RU2802211C2 - Anti-her3 conjugate antibody-drug - Google Patents

Abstract

FIELD: antitumor drugs.

SUBSTANCE: method of producing an antibody-drug conjugate includes the interaction of the compound (maleimid-N-yl)-(CH₂)n³-C(=O)-L²-L^P-NH-(CH₂)n¹-L^a-(CH₂)n²-C(=O)-(NH-DX) or (maleimide-N-yl)-(CH₂)n³-C(=O)-L²-L^P-(NH-DX) with an anti-HER3 antibody or a reactive derivative thereof; and conjugating the drug-linker fragment with the antibody in a manner to form a thioether bond at the disulfide bond site present in the hinge portion of the antibody, where n³ is an integer having a value of 2 to 8, L² represents -NH-(CH₂CH₂-O)n⁴-CH₂CH₂-C(=O)- or a single bond, where n⁴ is an integer having a value from 1 to 6, L^P represents a peptide residue consisting of 2–7 amino acids selected from phenylalanine, glycine, valine, lysine, citrulline, serine, glutamic acid and aspartic acid, n¹ is an integer having a value from 0 to 6, n² is an integer having a value from 0 to 5, L^a represents either -O- or a single bond. Also another option for obtaining an antibody-drug conjugate is disclosed.

EFFECT: group of inventions provides antitumor effect and safety.

26 cl, 30 dwg, 2 tbl, 23 ex

Description

[0001]

The present invention relates to an antibody drug conjugate comprising an anti-HER3 antibody and an antitumor drug conjugated to each other via a linker structure, which conjugate is useful as an antitumor drug.

Background of the invention

[0002]

An antibody-drug conjugate (ADC) containing a drug with cytotoxicity conjugated to an antibody that binds to an antigen expressed on the surface of cancer cells and is capable of being internalized by cells (an antibody that binds to an antigen is also capable of being internalized by cells) can deliver the drug selectively to the cancer cells and is expected to cause the drug to accumulate in the cancer cells and kill the cancer cells (see non-patent literature 1-3). As an ADC, Mylotarg (registered trademark; gemtuzumab ozogamicin), in which calicheamicin is conjugated to an anti-CD33 antibody, is approved as a therapeutic agent for acute myeloid leukemia. In addition, Adcetris (registered trademark; Brentuximab vedotin), in which auristatin E is conjugated to an anti-CD30 antibody, has recently been approved as a therapeutic agent for Hodgkin's lymphoma and anaplastic large cell lymphoma (see non-patent literature 4). Drugs contained in currently approved ADCs target either DNA or tubulin.

[0003]

With regard to antitumor low molecular weight compounds, camptothecin derivatives are known, compounds that inhibit topoisomerase I to exhibit an antitumor effect. Among them, an antitumor compound represented by the formula shown below (exatecan, chemical name: (1S,9S)-1-amino-9-ethyl-5-fluoro-2,3-dihydro-9-hydroxy-4-methyl-1H ,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-10,13(9H,15H)-dione), is a water-soluble camptothecin derivative (Patent Literature 1 and 2).

[0004]

[Chemical formula 1]

[0005]

Unlike irinotecan currently used clinically, this compound does not require enzyme activation to exert an antitumor effect. Moreover, compared with SN-38 as the main pharmaceutical active of irinotecan and topotecan also used in clinical setting, it has a stronger topoisomerase I inhibitory effect and higher in vitro cell-killing activity against various cancer cells. In particular, it exhibits an effect against cancer cells which are resistant to SN-38 or the like. as a result of P-glycoprotein expression. In addition, in a subcutaneously transplanted human tumor mouse model, it has shown a strong anti-tumor effect and thus has passed clinical trials, but is not yet on the market (see Non-Patent Literature 5-10). Whether or not exatecan is effective as an ADC remains unclear.

[0006]

DE-310 is a complex in which exatecan is conjugated to a biodegradable carboxymethyldextran polyalcohol polymer via a GGFG peptide spacer (Patent Literature 3). By converting exatecan into a polymeric prodrug form, the retention property of its high blood levels can be maintained, and also the high permeability to the tumor area is passively increased by exploiting the increased permeability of newly formed blood vessels in the tumor and the retention property in tumor tissues. As for DE-310, through the cleavage of the peptide spacer with an enzyme, exatecan and exatecan with amino-linked glycine are continuously released as the main active substance, and as a result, the pharmacokinetic properties are improved. According to various models of tumor evaluation in non-clinical studies, DE-310 was found to be more effective than exatecan administered alone, even though the total amount of exatecan contained in it is less than when exatecan was administered. separately. A clinical trial was conducted for DE-310 and effective cases were confirmed. There is also a report suggesting that the main active ingredient accumulates in tumors and not in normal tissues. However, there is also a report indicating that the accumulation of DE-310 and the main active ingredient in the tumor is not much different from the accumulation in normal tissues, and thus no passive targeting was observed in humans (see non-patent literature 11-14) . As a result, DE-310 has also not been commercialized and it remains unclear whether or not exatecan is effective as a drug for such targeting.

[0007]

Also known as a compound related to DE-310 is a complex in which a structural element represented as -NH-(CH ₂ ) ₄ -C(=O)- is inserted between the -GGFG-spacer and exatecan to form -GGFG- NH-(CH ₂ ) ₄ -C(=O)- used as a spacer structure (Patent Literature 4). However, the antitumor effect of this complex is generally unknown.

[0008]

Human epidermal growth factor receptor 3 (also known as HER3 and ErbB3) is a tyrosine protein kinase receptor kinase and belongs to the epidermal growth factor receptor (EGFR) tyrosine protein receptor kinase subfamily, which also includes HER1 (also known as EGFR), HER2, and HER4 (see non-patent literature 15-17). As with the prototype epidermal growth factor receptor, the HER3 transmembrane receptor consists of an extracellular ligand-binding domain (ECD), a dimerization domain in the ECD, a transmembrane domain, and a carboxy-terminal phosphorylation domain. HER1, HER2 and HER4 include an intracellular protein tyrosine kinase domain (TKD) in addition to these domains, while HER3 does not contain such a domain and thus is not capable of autophosphorylation.

Heregulin ligand (HRG) binds to the extracellular domain of HER3 and activates a receptor-mediated signaling pathway, promoting dimerization with other members of the human epidermal growth factor receptor (HER) family and transphosphorylation of its intracellular domain. Dimer formation of HER3 with other members of the HER family expands the signaling potential of HER3 and serves as a means not only for signal diversification, but also for signal amplification. For example, the HER2/HER3 heterodimer induces one of the most important mitogenic signals among members of the HER family. HER3 is overexpressed in some types of cancer such as breast, gastrointestinal, and pancreatic cancers. Interestingly, a relationship has been shown between HER2/HER3 expression and progression from a non-invasive stage to an invasive stage (see non-patent literature 18-20). Therefore, agents that interfere with HER3-mediated signaling are desirable. Anti-HER3 antibodies and their immunoconjugates have been described, for example, in Patent Literature 5-10, respectively.

List of reference documents

Patent Literature

[0009]

[Patent Literature 1] Japanese Patent Laid-Open No. 5-59061

[Patent Literature 2] Japanese Patent Laid-Open No. 8-337584

[Patent Literature 3] International Publication No. WO 1997/46260

[Patent Literature 4] International Publication No. WO 2000/25825

[Patent Literature 5] US Patent No. 5968511

[Patent Literature 6] US Patent No. 5480968

[Patent Literature 7] International Publication No. WO 2003/013602

[Patent Literature 8] International Publication No. WO 2007/077028

[Patent Literature 9] International Publication No. WO 2008/100624

[Patent Literature 10] International Publication No. WO 2012/019024

Non-Patent Literature

[0010]

[Non-Patent Literature 1] Ducry, L., et al., Bioconjugate Chem. (2010) 21, 5-13.

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

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

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

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

[Non-Patent Literature 6] Mitsui, I., et al., Jpn J. Cancer Res. (1995) 86, 776-786.

[Non-Patent Literature 7] Takiguchi, S., et al., Jpn J. Cancer Res. (1997) 88, 760-769.

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

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

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

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

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

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

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

[Non-Patent Literature 15] Plowman, et al., Proc. Natl. Acad. sci. U.S.A. (1990) 87, 4905-4909.

[Non-Patent Literature 16] Kraus et al., Proc. Natl. Acad. sci. U.S.A. (1989) 86, 9193-9197.

[Non-Patent Literature 17] Kraus et al., Proc. Natl. Acad. sci. U.S.A. (1993) 90, 2900-2094.

[Non-Patent Literature 18] Alimandi et al., Oncogene (1995) 10, 1813-1821.

[Non-Patent Literature 19] DeFazio et al., Int. J. Cancer (2000) 87, 487-498.

[Non-Patent Literature 20] Nadiu et al., Br. J. Cancer (1998) 78, 1385-1390.

The essence of the invention

Technical problem

[0011]

As for treating a tumor with an antibody, an insufficient anti-tumor effect can be observed even when the antibody recognizes the c antigen binds to tumor cells, and thus a more effective anti-tumor antibody is sometimes needed. In addition, many anti-tumor small molecule compounds have a safety problem such as side effect and toxicity even though the compounds have excellent anti-tumor effect. Therefore, the problem remains of achieving a higher therapeutic effect with a greater increase in safety. Thus, it is an object of the present invention to provide an antitumor drug having excellent therapeutic effect, which is excellent in terms of antitumor effect and safety.

Solution

[0012]

The present inventors believed that since the anti-HER3 antibody is an antibody that is capable of targeting tumor cells, that is, it is an antibody that has tumor cell recognition property, tumor cell binding property, internalization property in tumor cells, destructive activity against tumor cells. cells or the like, when exatecan as an antitumor compound is converted into an antibody-drug conjugate by conjugation with an antibody through a linker structural group, the antitumor compound can be more reliably delivered to tumor cells to specifically exhibit the antitumor effect of the compound in tumor cells, and thus , an anti-tumor effect can certainly occur, and an enhanced cytotoxic effect of the anti-HER3 antibody is also expected, and the dose of the anti-tumor compound can be reduced compared to the case of administering the compound alone, and thus the effect of the anti-tumor compound on normal cells can be reduced to achieve greater safety.

In this regard, the present inventors have created a linker with a specific structure, and they have been able to obtain an antibody-drug conjugate in which an anti-HER3 antibody and exatecan are conjugated to each other through a linker, and confirm the excellent antitumor effect exhibited by the conjugate, thus creating Thus, the present invention.

[0013]

In particular, the present invention relates to the following.

[1] An antibody-drug conjugate, wherein the antitumor compound represented by the following formula

[Chemical formula 2]

conjugated to an anti-HER3 antibody via a thioether bond, which is formed at the site of a disulfide bond present in the hinge portion of the anti-HER3 antibody, through a linker having the structure represented by the following formula:

-L ¹ -L ² -L ^P -NH-(CH ₂ )n ¹ -L ^a -(CH ₂ )n ² -C(=O)- or -L ¹ -L ² -L ^P -.

[0014]

Here, the anti-HER3 antibody is linked to the L ¹ terminus, the antitumor compound is linked to the carbonyl group of the -(CH ₂ )n ² -C(=O)- moiety or the C terminus of the L ^P , with the nitrogen atom of the amino group at position 1 as position binding.

In this formula, n ¹ is an integer having a value between 0 and 6,

n ² is an integer having a value from 0 to 5,

L ¹ is -(Succinimide-3-yl-N)-(CH ₂ )n ³ -C(=O)-,

where n ³ is an integer having a value from 2 to 8,

L ² is -NH-(CH ₂ CH ₂ -O)n ⁴ -CH ₂ CH ₂ -C(=O)- or a single bond,

where n ⁴ is an integer having a value from 1 to 6,

L ^P is a peptide residue consisting of 2-7 amino acids,

L ^a is -O- or a single bond,

-(Succinimide-3-yl-N)- has the structure represented by the following formula:

[Chemical formula 3]

which is linked to the anti-HER3 antibody at position 3 and linked at the nitrogen atom at position 1 to the methylene group in the linker structure containing this structure.

[0015]

The present invention further relates to each of the following.

[2] The antibody-drug conjugate according to [1], wherein the peptide residue L ^P is a peptide residue including an amino acid selected from phenylalanine, glycine, valine, lysine, citrulline, serine, glutamic acid, and aspartic acid.

