant, and the dissociation constant were calculated in the same manner as described above using the antibody obtained in 6-3-2.

Table 2 Using purified antibodies as binding activity of antibody samples

(Test Example 3) Cytotoxic activity of ADC

Human plasmacytoma NCI-H929, human Burkitt's lymphoma Ramos, human melanoma MeWo were purchased from the American Type Culture Collection. NCI-H929 cells and Ramos cell lines were seeded at 2×10 ³ cells/well, MeWo cell line was seeded at 1×10 ³ cells/well in 96-well plates, and each antibody and ADC diluted in medium were added. Concentrations: 0.1, 1, 10, 100, 1000 ng/mL, each n=2). The cells were cultured for 6 days in an incubator set at 37 ° C and 5% CO ₂ , 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 regression linearity. The results are shown in the table.

Cell survival rate (%) = 100 × (T-B) / (C-B)

T: ADC treatment of cell luminescence

C: average luminescence of untreated cell wells (n=2)

B: average luminescence of blank holes (n=2)

hM23H1L1, hM23H1L2 and hM23H2L2 did not exhibit in vitro cytotoxic activity, but their ADCs exhibited cytotoxic activity.

(Test Example 4) Antitumor effect of ADC (1)

Five-week-old female SCID mice (Charles River, Japan) were domesticated for 7 days under SPF conditioning prior to experimental use. The mice were fed a solid feed (FR-2, Funabashi Farms Co., Ltd.) and water was added with 1 to 5 ppm of chlorine. Ramos cells (8 × 10 ⁷ cells/mL) suspended in physiological saline were subcutaneously transplanted into the right axillary portion (day 0) of SCID mice at 100 μL/mouse. On the 10th day, the long diameter and the short diameter of the tumor were measured with an electronic 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 × long diameter (mm) × [short diameter (mm)] ²

On the day of grouping, the antibody or ADC was diluted to 0.3 mg/mL with ABS and administered intravenously at 10 mL/kg (3 mg/kg). Thereafter, the tumor diameter was measured twice a week. The change in tumor volume is shown in Figures 9-11.

Although hM23H1L1, hM23H1L2 and hM23H2L2 exhibited tumor growth inhibitory activity, their ADCs showed stronger antitumor activity than antibodies alone.

(Test Example 5) Antitumor effect of ADC (2)

Five-week-old female SCID mice (Charles River, Japan) were domesticated for 9 days under SPF conditioning prior to experimental use. The mice were fed a solid feed (FR-2, Funabashi Farms Co., Ltd.) and water was added with 1 to 5 ppm of chlorine. Ramos cells (5 × 10 ⁷ cells/mL) suspended in physiological saline were subcutaneously transplanted into the right axillary portion (day 0) of SCID mice at 100 μL/mouse. On the eleventh day, the long diameter and the short diameter of the tumor were measured with an electronic digital caliper (CD-15CX, Mitutoyo Corp.), and grouping was performed based on the tumor volume calculated according to the following formula (n=8).

Tumor volume (mm ³ ) = 1/2 × long diameter (mm) × [short diameter (mm)] ²

On the day of grouping, the antibody or ADC was diluted to 0.3 mg/mL with ABS and administered intravenously at 10 mL/kg (3 mg/kg). Thereafter, the tumor diameter was measured twice a week. The change in tumor volume is shown in Figure 12.

Although hM23-H1L1 exhibited tumor growth inhibitory activity, ADC showed stronger antitumor activity than antibody alone.

[sequence table non-keyword text]

SEQ ID NO: 1: Nucleotide sequence of cDNA encoding the variable region of the heavy chain of the M23 antibody

SEQ ID NO: 2: amino acid sequence of the variable region of the heavy chain of the M23 antibody

SEQ ID NO: 3: Nucleotide sequence of cDNA encoding the variable region of the light chain of the M23 antibody

SEQ ID NO: 4: amino acid sequence of the variable region of the light chain of the M23 antibody

SEQ ID NO: 5: Nucleotide sequence encoding human kappa chain secretion message and human kappa chain constant region

SEQ ID NO: 6: Nucleotide sequence encoding human heavy chain secretion information and human IgG1 constant region

