antibody, functional antibody fragment, polynucleotide, expression vector, host cells, methods for p

专利摘要:
The present invention addresses the problem of providing: an antibody with an internalizing activity binding to CDH6; an antibody-drug conjugate consisting of the antibody mentioned above and a drug with an anti-tumor activity; a medicament that uses the antibody-drug conjugate, said medicament having a therapeutic effect on tumors; a method for treating tumors using the antibody, the antibody-drug conjugate or the drug; etc. Provided: an anti-CDH6 antibody with internalizing activity; an antibody-drug conjugate consisting of the antibody mentioned above and a drug with an anti-tumor activity; and a drug and a method for treating tumors using the same.
公开号:BR112019023832A2
申请号:R112019023832-8
申请日:2018-05-14
公开日:2020-07-28
发明作者:Atsuko Saito；Tsuyoshi Hirata；Kensuke Nakamura
申请人:Daiichi Sankyo Company, Limited；
IPC主号:

专利说明:

[001] [001] The present invention relates to an anti-CDH6 antibody that binds to CDH6 and with an internalizing effect, a method for the production of the anti-CDH6 antibody, an antibody-drug conjugate comprising the antibody, an anti-tumor agent comprising the antibody-drug conjugate and the like. Fundamentals of Technique
[002] [002] Cadherins are glycoproteins present on the surface of cell membranes and function as cell-cell adhesion molecules through the calcium ion-dependent binding of their extracellular N-terminal domains or as signal molecules responsible for cell-cell interaction. Classic cadherins are in the cadherin superfamily and are single-pass transmembrane proteins composed of five extracellular domains (CE domains), a transmembrane region and an intracellular domain. Classical cadherins are classified in the type I family typified by E-cadherin and N-cadherin, and the type II family according to the homologies of their amino acid sequences.
[003] [003] Cadherin-6 (CDH6) is a single-pass transmembrane protein composed of 790 amino acids, classified in the type II cadherin family, and this protein has extracellular N-terminal and intracellular C-terminal domains. The human CDH6 gene was first cloned in 1995 (Non-Patent Literature 1), and its sequence can be referred to, for
[004] [004] CDH6 is specifically expressed in the brain or kidneys at the stage of development and has been reported to play an important role in the formation of the central nervous system circuit (Non-Patent Literature 2 and 3) and in the development of nephrons in the kidney (Literature Non-Patent 4 and 5). CDH6 expression in normal adult human tissues is located in kidney tubules, bile duct epithelial cells and the like.
[005] [005] Meanwhile, CDH6 is known to be specifically overexpressed at tumor sites in some cancers in human adults. The correlation of CDH6 expression with poor prognosis and its applicability as a tumor marker has been reported in relation to human renal cell carcinoma, particularly clear renal cell carcinoma (Non-Patent Literature 6 and 7). The high expression of CDH6 has also been reported in relation to human ovarian cancer (Non-Patent Literature 8). It has also been reported that CDH6 is involved in the epithelial-mesenchymal transition of human thyroid cancer (Non-Patent Literature 9). In addition, it has been reported that CDH6 is also expressed in human bile duct cancer and human small cell lung cancer (Non-Patent Literature 12 and 13).
[006] [006] Cancers are among the leading causes of death. Although the number of cancer patients is expected to increase with an aging population, treatment needs have not yet been sufficiently met. The problems with conventional chemotherapeutic drugs are as follows: due to their low selectivity, these chemotherapeutic drugs are toxic not only to tumor cells, but also to normal cells and, therefore, have adverse reactions; and chemotherapy drugs cannot be administered in sufficient quantities and, therefore, cannot produce their effects sufficiently. Thus, in recent years,
[007] [007] Antibodies are highly stable in the blood and specifically bind to their target antigens. For these reasons, a reduction in adverse reaction is expected, and a large number of antibody drugs have been developed for molecules highly expressed on the surface of cancer cells. One of the techniques that depend on the specific antigen-binding capacity of antibodies is to use an antibody-drug conjugate (ADC). ADC is a conjugate in which an antibody that binds to an antigen expressed on the surface of cancer cells and can internalize the antigen in the cell through binding is conjugated to a drug with cytotoxic activity. ADC can efficiently deliver the drug to cancer cells and, therefore, is expected to kill cells by accumulating the drug in cancer cells (Non-Patent Literature 10 and Patent Literature 1 and 2). With regard to ADC, for example, Adcetris (TM) (brentuximab vedotine) comprising an anti-CD30 monoclonal antibody conjugated to monomethyl auristatin E has been approved as a therapeutic drug for Hodgkin's lymphoma and large-cell anaplastic lymphoma. In addition, Kadcyla (TM) (trastuzumab emtansin) comprising an anti-HER2 monoclonal antibody conjugated to emtansin is used to treat progressive or recurrent HER2 positive breast cancer.
[008] [008] The characteristics of a target antigen suitable for ADC as an antitumor drug are as follows: the antigen is specifically highly expressed on the surface of cancer cells, but has low expression or is not expressed in normal cells; the antigen can be internalized in the cells; the antigen is not secreted from the cell surface; etc.
[009] [009] ADC comprising DM4 conjugated to an anti-CDH6 antibody that specifically binds to the EC 5 (EC5) domain of CDH6 is known as ADC targeting CDH6 (Patent Literature 3). Patent Literature Citation List
[0010] [0010] Patent Literature 1: WO2014 / 057687
[0011] [0011] Patent Literature 2: US2016 / 0297890
[0012] [0012] Patent Literature 3: WO2016 / 024195 Non-Patent Literature
[0013] [0013] Non-Patent Literature 1: Shimoyama Y, et al., Cancer Research, 2206-2211, 55, May 15, 1995
[0014] [0014] Non-Patent Literature 2: Inoue T, et al., Developmental Biology, 183-194, 1997
[0015] [0015] Non-Patent Literature 3: Osterhout J A, et al., Neuron, 632- 639, 71, Aug 25, 2011
[0016] [0016] Non-Patent Literature 4: Cho EA, et al., Development, 803-812, 125, 1998
[0017] [0017] Non-Patent Literature 5: Mah S P, et al., Developmental Biology, 38-53, 223, 2000
[0018] [0018] Non-Patent Literature 6: Paul R, et al., Cancer Research, 2741-2748, July 1, 57, 1997
[0019] [0019] Non-Patent Literature 7: Shimazui T, et al., Cancer, 963-968, 101 (5), Sep.1, 2004
[0020] [0020] Non-Patent Literature 8: Koebel M, et al., PLoS Medicine, 1749-1760, 5 (12), e232, Dec.2008
[0021] [0021] Non-Patent Literature 9: Gugnoni M, et al., Oncogene, 667-677, 36, 2017
[0022] [0022] Non-Patent Literature 10: Polakis P., Pharmacological Reviews, 3-19, 68, 2016
[0023] [0023] Non-Patent Literature 11: Peters C, et al., Bioscience Reports, 1-20, 35, 2015
[0024] [0024] Non-Patent Literature 12: Goeppert B, et al., Epigenetics, 780- 790, 11 (11), 2016
[0025] [0025] Non-Patent Literature 13: Yokoi S, et al., American Journal of Pathology, 207-216, 161, 1, 2002 Summary of the Invention Technical Problem
[0026] [0026] It is an object of the present invention to provide an antibody that specifically binds to CDH6 and with high internalizing activity, an antibody-drug conjugate comprising the antibody and with high antitumor activity, a pharmaceutical product comprising the antibody-drug conjugate and with therapeutic effects on a tumor, a method for treating a tumor using the antibody, the antibody-drug conjugate or the pharmaceutical product and the like. Solution to the Problem
[0027] [0027] The present inventors conducted intensive studies aimed at reaching the object described above and found that, surprisingly, an antibody that specifically binds to the extracellular domain 3 (in this description, also called omo EC3) of CDH6 has extremely internalizing activity high against cells
[0028] [0028] The present invention includes the following aspects of the invention:
[1] [1] an antibody that specifically binds to the amino acid sequence shown in SEQ ID NO: 4 and capable of internalizing that allows cell uptake, or a functional fragment of the antibody;
[2] [2] the antibody or functional fragment of the antibody according to [1], which has competitive inhibitory activity, for binding to the amino acid sequence shown in SEQ ID NO: 4, against at least any antibody selected from the group consisting of the following antibodies (1) to (5): (1) an antibody with a light chain consisting of the amino acid sequence at positions 21 to 233 in SEQ ID NO: 53 and a heavy chain consisting of the amino acid sequence at positions 20 to 471 in SEQ ID NO: 56, (2) an antibody with a light chain consisting of the amino acid sequence at positions 21 to 233 in SEQ ID NO: 61 and a heavy chain consisting of the amino acid sequence at positions 20 to 471 in SEQ ID NO: 69, (3) an antibody with a light chain consisting of the amino acid sequence at positions 21 to 233 in SEQ ID NO: 61 and a heavy chain consisting of the amino acid sequence at positions 20 to 471 in SEQ ID NO: 73, (4) an antibody with a light chain consisting of the sequence that of amino acids at positions 21 to 233 in SEQ ID NO: 65 and a
[3] [3] the antibody or functional fragment of the antibody according to [1] or [2], which comprises CDRL1, CDRL2 and CDRL3 in any combination selected from the group consisting of the following combinations (1) to (4): (1) CDRL1 consisting of the amino acid sequence shown in SEQ ID NO: 12, CDRL2 consisting of the amino acid sequence shown in SEQ ID NO: 13, and CDRL3 consisting of the amino acid sequence shown in SEQ ID NO: 14, (2) CDRL1 which consists of the amino acid sequence shown in SEQ ID NO: 22, CDRL2 which consists of the amino acid sequence shown in SEQ ID NO: 23, and CDRL3 which consists of the amino acid sequence shown in SEQ ID NO: 24, (3) CDRL1 which consists of the amino acid sequence shown in SEQ ID NO: 32, CDRL2 which consists of the amino acid sequence shown in SEQ ID NO: 33, and CDRL3 which consists of the amino acid sequence shown in SEQ ID NO: 34, and (4) CDRL1 that consists of the amino acid sequence shown in SEQ ID NO: 42, CDRL2 which consists of the sequence amino acid sequence shown in SEQ ID NO: 43, and CDRL3 consisting of the amino acid sequence shown in SEQ ID NO: 44, and CDRH1, CDRH2 and CDRH3 in any combination selected from the group consisting of the following combinations (5) to (9 ): (5) CDRH1 which consists of the amino acid sequence
[4] [4] the antibody or antibody functional fragment according to any one of [1] to [3], which comprises CDRL1, CDRL2 and CDRL3, and CDRH1, CDRH2 and CDRH3 in any combination selected from the group consisting of the following combinations (1) to (5): (1) CDRL1 which consists of the amino acid sequence shown in SEQ ID NO: 12, CDRL2 which consists of the amino acid sequence shown in SEQ ID NO: 13, and CDRL3 which consists of the amino acid sequence shown in SEQ ID NO: 14, and CDRH1 which consists of the amino acid sequence shown in SEQ ID NO: 17, CDRH2 which consists of the amino acid sequence shown in SEQ ID NO: 18, and
[5] [5] the antibody or the functional fragment of the antibody according to any one of [1] to [4], which is humanized;
[6] [6] the antibody or the antibody functional fragment according to any one of [1] to [5], which has any light chain variable region selected from the group consisting of the following variable regions (1) to (4): (1) a light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 63, (2) a light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 67, (3) a amino acid sequence with a sequence identity of at least 95% or more with the sequence of a different structural region from each CDR sequence in the amino acid sequences of (1) and (2), and (4) an amino acid sequence that comprises a deletion, substitution or addition of one or more amino acids in the sequence of a different structural region of each CDR sequence in the amino acid sequences from (1) to (3), and any heavy chain variable region selected from the group consisting of the following variable regions (5) to (9): (5) a heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 71, (6) a heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 75,
[7] [7] the antibody or antibody functional fragment according to any one of [1] to [6], which comprises a light chain variable region and a heavy chain variable region in any of the following combinations (1) a (4): (1) a light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 63 and a heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 71, (2) a light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 63 and a heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 75, (3) a light chain variable region which consists of the amino acid sequence shown in SEQ ID NO: 67 and a heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 75, and (4) a light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 63 and a vari heavy chain compound consisting of the amino acid sequence shown in SEQ
[8] [8] the antibody or the functional fragment of the antibody according to any one of [1] to [7], which has any of the following combinations (1) to (4): (1) a light chain consisting of the sequence of amino acids at positions 21 to 233 in SEQ ID NO: 61 and a heavy chain consisting of the sequence of amino acids at positions 20 to 471 in SEQ ID NO: 69, (2) a light chain consisting of the sequence of amino acids at positions 21 to 233 in SEQ ID NO: 61 and a heavy chain consisting of the amino acid sequence at positions 20 to 471 in SEQ ID NO: 73, (3) a light chain consisting of the amino acid sequence at positions 21 to 233 in SEQ ID NO: 65 and a heavy chain consisting of the amino acid sequence at positions 20 to 471 in SEQ ID NO: 73, and (4) a light chain consisting of the amino acid sequence at positions 21 to 233 in SEQ ID NO: 61 and a heavy chain consisting of the amino acid sequence at positions 20 to 471 in SEQ ID NO: 77;
[9] [9] the antibody or the functional fragment of the antibody according to [8], which has a light chain consisting of the amino acid sequence at positions 21 to 233 in SEQ ID NO: 61 and a heavy chain consisting of the amino acid sequence in positions 20 to 471 in SEQ ID NO: 69;
[10] [10] the antibody or the functional fragment of the antibody according to [8], which has a light chain consisting of the amino acid sequence at positions 21 to 233 in SEQ ID NO: 61 and a heavy chain consisting of the amino acid sequence in positions 20 to 471 in SEQ ID NO: 73;
[11] [11] the antibody or the functional fragment of the antibody according to [8], which has a light chain consisting of the amino acid sequence at positions 21 to 233 in SEQ ID NO: 65 and a heavy chain
[12] [12] the antibody or the functional fragment of the antibody according to [8], which has a light chain consisting of the amino acid sequence at positions 21 to 233 in SEQ ID NO: 61 and a heavy chain consisting of the amino acid sequence in positions 20 to 471 in SEQ ID NO: 77;
[13] [13] the functional fragment of the antibody according to any one of [1] to [12], wherein the functional fragment is selected from the group consisting of Fab, F (ab ') 2, Fab' and Fv;
[14] [14] a polynucleotide that encodes the antibody or the functional fragment of the antibody according to any one of [1] to [13];
[15] [15] the polynucleotide according to [14], which comprises polynucleotides in any combination selected from the group consisting of the following combinations (1) to (5): (1) a polynucleotide encoding a variable region of light chain comprising CDRL1 consisting of the amino acid sequence shown in SEQ ID NO: 12, CDRL2 consisting of the amino acid sequence shown in SEQ ID NO: 13 and CDRL3 consisting of the amino acid sequence shown in SEQ ID NO: 14, and a polynucleotide encoding a heavy chain variable region comprising CDRH1 which consists of the amino acid sequence shown in SEQ ID NO: 17, CDRH2 which consists of the amino acid sequence shown in SEQ ID NO: 18 and CDRH3 which consists of the amino acid sequence shown in SEQ ID NO: 19, (2) a polynucleotide encoding a light chain variable region comprising CDRL1 which consists of the amino acid sequence shown in SEQ ID NO: 22, CDRL2 which consists of the amino acid sequence shows SEQ ID NO: 23 and CDRL3 which consists of
[16] [16] the polynucleotide according to [14] or [15], which comprises a polynucleotide encoding a light chain consisting of the amino acid sequence at positions 21 to 233 in SEQ ID NO: 61 and a polynucleotide encoding a heavy chain which consists of the amino acid sequence at positions 20 to 471 in SEQ ID NO: 69;
[17] [17] the polynucleotide according to [14] or [15], which comprises a polynucleotide encoding a light chain consisting of the amino acid sequence at positions 21 to 233 in SEQ ID NO: 61 and a polynucleotide encoding a heavy chain consisting of the amino acid sequence at positions 20 to 471 in SEQ ID NO: 73;
[18] [18] the polynucleotide according to [14] or [15], which comprises a polynucleotide encoding a light chain consisting of the amino acid sequence at positions 21 to 233 in SEQ ID NO: 65 and a polynucleotide encoding a heavy chain consisting of the amino acid sequence at positions 20 to 471 in SEQ ID NO: 73;
[19] [19] the polynucleotide according to [14] or [15], which comprises a polynucleotide encoding a light chain consisting of the amino acid sequence at positions 21 to 233 in SEQ ID NO: 61 and a polynucleotide encoding a heavy chain consisting of the amino acid sequence at positions 20 to 471 in SEQ ID NO: 77;
[20] [20] an expression vector comprising the polynucleotide according to any one of [14] to [19];
[21] [21] host cells transformed with the expression vector according to [20];
[22] [22] host cells according to [21], wherein the host cells are eukaryotic cells;
[23] [23] a method for producing an antibody of interest or a functional fragment of the antibody, comprising the step of culturing host cells according to [21] or [22], and the step of collecting an antibody of interest or a functional antibody fragment from the culture obtained by the step mentioned above;
[24] [24] the antibody or the functional fragment of the antibody according to any one of [1] to [13], wherein the heavy chain or the light chain has undergone one or two or more modifications selected from the group consisting of linked glycosylation to N, O-linked glycosylation, N-terminal processing, C-terminal processing, deamidation, aspartic acid isomerization, methionine oxidation, addition of a methionine residue to the N-terminal, amidation of a proline residue, N-glutamine conversion - terminal or N-terminal glutamic acid in pyroglutamic acid, and a deletion of one or two amino acids from the carboxyl terminal;
[25] [25] the antibody according to [24], in which one or two amino acids are deleted from the carboxyl terminus of a heavy chain thereof;
[26] [26] the antibody according to [25], in which an amino acid is deleted from each of the carboxyl terminals of both heavy chains therein;
[27] [27] the antibody according to any one of [24] to [26], wherein a proline residue at the carboxyl terminus of a heavy chain thereof is additionally amidated;
[28] [28] the antibody or the functional fragment of the antibody according to any one of [1] to [13] and [24] to [27], where the modification of
[29] [29] an antibody-drug conjugate comprising the antibody or functional fragment of the antibody according to any one of [1] to [13] and [24] to [28] conjugated to a drug;
[30] [30] the antibody-drug conjugate according to [29], wherein the drug is an anti-tumor compound;
[31] [31] the antibody-drug conjugate according to [30], wherein the anti-tumor compound is an anti-tumor compound represented by the following formula: [Formula 1]
[32] [32] the antibody-drug conjugate according to any one of [29] to [31], wherein the antibody is conjugated to the drug via a linker with a structure selected from the group consisting of the following formulas (a) to (f): (a) - (Succinimid-3-yl-N) -CH2CH2-C (= O) -GGFG-NH-CH2CH2CH2-C (= O) -, (b) - (Succinimid-3-yl- N) -CH2CH2CH2CH2CH2-C (= O) -GGFG-NH-CH2CH2CH2-C (= O) -, (c) - (Succinimid-3-yl-N) -CH2CH2CH2CH2CH2-C (= O) -GGFG- NH- CH2-O-CH2-C (= O) -, (d) - (Succinimid-3-yl-N) -CH2CH2CH2CH2CH2-C (= O) -GGFG-NH-CH2CH2-O-CH2-C (= O) -, (e) - (Succinimid-3-yl-N) -CH2CH2-C (= O) -NH-CH2CH2O- CH2CH2O-CH2CH2-C (= O) -GGFG-NH-CH2CH2CH2-C (= O) - , and
[33] [33] the antibody-drug conjugate according to any one of [29] to [32], wherein the linker is represented by any formula selected from the group consisting of the following formulas (c), (d) and (e) : (c) - (Succinimid-3-yl-N) -CH2CH2CH2CH2CH2-C (= O) -GGFG-NH-CH2-O-CH2-C (= O) -, (d) - (Succinimid-3-yl -N) -CH2CH2CH2CH2CH2-C (= O) -GGFG- NH-CH2CH2-O-CH2-C (= O) -, and (e) - (Succinimid-3-yl-N) -CH2CH2-C (= O ) -NH-CH2CH2O- CH2CH2O-CH2CH2-C (= O) -GGFG-NH-CH2CH2CH2-C (= O) -;
[34] [34] the antibody-drug conjugate according to any one of [29] to [33], wherein the linker is represented by the following formula (c)
[35] [35] the antibody-drug conjugate according to any one of [29] to [34], which has a structure represented by the following formula: [Formula 3] where AB represents the antibody or the functional fragment of the antibody, n represents the average number of units of the drug-ligand structure conjugated to the antibody per antibody, and the antibody is connected to the linker via a sulfhydryl group derived from the antibody;
[36] [36] the antibody-drug conjugate according to any one of [29] to [34], which has a structure represented by the following formula:
[37] [37] the antibody-drug conjugate according to any one of [29] to [36], wherein the antibody is an antibody comprising a light chain and a heavy chain in any combination selected from the group consisting of the following combinations ( 1) to (4), or a functional fragment of the antibody: (1) a light chain consisting of the amino acid sequence at positions 21 to 233 in SEQ ID NO: 61 and a heavy chain consisting of the amino acid sequence at positions 20 to 471 in SEQ ID NO: 69, (2) a light chain consisting of the amino acid sequence at positions 21 to 233 in SEQ ID NO: 61 and a heavy chain consisting of the amino acid sequence at positions 20 to 471 in SEQ ID NO: 73, (3) a light chain consisting of the amino acid sequence at positions 21 to 233 in SEQ ID NO: 65 and a heavy chain consisting of the amino acid sequence at positions 20 to 471 in SEQ ID NO: 73, and (4) a light chain consisting of the amino acid sequence at positions 21 to 233 in SEQ ID NO: 61 and a heavy chain consisting of
[38] [38] the antibody-drug conjugate according to any one of [29] to [37], wherein the antibody is an antibody comprising a light chain consisting of the amino acid sequence at positions 21 to 233 in SEQ ID NO: 61 and a heavy chain consisting of the amino acid sequence at positions 20 to 471 in SEQ ID NO: 69, or a functional fragment of the antibody;
[39] [39] the antibody-drug conjugate according to any one of [29] to [37], wherein the antibody is an antibody comprising a light chain consisting of the amino acid sequence at positions 21 to 233 in SEQ ID NO: 61 and a heavy chain consisting of the amino acid sequence at positions 20 to 471 in SEQ ID NO: 77, or a functional fragment of the antibody;
[40] [40] the antibody-drug conjugate according to any one of [29] to [39], wherein the heavy chain or the light chain has undergone one or two or more modifications selected from the group consisting of N-linked glycosylation, O-linked glycosylation, N-terminal processing, C-terminal processing, deamidation, isomerization of aspartic acid, oxidation of methionine, addition of a methionine residue to the N-terminal, amidation of a proline residue, conversion of N-terminal glutamine or N-terminal glutamic acid in pyroglutamic acid, and a deletion of one or two amino acids from the carboxyl terminal;
[41] [41] the antibody-drug conjugate according to any of [29] to [40], wherein the average number of units of the selected antibody-conjugated drug-ligand structure is in the range of 1 to 10;
[42] [42] the antibody-drug conjugate according to [41], wherein the average number of units of the selected antibody-conjugated drug-ligand structure is in the range of 2 to 8;
[43] [43] the antibody-drug conjugate according to [42], in
[44] [44] the antibody-drug conjugate according to [43], wherein the average number of units of the selected antibody-conjugated drug-ligand structure is in the range 7 to 8;
[45] [45] a pharmaceutical composition comprising the antibody-drug conjugate according to any one of [29] to [44], a salt thereof or a hydrate of the conjugate or salt;
[46] [46] the pharmaceutical composition according to [45], which is an antitumor drug;
[47] [47] the pharmaceutical composition according to [46], wherein the tumor is a tumor that expresses CDH6;
[48] [48] the pharmaceutical composition according to [46] or [47], wherein the tumor is renal cell carcinoma, clear renal cell carcinoma, papillary renal cell carcinoma, ovarian cancer, serous adenocarcinoma of the ovary, cancer of thyroid, bile duct cancer, lung cancer, small cell lung cancer, glioblastoma, mesothelioma, uterine cancer, pancreatic cancer, Wilms' tumor or neuroblastoma;
[49] [49] a method for treating a tumor, which comprises administering any selected component of the antibody-drug conjugate as defined in any one of [29] to [44], a salt thereof and a hydrate of the conjugate or salt to an individual;
[50] [50] the treatment method according to [49], in which the tumor is a tumor that expresses CDH6;
[51] [51] the treatment method according to [49] or [50], in which the tumor is renal cell carcinoma, clear renal cell carcinoma, papillary renal cell carcinoma, ovarian cancer, serous adenocarcinoma of the ovary, cancer thyroid, bile duct cancer, lung cancer,
[52] [52] a method for treating a tumor, which comprises administering a pharmaceutical composition comprising at least one component selected from the antibody-drug conjugate as defined in any one of [29] to [44], a salt thereof and a hydrate of the conjugate or salt, and at least one antitumor drug to an individual, simultaneously, separately or continuously;
[53] [53] a method for producing an antibody-drug conjugate, comprising the step of reacting the antibody or the functional fragment of the antibody according to any one of [1] to [13] and [24] to [28], or an antibody or a functional fragment of the antibody obtained by the production method according to [23] with a drug-linker intermediate compound; and
[54] [54] a method for producing an antibody-drug conjugate, comprising the step of culturing the host cells according to [21] or [22], the step of collecting an antibody of interest or a functional fragment of the culture antibody obtained by the step mentioned above, and the step of reacting the antibody or functional fragment of the antibody obtained by the step mentioned above with a drug-binding intermediate compound. Advantageous Effects of the Invention
[0029] [0029] The characteristics of the anti-CDH6 antibody of the present invention are to specifically recognize the EC 3 (EC3) domain of CDH6 and to have high internalizing activity. An anti-CDH6 antibody-drug conjugate comprising the anti-CDH6 antibody of the present invention can be expected to be conjugated to a drug that exerts toxicity in cells by means of a ligand with a specific structure to achieve an excellent anti-tumor effect and safety by administration to patients with
[0030] [0030] [Figure 1] Figure 1 shows the results of flow cytometry analysis of the binding of four mouse anti-CDH6 monoclonal antibodies (clone # rG019, rG055, rG056 and rG061) or mouse IgG control to control cells or 293T cells transfected with hCDH6. The abscissa represents the fluorescence intensity of FITC, indicating the amount of antibody bound, and the ordinate represents the cell count.