[3] An antibody-drug conjugate according to [1] or [2], wherein L ^P is a peptide residue selected from the following group

-GGF-,

-DGGF-,

-(D-)D-GGF-,

-EGGF-,

-GGFG-,

-SGGF-,

-KGGF-,

-DGGFG-,

-GGFGG-,

-DDGGFG-,

-KDGGFG-, and

-GGFGGGF-;

(where "(D-)D" is D-aspartic acid).

[4] The antibody-drug conjugate according to [1] or [2], wherein ^LP is a peptide residue of 4 or 5 amino acids.

[5] The antibody-drug conjugate according to any one of [1] to [4], wherein L ^P is -GGFG- or -DGGFG-.

[6] The antibody-drug conjugate according to any one of [1] to [4], wherein L ^P is -GGFG-.

[0016]

[7] The antibody-drug conjugate according to any one of [1] to [6], wherein n ³ is an integer having a value of 2 to 5, and L ² is a single bond.

[8] The antibody drug conjugate according to any one of [1] to [7], wherein the linker is -L ¹ -L ² -L ^P -NH-(CH ₂ )n ¹ -L ^a -(CH ₂ )n ² -C(=O)-.

[9] The antibody-drug conjugate according to [8], wherein n ³ is an integer having a value of 2 to 5, L ² is -NH-(CH ₂ CH ₂ -O)n ⁴ -CH ₂ CH ₂ -C(=O)-, and n ⁴ is 2 or 4.

[10] The antibody-drug conjugate according to [8] or [9], wherein -NH-(CH ₂ )n ¹ -L ^a -(CH ₂ )n ² -C(=O)- is a partial a structure having a chain length of 4 or 7 atoms.

[11] The antibody-drug conjugate according to [8] or [9], wherein -NH-(CH ₂ )n ¹ -L ^a -(CH ₂ )n ² -C(=O)- is a partial a structure having a chain length of 5 or 6 atoms.

[12] The antibody-drug conjugate described in [10] or [11], wherein -NH-(CH ₂ )n ¹ -L ^a -(CH ₂ )n ² -C(=O)- is

-NH-CH ₂ CH ₂ -C (= O) -,

-NH-CH ₂ CH ₂ CH ₂ -C (= O) -,

-NH-CH ₂ CH ₂ CH ₂ CH ₂ -C (= O) -,

-NH-CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ -C (= O) -,

-NH-CH ₂ -O-CH ₂ -C(=O)- or

-NH-CH ₂ CH ₂ -O-CH ₂ -C(=O)-.

[13] The antibody-drug conjugate according to [12], wherein -NH-(CH ₂ )n ¹ -L ^a -(CH ₂ )n ² -C(=O)- is any of the following:

-NH-CH ₂ CH ₂ CH ₂ -C (= O) -,

-NH-CH ₂ -O-CH ₂ -C(=O)- or

-NH-CH ₂ CH ₂ -O-CH ₂ -C(=O)-.

[14] The antibody drug conjugate according to any one of [1] to [5], wherein the linker is -L ¹ -L ² -L ^P -.

[15] The antibody-drug conjugate according to [14], wherein L ^P is -DGGFG-.

[16] The antibody-drug conjugate according to [15], wherein n ³ is an integer having a value of 2 to 5, and L ² is a single bond.

[0017]

[17] The antibody-drug conjugate according to [1], wherein the drug-linker structural fragment in which the drug is linked to -L ¹ -L ² -L ^P -NH-(CH ₂ )n ¹ -L ^a -(CH ₂ )n ² -C(=O)- or -L ¹ -L ² -L ^P - is one drug-linker structure selected from the following group:

-(Succinimide-3-yl-N)-CH ₂ CH ₂ -C(=O)-GGFG-NH-CH ₂ CH ₂ -C(=O)-(NH-DX),

-(Succinimide-3-yl-N)-CH ₂ CH ₂ -C (= O) -GGFG-NH-CH ₂ CH ₂ CH ₂ -C (= O) - (NH-DX),

-(Succinimide-3-yl-N)-CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ -C (= O) -GGFG-NH-CH ₂ CH ₂ -C (= O) - (NH-DX),

-(Succinimide-3-yl-N)-CH ₂ CH ₂ CH _{2 CH 2 CH 2} -C (= O) -GGFG-NH-CH ₂ CH ₂ CH ₂ -C (= O) - (NH-DX) ,

-(Succinimide-3-yl-N)-CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ -C (= O) -GGFG-NH-CH ₂ CH ₂ CH 2 CH _{2 CH 2 -C} (= O) - ( NH-DX),

-(Succinimide-3-yl-N)-CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ -C (= O) -GGFG-NH-CH ₂ -O-CH ₂ -C (= O) - (NH-DX ),

-(Succinimide-3-yl-N)-CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ -C (= O) -GGFG-NH-CH ₂ CH ₂ -O-CH ₂ -C (= O) - (NH -DX),

-(Succinimid-3-yl-N)-CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ -C (= O) -DGGFG-NH-CH ₂ CH ₂ -C (= O) - (NH-DX),

-(Succinimide-3-yl-N)-CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ -C (= O) -DGGFG-NH-CH ₂ CH ₂ CH ₂ -C (= O) - (NH-DX) ,

-(Succinimide-3-yl-N)-CH ₂ CH ₂ CH 2 CH _{2 CH 2} -C (= O) -DGGFG-NH-CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ -C (= O) - ( NH-DX),

-(Succinimide-3-yl-N)-CH ₂ CH ₂ -C(=O)-NH-CH ₂ CH ₂ -O-CH ₂ CH ₂ -O-CH ₂ CH ₂ -C(=O)-GGFG -NH-CH ₂ CH ₂ -C (= O) - (NH-DX),

-(Succinimide-3-yl-N)-CH ₂ CH ₂ -C(=O)-NH-CH ₂ CH ₂ -O-CH ₂ CH ₂ -O-CH ₂ CH ₂ -C(=O)-GGFG -NH-CH ₂ CH ₂ CH ₂ -C (= O) - (NH-DX),

-(Succinimide-3-yl-N) -CH ₂ CH ₂ -C (= O) -NH-CH ₂ CH ₂ -O-CH ₂ CH ₂ -O-CH ₂ CH ₂ -O-CH ₂ CH ₂ - O-CH ₂ CH ₂ -C (= O) -GGFG-NH-CH ₂ CH ₂ -C (= O) - (NH-DX),

-(Succinimide-3-yl-N) -CH ₂ CH ₂ -C (= O) -NH-CH ₂ CH ₂ -O-CH ₂ CH ₂ -O-CH ₂ CH ₂ -O-CH ₂ CH ₂ - O-CH ₂ CH ₂ -C (= O) -GGFG-NH-CH ₂ CH ₂ CH ₂ -C (= O) - (NH-DX),

-(Succinimide-3-yl-N)-CH ₂ CH ₂ -C(=O)-GGFG-(NH-DX),

-(Succinimide-3-yl-N)-CH ₂ CH ₂ -C(=O)-DGGFG-(NH-DX),

-(Succinimide-3-yl-N)-CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ -C (= O) -GGFG-(NH-DX),

-(Succinimide-3-yl-N)-CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ -C(=O)-DGGFG-(NH-DX).

[0018]

In the above structures, -(Succinimide-3-yl-N)- has the structure represented by the following formula:

[Chemical formula 4]

which is linked to the anti-HER3 antibody at position 3 and linked to the methylene group in the linker structure containing it at the nitrogen atom at position 1,

-(NH-DX) is a group represented by the following formula, where the nitrogen atom of the amino group at position 1 is the binding position,

[Chemical Formula 5]

-GGFG- is the tetrapeptide residue -Gly-Gly-Phe-Gly-, and -DGGFG- is the pentapeptide residue -Asp-Gly-Gly-Phe-Gly-.

[0019]

[18] The antibody-drug conjugate described in [1], wherein the drug-linker structural fragment containing the drug linked to -L ¹ -L ² -L ^P -NH-(CH ₂ )n ¹ -L ^a -(CH ₂ )n ² -C(=O)-, is one drug-linker structure selected from the following group:

-(Succinimide-3-yl-N)-CH ₂ CH ₂ CH _{2 CH 2 CH 2} -C (= O) -GGFG-NH-CH ₂ CH ₂ CH ₂ -C (= O) - (NH-DX) ,

-(Succinimide-3-yl-N)-CH ₂ CH ₂ CH 2 CH _{2 CH 2 -C} (= O) -DGGFG-NH-CH ₂ CH ₂ CH ₂ -C (= O) - (NH-DX) ,

-(Succinimide-3-yl-N)-CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ -C (= O) -GGFG-NH-CH ₂ -O-CH ₂ -C (= O) - (NH-DX ),

-(Succinimide-3-yl-N)-CH ₂ CH ₂ -C(=O)-NH-CH ₂ CH ₂ -O-CH ₂ CH ₂ -O-CH ₂ CH ₂ -C(=O)-GGFG -NH-CH ₂ CH ₂ CH ₂ -C (= O) - (NH-DX),

-(Succinimide-3-yl-N)-CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ -C(=O)-DGGFG-(NH-DX).

[0020]

Here, -(Succinimide-3-yl-N)-, -(NH-DX), -GGFG- and -DGGFG- are as described above.

[0021]

[19] An antibody-drug conjugate comprising an antitumor compound represented by the following formula:

[Chemical formula 6]

conjugated to an anti-HER3 antibody via a thioether bond, which is formed at the disulfide bond site present in the hinge portion of the anti-HER3 antibody, through a linker having the structure represented by the following formula:

-L ¹ -L ² -L ^P -NH-(CH ₂ )n ¹ -L ^a -(CH ₂ )n ² -C(=O)-.

Here, the anti-HER3 antibody is linked to the L ¹ end, and the antitumor compound is linked to the carbonyl group of the -(CH ₂ )n ² -C(=O)- moiety.

In the formula, n ¹ is an integer having a value from 0 to 6,

n ² is an integer having a value from 0 to 5,

L ¹ is -(Succinimide-3-yl-N)-(CH ₂ )n ³ -C(=O)-,

where n ³ is an integer having a value from 2 to 8,

L ² is -NH-(CH ₂ CH ₂ -O)n ⁴ -CH ₂ CH ₂ -C(=O)- or a single bond,

where n ⁴ is an integer having a value from 1 to 6,

L ^P is a tetrapeptide residue -GGFG-,

L ^a is -O- or a single bond,

-(Succinimide-3-yl-N)- has the structure represented by the following formula:

[Chemical formula 7]

which is linked to the anti-HER3 antibody at position 3 and binds at the nitrogen atom at position 1 to the methylene group in the linker structure containing this structure.

[0022]

[20] The antibody-drug conjugate according to [19], wherein n ¹ is 3, n ² is 0, n ³ is 2, L ² is -NH-(CH ₂ CH ₂ -O) n ⁴ -CH ₂ CH ₂ -C(=O)-, n ⁴ is 2, and L ^a is a single bond, or

n ¹ is 1, n ² is 1, n ³ is 5, L ² is a single bond, and L ^a is -O-, or

n ¹ is 2, n ² is 1, n ³ is 5, L ² is a single bond, and L ^a is -O-.

[21] An antibody-drug conjugate according to [19] or [20], wherein n ³ is 2 or 5 and L ² is a single bond.

[22] An antibody-drug conjugate according to [19] or [20], wherein n ³ is 2 or 5, L ² is -NH-(CH ₂ CH ₂ -O)n ⁴ -CH ₂ CH ₂ -C(=O)-, and n ⁴ is 2 or 4.

[23] The antibody drug conjugate described in any one of [19]-[22], wherein -NH-(CH ₂ )n ¹ -L ^a -(CH ₂ )n ² -C(=O)- is yourself

-NH-CH ₂ CH ₂ CH ₂ -C (= O) -,

-NH-CH ₂ -O-CH ₂ -C(=O)- or

-NH-CH ₂ CH ₂ -O-CH ₂ -C(=O)-.