SEQ ID NO: 7: Nucleotide sequence of the chimeric M23 antibody heavy chain

SEQ ID NO: 8: Amino acid sequence of the chimeric M23 antibody heavy chain

SEQ ID NO: 9: Nucleotide sequence of chimeric M23 antibody light chain

SEQ ID NO: 10: Amino acid sequence of chimeric M23 antibody light chain

SEQ ID NO: 11: Nucleotide sequence of hM23-H1 heavy chain

SEQ ID NO: 12: amino acid sequence of hM23-H1 heavy chain

SEQ ID NO: 13: Nucleotide sequence of hM23-H2 heavy chain

SEQ ID NO: 14: amino acid sequence of hM23-H2 heavy chain

SEQ ID NO: 15: Nucleotide sequence of hM23-L1 light chain

SEQ ID NO: 16: Amino acid sequence of hM23-L1 light chain

SEQ ID NO: 17: Nucleotide sequence of hM23-L2 light chain

SEQ ID NO: 18: amino acid sequence of hM23-L2 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 the primer mG2aVR1

SEQ ID NO: 26: Nucleotide sequence of the 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: Nucleotide sequence of the primer M23H-R

SEQ ID NO: 31: Nucleotide sequence of the primer M23L-F

SEQ ID NO: 32: Nucleotide sequence of the primer M23L-R

SEQ ID NO: 33: Nucleotide sequence of the primer EG-Inf-F

SEQ ID NO: 34: Nucleotide sequence of the primer EG1-Inf-R

SEQ ID NO: 35: Nucleotide sequence of primer CM-LKF

SEQ ID NO: 36: Nucleotide sequence of the primer KCL-Inf-R

SEQ ID NO: 37: Nucleotide sequence of human CD98 heavy chain

SEQ ID NO: 38: amino acid sequence of human CD98 heavy chain

<110> First Three Co., Ltd.

<120> Anti-CD98 Antibody - Drug Conjugates

<130> PD20-9010W0

<150> JP2014-064591

<151> 2014-03-26

<160> 38

<170> PatentIn version 3.5

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Claims (37)