[0031] [0031] [Figure 2-1] Figure 2-1 shows the binding activity of four mouse anti-CDH6 monoclonal antibodies (rG019, rG055, rG056 and rG061) or mouse IgG2b negative control antibody against control cells or 293 cells transfected with full-length hCDH6. The abscissa represents the fluorescence intensity of FITC, indicating the amount of antibody bound, and the ordinate represents the cell count.
[0032] [0032] [Figure 2-2] Figure 2-2 shows the binding activity of four mouse anti-CDH6 monoclonal antibodies (rG019, rG055, rG056 and rG061) or mouse IgG control against control cells or transfected 293 cells with hCDH6 deleted in EC1. The abscissa represents the fluorescence intensity of FITC, indicating the amount of antibody bound, and the ordinate represents the cell count.
[0033] [0033] [Figure 2-3] Figure 2-3 shows the binding activity of four mouse anti-CDH6 monoclonal antibodies (rG019, rG055, rG056 and rG061) or mouse IgG control against control cells or transfected 293 cells with hCDH6 deleted in EC2. The abscissa represents the fluorescence intensity of FITC, indicating the amount of antibody bound, and the ordinate represents the cell count.
[0034] [0034] [Figure 2-4] Figure 2-4 shows the binding activity of four mouse anti-CDH6 monoclonal antibodies (rG019, rG055, rG056 and rG061) or mouse IgG control against control cells or transfected 293 cells with hCDH6 deleted in EC3. The abscissa represents the fluorescence intensity of FITC, indicating the amount of antibody bound, and the ordinate represents the cell count.
[0035] [0035] [Figure 2-5] Figure 2-5 shows the binding activity of four mouse anti-CDH6 monoclonal antibodies (rG019, rG055, rG056 and rG061) or mouse IgG control against control cells or transfected 293 cells with hCDH6 deleted in EC4. The abscissa represents the fluorescence intensity of FITC, indicating the amount of antibody bound, and the ordinate represents the cell count.
[0036] [0036] [Figure 2-6] Figure 2-6 shows the binding activity of four mouse anti-CDH6 monoclonal antibodies (rG019, rG055, rG056 and rG061) or mouse IgG control against control cells or transfected 293 cells with hCDH6 deleted in EC5. The abscissa represents the fluorescence intensity of FITC, indicating the amount of antibody bound, and the ordinate represents the cell count.
[0037] [0037] [Figure 3] Figure 3 shows the results of flow cytometry of the evaluation of CDH6 expression on the cell membrane surface of 4 types of human tumor cell lines (NIH human ovarian tumor cell lines: OVCAR-3, PA-1 and ES-2 and human tumor cell line from human kidney cells 786-O). The abscissa represents the fluorescence intensity of FITC, indicating the amount of antibody bound, and the ordinate represents the cell count.
[0038] [0038] [Figure 4] Figure 4 shows a graph in which the internalizing activity of 4 types of rat anti-CDH6 antibodies (rG019, rG055, rG056 and rG061) or rat IgG control was evaluated in NIH cells: OVCAR-3 and 786-O cells using Rat-ZAP anti-mouse IgG reagent
[0039] [0039] [Figure 5] Figure 5 shows the binding of the human chimeric anti-CDH6 antibody chG019 to human CDH6 and monkey CDH6. The abscissa represents the concentration of antibodies and the ordinate represents the amount of antibody bound based on the average fluorescence intensity.
[0040] [0040] [Figure 6-1] Figures 6-1 and 6-2 show the binding activity of four humanized hG019 antibodies (H01L02, H02L02, H02L03 and H04L02) or a negative control human IgG1 antibody against human CDH6, CDH6 monkeys, mouse CDH6 and mouse CDH6. The abscissa represents the concentration of antibodies and the ordinate represents the amount of antibody bound based on the average fluorescence intensity.
[0041] [0041] [Figure 6-2] Figures 6-1 and 6-2 show the binding activity of four humanized hG019 antibodies (H01L02, H02L02, H02L03 and H04L02) or negative control human IgG1 antibody against human CDH6, CDH6 of monkey, mouse CDH6 and rat CDH6. The abscissa represents the concentration of antibodies and the ordinate represents the amount of antibody bound based on the average fluorescence intensity.
[0042] [0042] [Figure 7-1] Figure 7-1 shows the binding activity of four humanized hG019 antibodies (H01L02, H02L02, H02L03 and H04L02), anti-CDH6 antibody NOV0712 or negative control antibody
[0043] [0043] [Figure 7-2] Figure 7-2 shows the binding activity of four humanized hG019 antibodies (H01L02, H02L02, H02L03 and H04L02), anti-CDH6 NOV0712 antibody or hIgG1 negative control antibody against control cells or 293α cells transfected with hCDH6 deleted in EC1. The abscissa represents the intensity of APC fluorescence, indicating the amount of antibody bound. The ordinate represents the cell count.
[0044] [0044] [Figure 7-3] Figure 7-3 shows the binding activity of four humanized hG019 antibodies (H01L02, H02L02, H02L03 and H04L02), anti-CDH6 NOV0712 antibody or hIgG1 negative control antibody against control cells or 293α cells transfected with hCDH6 deleted in EC2. The abscissa represents the intensity of APC fluorescence, indicating the amount of antibody bound. The ordinate represents the cell count.
[0045] [0045] [Figure 7-4] Figure 7-4 shows the binding activity of four humanized hG019 antibodies (H01L02, H02L02, H02L03 and H04L02), anti-CDH6 antibody NOV0712 or hIgG1 negative control antibody against control cells or 293α cells transfected with hCDH6 deleted in EC3. The abscissa represents the intensity of APC fluorescence, indicating the amount of antibody bound. The ordinate represents the cell count.
[0046] [0046] [Figure 7-5] Figure 7-5 shows the binding activity of four humanized hG019 antibodies (H01L02, H02L02, H02L03 and H04L02), anti-CDH6 antibody NOV0712 or hIgG1 negative control against control cells or 293α cells transfected with hCDH6 deleted in
[0047] [0047] [Figure 7-6] Figure 7-6 shows the binding activity of four humanized hG019 antibodies (H01L02, H02L02, H02L03 and H04L02), anti-CDH6 antibody NOV0712 or hIgG1 negative control against control cells or 293α cells transfected with hCDH6 deleted in EC5. The abscissa represents the intensity of APC fluorescence, indicating the amount of antibody bound. The ordinate represents the cell count.
[0048] [0048] [Figure 8] Figure 8 shows the results of flow cytometry examining the expression of human CDH6 in the 786-O / hCDH6 stable expression cell line and its parental cell line 786-O. The abscissa represents the fluorescence intensity of Alexa Fluor 647, indicating the amount of antibody bound, and the ordinate represents a cell count.
[0049] [0049] [Figure 9] Figure 9 shows the binding competition assay of four unlabeled humanized hG019 antibodies (H01L02, H02L02, H02L03 and H04L02), anti-CDH6 antibody NOV0712 or hIgG1 negative control using (a) NOV0712 marked or (b) H01L02 marked. The abscissa represents the final concentration of added unlabeled antibody and the ordinate represents the amount of antibody bound based on the average fluorescence intensity.
[0050] [0050] [Figure 10-1] Figure 10-1 shows a graph in which the internalizing activity of four humanized hG019 antibodies (H01L02, H02L02, H02L03 and H04L02), anti-CDH6 antibody NOV0712 and a negative control antibody was evaluated in NIH cells: OVCAR-3 using anti-human IgG reagent Hum-ZAP conjugated to a toxin (saporin) that inhibits protein synthesis, or goat anti-human IgG fragment F (ab ') 2, specific Fc fragment (gamma) not conjugated with the toxin as a negative control. The ordinate of the graph represents the ATP activity
[0051] [0051] [Figure 10-2] Figure 10-2 shows a graph in which the internalizing activity of four humanized hG019 antibodies (H01L02, H02L02, H02L03 and H04L02), anti-CDH6 antibody NOV0712 and a negative control antibody was evaluated in 786-O cells using Hum-ZAP anti-human IgG reagent conjugated to a toxin (saporin) that inhibits protein synthesis, or goat anti-human IgG fragment F (ab ') 2, specific Fc fragment (gamma ) not conjugated with the toxin as a negative control. The ordinate of the graph represents the ATP activity (RLU). A cell survival rate (%), calculated as a relative survival rate, when the number of live cells in a well supplemented with the negative control instead of Hum-ZAP was set to 100%, is shown below each graph.
[0052] [0052] [Figure 10-3] Figure 10-3 shows a graph in which the internalizing activity of four humanized hG019 antibodies (H01L02, H02L02, H02L03 and H04L02), anti-CDH6 antibody NOV0712 and a negative control antibody was evaluated in PA-1 cells using Hum-ZAP anti-human IgG reagent conjugated to a toxin (saporin) that inhibits protein synthesis, or goat anti-human IgG fragment F (ab ') 2, specific Fc fragment (gamma ) not conjugated with the toxin as a negative control. The ordinate of the graph represents the ATP activity (RLU). A cell survival rate (%), calculated as a relative survival rate, when the number of live cells in a well supplemented with the negative control instead of Hum-ZAP was set to 100%, is shown below each graph.
[0053] [0053] [Figure 11] Figure 11 shows the results of the evaluation of
[0054] [0054] [Figure 12] Figure 12 shows the in vivo antitumor effects of four humanized drug-hG019 conjugates (H01L02-DXd, H02L02-DXd, H02L03-DXd and H04L02-DXd) or NOV0712-DM4. The evaluation was carried out using animal models in which the CDH6-positive human renal cell tumor cell line 786-O was inoculated in immunodeficient mice. The abscissa represents the number of days and the ordinate represents the volume of the tumor. The error interval represents a standard error (SE) value.
[0055] [0055] [Figure 13] Figure 13 shows the in vivo antitumor effects of the humanized drug-hG019 conjugate H01L02-DXd or NOV0712-DM4 or NOV0712-DXd. The evaluation was carried out using animal models in which the human ovarian tumor cell line CDH6-positive PA-1 was inoculated in immunodeficient mice. The abscissa represents the number of days and the ordinate represents the volume of the tumor. The error interval represents an SE value.
[0056] [0056] [Figure 14] Figure 14 shows the anti-tumor effects in vivo of the humanized drug-hG019 conjugate H01L02-DXd or NOV0712-DM4. The evaluation was carried out using animal models in which the CDH6-positive human ovarian tumor cell line NIH: OVCAR-3 was inoculated in immunodeficient mice. The abscissa represents the number of days and the ordinate represents the volume of the tumor. The error interval represents an SE value.
[0057] [0057] [Figure 15] Figure 15 shows the in vivo antitumor effects of the humanized drug-hG019 conjugate H01L02-DXd or NOV0712-
[0058] [0058] [Figure 16] Figure 16 shows the anti-tumor effects in vivo of the humanized drug-hG019 conjugate H01L02-DXd or NOV0712-DM4. The evaluation was carried out using animal models in which the human ovarian tumor cell line CDH6-negative ES-2 was inoculated in immunodeficient mice. The abscissa represents the number of days and the ordinate represents the volume of the tumor. The error interval represents an SE value. Description of Modalities
[0059] [0059] In the following, preferred embodiments for carrying out the present invention will be described with reference to the drawings. It should be noted that the modalities described below merely illustrate the representative modalities of the present invention, and the scope of the present invention should not be strictly interpreted due to these examples.
[0060] [0060] In the present description, the term "cancer" is used to have the same meaning as the term "tumor".
[0061] [0061] In the present description, the term "gene" is used to include not only DNA, but also their mRNA and cDNA, and cRNA therefor.
[0062] [0062] In the present description, the term "polynucleotide" or "nucleotide" is used to have the same meaning as that of a nucleic acid and also includes DNA, RNA, a probe, an oligonucleotide and a primer. In the present description, the terms "polynucleotide" and "nucleotide" may be used interchangeably with each other, unless otherwise specified.
[0063] [0063] In the present description, the terms "polypeptide" and "protein"
[0064] [0064] In the present description, the term "cell" includes cells in an individual animal and cultured cells.
[0065] [0065] In the present description, the term "CDH6" can be used to have the same meaning as that of the CDH6 protein. In the present description, human CDH6 is also called "hCDH6".
[0066] [0066] In the present description, the term "cytotoxic activity" is used to mean that a pathological change is caused to the cells anyway. The term not only means direct trauma, but also all types of structural or functional damage to cells, such as DNA cleavage, formation of a basic dimer, chromosomal cleavage, damage to the cell's mitotic apparatus and reduced activity of various types of enzymes.
[0067] [0067] In the present description, the expression "exerting toxicity in the cells" is used to mean that the toxicity is presented in the cells anyway. The term not only means direct trauma, but also all types of structural, functional or metabolic influences caused to cells, such as DNA cleavage, formation of a basic dimer, chromosomal cleavage, damage to the cell's mitotic apparatus, reduction in activities of various types of enzymes and suppression of the effects of cell growth factors.
[0068] [0068] In the present description, the term "functional fragment of an antibody", also called "antigen-binding fragment of an antibody", is used to mean a partial fragment of the antibody with binding activity against an antigen and includes Fab , F (ab ') 2, scFv, a diabody, a linear antibody and a multispecific antibody formed from antibody fragments and the like. Fab ', which is a monovalent fragment of variable regions of antibody obtained by treating F (ab') 2 under reducing conditions, is also included in the binding fragment
[0069] [0069] In the present description, the term "epitope" is used to mean the partial peptide or partial three-dimensional structure of CDH6, to which a specific anti-CDH6 antibody binds. This epitope, which is the partial peptide described above from CDH6, can be determined by a method well known to one skilled in the art, such as an immunoassay. First, several partial structures of an antigen are produced. With regard to the production of such partial structures, a known technique of oligopeptide synthesis can be applied. For example, a series of polypeptides, in which CDH6 has been successively truncated to an appropriate length from the C-terminus or the N-terminus thereof, is produced by a genetic recombination technique well known to a person skilled in the art. After that, the reactivity of an antibody to these polypeptides is studied and the recognition sites are approximately determined. After that, other shorter peptides are synthesized, and their reactivity to these peptides can then be studied, in order to determine an epitope. When an antibody that binds to a membrane protein with a plurality of extracellular domains is directed to a three-dimensional structure composed of a plurality of domains as an epitope, the domain to which the antibody binds can be determined by modifying the amino acid sequence of a specific extracellular domain and thus modifying the three-dimensional structure. The epitope, which is
[0070] [0070] In the present description, the phrase "antibodies that bind to the same epitope" is used to mean antibodies that bind to a common epitope. If a second antibody binds to a partial peptide or to a partial three-dimensional structure to which a first antibody binds, it can be determined that the first antibody and the second antibody bind to the same epitope. Alternatively, by confirming that a second antibody competes with a first antibody for binding the first antibody to an antigen (that is, a second antibody interferes with the binding of a first antibody to an antigen), it can be determined that the first antibody and the second antibody bind to the same epitope, even if the specific sequence or structure of the epitope has not been determined. In the present description, the term "binding to the same epitope" refers to the case in which it is determined that the first antibody and the second antibody bind to a common epitope by either or both methods of determination. When a first antibody and a second antibody bind to the same epitope and, in addition, the first antibody has special effects, such as antitumor activity or internalizing activity, the second antibody can be expected to have the same activity as that of the first antibody .
[0071] [0071] In this description, the term “CDR” is used to mean a region that determines complementarity. It is known that the heavy chain and the light chain of an antibody molecule each have three CDRs. This CDR is also called the hypervariable region and is located in the variable regions of an antibody's heavy and light chain. These regions have a particularly highly variable primary structure and are separated into three locations in the primary structure of the polypeptide chain in each
[0072] [0072] In the present invention, the term "hybridization under stringent conditions" is used to mean that hybridization is carried out in the hybridization solution commercially available on the market ExpressHyb Hybridization Solution (manufactured by Clontech Laboratories, Inc.) at 68 ° C, or that hybridization is carried out under conditions in which hybridization is performed using a DNA immobilized filter in the presence of 0.7 to 1.0 M NaCl at 68 ° C, and the resultant is then washed at 68 ° C with a concentration of SSC solution 0.1 to 2 times (where 1 time the SSC concentration consists of 150 mM NaCl and 15 mM sodium citrate) for identification or equivalent conditions.
[0073] [0073] In this description, the term "one to several" is used to mean 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3 or 1 or 2.
[0074] [0074] Cadherins are glycoproteins present on the surface of cell membranes and function as cell-cell adhesion molecules through the calcium ion-dependent binding of their N-terminal extracellular domains or as signaling molecules responsible for cell-cell interaction. Classic cadherins are in the cadherin superfamily and are single-pass transmembrane proteins composed of five extracellular domains (CE domains), a transmembrane region and
[0075] [0075] CDH6 (cadherin-6) is a single-pass transmembrane protein composed of 790 amino acids, classified in the type II cadherin family, and this protein has extracellular N-terminal and intracellular C-terminal domains. The human CDH6 gene was first cloned in 1995 (Non-Patent Literature 1), and its sequence can be referred to, for example, in accession numbers NM_004932 and NP_004923 (NCBI).
[0076] [0076] The CDH6 protein used in the present invention can be purified directly from cells expressing CDH6 from a human or non-human mammal (for example, a rat, mouse or monkey) and can then be used, or a fraction of the membrane cell of the cells mentioned above can be prepared and can be used as the CDH6 protein. Alternatively, CDH6 can also be obtained by synthesizing it in vitro, or by allowing host cells to produce CDH6 by genetic manipulation. According to this genetic manipulation, the CDH6 protein can be obtained, specifically, by incorporating the CDH6 cDNA into a vector capable of expressing the CDH6 cDNA and then synthesizing the CDH6 in a solution containing necessary enzymes, substrate and energy materials for transcription and translation, or transforming host cells from other prokaryotes or eukaryotes, to allow them to express CDH6. In addition, cells that express CDH6 based on the genetic manipulation described above, or a cell line that expresses CDH6, can be used to present the CDH6 protein. Alternatively, the expression vector in which the CDH6 cDNA has been incorporated can be administered directly to an animal to be immunized, and CDH6 can be expressed in the body of the animal thus immunized.
[0077] [0077] In addition, a protein consisting of a sequence of amino acids comprising a substitution, deletion and / or addition of one or more amino acids in the amino acid sequence described above for CDH6, and
[0078] [0078] The human CDH6 protein has the amino acid sequence shown in SEQ ID NO: 1. The extracellular region of the human CDH6 protein is composed of extracellular domain 1 (in this description, also called EC1) with the amino acid sequence at the positions 54 to 159 in the amino acid sequence shown in SEQ ID NO: 1, extracellular domain 2 (in this description, also called EC2) with the amino acid sequence at positions 160 to 268 in the amino acid sequence shown in SEQ ID NO: 1, extracellular domain 3 (in this description, also called EC3) with the amino acid sequence at positions 269 to 383 in the amino acid sequence shown in SEQ ID NO: 1, extracellular domain 4 (in this description, also called EC4) with amino acid sequence at positions 384 to 486 in the amino acid sequence shown in SEQ ID NO: 1, and extracellular domain 5 (in this description, also called EC5) with the amino acid sequence at positions 487 to 608 in the sequence d and amino acids shown in SEQ ID NO: 1. The amino acid sequences from EC1 to EC5 are shown in SEQ ID NOs: 2 to 6, respectively (Table 1).
[0079] An example of the anti-CDH6 antibody of the present invention can include an anti-CDH6 antibody that recognizes an amino acid sequence comprising the amino acid sequence shown in SEQ ID NO: 4, and has internalizing activity. An example of the anti-CDH6 antibody of the present invention can include an anti-CDH6 antibody that specifically recognizes an amino acid sequence comprising the amino acid sequence shown in SEQ ID NO: 4, and has internalizing activity. An example of the anti-CDH6 antibody of the present invention can include an anti-CDH6 antibody that recognizes a sequence of
[0080] [0080] The anti-CDH6 antibody of the present invention can be derived from any species. Preferred examples of the species can include humans, monkeys, rats, mice and rabbits. When the anti-CDH6 antibody of the present invention is derived from a different species of humans, it is preferred to chimerize or humanize the anti-CDH6 antibody by a well known technique. The antibody of the present invention can be a polyclonal antibody or it can be a monoclonal antibody, and a monoclonal antibody is preferred.
[0081] [0081] The anti-CDH6 antibody of the present invention is an antibody that can target tumor cells. Specifically, the anti-CDH6 antibody of the present invention has the property of being able to recognize tumor cells, the property of being able to bind to tumor cells and / or the property of being internalized in tumor cells by cell uptake and the like. Accordingly, the anti-CDH6 antibody of the present invention can be conjugated to a compound with antitumor activity through a linker to prepare an antibody-drug conjugate.
[0082] [0082] The binding activity of an antibody against tumor cells can be confirmed by flow cytometry. The uptake of an antibody in
[0083] [0083] In the present description, the term "high internalization capacity" is used to mean that the survival rate (which is indicated by a ratio relative to the survival rate of cells without adding antibodies defined as 100%) of the cells that express CDH6 for which the antibody mentioned above and a saporin-labeled anti-mouse IgG antibody is preferably 70% or less and more preferably 60% or less.
[0084] [0084] The antibody-anti-tumor drug conjugate of the present invention comprises a conjugate compound that exerts an anti-tumor effect. Therefore, it is preferred, but not essential, that the antibody itself has an anti-tumor effect. In order to specifically and / or selectively exert the cytotoxicity of the antitumor compound in tumor cells, it is important and preferred that the antibody has the property of being internalized and transferred to tumor cells.
[0085] [0085] The anti-CDH6 antibody can be obtained by immunizing an animal with a polypeptide that serves as an antigen by a method generally performed in that field and then collecting and purifying a
[0086] [0086] The origin of the antigen is not limited to a human and, therefore, an animal can also be immunized with an antigen derived from a non-human animal, such as a mouse or a rat. In this case, an antibody applicable to a human's disease can be selected by examining the cross-reactivity of the binding of the obtained antibody to the heterologous antigen with the human antigen.
[0087] [0087] In addition, antibody-producing cells that produce an antibody against the antigen can be fused with myeloma cells according to a known method (for example, Kohler and Milstein, Nature (1975) 256, 495-497; and Kennet , R. ed., Monoclonal Antibodies, 365-367, Plenum Press, NY (1980)) to establish hybridomas in order to obtain a monoclonal antibody.
[0088] [0088] In the following, the method for obtaining an antibody against CDH6 will be specifically described. (1) Preparation of the antigen
[0089] [0089] The antigen can be obtained by allowing host cells to produce a gene that encodes the protein of the antigen according to genetic manipulation. Specifically, a vector capable of expressing the antigen gene is produced, and the vector is then introduced into the host cells, so that the gene is expressed in it and, subsequently, the expressed antigen can be purified. The antibody can also be obtained by a method of immunizing an animal with cells that express antigens based on the genetic manipulation described above, or a cell line that expresses the antigen.