[0023]

[24] The antibody-drug conjugate described in any one of [19]-[23], wherein the drug-linker structural fragment containing the drug linked to -L ¹ -L ² -L ^P -NH-(CH ₂ )n ¹ -L ^a -(CH ₂ )n ² -C(=O)-, is one drug-linker structure selected from the following group:

-(Succinimide-3-yl-N)-CH ₂ CH ₂ -C(=O)-GGFG-NH-CH ₂ CH ₂ -C(=O)-(NH-DX),

-(Succinimide-3-yl-N)-CH ₂ CH ₂ -C (= O) -GGFG-NH-CH ₂ CH ₂ CH ₂ -C (= O) - (NH-DX),

-(Succinimide-3-yl-N)-CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ -C (= O) -GGFG-NH-CH ₂ CH ₂ -C (= O) - (NH-DX),

-(Succinimide-3-yl-N)-CH ₂ CH ₂ CH _{2 CH 2 CH 2} -C (= O) -GGFG-NH-CH ₂ CH ₂ CH ₂ -C (= O) - (NH-DX) ,

-(Succinimide-3-yl-N)-CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ -C (= O) -GGFG-NH-CH ₂ CH ₂ CH 2 CH _{2 CH 2 -C} (= O) - ( NH-DX),

-(Succinimide-3-yl-N)-CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ -C (= O) -GGFG-NH-CH ₂ -O-CH ₂ -C (= O) - (NH-DX ),

-(Succinimide-3-yl-N)-CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ -C (= O) -GGFG-NH-CH ₂ CH ₂ -O-CH ₂ -C (= O) - (NH -DX),

-(Succinimide-3-yl-N)-CH ₂ CH ₂ -C(=O)-NH-CH ₂ CH ₂ -O-CH ₂ CH ₂ -O-CH ₂ CH ₂ -C(=O)-GGFG -NH-CH ₂ CH ₂ -C (= O) - (NH-DX),

-(Succinimide-3-yl-N)-CH ₂ CH ₂ -C(=O)-NH-CH ₂ CH ₂ -O-CH ₂ CH ₂ -O-CH ₂ CH ₂ -C(=O)-GGFG -NH-CH ₂ CH ₂ CH ₂ -C (= O) - (NH-DX),

-(Succinimide-3-yl-N) -CH ₂ CH ₂ -C (= O) -NH-CH ₂ CH ₂ -O-CH ₂ CH ₂ -O-CH ₂ CH ₂ -O-CH ₂ CH ₂ - O-CH ₂ CH ₂ -C(=O)-GGFG-NH-CH ₂ CH ₂ -C(=O)-(NH-DX);

-(Succinimide-3-yl-N) -CH ₂ CH ₂ -C (= O) -NH-CH ₂ CH ₂ -O-CH ₂ CH ₂ -O-CH ₂ CH ₂ -O-CH ₂ CH ₂ - O-CH ₂ CH ₂ -C (= O) -GGFG-NH-CH ₂ CH ₂ CH ₂ -C (= O) - (NH-DX),

[0024]

In the above, -(Succinimide-3-yl-N)- has the structure represented by the following formula:

[Chemical formula 8]

which is linked to the anti-HER3 antibody at position 3 and linked at the nitrogen atom at position 1 to the methylene group in the linker structure containing this structure.

-(NH-DX) is a group represented by the following formula, where the nitrogen atom of the amino group at position 1 represents the binding position:

[Chemical formula 9]

-GGFG- is the tetrapeptide residue -Gly-Gly-Phe-Gly-.

[0025]

[25] The antibody-drug conjugate described in any one of [19]-[23], wherein the drug-linker structural fragment containing the drug linked to -L ¹ -L ² -L ^P -NH-(CH ₂ )n ¹ -L ^a -(CH ₂ )n ² -C(=O)-, is one drug-linker structure selected from the following group:

-(Succinimide-3-yl-N)-CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ -C (= O) -GGFG-NH-CH ₂ -O-CH ₂ -C (= O) - (NH-DX ),

-(Succinimide-3-yl-N)-CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ -C (= O) -GGFG-NH-CH ₂ CH ₂ -O-CH ₂ -C (= O) - (NH -DX),

-(Succinimide-3-yl-N)-CH ₂ CH ₂ -C(=O)-NH-CH ₂ CH ₂ -O-CH ₂ CH ₂ -O-CH ₂ CH ₂ -C(=O)-GGFG -NH-CH ₂ CH ₂ CH ₂ -C(=O)-(NH-DX).

In the above, -(Succinimide-3-yl-N)-, -(NH-DX) and -GGFG- are as defined above.

[0026]

[26] An antibody-drug conjugate according to any one of [1] to [25], wherein the average number of conjugated units of the selected single drug-linker structure per antibody is between 1 and 10.

[27] An antibody-drug conjugate according to any one of [1] to [25], wherein the average number of conjugated units of the selected single drug-linker structure per antibody is between 2 and 8.

[28] The antibody-drug conjugate according to any one of [1] to [25], wherein the average number of conjugated units of the selected single drug-linker structure per antibody is between 3 and 8.

[0027]

[29] A drug comprising an antibody-drug conjugate according to any one of [1] to [28], a salt thereof, or a hydrate thereof.

[30] An antitumor drug and/or an anticancer drug, comprising the antibody-drug conjugate according to any one of [1] to [28], a salt thereof, or a hydrate thereof.

[31] An antitumor drug and/or an anticancer drug according to [30], which is used for the treatment of lung cancer, kidney cancer, urothelial cancer, colorectal cancer, prostate cancer, glioblastoma multiforme, ovarian cancer, pancreatic cancer, cancer breast, melanoma, liver cancer, bladder cancer, gastric cancer, gastrointestinal stromal tumor, cervical cancer, head and neck cancer, esophageal cancer, epidermoid cancer, peritoneal cancer, adult glioblastoma multiforme, hepatic cancer, hepatocellular carcinoma, colon cancer colon, rectal cancer, colon and rectal cancer, endometrial cancer, uterine cancer, salivary gland cancer, renal cancer, vulvar cancer, thyroid cancer, hepatic carcinoma, anal carcinoma, or penile cancer.

[32] A pharmaceutical composition comprising an antibody-drug conjugate according to any one of [1] to [28], a salt or hydrate thereof as an active ingredient, and a pharmaceutically acceptable ingredient for formulating the composition.

[33] The pharmaceutical composition according to [32], which is used for the treatment of lung cancer, kidney cancer, urothelial cancer, colorectal cancer, prostate cancer, glioblastoma multiforme, ovarian cancer, pancreatic cancer, breast cancer, melanoma, liver cancer , bladder cancer, gastric cancer, gastrointestinal stromal tumor, cervical cancer, head and neck cancer, esophageal cancer, epidermoid cancer, peritoneal cancer, adult glioblastoma multiforme, hepatic cancer, hepatocellular carcinoma, colon cancer, rectal cancer, colon and rectal cancer, endometrial cancer, uterine cancer, salivary gland cancer, renal cancer, vulvar cancer, thyroid cancer, hepatic carcinoma, anal carcinoma, or penile cancer.

[34] A method for treating a tumor and/or cancer, comprising administering an antibody-drug conjugate according to any one of [1] to [28], a salt thereof, or a hydrate thereof.

[35] A drug according to [29], an antitumor drug and/or an anticancer drug according to [30] or [31], a pharmaceutical composition according to [32] or [33], or a treatment method in accordance with paragraph [34], which are used for administration in combination with an additional drug.

[36] The pharmaceutical composition according to [32] or [33], further comprising another additional drug as an active ingredient.

[0028]

[35] A method for producing an antibody-drug conjugate, comprising reacting a compound represented by the following formula:

(maleimide-N-yl)-(CH ₂ )n ³ -C(=O)-L ² -L ^P -NH-(CH ₂ )n ¹ -L ^a -(CH ₂ )n ² -C(=O )-(NH-DX) or (maleimide-N-yl)-(CH ₂ )n ³ -C(=O)-L ² -L ^P -(NH-DX)

with an anti-HER3 antibody or a reactive derivative thereof; and conjugating the drug-linker fragment to the antibody in a manner to form a thioether bond at a disulfide bond site present in the hinge portion of the antibody.

[0029]

In the presented formula, n ³ is an integer having a value from 2 to 8,

L ² is -NH-(CH ₂ CH ₂ -O)n ⁴ -CH ₂ CH ₂ -C(=O)- or a single bond, where n ⁴ is an integer value from 1 to 6,

L ^P is a peptide residue consisting of 2-7 amino acids selected from phenylalanine, glycine, valine, lysine, citrulline, serine, glutamic acid and aspartic acid,

n ¹ is an integer having a value from 0 to 6,

n ² is an integer having a value from 0 to 5,

L ^a is -O- or a single bond,

(maleimid-N-yl)- is a group represented by the following formula and contains a nitrogen atom as a binding position.

[Chemical Formula 10]

-(NH-DX) is a group represented by the following formula, where the nitrogen atom of the amino group at position 1 represents the binding position:

[Chemical formula 11]

[0030]

[36] The production method described in [35], wherein the method of conjugating a drug-linker fragment with an anti-HER3 antibody is a method of recovering an antibody to be converted into a reactive derivative.

[0031]

[37] The production method described in [35] or [36], wherein the average number of conjugated units of the selected single drug-linker structure per antibody is between 1 and 10.

[38] The production method described in [35] or [36], wherein the average number of conjugated units of the selected single drug-linker structure per antibody is between 2 and 8.

[39] The production method described in [35] or [36], wherein the average number of conjugated units of the selected single drug-linker structure per antibody is between 3 and 8.

[40] An antibody-drug conjugate obtained by the production method according to any one of [35] to [39].

[0032]

[41] An antibody-drug conjugate prepared by forming a thioether bond at a disulfide bond site present in the hinge portion of an anti-HER3 antibody, wherein the anti-HER3 antibody is treated under reducing conditions and then reacted with a compound selected from the group of compounds represented by below:

(maleimid-N-yl)-CH ₂ CH ₂ -C (= O) -GGFG-NH-CH ₂ CH ₂ -C (= O) - (NH-DX),

(maleimid-N-yl)-CH ₂ CH ₂ CH ₂ -C (= O) -GGFG-NH-CH ₂ CH ₂ -C (= O) - (NH-DX),

(maleimid-N-yl)-CH ₂ CH ₂ CH ₂ CH ₂ -C (= O) -GGFG-NH-CH ₂ CH ₂ -C (= O) - (NH-DX),

(maleimid-N-yl)-CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ -C (= O) -GGFG-NH-CH ₂ CH ₂ -C (= O) - (NH-DX),

(maleimid-N-yl)-CH ₂ CH ₂ -C (= O) -GGFG-NH-CH ₂ CH ₂ CH ₂ -C (= O) - (NH-DX),

(maleimid-N-yl)-CH ₂ CH ₂ CH ₂ -C (= O) -GGFG-NH-CH ₂ CH ₂ CH ₂ -C (= O) - (NH-DX),

(maleimid-N-yl)-CH ₂ CH ₂ CH ₂ CH ₂ -C (= O) -GGFG-NH-CH ₂ CH ₂ CH ₂ -C (= O) - (NH-DX),

(maleimid-N-yl)-CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ -C (= O) -GGFG-NH-CH ₂ CH ₂ CH ₂ -C (= O) - (NH-DX),

(maleimid-N-yl)-CH ₂ CH ₂ -C (= O) -GGFG-NH-CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ -C (= O) - (NH-DX),

(maleimid-N-yl)-CH ₂ CH ₂ CH ₂ -C (= O) -GGFG-NH-CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ -C (= O) - (NH-DX),

(maleimid-N-yl)-CH ₂ CH ₂ CH ₂ CH ₂ -C (= O) -GGFG-NH-CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ -C (= O) - (NH-DX),

(maleimid-N-yl) -CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ -C (= O) -GGFG-NH-CH ₂ CH ₂ CH 2 CH ₂ CH _{2 -C} (= O) - (NH-DX ),

(maleimid-N-yl)-CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ -C (= O) -DGGFG-NH-CH ₂ CH ₂ -C (= O) - (NH-DX), (maleimide-N -yl) -CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ -C (= O) -DGGFG-NH-CH ₂ CH ₂ CH ₂ -C (= O) - (NH-DX),

(maleimid-N-yl)-CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ -C (= O) -DGGFG-NH-CH ₂ CH 2 CH ₂ CH ₂ CH _{2 -C} (= O) - (NH-DX ),

(maleimid-N-yl)-CH ₂ CH ₂ -C(=O)-GGFG-NH-CH ₂ -O-CH ₂ -C(=O)-(NH-DX),

(maleimid-N-yl)-CH ₂ CH ₂ CH ₂ -C(=O)-GGFG-NH-CH ₂ -O-CH ₂ -C(=O)-(NH-DX),

(maleimid-N-yl)-CH ₂ CH ₂ CH ₂ CH ₂ -C (= O) -GGFG-NH-CH ₂ -O-CH ₂ -C (= O) - (NH-DX),

(maleimid-N-yl)-CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ -C (= O) -GGFG-NH-CH ₂ -O-CH ₂ -C (= O) - (NH-DX),

(maleimid-N-yl)-CH ₂ CH ₂ -C (= O) -GGFG-NH-CH ₂ CH ₂ -O-CH ₂ -C (= O) - (NH-DX),