An anti-CD98 antibody-drug conjugate comprising an anti-CD98 antibody, a linker and a drug anti-CD98 antibody-drug conjugate or a pharmacologically acceptable salt thereof, wherein the linker is selected from the group consisting of the following formula Linker: -(succinimide-3-yl-N)-CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ -C(=O)-GGFG-NH-CH ₂ -O-CH ₂ -C(= O)-;-(Amber 醯imino-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-(amber succinimide-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)-, the drug is a compound represented by the following formula: The nitrogen atom of the amine group at position 1 of the drug binds to the carbonyl moiety of the linker, and the anti-CD98 anti-system binds to the amber quinone group of the linker. The antibody-drug conjugate of claim 1, or a pharmacologically acceptable salt thereof, wherein the average number of bindings per one antibody is in the range of 2 to 8. The antibody-drug conjugate of claim 1, or a pharmacologically acceptable salt thereof, wherein the average number of bindings per one antibody is in the range of 3 to 6. The antibody-drug conjugate of claim 2 or 3, or a pharmacologically acceptable salt thereof, wherein the average number of bindings per 1 antibody is determined by reverse phase chromatography (RPC). The antibody-drug conjugate of claim 1, or a pharmacologically acceptable salt thereof, wherein the number of binding of the drug per one antibody is 2, 4, 6, or 8. The antibody-drug conjugate according to any one of claims 1 to 5, wherein the linker is of the formula: -(succinimide-3-yl-N)-CH ₂ CH, or a pharmacologically acceptable salt thereof ₂ CH ₂ CH ₂ CH ₂ -C(=O)-GGFG-NH-CH ₂ -O-CH ₂ -C(=O)-. The antibody-drug conjugate according to any one of claims 1 to 5, wherein the linker is of the formula: -(succinimide-3-yl-N)-CH ₂ CH, or a pharmacologically acceptable salt thereof ₂ CH ₂ CH ₂ CH ₂ -C(=O)-GGFG-NH-CH ₂ CH ₂ -O-CH ₂ -C(=O)-. The antibody-drug conjugate of any one of claims 1 to 5, wherein the linker is of the formula: -(Amber succinimide-3-yl-N)-CH ₂ CH, or a pharmacologically acceptable salt thereof 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 )-. The antibody-drug conjugate of any one of claims 1 to 8, or a pharmacologically acceptable salt thereof, wherein the anti-CD98 anti-system and the amino acid residue of SEQ ID NO: 462-541 of SEQ ID NO: 38 The parts are combined. The antibody-drug conjugate according to any one of claims 1 to 9, wherein the anti-CD98 antibody comprises: or an amino acid sequence represented by SEQ ID NO: 19, or a pharmacologically acceptable salt thereof CDRH1 consisting of an amino acid sequence in which one or several amino acid residues are added, deleted or substituted; the amino acid sequence represented by SEQ ID NO: 20 or one or several amines in the amino acid sequence a CDRH2 consisting of an amino acid sequence to which a base acid residue is added, deleted or substituted; an amino acid sequence represented by SEQ ID NO: 21 or having one or several amino acid residues added to the amino acid sequence a CDRH3 consisting of a deleted or substituted amino acid sequence; an amino acid sequence represented by SEQ ID NO: 22 or an amine having one or more amino acid residues added, deleted or substituted in the amino acid sequence a CDR1 consisting of a base acid sequence; an amino acid sequence represented by SEQ ID NO: 23 or an amino acid sequence having one or several amino acid residues added, deleted or substituted in the amino acid sequence; CDRL2; and the amino acid sequence represented by SEQ ID NO: 24 or 1 or 1 in the amino acid sequence Two amino acid residues have been added, deleted or substituted amino acid sequence consisting of CDRL3; Also, it binds to the CD98 heavy chain. The antibody-drug conjugate according to any one of claims 1 to 9, wherein the anti-CD98 antibody comprises: CDRH1 consisting of the amino acid sequence represented by SEQ ID NO: 19; CDRH2 consisting of the amino acid sequence represented by No. 20; CDRH3 consisting of the amino acid sequence represented by SEQ ID NO: 21; CDRL1 consisting of the amino acid sequence represented by SEQ ID NO: 22; SEQ ID NO: 23 The CDRL2 composed of the amino acid sequence represented; and the CDRL3 composed of the amino acid sequence represented by SEQ ID NO: 24; and binds to the CD98 heavy chain. The antibody-drug conjugate according to any one of claims 1 to 11, wherein the anti-CD98 antibody comprises: amino acid residues 20 to 135 of SEQ ID NO: 8 The amino acid sequence constituted, which has at least 90% identity of a heavy chain variable region and/or an amino acid sequence consisting of amino acid residues 21 to 135 of SEQ ID NO: 10, A light chain variable region having at least 90% identity. The antibody-drug conjugate of any one of claims 1 to 11, or a pharmacologically acceptable salt thereof, wherein the anti-CD98 antibody comprises: a heavy chain comprising a heavy chain variable region selected from the group consisting of: (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% or more identity with respect to the amino acid sequence