[0090] [0090] Alternatively, the antibody can also be obtained, without using the antigenic protein, by incorporating the antigenic protein's cDNA into a
[0091] [0091] The anti-CDH6 antibody used in the present invention is not particularly limited. For example, an antibody specified by an amino acid sequence shown in the sequence listing of the present application can be used appropriately. The anti-CDH6 antibody used in the present invention is desirably an antibody with the following properties: (1) an antibody with the following properties: (a) specific binding to CDH6, and (b) having the activity of being internalized in cells that express CDH6 by binding to CDH6; (2) the antibody according to (1) above, wherein the CDH6 is human CDH6; or (3) the antibody according to (1) or (2) above, wherein the antibody specifically recognizes human CDH6 EC3 and has internalizing activity.
[0092] [0092] The method for obtaining the CDH6 antibody of the present invention is not particularly limited as long as an anti-CDH6 antibody can be obtained. It is preferred to use CDH6 while maintaining its conformation as an antigen.
[0093] [0093] A preferred example of the method for obtaining the antibody may include a method of immunizing DNA. The DNA immunization method is an approach that involves transfecting an individual animal (for example, mouse or rat) with an antigen expression plasmid and then expressing the antigen in the individual to induce immunity
[0094] [0094] Specific examples of obtaining a monoclonal antibody may include the following procedures: (a) the immune response can be induced by incorporating the CDH6 cDNA into an expression vector (for example, pcDNA3.1; Thermo Fisher Scientific Inc.) and directly administering the vector to an animal (for example, a rat or mouse) to be immunized by a method such as electroporation or a gene gun, in order to express CDH6 in the animal's body. The administration of the vector by electroporation or similar can be carried out one or more times, preferably a plurality of times, if
[0095] [0095] Examples of the method for measuring the antibody titer used herein may include, but are not limited to, flow cytometry and cellular ELISA.
[0096] [0096] Examples of the hybridoma strain thus established may include hybridomas producing anti-CDH6 antibodies rG019, rG055, rG056 and rG061. It should be noted that, in the present description, an antibody produced by the hybridoma producing anti-CDH6 antibody rG019 is called "rG019 antibody" or simply "rG019", an antibody produced by the hybridoma rG055 is called "rG055 antibody" or simply “RG055”, an antibody produced by the hybridoma rG056 is called an “rG056 antibody” or simply “rG056” and an antibody produced by the hybridoma
[0097] [0097] The light chain variable region of the rG019 antibody consists of the amino acid sequence shown in SEQ ID NO: 10. The amino acid sequence of the light chain variable region of the rG019 antibody is encoded by the nucleotide sequence shown in SEQ ID NO: 11. The light chain variable region of the rG019 antibody has CDRL1 which consists of the amino acid sequence shown in SEQ ID NO: 12, CDRL2 which consists of the amino acid sequence shown in SEQ ID NO: 13, and CDRL3 which consists of the amino acid sequence shown in SEQ ID NO: 14. The heavy chain variable region of the rG019 antibody consists of the amino acid sequence shown in SEQ ID NO: 15. The amino acid sequence of the heavy chain variable region of the rG019 antibody is encoded by the nucleotide sequence shown in SEQ ID NO: 16. The heavy chain variable region of the rG019 antibody has CDRH1 which consists of the amino acid sequence shown in SEQ ID NO: 17, CDRH2 which consists of the amino acid sequence of those shown in SEQ ID NO: 18, and CDRH3 which consists of the amino acid sequence shown in SEQ ID NO: 19. The antibody sequence rG019 is shown in Table 1.
[0098] [0098] The light chain variable region of the rG055 antibody consists of the amino acid sequence shown in SEQ ID NO: 20. The amino acid sequence of the light chain variable region of the rG055 antibody is encoded by the nucleotide sequence shown in SEQ ID NO: 21. The light chain variable region of the rG055 antibody has CDRL1 which consists of the amino acid sequence shown in SEQ ID NO: 22, CDRL2 which consists of the amino acid sequence shown in SEQ ID NO: 23, and CDRL3 which consists of the amino acid sequence shown in SEQ ID NO: 24. The heavy chain variable region of the rG055 antibody consists of the amino acid sequence shown in SEQ ID NO: 25. The amino acid sequence of the heavy chain variable region of the rG055 antibody is encoded by
[0099] [0099] The light chain variable region of the rG056 antibody consists of the amino acid sequence shown in SEQ ID NO: 30. The amino acid sequence of the light chain variable region of the rG056 antibody is encoded by the nucleotide sequence shown in SEQ ID NO: 31. The light chain variable region of the rG056 antibody has CDRL1 which consists of the amino acid sequence shown in SEQ ID NO: 32, CDRL2 which consists of the amino acid sequence shown in SEQ ID NO: 33, and CDRL3 which consists of the amino acid sequence shown in SEQ ID NO: 34. The heavy chain variable region of the rG056 antibody consists of the amino acid sequence shown in SEQ ID NO: 35. The amino acid sequence of the heavy chain variable region of the rG056 antibody is encoded by the nucleotide sequence shown in SEQ ID NO: 36. The heavy chain variable region of the rG056 antibody has CDRH1 which consists of the amino acid sequence shown in SEQ ID NO: 37, CDRH2 which consists of the amino acid sequence of those shown in SEQ ID NO: 38, and CDRH3 which consists of the amino acid sequence shown in SEQ ID NO: 39. The sequence of the rG056 antibody is shown in Table 1.
[00100] [00100] The light chain variable region of the rG061 antibody consists of the amino acid sequence shown in SEQ ID NO: 40. The amino acid sequence of the light chain variable region of the rG061 antibody is encoded by the nucleotide sequence shown in SEQ ID NO: 41. The light chain variable region of the rG061 antibody has CDRL1 which consists of the amino acid sequence shown in SEQ ID NO: 42, CDRL2 that
[00101] [00101] In addition, in the case where steps (a) to (h) in “2. Production of anti-CDH6 antibody ”above are performed again to independently obtain a monoclonal antibody separately and also in the case where a monoclonal antibody is obtained separately by other methods, an antibody with internalizing activity equivalent to that of the rG019 antibody, of the rG055 antibody, antibody rG056 or antibody rG061 can be obtained. An example of such an antibody may include an antibody that binds to the same epitope to which the rG019 antibody, the rG055 antibody, the rG056 antibody or the rG061 antibody bind. If a newly prepared monoclonal antibody binds to a partial peptide or to a partial three-dimensional structure to which the rG019 antibody, the rG055 antibody, the rG056 antibody or the rG061 antibody binds, it can be determined that the monoclonal antibody binds to the same epitope to which the rG019 antibody, the rG055 antibody, the rG056 antibody or the rG061 antibody binds. In addition, by confirming that the monoclonal antibody competes with the rG019 antibody, the rG055 antibody, the rG056 antibody or the rG061 antibody in binding the antibody to CDH6 (that is, the monoclonal antibody interferes with the binding of the rG019 antibody, of the rG055 antibody, the rG056 antibody or the
[00102] [00102] The antibody of the present invention also includes genetically recombinant antibodies that have been artificially modified for the purpose of reducing heterogeneous antigenicity to humans, such as a chimeric antibody, a humanized antibody and a human antibody, as well as the monoclonal antibody described above against CDH6 . These antibodies can be produced by known methods.
[00103] [00103] Example of a chimeric antibody may include antibodies in which a variable region and a constant region are heterologous to each other, such as a chimeric antibody formed by the conjugation of the variable region of a mouse or rat-derived antibody to a human-derived constant region ( see Proc. Natl. Acad. Sci. USA, 81, 6851-6855, (1984)).
[00104] [00104] Examples of the chimeric antibody derived from the rat human anti-CDH6 antibody include an antibody consisting of a light chain comprising the variable region of the light chain of each rat human anti-CDH6 antibody described in the present description (for example, the rG019 antibody, rG055 antibody, rG056 antibody or rG061 antibody) and a human derived constant region, and a heavy chain comprising the variable region of the heavy chain and a constant region
[00105] [00105] Other examples of the chimeric antibody derived from the human rat anti-CDH6 antibody include an antibody consisting of a light chain comprising a variable region of the light chain with a substitution of one to several residues, 1 to 3 residues, 1 or 2 residues, preferably 1 residue, of amino acids in the variable region of the light chain of each human anti-CDH6 antibody described in the present description (for example, the rG019 antibody, the rG055 antibody, the rG056 antibody or the rG061 antibody) with other amino acid residues , and a heavy chain comprising a variable region of the heavy chain with a substitution of one to several residues, 1 to 3 residues, 1 or 2 residues, preferably 1 residue, of amino acids in the variable region of the heavy chain thereof with other amino acid residues . This antibody can have any constant region derived from man.
[00106] [00106] Other examples of the chimeric antibody derived from the human rat anti-CDH6 antibody include an antibody consisting of a light chain comprising a variable region of the light chain with a substitution of 1 or 2 residues, preferably 1 residue, of amino acids in any 1 to 3 CDRs in the light chain variable region of each anti-human CDH6 antibody described in the present description (for example, the rG019 antibody, the rG055 antibody, the rG056 antibody or the rG061 antibody) with other amino acid residues, and a chain heavy comprising a variable region of the heavy chain with a substitution of 1 or 2 residues, preferably 1 residue, of amino acids in any 1 to 3 CDRs in the variable region of the heavy chain of the same by other amino acid residues. This antibody can have any constant region derived from man.
[00107] [00107] Examples of the chimeric antibody derived from the rG019 antibody include an antibody consisting of a light chain comprising a variable region of the light chain consisting of the sequence
[00108] [00108] Other examples of the chimeric antibody derived from the rG019 antibody include an antibody consisting of a light chain comprising a variable region of the light chain with a substitution of one to several residues, 1 to 3 residues, 1 or 2 residues, preferably 1 residue , of amino acids in the variable region of the light chain consisting of the amino acid sequence shown in SEQ ID NO: 10 with other amino acid residues, and a heavy chain comprising a variable region of the heavy chain with a substitution of one to several residues, 1 to 3 residues, 1 or 2 residues, preferably 1 residue, of amino acids in the variable region of the heavy chain consisting of the amino acid sequence shown in SEQ ID NO: 15 with other amino acid residues. This antibody can have any constant region derived from man.
[00109] [00109] Other examples of the chimeric antibody derived from the rG019 antibody include an antibody consisting of a light chain comprising a variable region of the light chain with a substitution of 1 or 2 residues (preferably 1 residue) of amino acids in 1 to 3 CDRs in the region light chain variable consisting of the amino acid sequence shown in SEQ ID NO: 10 with other amino acid residues, and a heavy chain comprising a heavy chain variable region with a substitution of 1 or 2 residues (preferably 1 residue) of amino acids in 1 to 3 CDRs in the variable region of the heavy chain consisting of the amino acid sequence shown in SEQ ID NO: 15 with other amino acid residues. This antibody can have any constant region derived from man.
[00110] [00110] Other examples of the chimeric antibody derived from
[00111] [00111] Specific examples of the chimeric antibody derived from the rG019 antibody include an antibody consisting of a light chain consisting of the full-length light chain amino acid sequence shown in SEQ ID NO: 53, and a heavy chain consisting of the amino acid sequence full length of the heavy chain shown in SEQ ID NO: 56. In the present description, this chimeric anti-human CDH6 antibody is called "chimeric G019 antibody", "chG019 antibody" or "chG019". The full length amino acid sequence of the chG019 antibody light chain is encoded by the nucleotide sequence shown in SEQ ID NO: 54, and the full length amino acid sequence of the chG019 antibody heavy chain is encoded by the nucleotide sequence shown in SEQ ID NO: 57.
[00112] The chG019 antibody light chain variable region amino acid sequence is identical to the rG019 antibody light chain variable region amino acid sequence and consists of the amino acid sequence shown in SEQ ID NO: 10. The antibody light chain chG019 has CDRL1 which consists of the amino acid sequence shown in SEQ ID NO: 12, CDRL2 which consists of the amino acid sequence shown in SEQ ID NO: 13, and CDRL3 which consists of the amino acid sequence shown in SEQ ID NO: 14, which are identical to the light chain CDRL1, CDRL2 and CDRL3,
[00113] [00113] The amino acid sequence of the chG019 antibody heavy chain variable region consists of the amino acid sequence shown in SEQ ID NO: 58. The chG019 antibody plotted chain has CDRH1 which consists of the amino acid sequence shown in SEQ ID NO: 17 , CDRH2 which consists of the amino acid sequence shown in SEQ ID NO: 60, and CDRH3 which consists of the amino acid sequence shown in SEQ ID NO: 60
[00114] [00114] The chG019 antibody sequence is shown in Table 1.
[00115] [00115] Examples of the chimeric antibody derived from the rat human anti-CDH6 antibody rG055 include a chimeric antibody consisting of a light chain comprising a variable region of the light chain consisting of the amino acid sequence shown in SEQ ID NO: 20, and a heavy chain comprising a heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 25. This antibody can have any constant region derived from man.
[00116] [00116] Examples of the chimeric antibody derived from the rat human anti-CDH6 antibody rG056 include a chimeric antibody consisting of a light chain comprising a region
[00117] [00117] Examples of the chimeric antibody derived from the rat human anti-CDH6 antibody rG061 include a chimeric antibody consisting of a light chain comprising a variable region of the light chain consisting of the amino acid sequence shown in SEQ ID NO: 40, and a heavy chain comprising a variable region of heavy chain consisting of the amino acid sequence shown in SEQ ID NO: 45. That antibody can have any constant region derived from man.
[00118] [00118] Examples of the humanized antibody may include an antibody formed by incorporating only complementarity determining regions (CDRs) into an antibody derived from human (see Nature (1986) 321, p. 522-525), an antibody formed by incorporating the amino acid residues of some structures, as well as CDR sequences, in a human antibody according to a CDR graft method (International Publication No. WO90 / 07861) and an antibody formed by modifying the amino acid sequences of some CDRs, maintaining the ability to bind to the antigen.
[00119] [00119] In the present description, the humanized antibody derived from the rG019 antibody, the rG055 antibody, the rG056 antibody, the rG061 antibody or the chG019 antibody is not limited to a specific humanized antibody, as long as the humanized antibody retains all 6 sequences of CDRs unique to the rG019 antibody, the rG055 antibody, the rG056 antibody, the rG061 antibody or the chG019 antibody and have internalizing activity. The amino acid sequences of some CDRs in that
[00120] [00120] Concrete examples of the humanized antibody of the chG019 antibody may include any given combination of: a light chain comprising a variable region of the light chain consisting of any amino acid sequence selected from the group consisting of (1) the amino acid sequence shown in SEQ ID NO: 63 or 67, (2) an amino acid sequence with an identity of at least 95% or more (preferably an amino acid sequence with a sequence identity of at least 95% or more with the sequence of a structural region other than each CDR sequence) with the amino acid sequence described above (1), and (3) an amino acid sequence comprising a deletion, substitution or addition of one or more amino acids in the amino acid sequence described above (1); and a heavy chain comprising a variable region of the heavy chain consisting of any amino acid sequence selected from the group consisting of (4) the amino acid sequence shown in SEQ ID NO: 71, 75 or 79, (5) an amino acid sequence with a identity of at least 95% or more (preferably an amino acid sequence with a sequence identity of at least 95% or more with the sequence of a different structural region of each CDR sequence) with the amino acid sequence described above (4), and (6) an amino acid sequence comprising a deletion, substitution or addition of one or more amino acids in the amino acid sequence described above (4).
[00121] Alternatively, an antibody with a humanized heavy chain or light chain and the other chain derived from a mouse antibody or a chimeric antibody can also be used. Examples of such an antibody may include any given combination of: a light chain comprising a variable region of the light chain consisting of any
[00122] [00122] The amino acid substitution in the present description is preferably a conservative amino acid substitution. Conservative amino acid substitution is a substitution that occurs within a group of amino acids associated with certain amino acid side chains. Preferred amino acid groups are as follows: acid 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 uncharged polar family = glycine, asparagine, glutamine, cysteine, serine, threonine and tyrosine. Other preferred amino acid groups are as follows: aliphatic hydroxy group = serine and threonine; amide-containing group = asparagine and glutamine; aliphatic group = alanine, valine, leucine and isoleucine; and aromatic group = phenylalanine, tryptophan and tyrosine. Such amino acid substitution is preferably carried out without impairing the properties of a substance that has the original amino acid sequence.
[00123] [00123] Examples of the antibody with a preferred combination of the light chains and heavy chains described above include an antibody consisting of a light chain with the amino acid sequence of the region
[00124] [00124] Other examples of the antibody with a preferred combination of the light and heavy chains described above include an antibody consisting of a light chain consisting of the amino acid sequence at positions 21 to 233 in the full length amino acid sequence of the light chain shown in SEQ ID NO: 61 (in this description, also called the full-length light chain amino acid sequence
[00125] [00125] By combining sequences that show a high identity with the heavy chain amino acid sequences and light chain amino acid sequences described above, it is possible to select an antibody with a biological activity equivalent to that of each of the antibodies described above. That identity is an identity of generally 80% or more, preferably 90% or more, more preferably 95% or more, and even more preferably 99% or more. In addition, also by combining amino acid sequences of a heavy chain and a light chain comprising a substitution, deletion or addition of one or more amino acid residues in relation to the amino acid sequence of a heavy chain
[00126] [00126] The identity between two types of amino acid sequences can be determined by aligning the sequences using the standard parameters of Clustal W version 2 (Larkin MA, Blackshields G, Brown NP, Chenna R, McGettigan PA, McWilliam H, Valentin F, Wallace IM, Wilm A, Lopez R, Thompson JD, Gibson TJ and Higgins DG (2007), "Clustal W and Clustal X version 2.0", Bioinformatics. 23 (21): 2947-2948).
[00127] [00127] It should be noted that in the hL02 full-length light chain amino acid sequence shown in SEQ ID NO: 61, the amino acid sequence consisting of the amino acid residues at positions 1 to 20 is the signal sequence, the sequence amino acid residues consisting of amino acid residues at positions 21 to 128 is the variable region and the amino acid sequence consisting of amino acid residues at positions 129 to 233 is the constant region. In the hL02 full length light chain nucleotide sequence shown in SEQ ID NO: 62, the nucleotide sequence consisting of the nucleotides at positions 1 to 60 encodes the signal sequence, the nucleotide sequence consisting of the nucleotides at positions 61 to 384 encodes the variable region and the nucleotide sequence consisting of the nucleotides at positions 385 to 699 encodes the constant region.
[00128] [00128] In the hL03 full-length light chain amino acid sequence shown in SEQ ID NO: 65, the amino acid sequence consisting of the amino acid residues in positions 1 to 20 is the signal sequence, the amino acid sequence consisting of the amino acid residues at positions 21 to 128 is the variable region and the amino acid sequence consisting of amino acid residues at positions 129 to 233 is the constant region. In the hL03 full-length light chain nucleotide sequence shown in SEQ ID NO: 66, the nucleotide sequence consisting of
[00129] [00129] In the hH01 full length heavy chain amino acid sequence shown in SEQ ID NO: 69, the amino acid sequence consisting of the amino acid residues in positions 1 to 19 is the signal sequence, the amino acid sequence consisting of the amino acid residues at positions 20 to 141 is the variable region and the amino acid sequence consisting of the amino acid residues at positions 142a to 471 is the constant region. In the hH01 full-length nucleotide sequence shown in SEQ ID NO: 70, the nucleotide sequence consisting of the nucleotides at positions 1 through 57 encodes the signal sequence, the nucleotide sequence consisting of the nucleotides at positions 58 through 423 encodes the variable region and the nucleotide sequence consisting of the nucleotides at positions 424 to 1413 encodes the constant region.
[00130] [00130] In the hH02 full length heavy chain amino acid sequence shown in SEQ ID NO: 73, the amino acid sequence consisting of the amino acid residues in positions 1 to 19 is the signal sequence, the amino acid sequence consisting of amino acid residues at positions 20 to 141 is the variable region and the amino acid sequence consisting of the amino acid residues at positions 142a to 471 is the constant region. In the hH02 full-length nucleotide sequence shown in SEQ ID NO: 74, the nucleotide sequence consisting of the nucleotides at positions 1 to 57 encodes the signal sequence, the nucleotide sequence consisting of the nucleotides at positions 58 to 423 encodes the variable region and the nucleotide sequence consisting of the nucleotides at positions 424 to 1413 encodes the constant region.
[00131] [00131] Following full length chain amino acids
[00132] [00132] In the present description, Tables 1-1 to 1-15 are also collectively called Table 1.
[00133] [00133] Other examples of the antibody of the present invention may include a human antibody that binds to CDH6. The human anti-CDH6 antibody means a human antibody with only the gene sequence of an antibody derived from human chromosomes. The human anti-CDH6 antibody can be obtained by a method using a human antibody-producing mouse with a human chromosomal fragment comprising the heavy chain and light chain genes 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. 10, pp. 69-73 (Kitagawa, Y., Matsuda, T. and Iijima, S. eds.), Kluwer Academic Publishers, 1999; Tomizuka, K. et al., Proc. Natl. Acad. Sci USA (2000) 97, p. 722-727; etc.).
[00134] [00134] This human antibody-producing mouse can be produced specifically using a genetically modified animal,
[00135] [00135] Otherwise, the human anti-CDH6 antibody can also be obtained by transforming eukaryotic cells with cDNA that encodes each of the heavy and light chains of a human antibody, or preferably with a vector comprising the cDNA, according to with techniques of genetic recombination, and then cultivate the transformed cells by producing a genetically modified human monoclonal antibody, so that the antibody can be obtained from the culture supernatant.
[00136] In this context, eukaryotic cells and, preferably, mammalian cells such as CHO cells, lymphocytes or myelomas can, for example, be used as a host.
[00137] [00137] In addition, a method for obtaining a human antibody derived from phage display that has been selected from a library of human antibodies is also known (see 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), pp. 427- 431; etc.).
[00138] [00138] For example, a phage display method can be applied, which comprises allowing the variable regions of a human antibody to express themselves as a single chain antibody (scFv) on the surface of the phages and then selecting a link from phage to an antigen (Nature Biotechnology (2005), 23, (9), p. 1105-1116).
[00139] [00139] By analyzing the phage gene that was selected due to its ability to bind to the antigen, the DNA sequences that encode the variable regions of a human antibody that binds to the antigen can be determined.
[00140] [00140] Once the DNA sequence of scFv binding to the antigen has been determined, an expression vector with the sequence mentioned above is produced and the expression vector produced is then introduced into an appropriate host and can be allowed to express it, obtaining thus a human antibody (International Publication No. WO92 / 01047, WO92 / 20791, WO93 / 06213, WO93 / 11236, WO93 / 19172, WO95 / 01438, and WO95 / 15388, Annu. Rev. Immunol (1994) 12, p. 433 -455, Nature Biotechnology (2005) 23 (9), pp. 1105-1116).
[00141] [00141] If a newly produced human antibody binds to a partial peptide or a partial three-dimensional structure to which any rat anti-human CDH6 antibody, chimeric anti-human CDH6 antibody or humanized anti-human CDH6 antibody described in present description (for example, the rG019 antibody, the rG055 antibody, the rG056 antibody, the rG061 antibody, the chG019 antibody, the H01L02 antibody, the H02L02 antibody, the H02L03 antibody or the H04L02 antibody), it can be determined that the human antibody binds to the same epitope to which the anti-human CDH6 antibody, the chimeric anti-human CDH6 antibody or the humanized anti-human CDH6 antibody binds. Alternatively, in confirming that the human antibody competes with the rat human anti-CDH6 antibody, the chimeric anti-human CDH6 antibody or the humanized anti-human CDH6 antibody described in the present description (for example, the rG019 antibody, the rG055 antibody, the rG056 antibody, the rG061 antibody, the chG019 antibody, the H01L02 antibody, the H02L02 antibody, the H02L03 antibody or the H04L02 antibody) in binding the antibody to CDH6 (for example, the human antibody interferes with binding
[00142] [00142] Chimeric antibodies, humanized antibodies or human antibodies obtained by the methods described above are evaluated for their binding activity against the antigen according to a known method, etc., so that a preferred antibody can be selected.
[00143] [00143] An example of another indicator for comparing the properties of antibodies may include the stability of an antibody. A differential scanning calorimeter (DSC) is a device capable of quickly and accurately measuring an average point of thermal denaturation (Tm), serving as a good indicator for the relative structural stability of a
[00144] [00144] The antibody of the present invention also includes a modification of an antibody.