(maleimid-N-yl)-CH ₂ CH ₂ CH ₂ -C (= O) -GGFG-NH-CH ₂ CH ₂ -O-CH ₂ -C (= O) - (NH-DX),

(maleimid-N-yl)-CH ₂ CH ₂ CH ₂ CH ₂ -C (= O) -GGFG-NH-CH ₂ CH ₂ -O-CH ₂ -C (= O) - (NH-DX),

(maleimid-N-yl)-CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ -C (= O) -GGFG-NH-CH ₂ CH ₂ -O-CH ₂ -C (= O) - (NH-DX) ,

(maleimid-N-yl) -CH ₂ CH ₂ -C (= O) -NH-CH ₂ CH ₂ -O-CH ₂ CH ₂ -O-CH ₂ CH ₂ -C (= O) -GGFG-NH- CH ₂ CH ₂ -C (= O) - (NH-DX),

(maleimid-N-yl)-CH ₂ CH ₂ -C (= O) -NH-CH ₂ CH ₂ -O-CH ₂ CH ₂ -O-CH ₂ CH ₂ O-CH ₂ CH ₂ -C (= O )-GGFG-NH-CH ₂ CH ₂ -C (= O) - (NH-DX),

(maleimid-N-yl)-CH ₂ CH ₂ -C (= O) -NH-CH ₂ CH ₂ -O-CH ₂ CH ₂ -O-CH ₂ CH ₂ -O-CH ₂ CH ₂ -O-CH ₂ CH ₂ -C (= O) -GGFG-NH-CH ₂ CH ₂ -C (= O) - (NH-DX),

(maleimid-N-yl) -CH ₂ CH ₂ -C (= O) -NH-CH ₂ CH ₂ -O-CH ₂ CH ₂ -O-CH ₂ CH ₂ -C (= O) -GGFG-NH- CH ₂ CH ₂ CH ₂ -C (= O) - (NH-DX),

(maleimid-N-yl)-CH ₂ CH ₂ -C (= O) -NH-CH ₂ CH ₂ -O-CH ₂ CH ₂ -O-CH ₂ CH ₂ -O-CH ₂ CH ₂ -C (= O)-GGFG-NH-CH ₂ CH ₂ CH ₂ -C (= O) - (NH-DX),

(maleimid-N-yl)-CH ₂ CH ₂ -C (= O) -NH-CH ₂ CH ₂ -O-CH ₂ CH ₂ -O-CH ₂ CH ₂ -O-CH ₂ CH ₂ -O-CH ₂ CH ₂ -C (= O) -GGFG-NH-CH ₂ CH ₂ CH ₂ -C (= O) - (NH-DX),

(maleimid-N-yl) -CH ₂ CH ₂ -C (= O) -NH-CH ₂ CH ₂ -O-CH ₂ CH ₂ -O-CH ₂ CH ₂ -C (= O) -GGFG-NH- CH ₂ -O-CH ₂ -C (= O) - (NH-DX),

(maleimid-N-yl)-CH ₂ CH ₂ -C (= O) -NH-CH ₂ CH ₂ -O-CH ₂ CH ₂ -O-CH ₂ CH ₂ -O-CH ₂ CH ₂ -C (= O)-GGFG-NH-CH ₂ -O-CH ₂ -C (= O) - (NH-DX),

(maleimid-N-yl)-CH ₂ CH ₂ -C (= O) -NH-CH ₂ CH ₂ -O-CH ₂ CH ₂ -O-CH ₂ CH ₂ -O-CH ₂ CH ₂ -O-CH ₂ CH ₂ -C(=O)-GGFG-NH-CH ₂ -O-CH ₂ -C(=O)-(NH-DX),

(maleimid-N-yl) -CH ₂ CH ₂ -C (= O) -NH-CH ₂ CH ₂ -O-CH ₂ CH ₂ -O-CH ₂ CH ₂ -C (= O) -GGFG-NH- CH ₂ CH ₂ -O-CH ₂ -C (= O) - (NH-DX),

(maleimid-N-yl)-CH ₂ CH ₂ -C (= O) -NH-CH ₂ CH ₂ -O-CH ₂ CH ₂ -O-CH ₂ CH ₂ -O-CH ₂ CH ₂ -C (= O)-GGFG-NH-CH ₂ CH ₂ -O-CH ₂ -C (= O) - (NH-DX),

(maleimid-N-yl)-CH ₂ CH ₂ -C (= O) -NH-CH ₂ CH ₂ O-CH ₂ CH ₂ O-CH ₂ CH ₂ O-CH ₂ CH ₂ O-CH ₂ CH ₂ - C(=O)-GGFG-NH-CH ₂ CH ₂ -O-CH ₂ -C(=O)-(NH-DX),

(maleimide-N-yl)-CH ₂ CH ₂ -C (= O) -GGFG-(NH-DX),

(maleimid-N-yl)-CH ₂ CH ₂ CH ₂ -C (= O) -GGFG- (NH-DX),

(maleimid-N-yl)-CH ₂ CH ₂ CH ₂ CH ₂ -C (= O) -GGFG- (NH-DX),

(maleimid-N-yl)-CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ -C (= O) -GGFG-(NH-DX),

(maleimide-N-yl)-CH ₂ CH ₂ -C (= O) -DGGFG-(NH-DX),

(maleimid-N-yl)-CH ₂ CH ₂ CH ₂ -C (= O) -DGGFG- (NH-DX),

(maleimid-N-yl)-CH ₂ CH ₂ CH ₂ CH ₂ -C(=O)-DGGFG-(NH-DX) or

(maleimid-N-yl)-CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ -C(=O)-DGGFG-(NH-DX).

[0033]

In the above compounds, (maleimide-N-yl)- represents a group represented by the following formula:

[Chemical Formula 12]

which contains a nitrogen atom as the binding position.

-(NH-DX) is a group represented by the following formula, wherein the nitrogen atom of the amino group at position 1 is the binding position.

[Chemical formula 13]

-GGFG- is the tetrapeptide residue -Gly-Gly-Phe-Gly-, and -DGGFG- is the pentapeptide residue -Asp-Gly-Gly-Phe-Gly-.

[0034]

[42] An antibody-drug conjugate obtained by forming a thioether bond at a disulfide bond site present in the hinge portion of an anti-HER3 antibody, and characterized in that the anti-HER3 antibody is treated under reducing conditions and then reacted with a compound selected from group of compounds below:

(maleimid-N-yl) -CH ₂ CH ₂ -C (= O) -NH-CH ₂ CH ₂ -O-CH ₂ CH ₂ -O-CH ₂ CH ₂ -C (= O) -GGFG-NH- CH ₂ CH ₂ CH ₂ -C (= O) - (NH-DX),

(maleimid-N-yl)-CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ -C(=O)-GGFG-NH-CH ₂ -O-CH ₂ -C(=O)-(NH-DX) or

(maleimid-N-yl)-CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ -C (= O) -GGFG-NH-CH ₂ CH ₂ -O-CH ₂ -C (= O) - (NH-DX) .

In the above compounds, (maleimide-N-yl)-, -(NH-DX) and -GGFG- are as defined above.

[0035]

[43] An antibody-drug conjugate according to [41] or [42], wherein the average number of conjugated units of the selected single drug-linker structure per antibody is between 1 and 10.

[44] An antibody-drug conjugate according to [41] or [42], wherein the average number of conjugated units of the selected single drug-linker structure per antibody is between 2 and 8.

[45] An antibody-drug conjugate according to [41] or [42], wherein the average number of conjugated units of the selected single drug-linker structure per antibody is between 3 and 8.

[46] A drug comprising an antibody-drug conjugate according to any one of [40] to [45], a salt thereof, or a hydrate thereof.

[47] An antitumor drug and/or an anticancer drug, comprising an antibody-drug conjugate according to any of [40] to [45], a salt thereof, or a hydrate thereof.

[48] An antitumor drug and/or an anticancer drug according to [47], which is used for the treatment of lung cancer, kidney cancer, urothelial cancer, colorectal cancer, prostate cancer, glioblastoma multiforme, ovarian cancer, pancreatic cancer, cancer breast, melanoma, liver cancer, bladder cancer, gastric cancer, gastrointestinal stromal tumor, cervical cancer, head and neck cancer, esophageal cancer, epidermoid cancer, peritoneal cancer, adult glioblastoma multiforme, hepatic cancer, hepatocellular carcinoma, colon cancer colon, rectal cancer, colon and rectal cancer, endometrial cancer, uterine cancer, salivary gland cancer, renal cancer, vulvar cancer, thyroid cancer, hepatic carcinoma, anal carcinoma, or penile cancer.

[49] A pharmaceutical composition comprising an antibody-drug conjugate according to any one of [40] to [45], a salt or hydrate thereof as an active ingredient, and a pharmaceutically acceptable ingredient for formulating the composition.

[50] The pharmaceutical composition according to [49], which is used for the treatment of lung cancer, kidney cancer, urothelial cancer, colorectal cancer, prostate cancer, glioblastoma multiforme, ovarian cancer, pancreatic cancer, breast cancer, melanoma, liver cancer , bladder cancer, gastric cancer, gastrointestinal stromal tumor, cervical cancer, head and neck cancer, esophageal cancer, epidermoid cancer, peritoneal cancer, adult glioblastoma multiforme, hepatic cancer, hepatocellular carcinoma, colon cancer, rectal cancer, cancer colon and rectal cancer, endometrial cancer, uterine cancer, salivary gland cancer, renal cancer, vulvar cancer, thyroid cancer, hepatic carcinoma, anal carcinoma, or penile cancer.

[51] A method for treating a tumor and/or cancer, comprising administering an antibody-drug conjugate according to any one of [40] to [45], a salt thereof, or a hydrate thereof.

[52] A drug according to [46], an antitumor drug and/or an anticancer drug according to [47] or [48], a pharmaceutical composition according to [49] or [50], or a treatment method in accordance with paragraph [51], which are used for administration in combination with an additional drug.

[53] The pharmaceutical composition according to [49] or [50], further comprising another additional drug as an active ingredient.

Useful effects of the invention

[0036]

By using an anti-HER3 antibody-drug conjugate containing the antitumor compound exatecan conjugated through a linker with a specific structure, excellent antitumor effect and safety can be achieved.

Brief description of the drawings

[0037]

[Fig. 1]

Fig. 1 shows the full length amino acid sequence of the heavy chain of the human anti-HER3 antibody U1-59 (SEQ ID NO: 583).

[Fig. 2]

Fig. 2 shows the full length amino acid sequence of the light chain of the human anti-HER3 antibody U1-59 (SEQ ID NO: 584).

[Fig. 3]

Fig. 3 shows the mean fluorescence intensity of HCC1569 treated with serial dilutions of U1-59 or each antibody drug conjugate. KD and Bmax values were calculated using GraphPad Prism software.

[Fig. 4]

A549 cells were cultured for 2 days with U1-59 or different antibody-drug conjugates. HER3 or phosphorylated HER3 was analyzed by Western blotting. pan-Actin was determined as a control for electrophoresis.

[Fig. 5]

Fig. 5 shows the average decrease in HER3 expression on the surface of HCC1569 cells treated with U1-59 or each antibody-drug conjugate (37°C ("C" stands for "degrees Celsius"), 1 hour).

[Fig. 6]

Fig. 6 shows the results of testing the inhibition of mitogenic or survival signals by each HER3 antibody-drug conjugate in a human breast cancer cell line (HCC1569). Fig. 6A shows cell growth or survival data obtained for an antibody-drug conjugate in the presence of 10% FBS. Data are presented as mean +/- standard deviation of three repetitions. The y-axis represents the luminescence value showing the ATP activity of each sample. The abscissa axis represents the concentration of each antibody-drug conjugate. Fig. 6B shows the percentage reduction in luminescence caused by treatment with the antibody-drug conjugate when the luminescence of the untreated group was determined to be 100%.

[Fig. 7]

Fig. 7 shows the results of testing the inhibition of mitogenic or survival signals by each HER3 antibody-drug conjugate in a human breast cancer cell line (MDA-MB 453). Fig. 7A shows cell growth or survival data obtained for an antibody-drug conjugate in the presence of 10% FBS. The y-axis represents the luminescence value showing the ATP activity of each sample. The abscissa axis represents the concentration of each antibody-drug conjugate. Data are presented as mean +/- standard deviation of three repetitions. Fig. 7B shows the percentage reduction in luminescence caused by treatment with the antibody-drug conjugate when the luminescence of the untreated group was determined to be 100%.