of (1); and (3) one or more amino acids of the amino acid sequence of (1) are deleted, a substituted or added amino acid sequence; and a light chain comprising a light chain variable region selected from the group consisting of: (4) amino acid residues 21 to 135 of SEQ ID NO: 16 or 18. a constituent amino acid sequence; (5) an amino acid sequence having at least 95% identity with respect to the amino acid sequence of (4); and (6) an amino acid sequence of (4), An amino acid sequence in which 1 or several amino acids are deleted, substituted or added. The antibody-drug conjugate of any one of claims 1 to 11, or a pharmacologically acceptable salt thereof, wherein the anti-CD98 antibody comprises: an amino acid residue comprising SEQ ID NO: 12 of Nos. 20 to 135 a light chain variable region consisting of a heavy chain variable region composed of an amino acid sequence and an amino acid sequence comprising amino acid residues 21 to 135 of SEQ ID NO: 16 Light chain. The antibody-drug conjugate of any one of claims 1 to 11, or a pharmacologically acceptable salt thereof, wherein the anti-CD98 antibody comprises: an amino acid residue comprising SEQ ID NO: 12 of Nos. 20 to 135 a light chain variable region consisting of a heavy chain variable region composed of an amino acid sequence and an amino acid sequence comprising amino acid residues 21 to 135 of SEQ ID NO: 18 Light chain. The antibody-drug conjugate of any one of claims 1 to 11, wherein the anti-CD98 antibody comprises an amino acid residue of SEQ ID NO: 20 to No. a light chain variable region consisting of a heavy chain variable region composed of an amino acid sequence and an amino acid sequence comprising amino acid residues 21 to 135 of SEQ ID NO: 18 Light chain. The antibody-drug conjugate according to any one of claims 1 to 11, wherein the anti-CD98 antibody comprises: amino acid residues 20 to 465 of SEQ ID NO: 12; The light chain composed of the heavy chain composed of the amino acid sequence and the amino acid sequence of amino acid residues 21 to 240 of SEQ ID NO: 16 is used. The antibody-drug conjugate according to any one of claims 1 to 11, wherein the anti-CD98 antibody comprises: amino acid residues 20 to 465 of SEQ ID NO: 12; The light chain composed of the heavy chain composed of the amino acid sequence and the amino acid sequence of amino acid residues 21 to 240 of SEQ ID NO: 18 is used. The antibody-drug conjugate of any one of claims 1 to 11, or a pharmacologically acceptable salt thereof, wherein the anti-CD98 antibody comprises: amino acid residues 20 to 465 of SEQ ID NO: 14 The light chain composed of the heavy chain composed of the amino acid sequence and the amino acid sequence of amino acid residues 21 to 240 of SEQ ID NO: 18 is used. An anti-CD98 antibody or an antigen-binding fragment thereof, which binds to a site comprising an amino acid residue of 462 to 541 of SEQ ID NO: 38. An anti-CD98 antibody according to claim 20, or an antigen-binding fragment of the antibody, comprising: an amino acid sequence represented by SEQ ID NO: 19 or having one or more amino acid residues added to the amino acid sequence, a CDRH1 consisting of a deleted or substituted amino acid sequence; an amino acid sequence represented by SEQ ID NO: 20 or an amine group having 1 or several amino acid residues added, deleted or substituted in the amino acid sequence CDRH2 consisting of an acid sequence; an amino acid sequence represented by SEQ ID NO: 21 or a CDRH3 consisting of an amino acid sequence in which one or several amino acid residues of the amino acid sequence are added, deleted or substituted ; an amino acid sequence represented by SEQ ID NO: 22 or a CDRL1 consisting of an amino acid sequence in which one or more amino acid residues are added, deleted or substituted in the amino acid sequence; The amino acid sequence represented by the amino acid sequence represented by the 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 represented by SEQ ID NO: 24. One or several amino acid residues in the sequence or in the amino acid sequence are added or deleted Or a substituted amino acid sequence consisting of CDRL3. The anti-CD98 antibody of claim 20 or 21 or an antigen-binding fragment thereof, comprising: CDRH1 consisting of the amino acid sequence represented by SEQ ID NO: 19; CDRH2 consisting of the amino acid sequence represented by SEQ ID NO: 20; CDRH3 consisting of the amino acid sequence represented by SEQ ID NO: 21; CDRL1 consisting of the amino acid sequence represented by SEQ ID NO: 22; sequence recognition The CDRL2 composed of the amino acid sequence represented by No. 23; and the CDRL3 composed of the amino acid sequence represented by SEQ ID NO: 24. The anti-CD98 antibody according to any one of claims 20 to 22, or the antigen-binding fragment of the antibody, comprising: an amino acid sequence consisting of amino acid residues 20 to 135 of SEQ ID NO: 8 An amino acid sequence having at least 90% identity and/or an amino acid sequence consisting of amino acid residues 21 to 135 of SEQ ID NO: 10, having at least 90% identity Light chain variable region. The anti-CD98 