[00145] [00145] The modification is used to mean the antibody of the present invention, which is chemically or biologically modified. Examples of such a chemical modification include the attachment of a chemical moiety to an amino acid backbone and the chemical modification of an N-linked or O-linked carbohydrate chain. Examples of such biological modification include antibodies undergoing a post-translational modification (for example , N or O-linked glycosylation, N-terminal or C-terminal processing, deamidation, isomerization of aspartic acid, methionine oxidation and conversion of N-terminal glutamine or N-terminal glutamic acid to pyroglutamic acid) and antibodies to the N-terminal at to which a methionine residue is added as a result of having been expressed using prokaryotic host cells. In addition, such modification should also include labeled antibodies to allow the detection or isolation of the antibody of the present invention or an antigen, for example
[00146] [00146] Furthermore, by regulating a modification of the sugar chain (glycosylation, defucosylation, etc.) that binds to the antibody of the present invention, the antibody-dependent cellular cytotoxic activity can be increased. As techniques for regulating the modification of the sugar chain of an antibody, those described in International Publications No. WO1999 / 54342, WO2000 / 61739 and WO2002 / 31140, etc., are known, although the techniques are not limited to them. The antibody of the present invention also includes antibodies against which the modification of the sugar chain mentioned above has been regulated.
[00147] [00147] Once an antibody gene is isolated, the gene can be introduced into an appropriate host to produce an antibody, using an appropriate combination of a host and an expression vector. A specific example of the antibody gene may be a combination of a gene encoding the antibody heavy chain sequence described in the present description and a gene encoding the antibody light chain sequence described herein. After host cell transformation, this heavy chain sequence gene and a light chain sequence gene can be inserted into a single expression vector, or these genes can instead be inserted into different expression vectors.
[00148] [00148] When eukaryotic cells are used as hosts, animal cells, plant cells or eukaryotic microorganisms can be used. In particular, examples of animal cells may include mammalian cells, such as COS cells, which are monkey cells (Gluzman, Y., Cell (1981) 23, p. 175-182, ATCC CRL-1650), fibroblasts of
[00149] [00149] When prokaryotic cells are used as hosts, Escherichia coli or Bacillus subtilis can be used, for example.
[00150] [00150] An antibody gene of interest is introduced into these cells for transformation, and the transformed cells are then cultured in vitro to obtain an antibody. In the culture mentioned above, there are cases where the yield is different depending on the sequence of the antibody and therefore it is possible to select an antibody, which is easily produced as a medicine, from antibodies with equivalent binding activity, using the yield as a indicator. Accordingly, the antibody of the present invention also includes an antibody obtained by the method described above to produce an antibody, which comprises a step of culturing the transformed host cells and a step of collecting an antibody of interest or a functional fragment of the antibody from culture obtained in the step mentioned above.
[00151] [00151] It is known that the lysine residue at the carboxyl terminal of the heavy chain of an antibody produced in cultured mammalian cells is excluded (Journal of Chromatography A, 705: 129-134 (1995)), and it is also known that the two amino acid residues at the carboxyl terminal of the heavy chain, glycine and lysine, are excluded and that the newly positioned proline residue at the carboxyl terminal is amidated (Analytical Biochemistry, 360: 75-83 (2007)). However, this deletion and modification of these heavy chain sequences does not influence the antigen binding activity and the effector function (complement activation, antibody-dependent cell cytotoxicity, etc.) of an antibody. Therefore, the antibody according to
[00152] [00152] Examples of the antibody isotype of the present invention can include IgG (IgG1, IgG2, IgG3 and IgG4). Among others, IgG1 and IgG4 are preferable.
[00153] [00153] Examples of the biological activity of an antibody can generally include the activity of binding the antigen, the activity of internalizing in cells that express an antigen by binding to the antigen, the activity of neutralizing the activity of an antigen, the activity of increasing the antigen activity, cell-dependent cytotoxic activity
[00154] [00154] The antibody obtained can be purified to a homogeneous state. For the separation and purification of the antibody, separation and purification methods used for common proteins can be used. For example, column chromatography, filtration, ultrafiltration, reloading, dialysis, preparative polyacrylamide gel electrophoresis and isoelectric focusing are suitably selected and combined with each other so that the antibody can be separated and purified (Strategies for Protein Purification and Characterization: A Laboratory Course Manual, Daniel R. Marshak et al. Eds., Cold Spring Harbor Laboratory Press (1996); and Antibodies: A Laboratory Manual. Ed Harlow and David Lane, Cold Spring Harbor Laboratory (1988)), although examples of separation and purification are not limited to them.
[00155] [00155] Examples of the chromatography may include affinity chromatography, ion exchange chromatography, hydrophobic chromatography, gel filtration chromatography, reverse phase chromatography and absorption chromatography.
[00156] [00156] These chromatographic techniques can be performed using liquid chromatography such as HPLC or FPLC.
[00157] [00157] Examples of the column used in affinity chromatography may include a protein A column and a protein G column. Examples of the column involving the use of protein A may include Hyper D, POROS and Sepharose F. F. (Pharmacia).
[00158] [00158] In addition, when using a carrier immobilized by antigen, the antibody can be purified using the binding activity of the antibody to the antigen.
[00159] [00159] The anti-CDH6 antibody obtained in item “2. Production of anti-CDH6 antibody ”above can be conjugated to a drug via a fraction of the linker structure to prepare a drug-anti-CDH6 antibody conjugate. The drug is not particularly limited as long as it has a substituent or a partial structure that can be connected to a linker structure. The anti-CDH6 drug-antibody conjugate can be used for various purposes, according to the drug conjugate. Examples of such a drug may include substances with antitumor activity, substances effective for blood diseases, substances effective for autoimmune diseases, anti-inflammatory substances, antimicrobial substances, antifungal substances, antiparasitic substances, antiviral substances and anti-anesthetic substances. (1) -1 Antitumor compound
[00160] [00160] An example using an anti-tumor compound as a compound to be conjugated to the drug-anti-CDH6 antibody conjugate of the present invention will be described below. The anti-tumor compound is not particularly limited as long as the compound has an anti-tumor effect and has a substituent or a partial structure that can be connected to a linker structure. After part or all of the ligand is cleaved in the tumor cells, the fraction of the anti-tumor compound is released so that the anti-tumor compound has an anti-tumor effect. As the ligand is cleaved in the connection position with the drug, the anti-tumor compound is released in its original structure to exert its original anti-tumor effect.
[00161] [00161] The anti-CDH6 antibody obtained in item “2. Production of anti-CDH6 antibody ”above can be conjugated to the anti-tumor compound through a fraction of the linker structure to prepare a drug-anti-CDH6 antibody conjugate.
[00162] [00162] As an example of the anti-tumor compound used in the present invention, exactecan, a camptothecin derivative ((1S, 9S) -1-amino-9-ethyl-5-fluoro-2,3-dihydro-9- hydroxy-4-methyl-1H, 12H-benzo [de] pyran [3 ', 4': 6,7] indolizine [1,2-b] quinoline-10,13 (9H, 15H) -dione represented by the formula a below) can preferably be used. [Formula 5]
[00163] [00163] The compound can be easily obtained by, for example, a method described in US Patent Publication No. US2016 / 0297890 or other known methods, and the amino group at position 1 can preferably be used as a connecting position to the structure of Link. In addition, exactecan can be released into tumor cells while part of the ligand is still attached to it. However, the compound has an excellent anti-tumor effect, even in this state.
[00164] [00164] As exactecan has a camptothecin structure, it is known that the balance changes to a structure with a formed lactone ring (closed ring) in an aqueous acidic medium (for example, in the order of pH 3), while the equilibrium changes to a structure with an open lactone ring (open ring) in a basic aqueous medium (for example, in the order of pH 10). A drug conjugate into which accuracy residues corresponding to a closed ring structure and an open ring structure are also expected to have an equivalent antitumor effect, is not
[00165] [00165] Other examples of the anti-tumor compound may include anti-tumor compounds described in the literature (Pharmacological Reviews, 68, p. 3-19, 2016). Specific examples thereof may include doxorubicin, calicheamicin, dolastatin 10, auristatins such as monomethyl auristatin E (MMAE) and monomethyl auristatin F (MMAF), maytansinoids such as DM1 and DM4, a pyrrolbenzodiazepine dimer SG2000 (SJG-136), a camptothecin derivative SN-38, duocarmicins such as CC-1065, amanitin, daunorubicin, mitomycin C, bleomycin, cyclocytidine, vincristine, vinblastine, methotrexate, platinum-based antitumor agents (cisplatin and derivatives thereof) and Taxol and derivatives thereof.
[00166] [00166] In the antibody-drug conjugate, the number of conjugated drug molecules per antibody molecule is a key factor that influences its efficacy and safety. The production of the antibody-drug conjugate is performed by specifying reaction conditions, such as the quantities of starting materials and reagents used for the reaction, in order to achieve a constant number of conjugated drug molecules. Unlike the chemical reaction of a low molecular weight compound, a mixture is usually obtained containing different numbers of conjugated drug molecules. The number of conjugated drug molecules per antibody molecule is defined and indicated as an average value, that is, the average number of conjugated drug molecules. Unless otherwise stated, that is, except when representing an antibody-drug conjugate with a specific number of conjugated drug molecules that is included in an antibody-drug mixture with a different number of conjugated drug molecules, the number of Drug molecules conjugated according to the present invention also mean a mean value as a rule. The number of exact molecules conjugated to
[00167] The linker structure that conjugates the drug to the anti-CDH6 antibody in the drug-anti-CDH6 antibody conjugate of the present invention will be described.
[00168] [00168] In the antibody-drug conjugate of the present application, the linker structure that conjugates the anti-CDH6 antibody to the drug is not particularly limited as long as the resulting antibody-drug conjugate can be used. The binding structure can be properly selected and used according to the purpose of use. An example of the linker structure can include a linker described in the known literature (Pharmacol Rev 68: 3-19, January 2016, Protein Cell DOI 10.1007 / s13238-016-0323-0, etc.). Additional specific examples may include VC (valine-citrulline), MC (maleimidocaproyl), SMCC (succinimidyl 4- (N-maleimidomethyl) cyclohexane-1-carboxylate), SPP (4- (2-pyridyldithium) pentanoate succinimidyl), SS (disulfide), SPDB (N-succinimidyl 4- (2-pyridyldithium) butyrate), SS / hydrazone, hydrazone and carbonate.
[00169] [00169] Another example may include a linker structure described in US Patent Publication No. US2016 / 0297890 (as an example, those described in paragraphs [0260] to [0289] thereof). Any structure
[00170] [00170] More preferred are the following: - (Succinimid-3-yl-N) -CH2CH2CH2CH2CH2-C (= O) -GGFG- NH-CH2-O-CH2-C (= O) -, - (Succinimid-3 -il-N) -CH2CH2CH2CH2CH2-C (= O) -GGFG- NH-CH2CH2-O-CH2-C (= O) -, and - (Succinimid-3-yl-N) -CH2CH2-C (= O) -NH-CH2CH2O- CH2CH2O-CH2CH2-C (= O) -GGFG-NH-CH2CH2CH2-C (= O) -.
[00171] [00171] Even more preferred are the following: - (Succinimid-3-yl-N) -CH2CH2CH2CH2CH2-C (= O) -GGFG-NH-CH2-O-CH2-C (= O) -, and
[00172] [00172] The antibody is connected to the - (Succinimid-3-il-N) terminal (for example, an opposite terminal (left terminal) to the terminal to which - CH2CH2CH2CH2CH2- is connected to “- (Succinimid-3-il- N) - CH2CH2CH2CH2CH2-C (= O) -GGFG-NH-CH2-O-CH2-C (= O) - ”), and the antitumor compound is connected to a terminal (the carbonyl group of CH2-O-CH2- C (= O) - at the right terminal in the example described above) opposite the terminal to which the antibody is attached to - (Succinimid-3-yl-N). “- (Succinimid-3-yl-N) -” has a structure represented by the following formula: [Formula 6]
[00173] [00173] Position 3 of this partial structure is the connection position with the anti-CDH6 antibody. This connection with the antibody at position 3 is distinguished by the formation of a thioether bond. The nitrogen atom at position 1 of that fraction of the structure is connected to the methylene carbon atom that is present in the binder, including the structure.
[00174] [00174] In the antibody-drug conjugate of the present invention with exactecan as a drug, a fraction of the drug-binding structure with any structure given below is preferred for conjugation to the antibody. For such fractions of the linker-drug structure, the average conjugated number per antibody can be from 1 to 10 and is preferably from 2 to 8, more preferably from 5 to 8, even more preferably from 7 to 8 and even more preferably 8. - (Succinimid-3-yl-N) -CH2CH2-C (= O) -GGFG-NH-CH2CH2CH2-C (= O) - (NH-DX), - (Succinimid-3-yl-N) -CH2CH2CH2CH2CH2-C (= O) -GGFG- NH-CH2CH2CH2-C (= O) - (NH-DX),
[00175] [00175] More preferred are the following: - (Succinimid-3-yl-N) -CH2CH2CH2CH2CH2-C (= O) -GGFG- NH-CH2-O-CH2-C (= O) - (NH-DX), - (Succinimid-3-yl-N) -CH2CH2CH2CH2CH2-C (= O) -GGFG- NH-CH2CH2-O-CH2-C (= O) - (NH-DX), and - (Succinimid-3-il- N) -CH2CH2-C (= O) -NH-CH2CH2O- CH2CH2O-CH2CH2-C (= O) -GGFG-NH-CH2CH2CH2-C (= O) - (NH-DX).
[00176] [00176] Even more preferred are the following: - (Succinimid-3-yl-N) -CH2CH2CH2CH2CH2-C (= O) -GGFG-NH-CH2-O-CH2-C (= O) - (NH-DX) , and - (Succinimid-3-yl-N) -CH2CH2-C (= O) -NH-CH2CH2O- CH2CH2O-CH2CH2-C (= O) -GGFG-NH-CH2CH2CH2-C (= O) - (NH- DX).
[00177] [00177] - (NH-DX) has a structure represented by the following formula: [Formula 7]
[00178] [00178] The antibody that can be used in the antibody-drug conjugate of the present invention is not particularly limited as long as it is an anti-CDH6 antibody with internalizing activity or a functional fragment of the antibody, as described in the section above “2. Production of anti-CDH6 antibody “and the Examples.
[00179] [00179] In the following, a typical method for producing the antibody-drug conjugate of the present invention will be described. It should be noted that, in the description below, the “compound number” shown in each reaction scheme is used to represent a compound. Specifically, each compound is called a "compound of formula (1)", "compound (1)" or similar. The same goes for the other number of compounds. (3) -1 Production method 1
[00180] [00180] The antibody-drug conjugate represented by the formula (1) given below, in which the anti-CDH6 antibody is connected to the linker structure via a thioether can be produced by reacting an antibody with a sulfhydryl group converted from a bond disulfide by reducing the anti-CDH6 antibody, with the compound (2), the compound (2) can be obtained by a known method (for example, obtained by a method described in the patent publication literature US2016 / 297890 (for example, a method described in paragraphs [0336] to [0374])). Such an antibody-drug conjugate can be produced by the following method, for example. [Expression 1] () where AB represents an antibody with a sulfhydryl group,
[00181] [00181] Among them, the most preferred are the following: - (Succinimid-3-yl-N) -CH2CH2CH2CH2CH2-C (= O) -GGFG- NH-CH2-O-CH2-C (= O) -, - ( Succinimid-3-yl-N) -CH2CH2CH2CH2CH2-C (= O) -GGFG-NH-CH2CH2-O-CH2-C (= O) -, and - (Succinimid-3-yl-N) -CH2CH2-C ( = O) -NH-CH2CH2O-
[00182] [00182] More preferred are the following: - (Succinimid-3-yl-N) -CH2CH2CH2CH2CH2-C (= O) -GGFG- NH-CH2-O-CH2-C (= O) -, and - (Succinimid- 3-yl-N) -CH2CH2-C (= O) -NH-CH2CH2O- CH2CH2O-CH2CH2-C (= O) -GGFG-NH-CH2CH2CH2-C (= O) -.
[00183] [00183] In the reaction scheme described above, the antibody-drug conjugate (1) can be understood to have a structure in which a fraction of the drug structure for the linker is linked to an antibody. However, this description is given for the sake of convenience and, in fact, there are many cases in which a plurality of the structure fractions mentioned above are connected to an antibody molecule. The same goes for explaining the production method described below.
[00184] [00184] Specifically, the antibody-drug conjugate (1) can be produced by reacting the compound (2) obtainable by a known method (for example, obtainable by a method described in the patent publication literature US2016 / 297890 (for example, obtainable by a method described in paragraphs [0336] to [0374])), with the antibody (3a) having a sulfhydryl group.
[00185] [00185] The antibody (3a) with a sulfhydryl group can be obtained by a method well known to one skilled in the art (Hermanson, GT, Bioconjugate Techniques, pp. 56-136, pp. 456-493, Academic Press (1996) ). Examples of the method may include, but are not limited to: Traut's reagent reacting with the antibody's amino group; N-succinimidyl S-acetylthioalkanoates reacting with the amino group of the antibody followed by reaction with hydroxylamine; 3- (pyridyldithio) N-succinimidyl propionate reacting with the antibody, followed by reaction with a reducing agent; the antibody reacting with a reducing agent, such as dithiothreitol, 2-mercaptoethanol or tris (2-carboxyethyl) phosphine hydrochloride (TCEP) to reduce the interchain disulfide bond in the antibody to form a sulfhydryl group.
[00186] [00186] Specifically, an antibody with partially or completely reduced interchain disulfide bonds can be obtained using 0.3 to 3 molar equivalents of TCEP as the reducing agent by interchain disulfide bond in the antibody and reacting the reducing agent with the antibody in a buffer solution containing a chelating agent. Examples of the chelating agent may include ethylene diaminetetraacetic acid (EDTA) and diethylene triamine pentacetic acid (DTPA). The chelating agent can be used at a concentration of 1 mM to 20 mM. A solution of sodium phosphate, sodium borate, sodium acetate or similar can be used as a buffer solution. As a specific example, the antibody (3a) with partially or completely reduced sulfhydryl groups can be obtained by reacting the antibody with TCEP at 4 ° C to 37 ° C for 1 to 4 hours.
[00187] [00187] It should be noted that, when carrying out a reaction of adding a sulfhydryl group to a fraction of ligand-drug, the fraction of ligand-drug can be conjugated by a thioether bond.
[00188] [00188] Then, using 2 to 20 molar equivalents of the compound (2) per antibody (3a) with a sulfhydryl group, the antibody-drug conjugate (1) in which 2 to 8 molecules of drug are conjugated per antibody can be produced. Specifically, a solution containing the compound (2) dissolved in it can be added to a buffer solution containing the antibody (3a) with a sulfhydryl group for the reaction. In this context, a solution of sodium acetate, sodium phosphate, sodium borate or similar can be used as a buffer solution. The pH of the reaction is 5 to 9 and, more preferably, the reaction can be carried out near pH 7. An organic solvent such as dimethyl sulfoxide (DMSO), dimethylformamide (DMF), dimethylacetamide (DMA) or N-methyl-2-pyrrolidone (NMP) can be used as a solvent to dissolve the compound (2). The reaction can be carried out by adding the solution containing the compound (2) dissolved in the organic solvent at 1 to 20% v / v to a buffer solution containing the antibody (3a) with a sulfhydryl group. THE
[00189] [00189] The antibody-drug conjugate produced (1) can be subjected to concentration, buffer exchange, purification and measurement of antibody concentration and the average number of conjugated drug molecules per antibody molecule, according to common procedures described below , to identify the antibody-drug conjugate (1). (4) -1 Common procedure A: Concentration of aqueous antibody solution or antibody-drug conjugate
[00190] [00190] To an Amicon Ultra container (50,000 MWCO, Millipore Corporation), a solution of an antibody or an antibody-drug conjugate was added, and the solution of the antibody or the antibody-drug conjugate was concentrated by centrifugation (2000 centrifugation). G to 3800 G for 5 to 20 minutes) using a centrifuge (Allegra X-15R, Beckman Coulter, Inc.) (4) -2 Common procedure B: Measurement of antibody concentration
[00191] [00191] Using a UV detector (Nanodrop 1000, Thermo Fisher Scientific Inc.), the measurement of the antibody concentration was performed according to the method defined by the manufacturer. In this regard, the absorption coefficient of 280 nm was used, differing between the antibodies (1.3 mLmg-1 cm-1 to 1.8 mLmg-1cm-1). (4) -3 Common procedure C: Changing buffer for antibody
[00192] [00192] One NAP-25 column (Cat. No. 17-0852-02, GE Healthcare
[00193] [00193] A NAP-25 column was equilibrated with any commercially available buffer solution, such as an acetate buffer containing sorbitol (5%) (10 mM, pH 5.5; called ABS in the present description). An aqueous reaction solution of the antibody-drug conjugate (approximately 2.5 mL) was applied to the NAP-25 column and, subsequently, elution was performed with the buffer solution in an amount defined by the manufacturer, to collect an antibody fraction. A gel filtration purification process, in which the collected fraction was applied again to the NAP-25 column and eluted with the buffer solution, was repeated a total of 2 or 3 times to obtain the antibody-drug conjugate, excluding unconjugated drug binder and low molecular weight compounds (tris (2-carboxyethyl) hydrochloride (TCEP), N-acetyl-L-cysteine (NAC) and dimethylsulfoxide). (4) -5 Common procedure E: Measurement of the concentration of antibodies in the antibody-drug conjugate and the average number of conjugated drug molecules per antibody molecule
[00194] [00194] The concentration of conjugated drug in the antibody-drug conjugate can be calculated by measuring the UV absorbance of an aqueous solution of the antibody-drug conjugate in two lengths of
[00195] [00195] The total absorbance at any wavelength is equal to the sum of the absorbance of all light-absorbing chemical species that are present in a system [absorbance additivity]. Therefore, based on the hypothesis that the molar absorption coefficients of the antibody and the drug do not vary between before and after the conjugation between the antibody and the drug, the concentration of antibodies and the concentration of drug in the antibody-drug conjugate are represented by the following equations. A280 = AD, 280 + AA, 280 = εD, 280CD + εA, 280CA Equation (1) A370 = AD, 370 + AA, 370 = εD, 370CD + εA, 370CA Equation (2)
[00196] [00196] In this context, A280 represents the absorbance of an aqueous solution of the antibody-drug conjugate at 280 nm, A370 represents the absorbance of an aqueous solution of the antibody-drug conjugate at 370 nm, AA, 280 represents the absorbance of the antibody at 280 nm, AA, 370 represents the absorbance of the antibody at 370 nm, AD, 280 represents the absorbance of a conjugate precursor at 280 nm, AD, 370 represents the absorbance of a conjugate precursor at 370 nm, εA, 280 represents the coefficient molar absorption of the antibody at 280 nm, εA, 370 represents the molar absorption coefficient of the antibody at 370 nm, εD, 280 represents the molar absorption coefficient of a conjugate precursor at 280 nm, εD, 370 represents the absorption coefficient molar of a conjugate precursor at 370 nm, CA represents the concentration of antibody in the antibody-drug conjugate, and CD represents the concentration of drug in the antibody-drug conjugate.
[00197] [00197] In this context, in relation to εA, 280, εA, 370, εD, 280, and εD, 370, preliminarily prepared values are used (values estimated based on the calculation or measurement values obtained by UV measurement of the compound). For example, εA, 280 can be estimated from the antibody's amino acid sequence by a known calculation method (Protein Science, 1995, vol. 4,
[00198] [00198] The average number of drug molecules conjugated per antibody molecule in the antibody-drug conjugate can also be determined by high performance liquid chromatography (HPLC) analysis using the following method, in addition to the “(4) -5 Procedure common E ”mentioned above. In the following, the method for measuring the average number of drug molecules conjugated by HPLC will be described when the antibody is conjugated to the drug linker by a disulfide bond. One skilled in the art is able to properly measure the average number of drug molecules conjugated by HPLC, depending on the manner of connection between the antibody and the drug ligand, with reference to this method. F-1. Sample preparation for HPLC analysis (Reduction of antibody-drug conjugate)
[00199] [00199] An antibody-drug conjugate solution (approximately 1 mg / mL, 60 µL) is mixed with an aqueous solution of dithiothreitol (DTT) (100 mM, 15 µL). When incubating the mixture at 37 ° C for 30 minutes, the disulfide bond between the light chain and the heavy chain of the
[00200] [00200] HPLC analysis is performed under the following measurement conditions. HPLC system: Agilent 1290 HPLC system (Agilent Technologies, Inc.) Detector: Ultraviolet absorption spectrometer (measurement wavelength: 280 nm Column: ACQUITY UPLC BEH Phenyl (2.1 × 50 mm, 1.7 µm, 130 angstroms; Waters Corp., P / N 186002884) Column temperature: 80 ° C Mobile phase A: Aqueous solution containing 0.10% trifluoroacetic acid (TFA) and 15% 2-propanol Mobile phase B: Acetonitrile solution containing 0.075% TFA and 15% 2-propanol Gradient program: 14% -36% (0 min-15 min), 36% -80% (15 min-17 min), 80% -14% (17 min -17.01 min.) And 14% (17.01 min-25 min) Sample injection: 10 µL F-3.