[Fig. 8]

Fig. 8 shows the results of testing the inhibition of mitogenic or survival signals by each HER3 antibody-drug conjugate in a human melanoma cell line (A375). Fig. 8A shows cell growth or survival data obtained for an antibody-drug conjugate in the presence of 10% FBS. The y-axis represents the luminescence value showing the ATP activity of each sample. The abscissa axis represents the concentration of each antibody-drug conjugate. Data are presented as mean +/- standard deviation of three repetitions. Fig. 8B shows the percentage reduction in luminescence caused by treatment with the antibody-drug conjugate when the luminescence of the untreated group was determined to be 100%.

[Fig. 9]

Fig. 9 shows the results of testing the inhibition of mitogenic or survival signals by each HER3 antibody-drug conjugate in a human colorectal cancer cell line (HT29). Fig. 9A shows cell growth or survival data obtained for an antibody-drug conjugate in the presence of 10% FBS. The y-axis represents the luminescence value showing the ATP activity of each sample. The abscissa axis represents the concentration of each antibody-drug conjugate. Data are presented as mean +/- standard deviation of three repetitions. Fig. 9B shows the percentage reduction in luminescence caused by treatment with the antibody-drug conjugate when the luminescence of the untreated group was determined to be 100%.

[Fig. 10]

Fig. 10 shows the results of testing the inhibition of mitogenic or survival signals by each HER3 antibody-drug conjugate in a human lung cancer cell line (A549). Fig. 10A shows cell growth or survival data obtained for an antibody-drug conjugate in the presence of 10% FBS. The y-axis represents the luminescence value showing the ATP activity of each sample. The abscissa axis represents the concentration of each antibody-drug conjugate. Data are presented as mean +/- standard deviation of three repetitions. Fig. 10B shows the percentage reduction in luminescence caused by treatment with the antibody-drug conjugate when the luminescence of the untreated group was determined to be 100%.

[Fig. eleven]

Fig. 11 shows the results of comparing the percentage of cell growth inhibition or survival between the antibody-drug conjugate (3) and the antibody-drug conjugate (4). The left chart shows the percentage of cell growth inhibition or survival obtained for the antibody-drug conjugate in the presence of 10% FBS. The y-axis represents the luminescence showing the ATP activity of each sample. The abscissa axis represents the concentration of each antibody-drug conjugate. Data are presented as mean +/- standard deviation of three repetitions. The right chart shows a comparison of the decrease in luminescence induced by antibody drug conjugate treatment between high drug load (HDL) and medium drug load (MDL) when the luminescence of the untreated group was determined to be 100%.

[Fig. 12]

Fig. 12 shows the results of comparing the percentage of cell growth inhibition or survival between the antibody-drug conjugate (10) and the antibody-drug conjugate (11). The left chart shows the percentage of cell growth inhibition or survival obtained for the antibody-drug conjugate in the presence of 10% FBS. The y-axis represents the luminescence showing the ATP activity of each sample. The abscissa axis represents the concentration of each antibody-drug conjugate. Data are presented as mean +/- standard deviation of three repetitions. The right chart shows a comparison of the decrease in luminescence induced by antibody drug conjugate treatment between high drug load (HDL) and medium drug load (MDL) when the luminescence of the untreated group was determined to be 100%.

[Fig. 13]

Fig. 13 shows the results of comparing the percentage of cell growth inhibition or survival between the antibody-drug conjugate (13) and the antibody-drug conjugate (14). The left chart shows the percentage of cell growth inhibition or survival obtained for the antibody-drug conjugate in the presence of 10% FBS. The y-axis represents the luminescence showing the ATP activity of each sample. The abscissa axis represents the concentration of each antibody-drug conjugate. Data are presented as mean +/- standard deviation of three repetitions. The right chart shows a comparison of the decrease in luminescence induced by antibody drug conjugate treatment between high drug load (HDL) and medium drug load (MDL) when the luminescence of the untreated group was determined to be 100%.

[Fig. 14]

Fig. 14 shows the results of an antitumor activity test against human breast cancer (HCC1569) using an antibody-drug conjugate (3), (10) or (13). The y-axis represents the mean tumor volume. The x-axis represents the number of days after cell transplantation. All values are given as mean +/- standard deviation. Initial tumor volume and initial mouse body weight were analyzed based on descriptive data (mean and standard deviation) using Microsoft Excel 2009.

[Fig. 15]

Fig. 15 shows the results of an antitumor activity test against human melanoma (HT-144) using an antibody-drug conjugate (3), (10) or (13). The y-axis represents the mean tumor volume. The x-axis represents the number of days after cell transplantation. All values are given as mean +/- standard deviation. Initial tumor volume and initial mouse body weight were analyzed based on descriptive data (mean and standard deviation) using Microsoft Excel 2009.

[Fig. 16]

Fig. 16 shows the results of an antitumor activity test against human breast cancer (MDA-MB-453) using an antibody-drug conjugate (3), (10) or (13). The y-axis represents the mean tumor volume. The x-axis represents the number of days from the introduction. All values are given as mean +/- standard deviation. Initial tumor volume and initial mouse body weight were analyzed based on descriptive data (mean and standard deviation) using Microsoft Excel 2009.

[Fig. 17]

Fig. 17 shows the results of an antitumor activity test against a colorectal cancer cell line (HT-29) using an antibody-drug conjugate (3), (10) or (13). The y-axis represents the mean tumor volume. The x-axis represents the number of days after administration. All values are given as mean +/- standard deviation. Initial tumor volume and initial mouse body weight were analyzed based on descriptive data (mean and standard deviation) using Microsoft Excel 2009.

[Fig. 18]

Fig. 18 shows the results of an antitumor activity test against a human lung cancer cell line (A549) using an antibody-drug conjugate (3), (10) or (13). The y-axis represents the mean tumor volume. The x-axis represents the number of days after cell transplantation. All values are given as mean +/- standard deviation. Initial tumor volume and initial mouse body weight were analyzed based on descriptive data (mean and standard deviation) using Microsoft Excel 2009.

[Fig. 19]

Fig. 19 shows the results of an antitumor activity test against a triple negative breast cancer cell line (MDA-MB-468) using an antibody-drug conjugate (13). The y-axis represents the mean tumor volume. The x-axis represents the number of days after cell transplantation. All values are given as mean +/- standard deviation. Initial tumor volume and initial mouse body weight were analyzed based on descriptive data (mean and standard deviation) using Microsoft Excel 2009.

[Fig. 20]

Fig. 20 shows the results of an antitumor activity test against a human luminal breast cancer cell line (MCF-7) using an antibody-drug conjugate (16a). The y-axis represents the mean tumor volume. The x-axis represents the number of days after cell transplantation. All values are given as mean +/- standard deviation. Initial tumor volume and initial mouse body weight were analyzed based on descriptive data (mean and standard deviation) using Microsoft Excel 2009.

[Fig. 21]

Fig. 21 shows the results of an antitumor activity test against a human melanoma cell line (WM-266-4) using an antibody-drug conjugate (16a). The y-axis represents the mean tumor volume. The x-axis represents the number of days after cell transplantation. All values are given as mean +/- standard deviation. Initial tumor volume and initial mouse body weight were analyzed based on descriptive data (mean and standard deviation) using Microsoft Excel 2009.

[Fig. 22]

Fig. 22 shows the results of an antitumor activity test against a human ovarian cancer cell line (OVCAR-8) using an antibody-drug conjugate (16a). The y-axis represents the mean tumor volume. The x-axis represents the number of days after cell transplantation. All values are given as mean +/- standard deviation. Initial tumor volume and initial mouse body weight were analyzed based on descriptive data (mean and standard deviation) using Microsoft Excel 2009.

[Fig. 23]

Fig. 23 shows the results of an antitumor activity test against a human bladder cancer cell line (SW-780) using an antibody-drug conjugate (16a). The y-axis represents the mean tumor volume. The x-axis represents the number of days after cell transplantation. All values are given as mean +/- standard deviation. Initial tumor volume and initial mouse body weight were analyzed based on descriptive data (mean and standard deviation) using Microsoft Excel 2009.

[Fig. 24]

Fig. 24 shows the results of an antitumor activity test against a human breast cancer cell line (MDA-MB-453) using an antibody-drug conjugate (16a). The y-axis represents the mean tumor volume. The x-axis represents the number of days after cell transplantation. All values are given as mean +/- standard deviation. Initial tumor volume and initial mouse body weight were analyzed based on descriptive data (mean and standard deviation) using Microsoft Excel 2009.

[Fig. 25]

Fig. 25 shows the results of an antitumor activity test against a human breast cancer cell line (MDA-MB-453) using an antibody-drug conjugate (16a). The y-axis represents the mean tumor volume. The x-axis represents the number of days after cell transplantation. All values are given as mean +/- standard deviation. Initial tumor volume and initial mouse body weight were analyzed based on descriptive data (mean and standard deviation) using Microsoft Excel 2009.

[Fig. 26]

Fig. 26 shows the results of an antitumor activity test against a human breast cancer cell line (JIMT-1) using an antibody-drug conjugate (15). The y-axis represents the mean tumor volume. The x-axis represents the number of days after cell transplantation. All values are given as mean +/- standard deviation. Initial tumor volume and initial mouse body weight were analyzed based on descriptive data (mean and standard deviation) using Microsoft Excel 2009.

[Fig. 27]

Fig. 27 shows the results of an antitumor activity test against a human lung cancer cell line (PC9) using an antibody-drug conjugate (16a). The y-axis represents the mean tumor volume. The x-axis represents the number of days after cell transplantation. All values are given as mean +/- standard deviation. Initial tumor volume and initial mouse body weight were analyzed based on descriptive data (mean and standard deviation) using Microsoft Excel 2009.

[Fig. 28]

Fig. 28 shows the results of an antitumor activity test against a triple negative human breast cancer cell line (MDA-MB-468) using an antibody-drug conjugate (16a). The y-axis represents the mean tumor volume. The x-axis represents the number of days after cell transplantation. All values are given as mean +/- standard deviation. Initial tumor volume and initial mouse body weight were analyzed based on descriptive data (mean and standard deviation) using Microsoft Excel 2009.

[Fig. 29]

Fig. 29 shows the results of an antitumor activity test against a human head and neck cancer cell line (Fadu) using an antibody-drug conjugate (16a). The y-axis represents the mean tumor volume. The x-axis represents the number of days after cell transplantation. All values are given as mean +/- standard deviation. Initial tumor volume and initial mouse body weight were analyzed based on descriptive data (mean and standard deviation) using Microsoft Excel 2009.

[Fig. thirty]

Fig. 30 shows the results of an antitumor activity test using a patient-derived human gastric cancer tumor part (NIBIO-G016) and an antibody-drug conjugate (16a). The y-axis represents the mean tumor volume. The x-axis represents the number of days after cell transplantation. All values are given as mean +/- standard deviation. Initial tumor volume and initial mouse body weight were analyzed based on descriptive data (mean and standard deviation) using Microsoft Excel 2009.

Description of Embodiments

[0038]

In the following, preferred embodiments of the present invention will be described with reference to the drawings. Meanwhile, the embodiments described below are presented as representative examples of embodiments of the present invention, and the scope of the present invention should not be narrowly interpreted based on these embodiments.

[0039]

The present invention provides a HER3 protein-drug binding conjugate. Preferably, the HER3 binding protein of the present invention is a scaffold protein having antibody-like binding activity, or an antibody, i. anti-HER3 antibody.

[0040]

The anti-HER3 antibody-drug conjugate of the present invention is an anti-tumor drug in which an anti-HER3 antibody is conjugated to an anti-tumor compound through a linker structure, and is described in detail below.

In the context of the present invention, the term "scaffold protein" as used herein means a polypeptide or protein with exposed surface regions where amino acid insertions, substitutions or deletions are highly tolerated. Examples of scaffold proteins that can be used in accordance with the present invention are protein A from Staphylococcus aureus, bilin-binding protein from Pieris brassicae or other lipocalins, ankyrin repeat proteins, and human fibronectin (for a review, see Binz and Pluckthun, Curr Opin Biotechnol, 16, 459-69). Construction of a scaffold protein can be thought of as grafting or integrating an affinity function onto or into a structural scaffold having a stable fold protein. The affinity function means the binding affinity for a protein according to the present invention. The framework may be structurally separable from amino acid sequences conferring binding specificity. Typically, proteins that may be suitable for the development of such artificial affinity reagents can be prepared using rational, or most commonly, combinatorial protein engineering techniques such as anti-HER3 panning, either a purified protein or a protein present on the cell surface to determine binding agents in an in vitro scaffold library, techniques that are known in the art (Skerra, J. Mol. Recog., 2000; Binz and Pluckthun, 2005). In addition, a scaffold protein having antibody-like binding activity can be derived from an acceptor polypeptide containing a scaffold domain, which may have grafted donor polypeptide binding domains to impart donor polypeptide binding specificity to the scaffold domain containing the acceptor polypeptide. These inserted binding domains may be, for example, the complementarity determining region (CDR) of an antibody, in particular an anti-HER3 antibody. Insertion can be carried out by various methods known to those skilled in the art, including, for example, polypeptide synthesis, nucleic acid synthesis of the encoded amino acid, as well as various recombinant methods well known to those skilled in the art.