antibody of any one of claims 20 to 22, or an antigen-binding fragment thereof, comprising: a heavy chain comprising a heavy chain variable region selected from the group consisting of: (1) SEQ ID NO: 12 or An amino acid sequence consisting of an amino acid residue of No. 20 to No. 135 of 14; (2) an amino acid sequence having at least 95% or more identity with respect to the amino acid sequence of (1); (3) an amino acid sequence in which one or more amino acids of the amino acid sequence of (1) are deleted, substituted or added; a light chain comprising a light chain variable region selected from the group consisting of: (4) an amino acid sequence consisting of amino acid residues 21 to 135 of SEQ ID NO: 16 or 18; (5) relative An amino acid sequence having at least 95% or more of the amino acid sequence of (4); and (6) an amino acid sequence of (4), wherein one or more amino acids are deleted, Amino acid sequence substituted or added. An anti-CD98 antibody according to any one of claims 20 to 22, or an antigen-binding fragment of the antibody comprising: an amino acid sequence comprising an amino acid residue of 20 to 135 of SEQ ID NO: 12; A light chain of a light chain variable region composed of a heavy chain of a heavy chain variable region and an amino acid sequence comprising amino acid residues 21 to 135 of SEQ ID NO: 16. An anti-CD98 antibody according to any one of claims 20 to 22, or an antigen-binding fragment of the antibody comprising: an amino acid sequence comprising an amino acid residue of 20 to 135 of SEQ ID NO: 12; A light chain of a light chain variable region composed of a heavy chain of a heavy chain variable region and an amino acid sequence comprising amino acid residues 21 to 135 of SEQ ID NO: 18. The anti-CD98 antibody according to any one of claims 20 to 22, or the antigen-binding fragment of the antibody, comprising: an amino acid sequence comprising amino acid residues 20 to 135 of SEQ ID NO: 14 A light chain of a light chain variable region composed of a heavy chain of a heavy chain variable region and an amino acid sequence comprising amino acid residues 21 to 135 of SEQ ID NO: 18. The anti-CD98 antibody according to any one of claims 20 to 22, or the antigen-binding fragment of the antibody, comprising: an amino acid sequence comprising amino acid residues 20 to 465 of SEQ ID NO: 12; A light chain of a light chain variable region composed of a heavy chain of a heavy chain variable region and an amino acid sequence comprising amino acid residues 21 to 240 of SEQ ID NO: 16. The anti-CD98 antibody according to any one of claims 20 to 22, or the antigen-binding fragment of the antibody, comprising: an amino acid sequence comprising amino acid residues 20 to 465 of SEQ ID NO: 12; A light chain of a light chain variable region composed of a heavy chain of a heavy chain variable region and an amino acid sequence comprising amino acid residues 21 to 240 of SEQ ID NO: 18. The anti-CD98 antibody according to any one of claims 20 to 22, or an antigen-binding fragment of the antibody, comprising: an amino acid sequence comprising an amino acid residue of Nos. 20 to 465 of SEQ ID NO: 14 A light chain of a light chain variable region composed of a heavy chain of a heavy chain variable region and an amino acid sequence comprising amino acid residues 21 to 240 of SEQ ID NO: 18. An antibody-drug conjugate or a pharmacologically acceptable salt thereof, which comprises the anti-CD98 antibody of any one of claims 20 to 30 or an antigen-binding fragment thereof. A pharmaceutical composition comprising the antibody-drug conjugate of any one of claims 1 to 19 and 31, or a pharmacologically acceptable salt thereof, or the anti-CD98 antibody according to any one of claims 20 to 30 or The antigen-binding fragment of the antibody serves as an active ingredient. The pharmaceutical composition of claim 32, which is for use in anti-tumor or anti-cancer. The pharmaceutical composition according to claim 33, wherein the tumor or cancer is lung cancer, kidney cancer, urothelial carcinoma, colorectal 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 myeloid leukemia, acute lymphocytic leukemia, chronic myelogenous leukemia or multiple myeloma. An antibody-drug conjugate according to any one of claims 1 to 19 and 31, or a pharmaceutically acceptable salt thereof, or an anti-CD98 antibody according to any one of claims 20 to 30, or an antigen-binding fragment thereof Its use is for the manufacture of pharmaceutical compositions. An antibody-drug conjugate according to any one of claims 1 to 19 and 31, or a pharmaceutically acceptable salt thereof, or an anti-CD98 antibody according to any one of claims 20 to 30, or an antigen-binding fragment thereof It is used for the treatment of tumors and/or cancer. A method for treating a tumor and/or a cancer, comprising the antibody-drug conjugate according to any one of claims 1 to 19 and 31, or a pharmacologically acceptable salt thereof, or as claimed in claims 20 to 30 A therapeutically effective amount of the anti-CD98 antibody or antigen-binding fragment of the antibody is administered to a mammal.