[00201] [00201] F-3-1. In comparison with the light (L0) and heavy (H0) chains of unconjugated antibodies, a light chain linked to the drug molecule (s) (light chain linked to the drug molecule (s) i: Li ) and a heavy chain linked to the drug molecule (s) (heavy chain linked to the drug molecule (s) i: Hi) show greater hydrophobicity in proportion to the number of conjugated drug molecules and therefore have a longer retention time. Therefore, these chains are eluted in the order of, for example, L0 and L1 or H0, H1, H2 and H3. Detection peaks can be assigned to any of L0, L1, H0, H1, H2 and H3 by comparing the
[00202] [00202] F-3-2. Since the drug ligand has UV absorption, the peak area values are corrected in response to the number of drug ligand molecules conjugated according to the following expression using the light or heavy chain molar absorption coefficients and the drug binder. [Expression 2] Corrected value of the peak area of the light chain linked to the drug molecule (s) i Peak area Light chain molar absorption coefficient Light chain molar absorption coefficient + The number of conjugated drug molecules (i) × Molar absorption coefficient of the drug binder [Expression 3] Corrected value of the peak area of the heavy chain linked to the drug molecule (s) i Peak area Molar absorption coefficient of the heavy chain Absorption coefficient molar weight of the heavy chain + The number of conjugated drug molecules (i) × Molar absorption coefficient of the drug ligand
[00203] [00203] In this context, a value estimated from the amino acid sequence of the light chain or heavy chain of each antibody by a known calculation method (Protein Science, 1995, vol. 4, 2411-2423) can be used as a coefficient of molar absorption (280 nm) of the antibody light or heavy chain. In the case of H01L02, a molar absorption coefficient of 31710 and a molar absorption coefficient of 79990 were used as estimated values for the light chain and heavy chain, respectively, according to the antibody's amino acid sequence. The actually measured molar absorption coefficient (280 nm) of a compound in which the maleimide group was converted to succinimide thioether by the reaction of each drug ligand with mercaptoethanol or N-acetylcysteine was used as
[00204] [00204] F-3-3. The peak area ratio (%) of each chain is calculated for the total of the corrected values of the peak areas according to the following expression. [Expression 4] Peak area ratio of the light chain attached to the drug molecule (s) i Ratio of the peak area of the heavy chain attached to the drug molecule (s) i ALi and AHi: Corrected values peak areas of Li and Hi, respectively
[00205] [00205] F-3-4. The average number of drug molecules conjugated per antibody molecule in the antibody-drug conjugate is calculated according to the following expression. Average number of conjugated drug molecules = (L0 x 0 peak area ratio + L1 x 1 peak area ratio + H0 x 0 peak area ratio + H1 x 1 peak area ratio + peak area ratio H2 x 2 + peak area ratio H3 x 3) / 100 x 2
[00206] [00206] It should be noted that, in order to guarantee the amount of the antibody-drug conjugate, a plurality of antibody-drug conjugates with almost the same average number of conjugated drug molecules (for example, on the order of ± 1), that were produced under similar conditions, can be mixed to prepare a new batch. In this case, the average number of drug molecules in the new batch is between the average number of drug molecules before mixing.
[00207] [00207] A specific example of the antibody-drug conjugate of
[00208] [00208] In this context, AB represents the anti-CDH6 antibody described in the present description, and the antibody is conjugated to the drug linker via a sulfhydryl group resulting from the antibody. In this context, n has the same meaning as the so-called DAR (drug to antibody ratio) and represents a drug to antibody to antibody ratio. Specifically, n represents the number of conjugated drug molecules per antibody molecule, which is a defined numerical value and indicated as an average value, i.e., the average number of conjugated drug molecules. In the case of the antibody-drug conjugate represented by [Formula 9] or [Formula 10] of the present invention, n can be 2 to 8 and is
[00209] [00209] An example of the antibody-drug conjugate of the present invention can include an antibody-drug conjugate with a structure represented by the formula [Formula 9] or [Formula 10] described above, wherein the antibody represented by AB comprises any antibody selected from among the group consisting of the following antibodies (a) to (g), or a functional fragment of the antibody, or a pharmacologically acceptable salt of the antibody-drug conjugate: (a) an antibody consisting of a light chain consisting of the amino acid sequence at positions 21 to 233 in the full-length light chain amino acid sequence shown in SEQ ID NO: 61 and a heavy chain consisting of the amino acid sequence at positions 20 to 471 in the full-length heavy chain amino acid sequence shown in SEQ ID NO: 69; (b) an antibody consisting of a light chain consisting of the amino acid sequence at positions 21 to 233 in the full length amino acid sequence of the light chain shown in SEQ ID NO: 61 and a heavy chain consisting of the amino acid sequence in positions 20 to 471 in the full-length heavy chain amino acid sequence shown in SEQ ID NO: 73; (c) an antibody consisting of a light chain consisting of the amino acid sequence at positions 21 to 233 in the full length amino acid sequence of the light chain shown in SEQ ID NO: 61 and a heavy chain consisting of the amino acid sequence in positions 20 to 471 in the full-length heavy chain amino acid sequence shown in SEQ ID NO: 77; (d) an antibody consisting of a light chain consisting of the sequence of amino acids at positions 21 to 233 in the sequence of
[00210] [00210] Like the anti-CDH6 antibody of the present invention or the functional fragment of the antibody described in the section above “2. Production of
[00211] [00211] In addition, the anti-CDH6 antibody of the present invention or the functional fragment of the antibody can be used in the detection of cells that express CDH6.
[00212] [00212] Furthermore, since the anti-CDH6 antibody of the present invention or the functional fragment of the antibody has internalizing activity, it can be applied as the antibody in an antibody-drug conjugate.
[00213] [00213] When a drug with antitumor activity, such as cytotoxic activity, is used as a drug, the drug-anti-CDH6 antibody conjugate of the present invention described in the section above “3. Drug-anti-CDH6 antibody conjugate ”and in the Examples is a conjugate of the anti-CDH6 antibody and / or the functional antibody fragment with internalizing activity and the drug with anti-tumor activity, as cytotoxic activity. Since this anti-CDH6 drug-antibody conjugate has antitumor activity against cancer cells that express CDH6, it can be used as a medicine and, in particular, as a therapeutic agent and / or prophylactic agent for cancer.
[00214] [00214] The drug-anti-CDH6 antibody conjugate of this
[00215] [00215] When the drug-anti-CDH6 antibody conjugate of the present invention has a basic group such as an amino group, it can form a pharmacologically acceptable acid addition salt, if desired. Examples of such an acid addition salt may include: hydrohalides, such as hydrochloride, hydrochloride, hydrobromide and iodhydrate; salts of inorganic acids, such as nitrate, perchlorate, sulfate and phosphate; lower alkanesulfonates, such as methanesulfonate, trifluoromethanesulfonate and ethanesulfonate; arylsulfonates such as benzenesulfonate and p-toluenesulfonate; salts of organic acids such as formate, acetate, trifluoroacetate, malate, fumarate, succinate, citrate, tartrate, oxalate and maleate; and amino acid salts, such as ornithine, glutamate and aspartate salt.
[00216] [00216] When the drug-anti-CDH6 antibody conjugate of the present invention has an acidic group such as a carboxy group, it can form a pharmacologically acceptable base addition salt, if desired. Examples of such a base addition salt may include: alkali metal salts, such as a sodium salt, a potassium salt and lithium salt; alkaline earth metal salts, such as a calcium salt and a magnesium salt; inorganic salts such as an ammonium salt; and organic amine salts, such as a dibenzylamine salt, a morpholine salt, a phenylglycine alkyl ester salt, an ethylenediamine salt, an N-methylglucamine salt, a diethylamine salt, a triethylamine salt, a salt of cyclohexylamine, a dicyclohexylamine salt, an N, N'-dibenzylethylenediamine salt, a diethanolamine salt, an N-benzyl-N- (2-phenylethoxy) amine salt, a piperazine salt, tetramethylammonium salt and a salt of
[00217] [00217] The present invention can also include a drug-anti-CDH6 antibody conjugate in which one or more atoms that make up the antibody-drug conjugate are replaced by isotopes of the atoms. There are two types of isotopes: radioisotopes and stable isotopes. Examples of the isotope may include hydrogen isotypes (2H and 3H), carbon isotopes (11C, 13C and 14C), nitrogen isotopes (13N and 15N), oxygen isotopes (15O, 17O and 18O) and fluorine isotopes (18F ). A composition comprising the antibody-drug conjugate labeled with that isotope is useful as, for example, a therapeutic agent, a prophylactic agent, a research reagent, an assay reagent, a diagnostic agent and a diagnostic imaging agent in vivo. Each isotope-labeled antibody-drug conjugate and mixtures of an isotope-labeled antibody-drug conjugates for any given reason are included in the present invention. The isotope-labeled antibody-drug conjugate can be produced, for example, using an isotope-tagged starting material, instead of a starting material for the production method of the present invention mentioned below, according to a method known in the art.
[00218] [00218] Cytotoxicity in vitro can be measured based on the activity of suppressing the proliferative responses of cells, for example. For example, a cancer cell line that overexpresses CDH6 is grown and the drug-anti-CDH6 antibody conjugate is added in different concentrations to the culture system. Thereafter, its suppressive activity against the formation of foci, the formation of colonies and the growth of spheroids can be measured. In this context, for example, using a cancer cell line derived from a renal cell tumor or ovarian tumor, cell growth inhibiting activity against renal cell tumor or
[00219] [00219] The in vivo therapeutic effects on cancer in an experimental animal can be measured, for example, by administering the drug-anti-CDH6 antibody conjugate to a nude mouse, in which a highly CDH6-expressing tumor cell line has been inoculated and measuring a change in cancer cells. In this context, for example, using an animal model derived from an immunodeficient mouse by inoculating cells derived from renal cell carcinoma, clear renal cell carcinoma, papillary renal cell carcinoma, ovarian cancer, serous ovarian adenocarcinoma or thyroid cancer, therapeutic effects on renal cell carcinoma, clear renal cell carcinoma, papillary renal cell carcinoma, ovarian cancer, serous ovarian adenocarcinoma or thyroid cancer can be measured.
[00220] [00220] The type of cancer to which the drug-anti-CDH6 antibody conjugate of the present invention is applied is not particularly limited as long as the cancer expresses CDH6 in the cancer cells to be treated. Examples of this may include renal cell carcinoma (eg, clear renal cell carcinoma or papillary renal cell carcinoma), ovarian cancer, serous ovarian adenocarcinoma, thyroid cancer, bile duct cancer, lung cancer (for example, cancer small cell lung cancer or non-small cell lung cancer), glioblastoma, mesothelioma, uterine cancer, pancreatic cancer, Wilms' tumor and neuroblastoma, although cancer is not limited to them as long as the cancer expresses CDH6. More preferred examples of cancer may include renal cell carcinoma (e.g., clear renal cell carcinoma and papillary renal cell carcinoma) and ovarian cancer.
[00221] [00221] The drug-anti-CDH6 antibody conjugate of the present invention can preferably be administered to a mammal, and more
[00222] [00222] A substance used in a pharmaceutical composition comprising the drug-anti-CDH6 antibody conjugate of the present invention can be suitably selected from pharmaceutical and other additives generally used in this field, in terms of the applied dose or applied concentration, and then used.
[00223] [00223] The drug-anti-CDH6 antibody conjugate of the present invention can be administered as a pharmaceutical composition comprising one or more pharmaceutically compatible components. For example, the pharmaceutical composition typically comprises one or more pharmaceutical carriers (for example, sterile liquids (for example, water and oil (including petroleum oil and oil of animal, plant or synthetic origin)) (for example, peanut oil , soybean oil, mineral oil and sesame oil))). Water is a more typical carrier when the pharmaceutical composition is administered intravenously. An aqueous saline solution, an aqueous dextrose solution and an aqueous glycerol solution can also be used as a liquid carrier, in particular, for an injection solution. Suitable pharmaceutical carriers are known in the art. If desired, the composition can also comprise a trace amount of a moisturizing agent, an emulsifying agent or a pH buffering agent. Examples of suitable pharmaceutical carriers are described in "Remington’s Pharmaceutical Sciences" by E. W. Martin. The prescription corresponds to a mode of administration.
[00224] [00224] Various dispensing systems are known and can be used to administer the drug-anti-CDH6 antibody conjugate of the present invention. Examples of the route of administration may include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous and subcutaneous routes. Administration can be done by injection or bolus injection,
[00225] [00225] According to a representative embodiment, the pharmaceutical composition is prescribed, as a pharmaceutical composition suitable for intravenous administration to a human, according to conventional procedures. The composition for intravenous administration is typically a solution in a sterile and isotonic aqueous buffer solution. If necessary, the medication may also contain a solubilizing agent and a local anesthetic to relieve pain in the injection area (for example, lidocaine). In general, the ingredients described above are supplied, separately or together in a mixture in unit dosage form, as a lyophilized powder or an anhydrous concentrate contained in a container that is obtained by sealing, for example, an ampoule or a sachet indicating the amount of active agent. When the drug is to be administered by injection, it can be administered using, for example, an injection vial containing sterile pharmaceutical grade water or saline. When the drug is to be administered by injection, an ampoule of water or sterile saline solution for injection can be provided so that the ingredients described above are mixed with each other before administration.
[00226] [00226] The pharmaceutical composition of the present invention can be a pharmaceutical composition comprising only the drug-anti-CDH6 antibody conjugate of the present application, or it can be a pharmaceutical composition comprising the drug-antibody anti-CDH6 conjugate and at least one other therapeutic agent. for cancer. The drug-anti-CDH6 antibody conjugate of the present invention can also be administered in conjunction with an additional therapeutic agent for cancer and thus can enhance an anti-cancer effect. The additional anticancer agent used for this purpose can be administered to an individual simultaneously
[00227] [00227] Such a pharmaceutical composition can be prepared as a formulation with a selected composition and a necessary purity in the form of a lyophilized formulation or a liquid formulation. The pharmaceutical composition prepared as a lyophilized formulation can be a formulation containing an appropriate pharmaceutical additive used in that field. Likewise, the liquid formulation can be prepared so that the liquid formulation contains various pharmaceutical additives used in this field.
[00228] [00228] The composition and concentration of the pharmaceutical composition also vary depending on the method of administration. With regard to the affinity of the anti-CDH6 drug-antibody conjugate comprised in the pharmaceutical composition of the present invention for the antigen, that is, the dissociation constant (Kd value) of the anti-CDH6 drug-antibody conjugate for the antigen, according to affinity increases (ie, the Kd value is low), the pharmaceutical composition can have medicinal effects, even if the applied dose is decreased. Consequently, the applied dose of the antibody-drug conjugate can also be determined by defining the applied dose based on the state of affinity of the antibody-drug conjugate for the antigen. When the antibody-drug conjugate of the present invention
[00229] [00229] Hereinafter, the present invention will be described specifically in the following examples. However, these examples are not intended to limit the scope of the present invention. In addition, these examples should not be interpreted to a limited extent by any means. It should be noted that, in the following examples, unless otherwise specified, individual operations related to genetic manipulation were performed according to the method described in “Molecular Cloning” (Sambrook, J., Fritsch, EF and Maniatis, T., published by Cold Spring Harbor Laboratory Press in 1989) or other methods described in experimental manuals used by those skilled in the art or, when commercially available reagents or kits were used, the examples were performed according to the instructions included in the commercially available products . In the present description, reagents, solvents and starting materials are readily available from commercially available sources, unless otherwise specified. [Example 1: Obtaining rat anti-human CDH6 antibody with internalizing activity] 1) -1 Construction of CDH6 expression vectors from humans, mice, rats and cynomolgus monkeys
[00230] [00230] Using a cDNA expression vector encoding the human CDH6 protein (NP_004923) (OriGene Technologies Inc.,
[00231] [00231] Using a cDNA expression vector encoding the mouse CDH6 protein (NP_031692) (OriGene Technologies Inc., MC221619), the cDNA was incorporated into a vector for expression in mammals according to a method known to one skilled in technique for producing the mouse CDH6 expression vectors pcDNA3.1-mCDH6 and p3xFLAG-CMV-9-mCDH6. The amino acid sequence of the mouse CDH6 ORF is shown in SEQ ID NO: 7.
[00232] [00232] Using each cDNA fraction of the cDNA expression vector encoding the mouse CDH6 protein (NP_037059) (OriGene Technologies Inc., RN211850), the cDNA was incorporated into a vector for expression in mammals according to a known method by one skilled in the art to produce the mouse CDH6 expression vectors pcDNA3.1-rCDH6 and p3xFLAG-CMV-9-rCDH6. The amino acid sequence of the rat CDH6 ORF is shown in SEQ ID NO: 8.
[00233] [00233] The cDNA encoding the cynomolgus monkey CDH6 protein was cloned with cDNA synthesized from the total RNA of the cynomolgus monkey kidney as a template using primer 1 (5'-CACCATGAGAACTTACCGCTACTTCTTGCTGCTC-3 ') (SEQ ID NO: 85 ) and primer 2 (5'-TTAGGAGTCTTTGTCACTGTCCACTCCTCC-3 ') (SEQ ID NO: 86). It was confirmed that the obtained sequence corresponded to the extracellular region of the CDH6 of the cynomolgus monkey (NCBI, XP_005556691.1). It was also confirmed that the sequence corresponded to the complete CDH6 sequence of the cynomolgus monkey (EHH54180.1) registered with the EMBL. The cDNA was incorporated into a vector for expression
[00234] [00234] The EndoFree Plasmid Giga kit (Qiagen N.V.) was used for mass production of the produced plasmid DNA. 1) -2 Immunization
[00235] [00235] For immunization, female WKY / Izm rats (Japan SLC, Inc.) were used. First, the lower limbs of each rat were pretreated with hyaluronidase (Sigma-Aldrich Co. LLC) and, subsequently, the human CDH6 expression vector pcDNA3.1-hCDH6 produced in Example 1) -1 was injected intramuscularly into the same locations. Subsequently, using ECM830 (BTX), in vivo electroporation was performed in the same locations, using a two-needle electrode. Approximately once every two weeks, the same electroporation in vivo was repeated and, subsequently, the lymph nodes or spleen were collected from the rat and then used in the production of hybridomas. 1) -3 Hybridoma production
[00236] [00236] Lymph node or spleen cells were fused with mouse myeloma SP2 / 0-ag14 cells (ATCC, No. CRL-1 581) according to electrical cell fusion, using an LF301 cell fusion unit (BEX Co ., Ltd.), and the cells were then suspended and diluted with ClonaCell-HY D selection medium (StemCell Technologies Inc.) and then cultured under conditions of 37 ° C and 5% CO2. Individual hybridoma colonies that appeared in the culture medium were collected as monoclonal hybridomas, then suspended in the ClonaCell-HY E selection medium (StemCell Technologies Inc.) and then grown under conditions of 37 ° C and 5% CO2. After moderate proliferation of cells, frozen stocks of individual hybridoma cells were produced, while the
[00237] [00237] 293α cells (a stable expression cell line derived from HEK293 cells that express αv integrin and β3 integrin) were prepared at 5 x 105 cells / ml in DMEM medium supplemented with 10% FBS. According to the transduction procedures for the use of Lipofectamine 2000 (Thermo Fisher Scientific Inc.), the DNA of pcDNA3.1-hCDH6 or pcDNA3.1-cynoCDH6 or pcDNA3.1 as a negative control was introduced in 293α cells, and the cells were dispensed in an amount of 100 µL / well in a 96-well plate (Corning Inc.). After that, the cells were grown under conditions of 37 ° C and 5% CO2 in DMEM medium supplemented with 10% FBS for 24 to 27 hours. The transfected cells obtained were used for Cell-ELISA in an adhesive state. 1) -4-2 Cell-ELISA
[00238] [00238] Culture supernatant from 293α cells transfected with the expression vector prepared in Example 1) -4-1 was removed and culture supernatant from each hybridoma was then added to 293α cells transfected with pcDNA3.1-hCDH6 or pcDNA3 .1-cynoCDH6 or pcDNA3.1. The cells were left to stand at 4 ° C for 1 hour. Well cells were washed once with PBS (+) supplemented with 5% FBS and then rabbit anti-mouse IgG-Peroxidase antibody (Sigma-Aldrich Co. LLC) which was diluted 500 times with PBS (+) supplemented with 5% FBS was added to the wells. The cells were left to stand at 4 ° C for 1 hour. The cells in the wells were washed three times with PBS (+) supplemented with 5% FBS and then
[00239] [00239] 293T cells were seeded in a 225 cm2 flask (Sumitomo Bakelite Co., Ltd.) at 5 × 104 cells / cm2, and the cells were then grown overnight under conditions of 37 ° C and 5% CO2 in DMEM medium supplemented with 10% FBS. PcDNA3.1-cynoCDH6 or pcDNA3.1 as a negative control was introduced into 293T cells using Lipofectamine 2000, and the cells were further cultured overnight under conditions of 37 ° C and 5% CO2. The 293T cells transfected with each vector were treated with TrypLE Express (Thermo Fisher Scientific Corp.) and the cells were washed with DMEM supplemented with 10% FBS and then suspended in PBS supplemented with 5% FBS. The cell suspension obtained was used in the analysis by flow cytometry.