[0041]

{Antibody}

In addition, the term "antibody" or "anti-HER3 antibody" as used herein means monoclonal antibody, polyclonal antibody, recombinant antibody, humanized antibody (Jones et al., Nature 321 (1986), 522-525; Riechmann et al., Nature 332 (1988), 323-329, and Presta, Curr. Op. Struct. Biol. 2 (1992), 593-596), chimeric antibody (Morrison et al., Proc. Natl. Acad. Sci. USA 81 (1984), 6851-6855), human antibody and fully human antibody, (Tomizuka, K. et al., Nature Genetics (1997) 16, p. 133-143; Kuroiwa, Y. et al. , Nucl Acids Res. (1998) 26, pp. 3447-3448 Yoshida, H. et al., Animal Cell Technology: Basic and Applied Aspects vol. 10, p. T. and Iijima, S. eds.), Kluwer Academic Publishers, 1999. Tomizuka, K. et al., Proc. Natl. Acad. Sci. USA (2000) 97, p.722-727, International Publication No. WO 2007/077028, etc.), a multispecific antibody (eg, a bispecific antibody) formed from at least two antibodies, or a fragment of such an antibody. The term "antibody fragment" includes any portion of the above antibodies, preferably their antigen-binding region or variable regions. Examples of antibody fragments include Fab fragments, Fab' fragments, F(ab') ₂ fragments, Fv fragments, diabodies (Hollinger et al., Proc. Natl. Acad. Sci. USA 90 (1993), 6444-6448), single chain molecules antibodies (Pluckthun in: The Pharmacology of Monoclonal Antibodies 113, Rosenburg and Moore, EDS, Springer Verlag, NY (1994), 269-315) and other fragments, provided they exhibit the desired HER3 binding ability.

[0042]

In addition, the term "antibody" or "anti-HER3 antibody" as used herein can include antibody-like molecules that contain engineered antibody sub-domains or naturally occurring antibody variants. These antibody-like molecules can be single-domain antibodies, such as VH-only or VL-only domains, either derived from natural sources such as camelids (Muyldermans et al., Reviews in Molecular Biotechnology 74, 277-302), or through in vitro display of libraries from human, camelid or other species (Holt et al., Trends Biotechnol., 21, 484-90).

[0043]

In accordance with the present invention, an "Fv fragment" is the minimum antibody fragment that contains a complete antigen-recognition and β-binding site. This region consists of a dimer of the variable domain with one heavy and one light chain in tight non-covalent association. It is in this configuration that the three CDR ^'s of each variable domain interact to define an antigen-binding site on the surface of the V _H -V _L dimer. Together, these six CDR ^'s confer antigen-binding specificity to the antibody. However, even one single variable domain (or half of the Fv, including only three antigen-specific CDR ^'s ) has the ability to recognize and bind to an antigen, although usually with less affinity than the entire binding site.

The "Fab fragment" also contains a light chain constant domain and a heavy chain first constant domain (CH1). A "Fab fragment" differs from a "Fab ¹ fragment" by the addition of several residues at the carboxy terminus of the heavy chain CH1 domain, including one or more cysteines from the antibody hinge region. The "F(ab') ₂ fragment" is initially formed as a pair of "Fab'fragments" with hinged cysteines between them. Methods for preparing such antibody fragments, such as papain or pepsin digestion, are known to those skilled in the art.

[0044]

In another preferred embodiment of the present invention, the anti-HER3 antibody of the present invention is an anti-HER3 antibody directed against the extracellular domain (ECD) of HER3.

[0045]

The anti-HER3 antibody used in the anti-HER3 antibody-drug conjugate of the present invention may be from any species. Preferred species examples may include humans, rats, mice and rabbits. An anti-HER3 antibody derived from a non-human species is preferably subjected to chimerization or humanization using a well known technique. The antibody of the present invention may be a polyclonal antibody or a monoclonal antibody, and preferably it is a monoclonal antibody.

The anti-HER3 antibody can be one that is capable of targeting tumor cells, that is, it has tumor cell recognition ability, tumor cell binding ability, internalization property in tumor cells, and cytotoxic activity against tumor cells, and the like. An anti-HER3 antibody can be conjugated to a compound having antitumor activity via a linker to form an antibody-drug conjugate.

The binding activity of an antibody against tumor cells can be confirmed by flow cytometry. Internalization of the antibody in tumor cells can be confirmed using (1) imaging assay of the antibody incorporated into the cells under a fluorescence microscope using a secondary antibody (fluorescently labeled) that binds to the therapeutic antibody (Cell Death and Differentiation (2008) 15, 751-761) , (2) assay measuring the amount of fluorescence incorporated into cells using a secondary antibody (fluorescently labeled) that binds to a therapeutic antibody (Molecular Biology of the Cell, Vol. 15, 5268-5282, December 2004), or (3) Mab -ZAP assay using an immunotoxin that binds to a therapeutic antibody, wherein the toxin is released upon incorporation into cells to inhibit cell growth (Bio Techniques 28: 162-165, January 2000). A recombinant complex protein based on the catalytic region of diphtheria toxin and G-protein can be used as an immunotoxin.

The antitumor activity of the antibody can be confirmedin vitroby determining inhibitory activity against cell growth. For example, a cancer cell line overexpressing a target protein for an antibody is cultured, and the antibody is added at different concentrations to the culture system to determine inhibitory activity against foci formation, colony formation, and spheroid growth. Antitumor activity can be confirmed in vivo, for example, by administering an antibody to a nude mouse with a transplanted tumor cell line strongly expressing the target protein, and determining the change in the cancer (tumor) cells.

Since the compound conjugated in the antibody-drug conjugate exhibits an antitumor effect, it is preferable, but not necessary, that the antibody itself has an antitumor effect. For specific and selective manifestation of cytotoxic activity by an antitumor compound specific and selective for tumor cells, it is important and also preferable that the antibody has the property of internalizing to migrate into tumor cells.

[0046]

An anti-HER3 antibody can be prepared using a method commonly used in the art, which includes immunizing animals with an antigenic polypeptide and collecting and purifying antibodies produced in vivo. The origin of the antigen is not limited to human origin, and animals can be immunized with an antigen originating from a non-human animal such as mouse, rat, and the like. In this case, the cross-reactivity of antibodies binding to the resulting heterologous antigen and human antigens can be tested to screen for an antibody useful for a human disease.

Alternatively, antibody-producing cells that produce antibodies against the antigen are fused with myeloma cells according to a method known in the art (e.g., Kohler and Milstein, Nature (1975) 256, p. 495-497; and Kennet, R ed., Monoclonal Antibodies, pp. 365-367, Plenum Press, N.Y. (1980)), to create hybridomas from which monoclonal antibodies can in turn be made.

The antigen can be obtained using genetic engineering methods to obtain host cells that produce a gene encoding an antigenic protein. In particular, vectors are prepared that allow the expression of a gene for such an antigen , and transferred into host cells so that the gene is expressed. The antigen expressed in this way can be purified. The antibody can also be produced using the method of immunizing animals with the genetically engineered antigen-expressing cells or antigen-expressed cell line described above.

Anti-HER3 antibody can be obtained using a procedure known in the art.

[0047]

The anti-HER3 antibody that can be used in the present invention is not particularly limited, and any of the antibodies having the properties described below is desirable, for example.

(1) An anti-HER3 antibody having the following properties:

(a) binds specifically to HER3, and/or

(b) has internalization activity in HER3-expressing cells through binding to HER3.

(2) An antibody according to (1), wherein the antibody binds to the extracellular domain of HER3.

(3) An antibody according to (1) or (2), wherein the antibody is a monoclonal antibody.

(4) An antibody according to any one of (1) to (3), wherein the antibody has antibody-dependent cell-mediated cytotoxic (ADCC) activity and/or complement-dependent cytotoxic (CDC) activity.

(5) An antibody according to any one of (1) to (4), wherein the antibody is a mouse monoclonal antibody, a chimeric monoclonal antibody, a humanized monoclonal antibody, or a human or fully human (monoclonal) antibody.

(6) An antibody according to any one of (1) to (5), wherein the antibody is a humanized monoclonal antibody comprising a heavy chain comprising the amino acid sequence shown in SEQ ID NO: 1 and a light chain comprising the amino acid sequence, shown in SEQ ID NO: 2.

(7) An antibody according to any of (1) to (6), wherein the antibody does not contain a lysine residue at the carboxy terminus of the heavy chain.

(8) An antibody according to (7), wherein the antibody comprises a heavy chain variable region represented by the amino acid sequence shown in SEQ ID NO: 70 and a light chain variable region represented by the amino acid sequence shown in SEQ ID NO: 72.

(9) An antibody obtained by the method for producing an antibody according to any one of (1) to (8), wherein the method includes the steps of: culturing a host cell transformed with an expression vector comprising a polynucleotide encoding an antibody; and harvesting the antibody of interest from the cultures obtained in the previous step.

[0048]

Next, the anti-HER antibody used in the invention will be described.

In the description of the present invention, the terms "cancer" and "tumor" are used interchangeably.

In the description of the present invention, the term "gene" includes not only DNA, but also its mRNA.

In the description of the present invention, the term "polynucleotide" is used interchangeably with nucleic acid, and also includes DNA, RNA, probes, oligonucleotides and primers.

In the description of the present invention, the terms "polypeptide" and "protein" are used interchangeably.

In the description of the present invention, the term "cell" also includes cells from a single animal and cultured cells.

In the description of the present invention, the term "HER3" is used interchangeably with the term "HER3 protein".

In the description of the present invention, the term "CDR" refers to complementarity determining region (CDR). An antibody molecule is known to have three complementarity determining regions (CDRs) in each light and heavy chain. CDRs, also referred to as hypervariable domains, are present in the variable regions of the heavy and light chains of an antibody. These sites have a highly variable primary structure, and are separated at three positions in the respective primary structures of the heavy and light polypeptide chains. In the present specification, antibody CDRs are represented as CDRH1, CDRH2, and CDRH3 of the amino-terminus of the heavy chain amino acid sequence, with respect to heavy chain CDRs, and are represented as CDRL1, CDRL2, and CDRL3 of the amino-terminus of the light chain amino acid sequence, with respect to light chain CDRs. These sites are in close proximity to each other in the spatial structure and determine the specificity for the antigen to which the antibody will bind.

In the specification of the present invention, the phrase "stringent hybridization" refers to hybridization at 68°C in the commercially available ExpressHyb Hybridization Solution (manufactured by Clontech, Inc.), or to identifiable hybridization under conditions involving hybridization at 68°C. in the presence of 0.7-1.0 M NaCl using a filter containing DNA immobilized on it, followed by washing at 68°C using 0.1-2 x SSC solution (1 x SSC solution consists of 150 mM NaCl and 15 mM sodium citrate), or to hybridization under conditions equivalent to those.

[0049]

1. HER3

Human epidermal growth factor receptor 3 (HER3, also known as ErbB3) is a receptor tyrosine protein kinase and belongs to the epidermal growth factor receptor (EGFR) receptor tyrosine protein kinase subfamily, which also includes HER1 (also known as EGFR), HER2, and HER4. HER3 is a transmembrane receptor and consists of an extracellular ligand-binding domain (ECD), a dimerization domain in the ECD, a transmembrane domain, an intracellular protein tyrosine kinase domain (TKD), and a C-terminal phosphorylation domain. HER3 has been found to be overexpressed in certain types of cancer such as breast, gastrointestinal and pancreatic cancers. A relationship has been shown between HER2/HER3 expression and progression from a non-invasive stage to an invasive stage.