[00240] [00240] The specificity of binding to CDH6 monkey cynomolg of an antibody produced from hybridomas producing antibody binding to human CDH6 and CDH6 monkey cynomolg which were selected by Cell-ELISA in Example 1) -4 was further confirmed by flow cytometry. The suspension of the transient expression 293T cells prepared in Example 1) -5-1 was centrifuged and the supernatant was then removed. After that, the cells were suspended by adding the culture supernatant from each hybridoma. The cells were left to stand at 4 ° C for 1 hour. The cells were washed twice with PBS supplemented with 5% FBS, and then the cells were suspended by the addition of anti-rat FITC IgG conjugate (Sigma-Aldrich Co. LLC) which was diluted 500 times with PBS supplemented with 5 % of FBS. The cells were left to stand at 4 ° C for 1 hour. The cells were washed twice with PBS supplemented with 5% FBS and then resuspended in PBS supplemented with 5% FBS and 2 µg / ml of 7-aminoactinomycin D (Molecular Probes, Inc.), followed by detection using a flow cytometer (FC500; Beckman Coulter, Inc.). The data were analyzed using FlowJo (Tree Star, Inc.). After the dead cells were removed from the analysis by blocking cells positive for 7-aminoactinomycin D, a histogram of the FITC fluorescence intensity of the living cells was generated. Hybridomas that produce antibodies that specifically bind to the CDH6 of the cynomolgus monkey expressed on the cell membrane surface were selected based on the results in which the antibody histogram shifted to the side of strong fluorescence intensity in the 293T cells transfected with pcDNA3.1- cynoCDH6 compared to 293T cells transfected with the pcDNA3.1 control. 1) -6 Determination of the isotype of the mouse monoclonal antibody
[00241] [00241] Clones rG019, rG055, rG056 and rG061 suggested to specifically and strongly bind human CDH6 and monkey CDH6 were selected from the hybridomas producing rat anti-CDH6 antibody selected in Example 1) -5, and the isotype of each antibody was identified. The subclass of the heavy chain and the type of antibody light chain were determined using a RAT MONOCLONAL ANTIBODY ISOTYPING TEST KIT (DS Pharma Biomedical Co., Ltd.). As a result, it was confirmed that all of these 4 clones rG019, rG055, rG056 and rG061 had an IgG2b subclass heavy chain and a κ chain type light chain. 1) -7 Preparation of rat human anti-CDH6 antibody 1) -7-1 Production of culture supernatant
[00242] [00242] Monoclonal anti-rat human CDH6 antibodies were purified from hybridoma culture supernatants. First, the volume of each hybridoma producing mouse monoclonal anti-CDH6 antibody was sufficiently increased with the ClonaCell-HY E Selection Medium (StemCell Technologies Inc.) and subsequently the medium was exchanged with Hybridoma SFM (Thermo Fisher Scientific Corp .) to which 20% FBS Ultra Low IgG (Thermo Fisher Scientific Corp.) was added. After that, the hybridoma was cultured for 4 to 5 days. The resulting culture supernatant was collected and the insoluble matter was removed by passing through a 0.8 µm filter and through a 0.2 µm filter. 1) -7-2 Purification of rat anti-CDH6 antibody
[00243] [00243] An antibody (rat anti-CDH6 antibody (rG019, rG055, rG056 or rG061)) was purified from the hybridoma culture supernatant prepared in Example 1) -7-1 according to Protein affinity chromatography G. The antibody was adsorbed onto a Protein G column (GE Healthcare Biosciences Corp.), the column was then washed with PBS and the antibody was then eluted with an aqueous solution of glycine / HCl
[00244] [00244] The human CDH6 binding activity of the anti-rat CDH6 antibody produced in Example 1) -7 was assessed by flow cytometry. Using Lipofectamine 2000 (Thermo Fisher Scientific Inc.), the pcDNA3.1-hCDH6 produced in Example 1) -1 was transiently introduced into 293T cells (ATCC). The cells were grown overnight under conditions of 37 ° C and 5% CO2 and then a cell suspension was prepared. The suspension of the transfected 293T cells was centrifuged and the supernatant was then removed. Subsequently, the cells were suspended by adding each of the 4 monoclonal anti-mouse CDH6 antibodies (clone #: rG019, rG055, rG056 and rG061), which were prepared in Example 1) -7 or rat IgG control (R&D) Systems, Inc.) (final concentration: 10 ng / ml). The cells were left to stand at 4 ° C for 1 hour. The cells were washed twice with PBS supplemented with 5% FBS and then suspended by the addition of rabbit anti-IgG (whole molecule) -FITC antibody (Sigma-Aldrich Co. LLC) which was diluted 50 times with PBS supplemented with 5% FBS. The cells were left to stand at 4 ° C for 1 hour. The cells were washed twice with PBS supplemented with 5% FBS, followed by
[00245] [00245] The full-length extracellular region of human CDH6 has five extracellular domains, EC1 (SEQ ID NO: 2), EC2 (SEQ ID NO: 3), EC3 (SEQ ID NO: 4), EC4 (SEQ ID NO: 5), and EC5 (SEQ ID NO: 6). A gene to be expressed so that each of the five EC domains can be excluded from full-length human CDH6 was synthesized by GeneArt and incorporated into the p3xFLAG-CMV-9 vectors for expression in mammals (Sigma-Aldrich Co. LLC) accordingly with a method known to one skilled in the art, in order to produce an expression vector for each domain deletion mutant without any from EC1 to EC5. 2) -2-2 Epitope analysis of rat anti-CDH6 antibody by flow cytometry using domain deletion mutant
[00246] [00246] The epitopes to which human anti-CDH6 antibodies of
[00247] [00247] In order to select a CDH6 positive human tumor cell line for use in evaluating the obtained antibodies, the CDH6 expression information was retrieved from a known database, and the CDH6 expression on the cell membrane surface was assessed by flow cytometry. The NIH human ovarian tumor cell lines: OVCAR-3, PA-1 and ES-2 and the 786-O human kidney cell tumor cell line (all obtained from ATCC) were grown under conditions of 37 ° C and 5 ° C. % CO2 and subsequently a cell suspension was prepared. The cells were centrifuged and the supernatant was then removed. Subsequently, the cells were suspended by the addition of a commercially available anti-human CDH6 antibody (MABU2715, R&D Systems, Inc.) or mouse IgG1 (BD Pharmingen) as a negative control (final concentration: 50 µg / mL). The cells were left to stand at 4 ° C for 1 hour. The cells were washed twice with PBS supplemented with 5% FBS and then suspended by the addition of Fragment F (ab ') 2 of anti-immunoglobulins
[00248] [00248] The internalizing activity of the rat anti-CDH6 antibodies was evaluated using a rat anti-IgG reagent Rat-ZAP (Advanced Targeting Systems) conjugated to a toxin (saporin) that inhibits protein synthesis. Specifically, the NIH: OVCAR-3 positive CDH6 human ovarian tumor cell line was seeded at 4 x 103 cells / well in a 96-well plate and then grown overnight under conditions of 37 ° C and 5 ° C. % CO2. The 786-O human CDH6 positive renal cell tumor cell line (ATCC) was seeded at 1 x 103 cells / well in a 96-well plate and then grown overnight. The next day, each mouse anti-CDH6 antibody (final concentration: 1 nM) or
[00249] [00249] In order to amplify the cDNA encoding each variable region of rG019, the total RNA was prepared from G019 using the TRIzol reagent (Ambion, Inc.). 3) -1-2 Amplification of the cDNA encoding the variable region of the rG019 heavy chain by 5'-RACE PCR and determination of the nucleotide sequence
[00250] [00250] The cDNA encoding the variable region of the heavy chain was amplified using approximately 1 µg of the total RNA prepared in Example 3) -1-1 and a SMARTer RACE cDNA Amplification Kit (Clontech Laboratories, Inc.). As primers used to amplify the cDNA of the variable region of the heavy chain gene rG019 according to the PCR, UPM (Universal Primer A Mix: included in the SMARTer RACE cDNA amplification kit) and primers designed from the sequences of the regions were used constants of known heavy chains of mice.
[00251] The cDNA encoding the variable region of the heavy chain amplified by PCR 5'-RACE was cloned into a plasmid, and then the nucleotide sequence of the cDNA of the variable region of the heavy chain was subjected to sequence analysis.
[00252] [00252] The determined nucleotide sequence of the cDNA encoding the variable region of the rG019 heavy chain is shown in SEQ ID NO: 16, and the amino acid sequence thereof is shown in SEQ ID NO:
[00253] [00253] Amplification and sequencing were performed by the same method as that applied in Example 3) -1-2. However, as primers used to amplify the cDNA of the variable region of the light chain gene rG019 according to the PCR, UPM (Universal Primer A Mix: included in the SMARTer RACE cDNA amplification kit) and primers designed from sequences of known rat light chain constant regions.
[00254] [00254] The determined nucleotide sequence of the cDNA encoding the variable region of the rG019 light chain is shown in SEQ ID NO: 11, and the amino acid sequence thereof is shown in SEQ ID NO: 10. 3) -2 Amplification and sequencing of the variable region of the heavy chain of
[00255] [00255] The sequences were determined by the same method as that applied in Example 3) -1.
[00256] [00256] The determined nucleotide sequence of the cDNA encoding the variable region of the rG055 heavy chain is shown in SEQ ID NO: 26, and the amino acid sequence thereof is shown in SEQ ID NO:
[00257] [00257] The sequences were determined by the same method as applied in Example 3) -1.
[00258] [00258] The determined nucleotide sequence of the cDNA encoding the variable region of the rG056 heavy chain is shown in SEQ ID NO: 36, and the amino acid sequence thereof is shown in SEQ ID NO:
[00259] [00259] The sequences were determined by the same method as applied in Example 3) -1.
[00260] [00260] The determined nucleotide sequence of the cDNA encoding the variable region of the rG061 heavy chain is shown in SEQ ID NO: 46, and the amino acid sequence thereof is shown in SEQ ID NO:
[00261] [00261] A fragment of approx. 5.4 kb, which was obtained by digesting the plasmid pcDNA3.3-TOPO / LacZ (Invitrogen Corp.) with the restriction enzymes XbaI and PmeI, was linked to a DNA fragment that comprises a DNA sequence (SEQ ID NO : 50) encoding a human light chain signal sequence and a human κ chain constant region, using an In-Fusion Advantage PCR cloning kit (Clontech Laboratories, Inc.), to produce pcDNA3.3 / LK.
[00262] [00262] A neomycin expression unit was removed from pcDNA3.3 / LK to construct pCMA-LK. 4) -1-2 Construction of the chimeric and humanized IgG1 heavy chain expression vector pCMA-G1
[00263] [00263] A DNA fragment, which was obtained by digesting pCMA-LK with XbaI and PmeI to remove the DNA sequence encoding the light chain signal sequence and the human κ chain constant region, was ligated to a fragment of DNA comprising a DNA sequence (SEQ ID NO: 51) encoding a human heavy chain signal sequence and a human IgG1 constant region, using an In-Fusion Advantage PCR cloning kit (Clontech Laboratories, Inc.), to build pCMA-G1. 4) -1-3 Construction of the heavy chain expression vector chG019
[00264] [00264] A DNA fragment from nucleotide positions 36 to 440 in the chG019 heavy chain nucleotide sequence shown in SEQ ID NO: 57 has been synthesized (GENEART). Using an In-Fusion HD PCR cloning kit (Clontech Laboratories, Inc.), the synthesized DNA fragment was
[00265] [00265] A DNA fragment comprising a DNA sequence (SEQ ID NO: 52) encoding the chG019 light chain has been synthesized (GENEART). Using an In-Fusion HD PCR cloning kit (Clontech Laboratories, Inc.), the synthesized DNA fragment was ligated into a DNA fragment, which was obtained by digesting pCMA-LK with XbaI and PmeI to remove the DNA sequence which encodes the signal sequence of the light chain and the constant region of the human κ chain thereof, in order to construct a chG019 light chain expression vector. 4) -2 Production and purification of the human chimeric anti-CDH6 antibody chG019 4) -2-1 Production of chG019
[00266] [00266] According to the manual, FreeStyle 293F cells (Invitrogen Corp.) were cultured and passed. 1.2 × 109 FreeStyle 293F cells (Invitrogen Corp.) in the logarithmic growth phase were seeded in a 3 L Fernbach Erlenmeyer flask (Corning Inc.), then diluted with FreeStyle 293 expression medium (Invitrogen Corp.) at 2, 0 × 106 cells / ml. To 40 ml of Opti-Pro SFM medium (Invitrogen Corp.), 0.24 mg of the heavy chain expression vector, 0.36 mg of the light chain expression vector and 1.8 mg of polyethyleneimine (Polyscience # 24765), and the obtained mixture was stirred gently. After incubation for 5 minutes, the mixture was added to FreeStyle 293F cells. The cells were cultured by shaking at 90 rpm in an 8% CO2 incubator at 37 ° C for 4 hours and then 600 mL of EX-CELL VPRO (SAFC) medium
[00267] [00267] An antibody was purified from the culture supernatant obtained in Example 4) -2-1 by a one-step process according to rProtein A affinity chromatography. The culture supernatant was applied to a column that was stuffed with MabSelectSuRe (GE Healthcare Biosciences Corp.) equilibrated with PBS, and then the column was washed with PBS in an amount of two or more times the column volume. Subsequently, the antibody was eluted with a 2 M arginine hydrochloride solution (pH 4.0), so that a fraction containing an antibody was collected. The fraction was dialyzed (Thermo Fisher Scientific Inc., Slide-A-Lyzer dialysis cassette), so that the buffer was replaced with HBSor (25 mM histidine / 5% sorbitol, pH 6.0). Using a UF VIVASPIN20 centrifugal filter device (molecular weight limit: UF10K, Sartorius Inc.), the antibody was concentrated, so that the IgG concentration was adjusted to 5 mg / ml or more. Finally, the antibody was filtered through a Minisart-Plus filter (Sartorius Inc.) to obtain a purified sample. 4) -3 Evaluation of the binding activity of the human chimeric anti-CDH6 antibody chG019
[00268] [00268] The CDH6 binding activity of the human chimeric anti-CDH6 antibody chG019 purified in 4) -2 was confirmed by flow cytometry. Using Lipofectamine 2000, pcDNA3.1-hCDH6 or pcDNA3.1- cynoCDH6 produced in Example 1) -1 or pcDNA3.1 was introduced transiently into 293α cells. The cells were grown overnight
[00269] [00269] The molecular modeling of the chG019 variable regions explored a method known as homology modeling (Methods in Enzymology, 203, 121-153, (1991)). The commercially available three-dimensional protein structure analysis program BioLuminate (manufactured by Schrodinger, LLC) was employed using a template (PDB ID: 2I9L) registered with the Protein Data Bank (Nuc. Acid Res.
[00270] [00270] chG019 was humanized by CDR grafting (Proc. Natl. Acad. Sci. USA 86, 10029-10033 (1989)). The consensus sequences of subgroup 1 of the human gamma chain and of subgroup 1 of the kappa chain are determined by KABAT et al. (Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service National Institutes of Health, Bethesda, MD. (1991)) had a high identity with the chG019 structural regions and, based on this, were selected as acceptors for the heavy chain and the light chain, respectively. Donor residues to be grafted into acceptors were selected by analyzing three-dimensional models with reference, for example, to the criteria given by Queen et al. (Proc. Natl. Acad. Sci. USA 86, 10029-10033 (1989)). 5) -2 Humanization of the heavy chain chG019
[00271] [00271] Three heavy chains thus designed were named hH01, hH02 and hH04. The full length amino acid sequence of the hH01 heavy chain is shown in SEQ ID NO: 69. The nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 69 is shown in SEQ ID NO: 70. The amino acid sequence of full length of the hH02 heavy chain is shown in SEQ ID NO: 73. The nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 73 is shown in SEQ ID NO: 74. the full length amino acid sequence of the heavy chain hH04 is shown in SEQ ID NO: 77. The nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 77 is shown in SEQ ID NO: 78. 5) -3 Humanization of the light chain chG019
[00272] [00272] Two light chains so designed were named hL02 and hL03. The full length amino acid sequence of the hL02 light chain
[00273] [00273] An antibody consisting of hH01 and hL02 has been called "antibody H01L02" or "H01L02". An antibody consisting of hH02 and hL02 has been termed "H02L02 antibody" or "H02L02". An antibody consisting of hH02 and hL03 has been termed "H02L03 antibody" or "H02L03". An antibody consisting of hH04 and hL02 has been termed "antibody H04L02" or "H04L02". 5) -5 Humanized anti-CDH6 antibody expression 5) -5-1 Construction of the humanized hG019 heavy chain expression vector 5) -5-1-1 Construction of the humanized hG019- H01 heavy chain expression vector
[00274] A DNA fragment from nucleotide positions 36 to 440 in the humanized hG019-H01 heavy chain nucleotide sequence shown in SEQ ID NO: 70 has been synthesized (GENEART). A humanized hG019-H01 heavy chain expression vector was constructed by the same method as that applied in Example 4) -1-3. 5) -5-1-2 Construction of the humanized heavy chain expression vector of the hG019- H02 type
[00275] [00275] A DNA fragment from nucleotide positions 36 to 440 in the humanized hG019-H02 heavy chain nucleotide sequence shown in SEQ ID NO: 74 has been synthesized (GENEART). A humanized hG019-H02 heavy chain expression vector was constructed
[00276] [00276] A DNA fragment from nucleotide positions 36 to 440 in the humanized hG019-H04 heavy chain nucleotide sequence shown in SEQ ID NO: 78 has been synthesized (GENEART). A humanized hG019-H04 heavy chain expression vector was constructed by the same method as that applied in Example 4) -1-3. 5) -5-2 Construction of the humanized hG019 light chain expression vector 5) -5-2-1 Construction of the humanized hG019-L02 light chain expression vector
[00277] [00277] A DNA fragment comprising a DNA sequence encoding the humanized hG019-L02 light chain variable region from nucleotide positions 37 to 399 in the humanized hG019-L02 light chain nucleotide sequence shown in SEQ ID NO: 62 has been synthesized (GENEART). Using an In-Fusion HD PCR cloning kit (Clontech Laboratories, Inc.), the synthesized DNA fragment was inserted into a pCMA-LK site that was cleaved with the restriction enzyme BsiWI in order to construct an expression vector of a humanized hG019-L02 type light chain. 5) -5-2-2 Construction of the hG019-L03 type humanized light chain expression vector
[00278] [00278] A DNA fragment comprising a DNA sequence encoding the humanized hG019-L03 type light chain variable from nucleotide positions 37 to 399 in the humanized hG019-L03 type light chain nucleotide shown in SEQ ID NO: 66 has been synthesized (GENEART). A humanized hG019-L03 light chain expression vector was constructed by the same method as the
[00279] [00279] The antibodies were produced by the same method as that applied in Example 4) -2-1. H01L02, H02L02, H02L03 and H04L02 were produced by the combination of the heavy chain and the light chain shown in Example 5) -4. 5) -5-3-2 Two-step purification of H01L02, H02L02, H02L03 and H04L02
[00280] [00280] The antibody was purified from the culture supernatant obtained in Example 5) -5-3-1 by a two-step process, that is, by affinity chromatography with rProtein A and ceramic hydroxyapatite. The culture supernatant was applied to a column that was filled with MabSelectSuRe (manufactured by GE Healthcare Biosciences Corp.) equilibrated with PBS, and then the column was washed with PBS in an amount of two or more times the column volume. Subsequently, the antibody was eluted using a 2 M arginine hydrochloride solution (pH 4.0). A fraction containing the antibody was dialyzed (Thermo Fisher Scientific Inc., Slide-A-Lyzer dialysis cassette), so that the buffer was replaced with PBS. The antibody solution was diluted 5 times with a 5 mM sodium phosphate buffer / 50 mM MES / pH 7.0 and then applied to a ceramic hydroxyapatite column (Bio-Rad Laboratories, Inc., Hydroxyapatite column Bio-Scale CHT Type 1) that was equilibrated with a buffer of 5 mM NaPi / 50 mM MES / 30 mM NaCl / pH 7.0. Elution was performed on a linear sodium chloride concentration gradient, so that a fraction containing an antibody was collected. This fraction was dialyzed (Thermo Fisher Scientific Inc., Slide-A-Lyzer dialysis cassette), so that the buffer was replaced by HBSor (25 mM histidine / 5% sorbitol, pH 6.0). The antibody was concentrated with a UF VIVASPIN20 centrifugal filter device (molecular weight limit: UF10K, Sartorius
[00281] [00281] The anti-CDH6 antibody NOV0712 used in the Examples was produced with reference to the full length heavy chain and full length light chain amino acid sequences (SEQ ID NO: 235 and SEQ ID NO: 234, respectively, in Publication International No. WO 2016/024195) of NOV0712 described in International Publication No. WO 2016/024195. Reference example 1) -1 Anti-CDH6 antibody NOV0712 Reference example 1) -1-1 Construction of the heavy chain expression vector of the anti-CDH6 antibody NOV0712
[00282] [00282] A DNA fragment encoding the NOV0712 heavy chain variable region of nucleotide positions 36 to 428 in the NOV0712 heavy chain nucleotide sequence shown in SEQ ID NO: 84 has been synthesized (GENEART). A NOV0712 heavy chain expression vector was constructed by the same method as that applied in Example 4) -1-3. The NOV0712 heavy chain amino acid sequence expressed by the NOV0712 heavy chain expression vector is shown in SEQ ID NO: 83. In the amino acid sequence shown in SEQ ID NO: 83, the amino acid sequence consisting of the amino acid residues at the positions 1 to 19 is a signaling sequence. Reference example 1) -1-2 Construction of the anti-CDH6 antibody light chain expression vector NOV0712
[00283] [00283] A DNA fragment comprising a DNA sequence encoding the variable region of the light chain NOV0712 from nucleotide positions 37 to 405 in the nucleotide sequence of the light chain NOV0712 shown in SEQ ID NO: 82 has been synthesized (GENEART) . a
[00284] [00284] NOV0712 was produced by the same method as applied in Example 4) -2-1. Reference example 1) -2-2 One-step purification of anti-CDH6 antibody NOV0712
[00285] [00285] The anti-CDH6 antibody NOV0712 was purified from the culture supernatant obtained in Reference Example 1) -2-1 by the same method as applied in Example 4) -2-2 (antibody concentration: 5 mg / l HBSor). [Example 6: In vitro evaluation of humanized hG019 and NOV0712] 6) -1 Evaluation of humanized hG019 binding activity 6) -1-1 Human CDH6 antigen binding capacity of humanized hG019
[00286] [00286] The dissociation constant between the antibody and the antigen (recombinant human chimera CDH6 Fc His, R&D Systems, Inc.) was measured using Biacore T200 (GE Healthcare Biosciences Corp.), according to a capture method, which it comprises capturing the antigen as a ligand with the immobilized anti-His antibody and then measuring the dissociation constant using an antibody as an analyte. Approximately 1000 RU of the antihistidine antibody (His capture kit, GE Healthcare Biosciences Corp.) was covalently linked to the CM5 sensor chip (GE Healthcare Biosciences Corp.) by the amine coupling method. The antibody
[00287] [00287] Using Lipofectamine 2000 (Thermo Fisher Scientific Inc.), pcDNA3.1-hCDH6, pcDNA3.1-cynoCDH6, p3xFLAG-CMV-9-mCDH6 or p3xFLAG-CMV-9-rCDH6 produced in Example 1) -1 was introduced transiently in 293α cells. The cells were grown overnight under conditions of 37 ° C and 5% CO2 and then a cell suspension was prepared. Non-transfected 293α cells were used as a negative control. The suspension of 293α cells produced as described above was centrifuged and the supernatant was then removed. After that, the cells were suspended by adding each of the 4 hG019 antibodies
[00288] [00288] Using Lipofectamine 2000 (Thermo Fisher Scientific Inc.),
[00289] [00289] The 786-O / hCDH6 stable expression cell line was produced by infecting 786-O cells (ATCC) with a recombinant retrovirus for full-length human CDH6 expression. A human CDH6 expression retrovirus vector (pQCXIN-hCDH6) was produced using a cDNA expression vector that encodes the human CDH6 protein (NP_004923) (OriGene Technologies Inc., RC217889) and incorporates the cDNA into the pQCXIN retrovirus vector ( Clontech Laboratories, Inc.) according to a method known to one skilled in the art. Using FuGene HD (Promega Corp.), pQCXIN-hCDH6 was transiently introduced into the RetroPack PT67 retrovirus packaging cells (Clontech Laboratories, Inc.). After 48 hours, a
[00290] [00290] The marked H01L02 and the marked NOV0712 were produced using an Alexa Fluor 488 Monoclonal Antibody Marking Kit (Thermo Fisher Scientific Inc.). The cell suspension of the 786-O / hCDH6 stable expression cell line produced in 6) -2-2-1 was centrifuged and the
[00291] [00291] The internalizing activity of humanized hG019 and NOV0712 was evaluated using a Hum-ZAP anti-human IgG reagent (Advanced Targeting Systems) conjugated to a toxin (saporin) that inhibits protein synthesis. Specifically, the NIH: OVCAR-3 positive CDH6 human ovarian tumor cell line was seeded at 4 x 103 cells / well in a 96-well plate and then grown overnight under conditions of 37 ° C and 5 ° C. % CO2. The 786-O human CDH6 positive renal cell tumor cell line (ATCC) was seeded at 1 x 103 cells / well in a 96-well plate and then grown overnight. The CDH6 PA-1 positive human ovarian tumor cell line (ATCC) was seeded at 1 x 103 cells / well in a 96-well plate and then grown overnight under conditions of 37 ° C and 5% CO2. The following day, each anti-CDH6 antibody (final concentration: 1 nM) or human IgG1 antibody (Calbiochem) as a negative control antibody was added to the plate. Hum-ZAP (final concentration: 0.5 nM) or goat anti-human IgG of fragment F (ab ') 2, specific for fragment Fc (gamma) (Jackson ImmunoResearch Laboratories, Inc.) not conjugated with the toxin (concentration final: 0.5 nM) as a negative control was additionally added to the plate, and the cells were cultured under conditions of 37 ° C and 5% CO2 for 3 days. The number of live cells was measured by quantifying ATP activity (RLU) using CellTiter-Glo (TM) luminescent cell viability assay. In this evaluation, Hum-ZAP is absorbed by the cells in a manner dependent on the internalization activity of the anti-
[00292] [00292] Antibody reduction: The H01L02 produced in Example 5 was adjusted to 9.85 mg / mL with PBS6.0 / EDTA using common procedures B (using 1.53 mLmg-1cm-1 as the 280 nm absorption coefficient) and C described in production method 1. For this solution (5.7 mL), a 10 mM aqueous solution of TCEP (Tokyo Chemical Industry Co., Ltd.) (0.221 mL; 6.0 equivalents per antibody molecule) and a 1 M aqueous solution of dipotassium hydrogen phosphate (Nacalai Tesque, Inc .; 0.0855 mL) were added. After confirming that the solution had a pH within 7.0 ± 0.1, the interchain disulfide bond in the antibody was reduced by incubating the solution at 37 ° C for 2 hours.
[00293] [00293] Conjugation between antibody and drug ligand: The solution described above was incubated at 15 ° C for 10 minutes. Subsequently, a 10 mM solution of N- [6- (2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl) hexanoyl] glycylglycyl-L-phenylalanyl-N- (2 - {[ (1S, 9S) -9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H, 12H-benzo [ de] pyran [3 ', 4': 6,7] indolizine [1,2-b] quinolin-1-yl] amino} -2-oxoethoxy) methyl] glycinamide in dimethyl sulfoxide (0.386 ml; 10 equivalents per antibody molecule ) was added to it, and the mixture obtained was
[00294] [00294] Purification: The solution described above was purified by the common procedure D described in production method 1 to obtain 19 ml of a solution containing the antibody-drug conjugate of the title "H01L02-ADC".
[00295] [00295] Characterization: Using the common procedure E (using εD, 280 = 5440 and εD, 370 = 21240) described in production method 1, the following characteristic values were obtained.
[00296] [00296] Antibody concentration: 2.26 mg / mL, antibody yield: 42.9 mg (76%), average number of conjugated drug molecules (n) per antibody molecule measured by the common procedure E: 5.9 , and average number of conjugated drug molecules (n) per antibody molecule measured by the common procedure F: 7.7. 7) -2 Production of antibody-drug conjugate H02L02-DXd Step 1: Antibody-drug conjugate (2) [Formula 12] H02L02 工程 1 1 Step
[00297] [00297] Antibody reduction: The H02L02 produced in Example 5 was adjusted to 9.95 mg / mL with PBS6.0 / EDTA using common procedures B (using 1.51 mLmg-1cm-1 as the absorption coefficient of 280 nm) and C described in production method 1. For this solution (5.7 mL), a 10 mM aqueous solution of TCEP (Tokyo Chemical Industry Co., Ltd.) (0.234 mL; 6.0 equivalents per antibody molecule) and a 1 M aqueous solution of dipotassium hydrogen phosphate (Nacalai Tesque, Inc .; 0.0855 mL) were added. After confirming that the solution had a pH within 7.0 ± 0.1, the interchain disulfide bond in the antibody was reduced by incubating the solution at 37 ° C for 2 hours.
[00298] [00298] Conjugation between antibody and drug ligand: The solution described above was incubated at 15 ° C for 10 minutes. Subsequently, a 10mM solution of N- [6- (2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl) hexanoyl] glycylglycyl-L-phenylalanyl-N- (2 - {[( 1S, 9S) -9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H, 12H-benzo [de ] pyran [3 ', 4': 6,7] indolizine [1,2-b] quinolin-1-yl] amino} -2-oxoethoxy) methyl] glycinamide in dimethylsulfoxide (0.389 ml; 10 equivalents per antibody molecule) was added to it, and the obtained mixture was incubated at 15 ° C for 1 hour to conjugate the drug ligand to the antibody. Subsequently, an aqueous solution of 100 mM NAC (Sigma-Aldrich Co. LLC) (0.0350 mL; 9 equivalents per antibody molecule) was added to it, and the obtained mixture was stirred at room temperature for 20 minutes to finish the drug binder reaction.
[00299] [00299] Purification: The solution described above was purified by the common procedure D described in production method 1 to obtain 19 ml of a solution containing the antibody-drug conjugate of the title "H02L02-ADC".
[00300] [00300] Characterization: Using the common procedure E (using εD, 280 = 5440 and εD, 370 = 21240) described in production method 1, the following
[00301] [00301] Antibody concentration: 2.61 mg / mL, antibody yield: 49.6 mg (87%), average number of conjugated drug molecules (n) per antibody molecule measured by the common procedure E: 5.9 , and average number of conjugated drug molecules (n) per antibody molecule measured by the common procedure F: 7.6. 7) -3 Production of antibody-drug conjugate H02L03-DXd Step 1: Antibody-drug conjugate (3) [Formula 13] THE O O O H H N N N O N N N O O H H H
[00302] [00302] Antibody reduction: The H02L03 produced in Example 5 was adjusted to 9.86 mg / mL with PBS6.0 / EDTA using common procedures B (using 1.53 mLmg-1cm-1 as the 280 nm absorption coefficient) and C described in production method 1. For this solution (5.7 mL), a 10 mM aqueous solution of TCEP (Tokyo Chemical Industry Co., Ltd.) (0.270 mL; 7.0 equivalents per antibody molecule) and a 1 M aqueous solution of dipotassium hydrogen phosphate (Nacalai Tesque, Inc .; 0.0855 mL) were added. After confirming that the solution had a pH within 7.0 ± 0.1, the interchain disulfide bond in the antibody was reduced by incubating the solution at 37 ° C for 2 hours.
[00303] [00303] Conjugation between antibody and drug ligand: The solution described above was incubated at 15 ° C for 10 minutes. Subsequently, a 10 mM solution of N- [6- (2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl) hexanoyl] glycylglycyl-L-phenylalanyl-N - [(2- { [(1S, 9S) -9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H, 12H-benzo [de] pyran [3 ', 4': 6,7] indolizino [1,2-b] quinolin-1-yl] amino} -2-oxoethoxy) methyl] glycinamide in dimethyl sulfoxide (0.386 ml; 10 equivalents per molecule of antibody) was added to it, and the obtained mixture was incubated at 15 ° C for 1 hour to conjugate the drug ligand to the antibody. Subsequently, an aqueous solution of 100 mM NAC (Sigma-Aldrich Co. LLC) (0.0347 mL; 9 equivalents per antibody molecule) was added to it, and the obtained mixture was stirred at room temperature for 20 minutes to finish the drug binder reaction.
[00304] [00304] Purification: The solution described above was purified by the common procedure D described in production method 1 to obtain 19 ml of a solution containing the antibody-drug conjugate of the title "H01L02-ADC".
[00305] [00305] Characterization: Using the common procedure E (using εD, 280 = 5440 and εD, 370 = 21240) described in production method 1, the following characteristic values were obtained.
[00306] [00306] Antibody concentration: 2.71 mg / mL, antibody yield: 51.4 mg (91%), average number of conjugated drug molecules (n) per antibody molecule measured by the common procedure E: 5.7 , and average number of conjugated drug molecules (n) per antibody molecule measured by the common procedure F: 7.6. 7) -4 Production of antibody-drug conjugate H04L02-DXd Step 1: Antibody-drug conjugate (4) [Formula 14]
[00307] [00307] Antibody reduction: The H04L02 produced in Example 5 was adjusted to 9.86 mg / mL with PBS6.0 / EDTA using common procedures B (using 1.53 mLmg-1cm-1 as the absorption coefficient of 280 nm) and C described in production method 1. For this solution (5.7 mL), a 10 mM aqueous solution of TCEP (Tokyo Chemical Industry Co., Ltd.) (0.232 mL; 6.0 equivalents per antibody molecule) and a 1 M aqueous solution of dipotassium hydrogen phosphate (Nacalai Tesque, Inc .; 0.0855 mL) were added. After confirming that the solution had a pH within 7.0 ± 0.1, the interchain disulfide bond in the antibody was reduced by incubating the solution at 37 ° C for 2 hours.
[00308] [00308] Conjugation between antibody and drug ligand: The solution described above was incubated at 15 ° C for 10 minutes. Subsequently, a 10 mM solution of N- [6- (2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl) hexanoyl] glycylglycyl-L-phenylalanyl-N - [(2- { [(1S, 9S) -9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H, 12H-benzo [de] pyran [3 ', 4': 6,7] indolizino [1,2-b] quinolin-1-yl] amino} -2-oxoethoxy) methyl] glycinamide in dimethyl sulfoxide (0.386 ml; 10 equivalents per molecule of antibody) was added to it, and the obtained mixture was incubated at 15 ° C for 1 hour to conjugate the drug ligand to the antibody.
[00309] [00309] Purification: The solution described above was purified by the common procedure D described in production method 1 to obtain 19 ml of a solution containing the antibody-drug conjugate of the title "H04L02-ADC".
[00310] [00310] Characterization: Using the common procedure E (using εD, 280 = 5440 and εD, 370 = 21240) described in production method 1, the following characteristic values were obtained.
[00311] [00311] Antibody concentration: 2.56 mg / mL, antibody yield: 48.7 mg (87%), average number of conjugated drug molecules (n) per antibody molecule measured by the common procedure E: 5.8 , and average number of conjugated drug molecules (n) per antibody molecule measured by the common procedure F: 7.6. [Reference example 2: Production of drug-NOV0712 conjugate] Reference example 2) -1 Production of antibody-drug conjugate NOV0712-DM4 Antibody-drug conjugate (5)
[00312] [00312] Conjugation between antibody and drug ligand: The NOV0712 produced in Reference Example 1 was adjusted to 9.7 mg / mL with 20 mM HEPES8.1 (HEPES, 1 M Buffer Solution (20 mL) manufactured by Life Technologies Corp had the pH adjusted to 8.1 with 1 M sodium hydroxide and then brought to 1 L with distilled water) using common procedures B (using 1.51 mLmg-1cm-1 as the absorption coefficient at 280 nm) and C described in production method 1. The solution was incubated at 20 ° C for 10 minutes. Subsequently, a 10 mM solution of 1- (2,5-dioxopyrrolidin-1-yloxy) -1-oxo-4- (pyridin-2-ildisulfanyl) butane-2-sulfonic acid
[00313] [00313] Purification: The solution described above was purified by the common procedure D described in production method 1 to obtain 28 ml of a solution containing the antibody-drug conjugate of the title “NOV0712-DM4”.
[00314] [00314] Characterization: Using the common procedure E (using εA, 280 = 200500, εA, 252 = 76295, εD, 280 = 43170, and εD, 252 = 23224) described in production method 1, the following characteristic values were obtained .
[00315] [00315] Antibody concentration: 2.58 mg / mL, antibody yield: 72.2 mg (93%), and average number of conjugated drug molecules (n) per antibody molecule measured by the common procedure E: 3, 0. Reference Example 2) -2 Production of antibody-drug conjugate NOV0712-DXd Step 1: Antibody-drug conjugate (6)
[00316] [00316] Antibody reduction: NOV0712 produced in Reference Example 1 was adjusted to 9.26 mg / mL with PBS6.0 / EDTA using common procedures B (using 1.5 mLmg-1cm-1 as the absorption coefficient of 280 nm) and C described in production method 1. For this solution (6.6 mL), a 10 mM aqueous solution of TCEP (Tokyo Chemical Industry Co., Ltd.) (0.254 mL; 6.0 equivalents per molecule of antibody) and a 1 M aqueous solution of dipotassium hydrogen phosphate (Nacalai Tesque, Inc .; 0.0990 mL) were added. After confirming that the solution had a pH within 7.0 ± 0.1, the interchain disulfide bond in the antibody was reduced by incubating the solution at 37 ° C for 2 hours.
[00317] [00317] Conjugation between antibody and drug ligand: The solution described above was incubated at 15 ° C for 10 minutes. Subsequently, a 10 mM solution of N- [6- (2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl) hexanoyl] glycylglycyl-L-phenylalanyl-N - [(2- { [(1S, 9S) -9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H, 12H-benzo [de] pyran [3 ', 4': 6,7] indolizino [1,2-b] quinolin-1-yl] amino} -2-oxoethoxy) methyl] glycinamide in dimethylsulfoxide (0.381 ml; 9 equivalents per molecule antibody) was added to it, and the mixture obtained was
[00318] [00318] Purification: The solution described above was purified by the common procedure D described in production method 1 to obtain 23.5 ml of a solution containing the antibody-drug conjugate of the title “NOV0712-ADC”.
[00319] [00319] Characterization: Using the common procedure E (using εD, 280 = 5440 and εD, 370 = 21240) described in production method 1, the following characteristic values were obtained.
[00320] [00320] Antibody concentration: 2.26 mg / mL, antibody yield: 56.4 mg (92%), average number of conjugated drug molecules (n) per antibody molecule measured by the common procedure E: 6.4 , and mean number of conjugated drug molecules (n) per antibody molecule measured by the common procedure F: 7.8. [Reference example 3: Production of H01L02-DM4] Reference example 3) -1 Production of antibody-drug conjugate H01L02-DM4 Antibody-drug conjugate (7)
[00321] [00321] Conjugation between antibody and drug ligand: The H01L02 produced in Example 5 was adjusted to 9.8 mg / mL with 20 mM HEPES8.1 (HEPES, 1 M Buffer Solution (20 mL) manufactured by Life Technologies Corp. had the pH adjusted to 8.1 with 1 M sodium hydroxide and then brought to 1 L with distilled water) using common procedures B (using 1.53 mLmg-1cm-1 as the absorption coefficient at 280 nm) and C described in the method of production 1. The solution was incubated at 20 ° C for 10 minutes. Subsequently, a 10 mM solution of 1- (2,5-dioxopyrrolidin-1-
[00322] [00322] Purification: The solution described above was purified by the common procedure D described in production method 1 to obtain 3.5 ml of a solution containing the antibody-drug conjugate of the title "H01L02-DM4".
[00323] [00323] Characterization: Using the common procedure E (using εA, 280 = 223400, εA, 252 = 85646, εD, 280 = 4317, and εD, 252 = 23224) described in production method 1, the following characteristic values were obtained .
[00324] [00324] Antibody concentration: 1.97 mg / mL, antibody yield: 6.90 mg (88%), and average number of conjugated drug molecules (n) per antibody molecule measured by the common procedure E: 3, 6. [Example 8: Evaluation of the in vitro activity of the antibody-drug conjugate] 8) -1 Evaluation of the in vitro cell growth inhibiting activity of the antibody-drug conjugate against CDH6 positive human tumor cell lines
[00325] [00325] The CDH6 PA-1 positive human ovary tumor cell line was seeded on a 96-well plate at 2 x 103 cells / 100 µL / well in MEM medium supplemented with 10% FBS, and the
[00326] [00326] The anti-tumor effects of antibody-drug conjugates were evaluated using animal models derived from immunodeficient mice by inoculating CDH6 positive human tumor cell lines. BALB / c nude mice aged four to five weeks (CAnN.Cg-Foxnl [nu] / CrlCrlj [Foxnlnu / Foxnlnu], Charles River Laboratories Japan Inc.) and SCID mice (CB17 / Icr- Prkdc [scid] / CrlCrlj , Charles River Laboratories Japan Inc.) were acclimatized for 3 days or more under SPF conditions prior to use in the experiment. The mice were fed a sterile solid diet (FR-2, Funabashi Farms Co., Ltd) and received sterile tap water (which was prepared by adding a 5 to 15 ppm sodium hypochlorite solution to the tap water). The long diameter and the short diameter of the inoculated tumor were measured twice a week using digital calipers
[00327] [00327] Each antibody-drug conjugate was diluted with ABS buffer (10 mM acetate buffer, 5% sorbitol, pH 5.5) (Nacalai Tesque, Inc.), and the dilution was administered intravenously at the dose shown in each example to the tail of each mouse. The ABS buffer was administered in the same manner as above to a control group (vehicle group). Six mice were used per group in the experiment. 9) -1 Antitumor effect - (1)
[00328] [00328] The 786-O (ATCC) tumor cell line of CDH6 positive human kidney cells, whose expression of CDH6 was confirmed in Example 2) -3-1, was suspended in Matrigel (Corning Inc.) and the cell suspension was inoculated subcutaneously in a dose of 5 × 106 cells in the region of the right flank of each male SCID mouse (day 0). On day 18, the mice were grouped randomly. On the day of grouping, each of the 4 antibody-drug conjugates (clone names: H01L02-DXd, H02L02-DXd, H02L03-DXd and H04L02-DXd) produced in Example 7 or NOV0712-DM4 produced in Reference Example 2 were administered intravenously at a dose of 3 mg / kg at the tail of each mouse. The results are shown in Figure 12. The abscissa represents the number of days and the ordinate represents the tumor volume. The error interval represents an SE value.
[00329] [00329] NOV0712-DM4 had no significant antitumor effect in this tumor model. All 4 antibody-drug conjugates produced in Example 7 decreased the tumor volume after administration, exerted significant tumor regression and sustained the tumor regression effect for 24 days after administration (Figure 12).
[00330] The CDH6 positive human ovarian tumor cell line PA-1 (ATCC) positive for CDH6, whose expression of CDH6 was confirmed in Example 2) -3-1, was suspended in Matrigel (Corning Inc.) and the cell suspension was inoculated subcutaneously in a dose of 8.5 × 106 cells in the region of the right flank of each female nude mouse (day 0). On day 11, the mice were randomly grouped. On the day of grouping, the antibody-drug conjugate H01L02-DXd produced in Example 7, or NOV0712-DM4 or NOV0712-DXd produced in Reference Example 2 was administered intravenously at doses of 1 or 3 mg / kg at the tail of each mouse. The results are shown in Figure 13. The abscissa represents the number of days and the ordinate represents the tumor volume. The error interval represents an SE value.
[00331] [00331] NOV0712-DM4 had no antitumor effect in any of the doses of 1 and 3 mg / kg in this tumor model. On the other hand, H01L02-DXd significantly decreased tumor volume after administration at doses of 1 and 3 mg / kg and exerted a tumor regression effect (Figure 13). The H01L02 antibody obtained in the present description and the NOV0712 antibody were conjugated to the same drug DXd and the medicinal effects of the resulting samples were compared. As a result, H01L02-DXd had a stronger antitumor effect than NOV0712-DXd at doses of 1 and 3 mg / kg. Specifically, it has been demonstrated that the H01L02 antibody of the present invention is a superior antibody to antibody-drug conjugates as antitumor agents to the NOV0712 antibody (Figure 13). 9) -3 Antitumor effect - (3)
[00332] The NIH: OVCAR-3 (ATCC) CDH6 positive human ovary tumor cell line, whose expression of CDH6 was confirmed in Example 2) -3-1, was suspended in Matrigel (Corning Inc.) and
[00333] [00333] NOV0712-DM4 had no anti-tumor effect at a dose of 1 mg / kg and had an anti-tumor effect at a dose of 3 mg / kg, although tumor growth was observed two weeks after administration. On the other hand, H01L02-DXd significantly suppressed the increase in tumor volume after administration at doses of 1 and 3 mg / kg, and sustained, particularly, at the dose of 3 mg / kg, the tumor growth inhibiting effect for a long period of 31 days after administration (Figure 14).
[00334] [00334] The tumor growth-inhibiting effect of NOV0712-DM4 produced in Reference Example 2 or H01L02-DM4 produced in Reference Example 3 was evaluated in the same manner as above using PA-1 cells. H01L02-DM4 decreased the tumor volume even more than NOV0712-DM4. Thus, the H01L02 antibody of the present invention is superior as an antibody to antibody-drug conjugates acting as anti-tumor agents compared to the NOV0712 antibody. 9) -4 Antitumor effect - (4)
[00335] [00335] The 786-O (ATCC) tumor cell line of CDH6 positive human kidney cells, whose expression of CDH6 was confirmed in Example 2) -3-1, was suspended in Matrigel (Corning Inc.) and the cell suspension was inoculated subcutaneously at a dose of 5 × 106
[00336] [00336] NOV0712-DM4 had no significant antitumor effect in any of the doses of 1 and 3 mg / kg in this tumor model. On the other hand, H01L02-DXd decreased the tumor volume after administration at doses of 1 and 3 mg / kg, and exerted, particularly, at the dose of 3 mg / kg, significant tumor regression, and sustained the regression effect tumor for 20 days after administration (Figure 15). 9) -5 Antitumor effect - (5)
[00337] [00337] The human CDH6 negative ES-2 tumor cell line (ATCC), whose absence of CDH6 expression was confirmed in Example 2) -3-1, was suspended in physiological saline, and the cell suspension was inoculated subcutaneously at a dose of 1 × 106 cells in the region of the right flank of each female nude mouse (day 0). On day 7, the mice were randomly grouped. On the day of grouping, the H01L02-DXd antibody-drug conjugate produced in Example 7, or NOV0712-DM4 produced in Reference Example 2 were administered intravenously at doses of 1 or 3 mg / kg at the tail of each mouse. The results are shown in Figure 16. The abscissa represents the number of days and the ordinate represents the tumor volume. The error interval represents an SE value.
[00338] [00338] In this tumor model that does not express CDH6, H01L02- DXd and NOV0712-DM4 did not show antitumor effect in any of the
[00339] [00339] The present invention provides an anti-CDH6 antibody with internalizing activity and an antibody-drug conjugate comprising the antibody. The antibody-drug conjugate can be used as a therapeutic drug for cancer and the like.

权利要求:
Claims (1)
[1]
1/18
1. Antibody, characterized by the fact that it specifically binds to the amino acid sequence shown in SEQ ID NO: 4 and with internalization capacity that allows cell uptake, or a functional fragment of the antibody.
2. Antibody or functional fragment of the antibody according to claim 1, characterized by the fact that it has competitive inhibitory activity, for binding to the amino acid sequence shown in SEQ ID NO: 4, against at least any antibody selected from the group consisting of following antibodies (1) to (5): (1) an antibody with a light chain consisting of the amino acid sequence at positions 21 to 233 in SEQ ID NO: 53 and a heavy chain consisting of the amino acid sequence at positions 20 to 471 in SEQ ID NO: 56, (2) an antibody with a light chain consisting of the amino acid sequence at positions 21 to 233 in SEQ ID NO: 61 and a heavy chain consisting of the amino acid sequence at positions 20 to 471 in SEQ ID NO: 69, (3) an antibody with a light chain consisting of the amino acid sequence at positions 21 to 233 in SEQ ID NO: 61 and a heavy chain consisting of the amino acid sequence at positions 20 to 471 in SEQ ID NO: 73, (4) an antibody with a the light chain consisting of the amino acid sequence at positions 21 to 233 in SEQ ID NO: 65 and a heavy chain consisting of the amino acid sequence at positions 20 to 471 in SEQ ID NO: 73, and (5) an antibody with a light chain consisting of the amino acid sequence at positions 21 to 233 in SEQ ID NO: 61 and a heavy chain consisting of the amino acid sequence at positions 20 to
2/18 471 in SEQ ID NO: 77.
3. Antibody or functional fragment of the antibody according to claim 1 or 2, characterized by the fact that it comprises CDRL1, CDRL2 and CDRL3 in any combination selected from the group consisting of the following combinations (1) to (4): (1) CDRL1 consisting of the amino acid sequence shown in SEQ ID NO: 12, CDRL2 consisting of the amino acid sequence shown in SEQ ID NO: 13, and CDRL3 consisting of the amino acid sequence shown in SEQ ID NO: 14, (2) CDRL1 which consists of the amino acid sequence shown in SEQ ID NO: 22, CDRL2 which consists of the amino acid sequence shown in SEQ ID NO: 23, and CDRL3 which consists of the amino acid sequence shown in SEQ ID NO: 24, (3) CDRL1 which consists of the amino acid sequence shown in SEQ ID NO: 32, CDRL2 which consists of the amino acid sequence shown in SEQ ID NO: 33, and CDRL3 which consists of the amino acid sequence shown in SEQ ID NO: 34, and (4) CDRL1 that consists of the amino acid sequence shown in SEQ ID NO: 42, CDRL2 consisting of the amino acid sequence shown in SEQ ID NO: 43, and CDRL3 consisting of the amino acid sequence shown in SEQ ID NO: 44, and CDRH1, CDRH2 and CDRH3 in any selected combination from the group consisting of combinations (5) to (9): (5) CDRH1 which consists of the amino acid sequence shown in SEQ ID NO: 17, CDRH2 which consists of the amino acid sequence shown in SEQ ID NO: 18, and CDRH3 which consists of the sequence of amino acids shown in SEQ ID NO: 19, (6) CDRH1 which consists of the amino acid sequence shown in SEQ ID NO: 27, CDRH2 which consists of the
3/18 amino acids shown in SEQ ID NO: 28, and CDRH3 which consists of the amino acid sequence shown in SEQ ID NO: 29, (7) CDRH1 which consists of the amino acid sequence shown in SEQ ID NO: 37, CDRH2 which consists of amino acid sequence shown in SEQ ID NO: 38, and CDRH3 which consists of the amino acid sequence shown in SEQ ID NO: 39, (8) CDRH1 which consists of the amino acid sequence shown in SEQ ID NO: 47, CDRH2 which consists of the sequence amino acid shown in SEQ ID NO: 48, and CDRH3 which consists of the amino acid sequence shown in SEQ ID NO: 49, and (9) CDRH1 which consists of the amino acid sequence shown in SEQ ID NO: 17, CDRH2 which consists of the sequence amino acid shown in SEQ ID NO: 60, and CDRH3 which consists of the amino acid sequence shown in SEQ ID NO: 19.
Antibody or functional fragment of the antibody according to any one of claims 1 to 3, characterized in that it comprises CDRL1, CDRL2 and CDRL3, and CDRH1, CDRH2 and CDRH3 in any combination selected from the group consisting of the following combinations (1 ) to (5): (1) CDRL1 consisting of the amino acid sequence shown in SEQ ID NO: 12, CDRL2 consisting of the amino acid sequence shown in SEQ ID NO: 13, and CDRL3 consisting of the amino acid sequence shown in SEQ ID NO: 14, and CDRH1 which consists of the amino acid sequence shown in SEQ ID NO: 17, CDRH2 which consists of the amino acid sequence shown in SEQ ID NO: 18, and CDRH3 which consists of the amino acid sequence shown in SEQ ID NO: 19, (2) CDRL1 consisting of the amino acid sequence shown in SEQ ID NO: 22, CDRL2 consisting of the sequence of
4/18 amino acids shown in SEQ ID NO: 23, and CDRL3 which consists of the amino acid sequence shown in SEQ ID NO: 24, and CDRH1 which consists of the amino acid sequence shown in SEQ ID NO: 27, CDRH2 which consists of the sequence of amino acids shown in SEQ ID NO: 28, and CDRH3 which consists of the amino acid sequence shown in SEQ ID NO: 29, (3) CDRL1 which consists of the amino acid sequence shown in SEQ ID NO: 32, CDRL2 which consists of the amino acid sequence shown in SEQ ID NO: 33, and CDRL3 which consists of the amino acid sequence shown in SEQ ID NO: 34, and CDRH1 which consists of the amino acid sequence shown in SEQ ID NO: 37, CDRH2 which consists of the amino acid sequence shown in SEQ ID NO: 38, and CDRH3 which consists of the amino acid sequence shown in SEQ ID NO: 39, (4) CDRL1 which consists of the amino acid sequence shown in SEQ ID NO: 42, CDRL2 which consists of the amino acid sequence shown in SEQ ID NO: 43, and CDRL3 consisting of the amino acid sequence those shown in SEQ ID NO: 44, and CDRH1 which consists of the amino acid sequence shown in SEQ ID NO: 47, CDRH2 which consists of the amino acid sequence shown in SEQ ID NO: 48, and CDRH3 which consists of the amino acid sequence shown in SEQ ID NO: 49, and (5) CDRL1 which consists of the amino acid sequence shown in SEQ ID NO: 12, CDRL2 which consists of the amino acid sequence shown in SEQ ID NO: 13, and CDRL3 which consists of the amino acid sequence shown in SEQ ID NO: 14, and CDRH1 which consists of the amino acid sequence shown in SEQ ID NO: 17, CDRH2 which consists of the amino acid sequence shown in SEQ ID NO: 60, and CDRH3 which consists of the amino acid sequence shown in SEQ ID NO :
5/18
19.