The HER3 protein used in the present invention can be used after direct purification from HER3-expressing human cells or non-human mammalian cells (rat, mouse, etc.), or can be used by obtaining cell membrane cell fractions. Alternatively, HER3 can be synthesized in vitro or can be obtained from host cells by genetic engineering. In genetic engineering techniques, for example, HER3 cDNA is inserted into vectors that allow its expression, and HER3 can then be expressed by synthesis in a solution containing the enzymes necessary for transcription and translation, substrates and energetic substances, or by transformation of other host prokaryotic cells or eukaryotic host cells to produce protein. Alternatively, the genetically engineered HER3 expressing cells described above or a cell line expressing HER3 can be used as the HER3 protein.

The RNA sequence, cDNA sequence and amino acid sequence of HER3 are available in a public database and can be listed, for example, under accession numbers such as AAA35979 (precursor including signal sequence consisting of amino-terminal 19 amino acid residues), M34309 (NCBI) , For example.

The above amino acid sequence of HER3 consists of an amino acid sequence subjected to substitutions, deletions, additions and/or insertions of at least one amino acid, and proteins having a biological activity equivalent to that of this protein are also included in HER3.

[0050]

2. Obtaining anti-HER3 antibodies

An anti-HER3 antibody of the present invention can be prepared using a method commonly used in the art, which includes immunizing an animal with HER3 or an arbitrary polypeptide selected from the HER3 amino acid sequence, and collecting and purifying the antibody produced in vivo. The species of HER3 that can be used as an antigen is not limited to a human, and an animal can be immunized with HER3 derived from a non-human animal such as a mouse or a rat. In this case, by examining cross-reactivity between an antibody that binds to the obtained heterologous HER3 and human HER3, an antibody applicable to a human disease can be selected.

In addition, a monoclonal antibody can be obtained from a hybridoma obtained by fusing antibody-producing cells that produce anti-HER3 antibody with myeloma cells according to a known method (e.g., Kohler and Milstein, Nature, (1975) 256, pp. 495- 497; Kennet, R. ed., Monoclonal Antobodies, pp. 365-367, Plenum Press, N.Y. (1980)).

HER3 that can be used as an antigen can be obtained by genetically engineering expression of the HER3 gene in a host cell.

Specifically, a vector capable of expressing the HER3 gene is obtained, and the resulting vector is transfected into a host cell to express the gene, and then the expressed HER3 is purified.

Also, engineered HER3 expressing cells or a cell line expressing HER3 can be used as the HER3 protein. The following describes in detail the method for obtaining antibodies against HER3.

[0051]

(1) Obtaining an antigen

Examples of an antigen that can be used to make an anti-HER3 antibody include HER3, a polypeptide consisting of a partial amino acid sequence including at least 6 consecutive HER3s, and a derivative obtained by adding a specified amino acid sequence or carrier.

HER3 can be purified directly from human tumor tissues or tumor cells and then used. In addition, HER3 can be obtained by its in vitro synthesis or by its production in the host cell using genetic engineering techniques.

With regard to genetic engineering, for example, after inserting HER3 cDNA into a vector capable of expressing HER3 cDNA, HER3 can be obtained by synthesizing it in a solution containing an enzyme, substrate, and an energy substance necessary for transcription and translation, or by expressing HER3 in another prokaryotic or eukaryotic transformed host cell.

In addition, the antigen can also be obtained as a secretory protein by expressing a fusion protein obtained by ligating the extracellular domain of HER3, which is a membrane protein, with an antibody constant region in an appropriate host-vector system.

HER3 cDNA can be obtained, for example, by the so-called PCR method, in which a polymerase chain reaction (referred to as "PCR"; see Saiki, R. K., et al., Science, (1988) 239, pp. 487-489) is carried out using a cDNA library expressing the HER3 cDNA as a template; and primers that specifically amplify the HER3 cDNA.

As an example of in vitro polypeptide synthesis, for example, but not limited to, the Rapid Translation System (RTS), manufactured by Roche Diagnostics, Inc.

Examples of prokaryotic host cells include Escherichia coli and Bacillus subtilis. To transform the host cells with the target genome, the host cells are transformed with a plasmid vector comprising a replicon, i. an origin of replication derived from a species compatible with the host, and a regulatory sequence. In addition, the vector preferably contains a sequence capable of conferring phenotypic selectivity on the transformed cell.

Examples of eukaryotic host cells include vertebrate cells, insect cells, and yeast cells. As vertebrate cells, for example, monkey COS cells (Gluzman, Y., Cell, (1981) 23, pp. 175-182, ATCC CRL-1650; ATCC: American Type Culture Collection), NIH3T3 mouse fibroblasts (ATCC No. . CRL-1658) and dihydrofolate reductase-deficient strains (Urlaub, G. And Chasin, L. A., Proc. Natl. Acad. Sci. USA (1980) 77, pp. 4126-4220) of Chinese hamster ovary cells (CHO cells; ATCC: CCL-61); and the like, however, cells are not limited to this.

The transformant thus obtained can be cultured according to the method commonly used in the art, and by culturing the transformant, the target polypeptide is produced intracellularly or extracellularly.

A suitable culture medium can be selected from various conventionally used culture media depending on the host cells used. If Escherichia coli is used, for example, LB medium supplemented with an antibiotic such as ampicillin or IPMG may be used if necessary.

The recombinant protein produced by the transformant intracellularly or extracellularly by such culturing can be isolated and purified by any of the various known separation methods based on the physical or chemical property of the protein.

Specific examples of methods include treatment with a conventional protein precipitation agent, ultrafiltration, various types of liquid chromatography such as molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography and affinity chromatography, dialysis, and a combination of such methods.

In addition, by attaching a label of six histidine residues to the recombinant protein to be expressed, the protein can be efficiently purified on a nickel affinity chromatography column. Alternatively, by attaching the IgG Fc region to the recombinant protein being expressed, the protein can be efficiently purified on a protein A column.

By combining the methods described above, a large amount of the desired polypeptide can be easily obtained in high yield and high purity.

The transformant described above, as such, can also be used as an antigen. It is also possible to use a cell line expressing HER3 as an antigen. Examples of such a cell line include, but are not limited to, these cell lines, as long as they express HER3.

[0052]

(2) Preparation of anti-HER3 monoclonal antibody

Examples of an antibody that specifically binds to HER3 include a monoclonal antibody that specifically binds to HER3, and the method for producing the antibody is described below.

To obtain a monoclonal antibody, as a rule, the following technological steps are required:

(a) purifying the biopolymer used as an antigen or obtaining cells expressing the antigen;

(b) obtaining antibody-producing cells by immunizing the animal with an antigen injection, blood collection, analysis of antibody titer to determine the time of removal of the spleen;

(c) obtaining myeloma cells (hereinafter referred to as "myeloma");

(d) fusion of antibody-producing cells with myeloma;

(e) screening for a group of hybridomas producing the desired antibody;

(f) separation of hybridomas into individual cell clones (cloning);

(g) optionally, culturing the hybridoma or growing the hybridoma-implanted animal to obtain a large amount of the monoclonal antibody;

(h) examination of the thus obtained monoclonal antibody for biological activity and binding specificity, or analysis of the antibody for properties as a labeled reagent; and so on.

Next, a method for producing a monoclonal antibody by following the steps described above will be described in detail, however, the method is not limited thereto, and, for example, antibody-producing cells other than spleen and myeloma cells can be used.

[0053]

(a) Antigen purification

As an antigen, HER3 obtained by the method described above or a partial peptide thereof can be used.

In addition, a membrane fraction obtained from recombinant cells expressing HER3, or recombinant cells expressing HER3 themselves, as well as a partial peptide of the protein of the present invention, chemically synthesized by a method known to those skilled in the art, can also be used as an antigen.

In addition, a cell line expressing HER3 can also be used as an antigen.

[0054]

(b) Obtaining antibody-producing cells

The antigen obtained in step (a) is mixed with an adjuvant such as complete or incomplete Freund's adjuvant or an excipient such as aluminum potassium sulfate, and the resulting mixture is used as an immunogen to immunize an experimental animal. In an alternative method, the experimental animal is immunized with antigen-expressing cells as an immunogen. As an experimental animal, you can fearlessly use any animal used in a known method for obtaining hybridomas. Specifically, for example, a mouse, a rat, a goat, a sheep, a cow, a horse or the like can be used. However, from the viewpoint of ease of availability of myeloma cells for fusion with the extracted antibody-producing cells, it is preferable to use a mouse or a rat as an animal for immunization.

In addition, the mouse or rat strain that can be used is not particularly limited, and in the case of a mouse, for example, various strains such as A, AKR, BALB/c, BDP, BA, CE, C3H, 57BL, C57BL, C57L, DBA, FL, HTH, HT1, LP, NZB, NZW, RF, R III, SJL, SWR, WB and 129 and the like, and in the case of a rat, for example Wistar, Low, Lewis, Sprague, Dawley, ACI, BN, Fischer, etc.

These mice and rats are commercially available from experimental animal manufacturers/distributors such as CLEA Japan, Inc. and Charles River Laboratories Japan, Inc.

When considering the compatibility of the fusion with the myeloma cells described below, in the case of the mouse, the BALB/c strain, and in the case of the rat, the Wistar and Low strains are particularly preferred as the immunization animal.

In addition, in view of the antigenic homology between a human and a mouse, it is also preferable to use a mouse having a reduced biological function for removing autoantibodies, that is, a mouse with an autoimmune disease.

The age of such a mouse or rat at the time of immunization is preferably 5-12 weeks, more preferably 6-8 weeks.

To immunize an animal with HER3 or its recombinant, for example, a known method can be used, as described in detail in, for example, Weir, D. M., Handbook of Experimental Immunology Vol. I. II. III., Blackwell Scientific Publications, Oxford (1987); Kabat, E. A. and Mayer, M. M., Experimental Immunochemistry, Charles C Thomas Publisher Springfield, Illinois (1964), or the like.

Of these immunization methods, the specific method preferred in the present invention is, for example, the following.

Namely, first, a fraction of a membrane protein serving as an antigen or a cell in which the expression of the antigen is caused is intradermally or intraperitoneally administered to an animal. However, a combination of both routes of administration is preferable in order to improve the efficiency of immunization, and when intradermal administration is carried out in the first half and intraperitoneal administration is carried out in the next half or only as the last administration, the efficiency of immunization is especially improved.

The antigen administration schedule varies depending on the type of animal to be immunized, the individual, or the like. However, in general, an administration regimen in which the antigen administration frequency is 3 to 6 times and a dosing interval is 2 to 6 weeks is preferred, and an administration regimen in which the antigen administration frequency is 3 to 4 times is more preferable. and the dosing interval is 2 to 4 weeks.

In addition, the dose of the antigen varies depending on the type of animal, individual or the like, however, the dose is generally set at 0.05-5 mg, preferably about 0.1-0.5 mg.

Booster immunization is carried out 1-6 weeks, preferably 1-4 weeks, more preferably 1-3 weeks after the introduction of the antigen described above. When the immunogen is a cell, 1'10 ⁶ to 1'10 ⁷ cells are used.

The dose of antigen at the time of the booster immunization varies depending on the type or size of the animal or the like, however, in the case of a mouse, for example, the dose is generally set at 0.05-5 mg, preferably 0. 1-0.5 mg, more preferably about 0.1-.2 mg. When the immunogen is a cell, 1'10 ⁶ to 1'10 ⁷ cells are used.

Spleen cells or lymphocytes, including antibody-producing cells, are aseptically isolated from the immunized animal 1-10 days, preferably 2-5 days, more preferably 2-3 days after booster immunization. At this time, the antibody titer is measured, and if an animal having a sufficiently elevated antibody titer is used as a source of antibody-producing cells, the subsequent procedure can be carried out more efficiently.

Examples of the antibody titer measurement method that can be used in the present invention include the RIA method and the ELISA method, but the method is not limited thereto. For example, if ELISA is used, the measurement of antibody titer in the present invention can be performed in accordance with the procedures described below.

First, the purified or partially purified antigen is adsorbed onto a solid phase surface, such as a 96-well ELISA plate, and the solid phase surface, which does not contain any adsorbed antigen, is coated with a protein not bound to the antigen in any way, such as bovine serum albumin (BSA) . After washing the surface, the surface is brought into contact with a serially diluted sample (eg mouse serum) as the primary antibody to allow the antibody contained in the sample to bind to the antigen.

Then, an enzyme-labeled anti-mouse antibody is added as a secondary antibody and allowed to bind to the mouse antibody. After washing, an enzyme substrate is added, and a change in absorbance which occurs due to color development induced by decomposition of the substrate or the like is measured, and an antibody titer is calculated based on the results.