Antibody or functional fragment of the antibody according to any one of claims 1 to 4, characterized in that it is humanized.
6. Antibody or functional antibody fragment according to any one of claims 1 to 5, characterized by the fact that it has any light chain variable region selected from the group consisting of the following variable regions (1) to (4): (1 ) a light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 63, (2) a light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 67, (3) a sequence of amino acids with a sequence identity of at least 95% or more with the sequence of a different structural region of each CDR sequence in the amino acid sequences of (1) and (2), and (4) an amino acid sequence comprising a deletion, substitution or addition of one or more amino acids in the sequence of a different structural region of each CDR sequence in the amino acid sequences from (1) to (3), and any heavy chain variable region selected from the group consisting of the following s variable regions (5) to (9): (5) a heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 71, (6) a heavy chain variable region consisting of the shown amino acid sequence in SEQ ID NO: 75, (7) a heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 79, (8) an amino acid sequence with a homology of
6/18 sequence of at least 95% or more with the sequence of a different structural region of each CDR sequence in the amino acid sequences from (5) to (7), and (9) an amino acid sequence that comprises a deletion, substitution or addition of one or more amino acids in the sequence of a different structural region of each CDR sequence in the amino acid sequences from (5) to (8).
Antibody or functional antibody fragment according to any one of claims 1 to 6, characterized in that it comprises a light chain variable region and a heavy chain variable region in any of the following combinations (1) to (4 ): (1) a light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 63 and a heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 71, (2) a region light chain variable consisting of the amino acid sequence shown in SEQ ID NO: 63 and a heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 75, (3) a light chain variable region consisting of amino acid sequence shown in SEQ ID NO: 67 and a heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 75, and (4) a light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 63 and a heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 79.
8. Antibody or functional fragment of the antibody according to any one of claims 1 to 7, characterized by the fact that
7/18 has any of the following combinations (1) to (4): (1) a light chain consisting of the amino acid sequence at positions 21 to 233 in SEQ ID NO: 61 and a heavy chain consisting of the amino acid sequence at positions 20 to 471 in SEQ ID NO: 69, (2) a light chain consisting of the amino acid sequence at positions 21 to 233 in SEQ ID NO: 61 and a heavy chain consisting of the amino acid sequence at positions 20 to 471 in SEQ ID NO: 73, (3) a light chain consisting of the amino acid sequence at positions 21 to 233 in SEQ ID NO: 65 and a heavy chain consisting of the amino acid sequence at positions 20 to 471 in SEQ ID NO: 73, and (4) a light chain consisting of the amino acid sequence at positions 21 to 233 in SEQ ID NO: 61 and a heavy chain consisting of the amino acid sequence at positions 20 to 471 in SEQ ID NO: 77.
Antibody or functional fragment of the antibody according to claim 8, characterized by the fact that it has a light chain consisting of the amino acid sequence at positions 21 to 233 in SEQ ID NO: 61 and a heavy chain consisting of the amino acids at positions 20 to 471 in SEQ ID NO: 69.
10. Antibody or functional fragment of the antibody according to claim 8, characterized by the fact that it has a light chain consisting of the amino acid sequence at positions 21 to 233 in SEQ ID NO: 61 and a heavy chain consisting of the amino acids at positions 20 to 471 in SEQ ID NO: 73.
11. Antibody or functional fragment of the antibody according to claim 8, characterized by the fact that it has a light chain consisting of the amino acid sequence at positions 21 to 233 in SEQ ID NO: 65 and a heavy chain consisting of the amino acids at positions 20 to 471 in SEQ ID NO: 73.
12. Antibody or functional fragment of the antibody according to
8/18 with claim 8, characterized by the fact that it has a light chain consisting of the amino acid sequence at positions 21 to 233 in SEQ ID NO: 61 and a heavy chain consisting of the amino acid sequence at positions 20 to 471 in SEQ ID NO: 77.
13. Functional fragment of the antibody as defined in any of claims 1 to 12, characterized in that the functional fragment is selected from the group consisting of Fab, F (ab ') 2, Fab' and Fv.
14. Polynucleotide, characterized by the fact that it encodes the antibody or the functional fragment of the antibody as defined in any one of claims 1 to 13.
Polynucleotide according to claim 14, characterized in that it comprises polynucleotides in any combination selected from the group consisting of the following combinations (1) to (5): (1) a polynucleotide that encodes a variable region of light chain that comprises CDRL1 which consists of the amino acid sequence shown in SEQ ID NO: 12, CDRL2 which consists of the amino acid sequence shown in SEQ ID NO: 13 and CDRL3 which consists of the amino acid sequence shown in SEQ ID NO: 14, and a polynucleotide that encodes a variable region of heavy chain comprising CDRH1 which consists of the amino acid sequence shown in SEQ ID NO: 17, CDRH2 which consists of the amino acid sequence shown in SEQ ID NO: 18 and CDRH3 which consists of the amino acid sequence shown in SEQ ID NO: 19, (2) a polynucleotide encoding a light chain variable region comprising CDRL1 that consists of the amino acid sequence shown in SEQ ID NO: 22, CDRL2 that consists of ste in the amino acid sequence shown in SEQ ID NO: 23 and CDRL3 which consists of
9/18 amino acid sequence shown in SEQ ID NO: 24, and a polynucleotide that encodes a heavy chain variable region comprising CDRH1 that consists of the amino acid sequence shown in SEQ ID NO: 27, CDRH2 that consists of the amino acid sequence shown in SEQ ID NO: 28 and CDRH3 which consists of the amino acid sequence shown in SEQ ID NO: 29, (3) a polynucleotide that encodes a light chain variable region comprising CDRL1 which consists of the amino acid sequence shown in SEQ ID NO: 32, CDRL2 consisting of the amino acid sequence shown in SEQ ID NO: 33 and CDRL3 consisting of the amino acid sequence shown in SEQ ID NO: 34, and a polynucleotide encoding a heavy chain variable region comprising CDRH1 consisting of the sequence amino acid shown in SEQ ID NO: 37, CDRH2 which consists of the amino acid sequence shown in SEQ ID NO: 38 and CDRH3 which consists of the amino acid sequence shown in SEQ ID NO: 39, (4) a polynuc leotide encoding a light chain variable region comprising CDRL1 consisting of the amino acid sequence shown in SEQ ID NO: 42, CDRL2 consisting of the amino acid sequence shown in SEQ ID NO: 43 and CDRL3 consisting of the amino acid sequence shown in SEQ ID NO: 44, and a polynucleotide that encodes a variable region of heavy chain comprising CDRH1 which consists of the amino acid sequence shown in SEQ ID NO: 47, CDRH2 which consists of the amino acid sequence shown in SEQ ID NO: 48 and CDRH3 consisting of the amino acid sequence shown in SEQ ID NO: 49, and (5) a polynucleotide encoding a light chain variable region comprising CDRL1 which consists of the amino acid sequence shown in SEQ ID NO: 12, CDRL2 which consists of the sequence
10/18 amino acids shown in SEQ ID NO: 13 and CDRL3 consisting of the amino acid sequence shown in SEQ ID NO: 14, and a polynucleotide encoding a heavy chain variable region comprising CDRH1 consisting of the amino acid sequence shown in SEQ ID NO: 17, CDRH2 which consists of the amino acid sequence shown in SEQ ID NO: 60 and CDRH3 which consists of the amino acid sequence shown in SEQ ID NO: 19.
16. A polynucleotide according to claim 14 or 15, characterized in that it comprises a polynucleotide encoding a light chain consisting of the amino acid sequence at positions 21 to 233 in SEQ ID NO: 61 and a polynucleotide encoding a heavy chain consisting of the amino acid sequence at positions 20 to 471 in SEQ ID NO: 69.
17. Polynucleotide according to claim 14 or 15, characterized in that it comprises a polynucleotide encoding a light chain consisting of the amino acid sequence at positions 21 to 233 in SEQ ID NO: 61 and a polynucleotide encoding a heavy chain consisting of the amino acid sequence at positions 20 to 471 in SEQ ID NO: 73.
A polynucleotide according to claim 14 or 15, characterized in that it comprises a polynucleotide that encodes a light chain consisting of the amino acid sequence at positions 21 to 233 in SEQ ID NO: 65 and a polynucleotide that encodes a heavy chain consisting of the amino acid sequence at positions 20 to 471 in SEQ ID NO: 73.
19. Polynucleotide according to claim 14 or 15, characterized in that it comprises a polynucleotide that encodes a light chain consisting of the amino acid sequence at positions 21 to 233 in SEQ ID NO: 61 and a polynucleotide that encodes a heavy chain
11/18 consisting of the amino acid sequence at positions 20 to 471 in SEQ ID NO: 77.
20. Expression vector, characterized by the fact that it comprises the polynucleotide as defined in any one of claims 14 to 19.
21. Host cells, characterized by the fact that they are transformed with the expression vector as defined in claim 20.
22. Host cells according to claim 21, characterized in that the host cells are eukaryotic cells.
23. Method for producing an antibody of interest or a functional fragment of the antibody, characterized in that it comprises the step of culturing the host cells as defined in claim 21 or 22, and the step of collecting an antibody of interest or a fragment of the culture antibody obtained by the step mentioned above.
24. Antibody or functional fragment of the antibody according to any of claims 1 to 13, characterized in that the heavy chain or the light chain has undergone one or two or more modifications selected from the group consisting of N-linked glycosylation, O-linked glycosylation, N-terminal processing, C-terminal processing, deamidation, isomerization of aspartic acid, methionine oxidation, addition of a methionine residue to the N-terminal, amidation of a proline residue, conversion of N-terminal glutamine or N-terminal glutamic acid in pyroglutamic acid, and a deletion of one or two amino acids from the carboxyl terminal.
25. The antibody according to claim 24, characterized by the fact that one or two amino acids are deleted from the carboxyl terminus of a heavy chain thereof.
26. Antibody according to claim 25, characterized by the fact that an amino acid is deleted from each of the terminals
12/18 carboxyl of both heavy chains of the same.
27. The antibody according to any one of claims 24 to 26, characterized in that a proline residue at the carboxyl terminus of a heavy chain is additionally amidated.
28. Antibody or functional fragment of the antibody according to any one of claims 1 to 13 or 24 to 27, characterized in that the modification of the sugar chain is regulated to enhance the antibody-dependent cellular cytotoxic activity.
29. Antibody-drug conjugate, characterized in that it comprises the antibody or the functional fragment of the antibody as defined in any one of claims 1 to 13 or 24 to 28 conjugated to a drug.
30. Antibody-drug conjugate according to claim 29, characterized in that the drug is an anti-tumor compound.
31. Antibody-drug conjugate according to claim 30, characterized in that the anti-tumor compound is an anti-tumor compound represented by the following formula: [Formula 1]
32. Antibody-drug conjugate according to any of claims 29 to 31, characterized in that the antibody is conjugated to the drug through a linker with a structure selected from the group consisting of the following formulas (a) to (f ): (a) - (Succinimid-3-yl-N) -CH2CH2-C (= O) -GGFG-NH-
13/18 CH2CH2CH2-C (= O) -, (b) - (Succinimid-3-yl-N) -CH2CH2CH2CH2CH2-C (= O) -GGFG-NH-CH2CH2CH2-C (= O) -, (c) - (Succinimid-3-yl-N) -CH2CH2CH2CH2CH2-C (= O) -GGFG- NH-CH2-O-CH2-C (= O) -, (d) - (Succinimid-3-yl-N) - CH2CH2CH2CH2CH2-C (= O) -GGFG-NH-CH2CH2-O-CH2-C (= O) -, (e) - (Succinimid-3-yl-N) -CH2CH2-C (= O) -NH-CH2CH2O - CH2CH2O-CH2CH2-C (= O) -GGFG-NH-CH2CH2CH2-C (= O) -, and (f) - (Succinimid-3-yl-N) -CH2CH2-C (= O) -NH-CH2CH2O - CH2CH2O-CH2CH2O-CH2CH2O-CH2CH2-C (= O) -GGFG-NH-CH2CH2CH2- C (= O) -, in which the antibody is connected to the - (Succinimid-3-yl-N) terminal, the compound antitumor is connected to the carbonyl group of the fraction - CH2CH2CH2-C (= O) - of (a), (b), (e) or (f), of the CH2-O-CH2-C (= O) - of ( c) or the CH2CH2-O-CH2-C (= O) - of (d) fraction with the nitrogen atom of the amino group in position 1 as a connecting position, GGFG represents an amino acid sequence consisting of glycine-glycine -phenylalanine- glycine linked via peptide bonds, and - (Succinimid-3-yl-N) - has a structure represented by the following inte formula: [Formula 2] which is connected to the antibody in position 3 of the same and is connected to a methylene group in the linker structure that contains this structure in the nitrogen atom in position 1.
33. Antibody-drug conjugate according to any
14/18 one of claims 29 to 32, characterized by the fact that the binder is represented by any formula selected from the group consisting of the following formulas (c), (d) and (e): (c) - (Succinimid-3 -il-N) -CH2CH2CH2CH2CH2-C (= O) -GGFG- NH-CH2-O-CH2-C (= O) -, (d) - (Succinimid-3-yl-N) -CH2CH2CH2CH2CH2-C (= O) -GGFG- NH-CH2CH2-O-CH2-C (= O) -, and (e) - (Succinimid-3-yl-N) -CH2CH2-C (= O) -NH-CH2CH2O- CH2CH2O-CH2CH2 -C (= O) -GGFG-NH-CH2CH2CH2-C (= O) -.
34. Antibody-drug conjugate according to any one of claims 29 to 33, characterized in that the linker is represented by the following formula (c) or (e): (c) - (Succinimid-3-yl-N) -CH2CH2CH2CH2CH2-C (= O) -GGFG- NH-CH2-O-CH2-C (= O) -, and (e) - (Succinimid-3-yl-N) -CH2CH2-C (= O) -NH -CH2CH2O- CH2CH2O-CH2CH2-C (= O) -GGFG-NH-CH2CH2CH2-C (= O) -.
35. Antibody-drug conjugate according to any one of claims 29 to 34, characterized in that it has a structure represented by the following formula: [Formula 3] in which AB represents the antibody or the functional fragment of the antibody, n represents the average number of units in the
15/18 drug-ligand conjugated to the antibody by antibody, and the antibody is connected to the ligand through a sulfhydryl group derived from the antibody.
36. Antibody-drug conjugate according to any one of claims 29 to 34, characterized in that it has a structure represented by the following formula: [Formula 4] where AB represents the antibody or the functional fragment of the antibody, n represents the average number of units of the drug-ligand structure conjugated to the antibody per antibody, and the antibody is connected to the ligand through a sulfhydryl group derived from the antibody.
37. Antibody-drug conjugate according to any of claims 29 to 36, characterized in that the antibody is an antibody comprising a light chain and a heavy chain in any combination selected from the group consisting of the following combinations (1) to (4), or a functional fragment of the antibody: (1) a light chain consisting of the amino acid sequence at positions 21 to 233 in SEQ ID NO: 61 and a heavy chain consisting of the amino acid sequence at positions 20 to 471 in SEQ ID NO: 69, (2) a light chain consisting of the amino acid sequence at positions 21 to 233 in SEQ ID NO: 61 and a heavy chain consisting of the amino acid sequence at positions 20 to 471 in SEQ ID NO: 73,
16/18 (3) a light chain consisting of the amino acid sequence at positions 21 to 233 in SEQ ID NO: 65 and a heavy chain consisting of the amino acid sequence at positions 20 to 471 in SEQ ID NO: 73, and ( 4) a light chain consisting of the amino acid sequence at positions 21 to 233 in SEQ ID NO: 61 and a heavy chain consisting of the amino acid sequence at positions 20 to 471 in SEQ ID NO: 77.
38. Antibody-drug conjugate according to claim 37, characterized in that the antibody is an antibody that comprises a light chain consisting of the amino acid sequence at positions 21 to 233 in SEQ ID NO: 61 and a heavy chain that it consists of the amino acid sequence at positions 20 to 471 in SEQ ID NO: 69, or a functional fragment of the antibody.
39. Antibody-drug conjugate according to claim 37, characterized in that the antibody is an antibody comprising a light chain consisting of the amino acid sequence at positions 21 to 233 in SEQ ID NO: 61 and a heavy chain which it consists of the amino acid sequence at positions 20 to 471 in SEQ ID NO: 77, or a functional fragment of the antibody.
40. Antibody-drug conjugate according to any one of claims 29 to 39, characterized in that the heavy chain or the light chain has undergone one or two or more modifications selected from the group consisting of N-linked glycosylation, linked glycosylation a O, N-terminal processing, C-terminal processing, deamidation, aspartic acid isomerization, methionine oxidation, addition of a methionine residue to the N-terminal, amidation of a proline residue, conversion of N-terminal glutamine or glutamic acid N-terminal in pyroglutamic acid, and a deletion of one or two amino acids from the carboxyl terminal.
41. Antibody-drug conjugate according to any
17/18 one of claims 29 to 40, characterized by the fact that the average number of units of the selected drug-ligand structure conjugated by antibody is in the range of 1 to 10.
42. Antibody-drug conjugate according to claim 41, characterized in that the average number of units of the selected antibody-conjugated drug-ligand structure is in the range of 2 to 8.
43. Antibody-drug conjugate according to claim 42, characterized by the fact that the average number of units of the selected antibody-conjugated drug-ligand structure is in the range of 5 to 8.
44. Antibody-drug conjugate according to claim 43, characterized by the fact that the average number of units of the selected antibody-conjugated drug-ligand structure is in the range 7 to 8.
45. Pharmaceutical composition, characterized in that it comprises the antibody-drug conjugate as defined in any of claims 29 to 44, a salt thereof or a hydrate of the conjugate or salt.
46. Pharmaceutical composition according to claim 45, characterized by the fact that it is an anti-tumor drug.
47. Pharmaceutical composition according to claim 46, characterized by the fact that the tumor is a tumor that expresses CDH6.
48. Pharmaceutical composition according to claim 46 or 47, characterized in that the tumor is renal cell carcinoma, renal clear cell carcinoma, renal papillary cell carcinoma, ovarian cancer, serous adenocarcinoma of the ovary, thyroid cancer , bile duct cancer, lung cancer, small cell lung cancer, glioblastoma, mesothelioma, uterine cancer, pancreatic cancer,
18/18 Wilms tumor or neuroblastoma.
49. Use of any selected component of the antibody-drug conjugate as defined in any of claims 29 to 44, a salt thereof and a hydrate of the conjugate or salt, characterized in that it is for the manufacture of a medicament to treat a tumor in an individual.
50. Use according to claim 49, characterized by the fact that the tumor is a tumor that expresses CDH6.
51. Use according to claim 49 or 50, characterized in that the tumor is renal cell carcinoma, clear cell renal carcinoma, papillary renal cell carcinoma, ovarian cancer, serous ovarian adenocarcinoma, thyroid cancer, bile duct cancer, lung cancer, small cell lung cancer, glioblastoma, mesothelioma, uterine cancer, pancreatic cancer, Wilms' tumor or neuroblastoma.
52. Use of a pharmaceutical composition comprising at least one component selected from the antibody-drug conjugate as defined in any of claims 29 to 44, a salt thereof and a hydrate of the conjugate or salt, characterized in that it is for manufacture of a drug to treat a tumor in an individual, where the drug is administered simultaneously, separately or continuously with at least one antitumor drug.
53. Method for producing an antibody-drug conjugate, characterized in that it comprises the step of reacting the antibody or the functional fragment of the antibody as defined in any one of claims 1 to 13 or 24 to 28, or an antibody or fragment functional value of the antibody obtained by the production method as defined in claim 23 with a drug-linker intermediate compound.
Cell count Cell count Figure 1
Petition 870200000169, of 01/02/2020, p. 190/216 Cell count Cell count 1/25
Cell count
Figure 2-1
Control cell or cell transfected with full length hCDH6
Cell count Cell count Cell count Cell count
Cell count
Figure 2-2
Control cell or cell transfected with deleted EC1 hCDH6
Cell count Cell count
Cell count Cell count Cell count
Figure 2-3
Control cell or cell transfected with deleted ECCD hCDH6
Cell count Cell count
Cell count Cell count Cell count
Figure 2-4
Control cell or cell transfected with deleted EC3 hCDH6
Cell count Cell count
Cell count Cell count Cell count
Figure 2-5
Control cell or cell transfected with deleted EC4 hCDH6
Cell count Cell count
Cell count Cell count Cell count
Figure 2-6
Control cell or cell transfected with deleted ECCD hCDH6
Cell count Cell count
Cell count Cell count
Cell count
Figure 3
Petition 870200000169, of 01/02/2020, p. 197/216 Cell count Cell count 8/25
Cell count Cell count
Figure 4
ATP activity (RLU)
Negative control
Rat IgG2b
Antibody clone name
NIH cell survival rate: OVCAR-3 using Rat-ZAP (%)
Mouse IgG2b ATP Activity (RLU)
Negative control
Rat IgG2b
Antibody clone name 786-O cell survival rate using Rat-ZAP (%) rat IgG2b
Figure 5
Transfected 293α cell
(Control)
ChG019 Antibody
Figure 6-1
Control
Antibody (nM)
Control
Antibody (nM)
Control
Antibody (nM)
Figure 6-2
Control
Antibody (nM)
hIgG1 control
Control
Antibody (nM)
Figure 7-1
Control cell or cell transfected with full length hCDH6
Cell count Cell count
Cell count Cell count
Cell count Cell count
Figure 7-2
Control cell or cell transfected with deleted EC1 hCDH6 Cell count
Cell count
Cell count Cell count Cell count
Cell count
Figure 7-3
Control cell or cell transfected with deleted ECCD hCDH6 Cell count
Cell count
Cell count Cell count
Cell count
Cell count
Figure 7-4
Control cell or cell transfected with deleted EC3 hCDH6
Cell count Cell count
Cell count Cell count
Cell count
Cell count
Figure 7-5
Control cell or cell transfected with deleted EC4 hCDH6 Cell count
Cell count
Cell count Cell count
Cell count
Cell count
Figure 7-6 Control cell or cell transfected with deleted ECCD hCDH6 Cell count
Cell count
Cell count Cell count
Cell count
Cell count
Figure 8
Parental cell line Stable expression cell line Cell count Cell count
Figure 9
Competition test for binding to NOV0712 pretreated cell marked with Alexa 488
Unlabeled antibody (nM)
Competition test for binding to H01L02 pretreated cell labeled with Alexa 488
Unlabeled antibody (nM)
Figure 10-1 ATP activity (RLU)
Negative control
Human IgG1
Antibody clone name
NIH cell survival rate: OVCAR-3 using Hum-ZAP (%)
Human IgG1
Figure 10-2 ATP activity (RLU)
Negative control
Human IgG1
Antibody clone name
Cell survival rate of 786-O using Hum-ZAP
Human IgG1
Figure 10-3
ATP activity (RLU)
Negative control
Human IgG1
Antibody clone name
Cell survival rate of PA-1 using Hum-ZAP (%)
Human IgG1
Figure 11 Cell survival rate (%)
Antibody-drug conjugate (nM)
Figure 12
ABS plug Tumor volume (mm³)
Number of days after administration
Figure 13 Tumor volume (mm³)
Plug
Number of days after administration
Figure 16 Tumor volume (mm³)
ABS plug
Number of days after administration

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法律状态:
2021-11-03| B350| Update of information on the portal [chapter 15.35 patent gazette]|

优先权:

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申请号 | 申请日 | 专利标题

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JP2017-096749|2017-05-15|

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JP2017096749|2017-05-15|

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PCT/JP2018/018572|WO2018212136A1|2017-05-15|2018-05-14|Anti-cdh6 antibody and anti-cdh6 antibody-drug conjugate|

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