Isolation of antibody-producing cells from spleen cells or lymphocytes of an immunized animal can be carried out in accordance with a known method (for example, Kohler et al., Nature (1975), 256, p. 495; Kohler et al., Eur. J. Immunol. ( 1977), 6, p. 511; Milstein et al., Nature (1977), 266, p. 550; Walsh, Nature (1977), 266, p. 495). For example, in the case of spleen cells, a general method can be used in which antibody-producing cells are isolated by homogenizing the spleen to obtain cells through filtration using a stainless steel sieve and suspending the cells in Minimum Essential Needle's Medium (MEM).

[0055]

(c) Obtaining myeloma cells (hereinafter referred to as "myeloma")

Myeloma cells that can be used for cell fusion are not particularly limited, and suitable cells can be selected from known cell lines. However, considering the convenience when the hybridoma is selected from fused cells, it is preferable to use an HGPRT (hypoxanthine guanine phosphoribosyltransferase) deficient strain whose selection procedure is well known.

More specifically, examples of an HGPRT-deficient strain include X63-Ag8(X63), NS1-ANS/1(NS1), P3X63-Ag8.U1(P3U1), X63-Ag8.653(X63.653), SP2/0-Ag14 (SP2/0), MPC11-45.6TG1.7(45.6TG), FO, S149/5XXO and BU.1 from mice; 210.RSY3.Ag.1.2.3(Y3) obtained from a rat; and U266AR(SKO-007), GM1500*GTG-A12(GM1500), UC729-6, LICR-LOW-HMy2(HMy2) and 8226AR/NIP4-1(NP41) from humans. These HGPRT-deficient strains are available, for example, from ATCC or the like.

These cell strains are subcultured in a suitable medium such as 8-azaguanine medium (medium prepared by adding 8-azaguanine to RPMI 1640 medium supplemented with glutamine, 2-mercaptoethanol, gentamicin and fetal bovine serum (hereinafter referred to as "FBS")) , Iskov's Modified Dulbecco's Medium (IMDM) or Dulbecco's Modified Eagle's Medium (DMEM). In this case, 3-4 days before cell fusion is performed, the cells are subcultured in a normal medium (eg, ASF104 medium (manufactured by Ajinomoto Co., Ltd.) containing 10% FCS) to provide at least 2 × 10 ⁷ cells per day cell fusion.

[0056]

(d) Cell fusion

Fusion between antibody-producing cells and myeloma cells can be suitably carried out according to a known method (Weir, D. M. Handbook of Experimental Immunology Vol. I. II. III., Blackwell Scientific Publications, Oxford (1987); Kabat, E. A. and Mayer, M. M., Experimental Immunochemistry, Charles C Thomas Publisher, Springfield, Illinois (1964), etc.) under such conditions that the percentage of cell survival is not greatly reduced.

As such a method, for example, a chemical method in which antibody-producing cells and myeloma cells are mixed in a solution containing a polymer such as polyethylene glycol at a high concentration, a physical method using electrical stimulation, or the like can be used. Of these methods, a specific example of a chemical method is described below.

Namely, in the case where polyethylene glycol is used in a solution containing a polymer at a high concentration, antibody-producing cells and myeloma cells are mixed in a solution of polyethylene glycol having a molecular weight of 1500-6000, more preferably 2000-4000, at a temperature of 30 to 40°C. C, preferably from 35 to 38°C, for 1-10 minutes, preferably 5-8 minutes.

[0057]

(e) Selection of hybridoma group

The selection method for hybridomas obtained by the above-described cell fusion is not specifically limited. The HAT (hypoxanthine, aminopterine, thymidine) method of choice is usually used (Kohler et al., Nature (1975), 256, p. 495; Milstein et al., Nature (1977), 266, p. 550).

This method is effective when hybridomas are obtained using HGPRT-deficient myeloma cells that cannot survive in the presence of aminopterin. That is, by culturing unfused cells and hybridomas in HAT medium, only aminopterin-resistant hybridomas are selectively allowed to survive and proliferate.

[0058]

(f) Division into individual cell clones (cloning)

As a cloning method for hybridomas, a known method such as the methylcellulose method, the soft agarose method, or the serial dilution method can be used (see, for example, Barbara, B. M. and Stanley, M. S.: Selected Methods in Cellular Immunology, W. H. Freeman and Company, San Francisco (1980)). Of these methods, a three-dimensional culture method such as a methylcellulose method is particularly preferable. For example, a group of hybridomas obtained by cell fusion is suspended in a methylcellulose medium such as ClonaCell-HY Selection Medium D (manufactured by StemCell Technologies, Inc., #03804) and cultured. Then the hybridomas formed colonies are harvested, thus monoclonal hybridomas can be obtained. The collected appropriate hybridoma colonies are cultured, and the hybridoma which is confirmed to have a stable antibody titer in the resulting hybridoma culture supernatant is selected as the HER3 monoclonal antibody-producing hybridoma strain.

[0059]

(g) Obtaining a monoclonal antibody by culturing hybridoma

By culturing the thus selected hybridoma, the monoclonal antibody can be efficiently obtained. However, it is preferable to screen for a hybridoma that produces the target monoclonal antibody prior to culture.

A known method can be used for such screening.

Measurement of antibody titer in the present invention can be carried out, for example, by the ELISA method, which is explained in paragraph (b) described above.

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

After cloning is completed, the HT medium is replaced with normal medium and the hybridoma is cultured.

Large-scale cultivation is carried out by rotating the culture using a large culture vessel or by centrifuging the culture. From the supernatant obtained by large-scale culture, a monoclonal antibody that specifically binds to the protein of the present invention can be obtained by purification using a method known to those skilled in the art, such as gel filtration.

Then, the hybridoma is administered by injection into the abdominal cavity of the mouse of the same strain as the hybridoma (for example, BALB/c described above) or Nu/Nu mice to proliferate the hybridoma, whereby ascites containing a large amount of the monoclonal antibody of the present invention can be obtained.

In the case where the hybridoma is introduced into the abdominal cavity, if a mineral oil such as 2,6,10,14-tetramethylpentadecane (pristane) is administered 3 to 7 days before, more ascites can be obtained.

For example, an immunosuppressant is pre-administered by intra-abdominal injection of the same strain as the hybridoma to inactivate T cells. 20 days later, 10 ^{6 -} 10 ⁷ hybridoma cells are suspended in serum-free medium (0.5 ml) and the suspension is injected into the abdominal cavity of the mouse. Typically, when the abdominal cavity becomes enlarged and filled with ascites, the ascites are collected from the mouse. This method can produce a monoclonal antibody at a concentration that is about 100 times or more higher than the concentration in the culture solution.

A monoclonal antibody obtained by the method described above can be purified by the method described, for example, in Weir, D. M., Handbook of Experimental Immunology Vol. I. II. III., Blackwell Scientific Publications, Oxford (1987).

The monoclonal antibody thus obtained has a high antigen specificity for HER3.

[0060]

(h) Monoclonal antibody assay

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

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

The Ouchterlony method is simple, but when the concentration of the monoclonal antibody is low, a condensation procedure is necessary.

On the other hand, when the ELISA method or the RIA method is used, by directly reacting the culture supernatant with the antigen-adsorbed solid phase and using antibodies corresponding to different immunoglobulin isotypes and subclasses as secondary antibodies, the isotype and subclass of the monoclonal antibody can be identified.

In addition, a commercially available identification kit (eg, Mouse Typer Kit, manufactured by Bio-Rad Laboratories, Inc.) or the like can also be used as an easier method.

In addition, protein quantitation can be performed by the Folin-Lowry method and the calculation method based on absorbance at 280 nm (1.4 (OD 280)=Immunoglobulin 1 mg/ml).

In addition, even when a monoclonal antibody is obtained separately and independently by carrying out steps (a) to (h) in item (2) again, an antibody having a cytotoxic activity that is equivalent to that of an anti-HER3 antibody can be obtained. One example of such an antibody is an antibody that binds to the same epitope as an anti-HER3 antibody. If a newly generated monoclonal antibody binds to the partial peptide or partial tertiary structure to which the anti-HER3 antibody binds, it can be determined that the monoclonal antibody binds to the same epitope as the anti-HER3 antibody. In addition, by confirming that the monoclonal antibody competes with the anti-HER3 antibody for binding to HER3 (i.e., the monoclonal antibody interferes with binding between the anti-HER3 antibody and HER3), it can be determined that the monoclonal antibody binds to the same epitope as the anti-HER3 antibody. -HER3 antibody, even if the specific sequence or structure of the epitope has not been determined. When the epitope is confirmed to be the same, it can be safely expected that the monoclonal antibody has the same antigen binding affinity and biological activity as an anti-HER3 antibody.

[0061]

(3) Other antibodies

The antibody of the present invention includes not only the anti-HER3 monoclonal antibody described above, but also a recombinant antibody artificially modified to reduce heterologous antigenicity for a human, such as a chimeric antibody, a humanized antibody, and a human antibody. These antibodies can be obtained using a known method.

As a chimeric antibody, an antibody in which the variable and constant regions of an antibody are from different species can be exemplified, such as a chimeric antibody in which a mouse or rat-derived antibody variable region is linked to a human-derived antibody constant region (see Proc Natl Acad Sci USA 81 6851-6855 (1984)).

As a humanized antibody, there can be exemplified an antibody obtained by integrating only a complementarity determining region (CDR) into an antibody of human origin (see Nature (1986) 321, pp. 522-525), and an antibody obtained by grafting a portion of amino acids framework residues, as well as the CDR sequence to a human antibody by CDR grafting (International Publication No. WO 90/07861).

The term "multiple" as used herein refers to 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, or 1 or 2.

[0062]

In accordance with the present invention, it should be understood that the amino acid sequence of the binding protein of the present invention is not limited to twenty conventional amino acids (see Immunology - A Synthesis ( ^2nd Edition, ES Golub and DR Gren, Eds., Sinauer Associates, Sunderland, Mass. (1991)), which is incorporated herein by reference). For example, amino acids may include stereoisomers (eg, D-amino acids) of the twenty traditional amino acids, non-naturally occurring amino acids such as alpha, alpha-disubstituted amino acids, N-alkyl amino acids, lactic acid, and other non-traditional amino acids. Examples of non-traditional amino acids that may also be suitable components for the binding protein of the present invention include: 4-hydroxyproline, gamma-carboxyglutamate, epsilon-N,N,N-trimethyllysine, epsilon-N-acetyl lysin, O-phosphoserine, N-acetylserine , N-formylmethionine, 3-methylhistidine, 5-hydroxylysine, sigma-N-methylarginine and other similar amino acids and imino acids, for example, 4-hydroxyproline.

[0063]

As an amino acid substitution in the present specification, a conservative amino acid substitution is preferred. A conservative amino acid substitution refers to a substitution occurring in a group of amino acids belonging to the amino acid side chains. Preferred amino acid groups are as follows: acid group (aspartic acid and glutamic acid); main group (lysine, arginine and histidine); non-polar group (alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine and tryptophan); and the uncharged polar family (glycine, asparagine, glutamine, cysteine, serine, threonine, and tyrosine). More preferred amino acid groups are as follows: aliphatic hydroxy group (serine and threonine); an amide-containing group (asparagine and glutamine); aliphatic group (alanine, valine, leucine and isoleucine); and an aromatic group (phenylalanine, tryptophan and tyrosine). Such an amino acid substitution is preferably carried out within limits that do not impair the properties of the substance having the original amino acid sequence. When the heavy and light chains of the antibodies of the present invention contain glutamate as the N-terminal amino acid, it can be cyclized (in the form of pyroglutamate). In the present invention, such pyroglutamate does not differ from normal glutamine in amino acid sequences. In the heavy and light chains of an antibody of the present invention, the cysteine may be in the form of cysteinyl. In the present invention, such a cysteinyl form does not differ from normal cysteine in amino acid sequences.

[0064]

In addition, the antibody of the present invention includes a human antibody that binds to HER3. An anti-HER3 human antibody refers to a human antibody having only the antibody gene sequence derived from the human chromosome. An anti-HER3 human antibody can be produced by a method using a human antibody-producing mouse having a fragment of a human chromosome comprising the human antibody heavy and light chain genes (see Tomizuka, K. et al., Nature Genetics (1997) 16, pp. 133- 143; Kuroiwa, Y. et al., Nucl. Acids Res. (1998) 26, pp. 3447-3448; Yoshida, H. et al., Animal Cell Technology: Basic and Applied Aspects vol. 10, pp. 69- 73 (Kitagawa, Y., Matuda, T. and Iijima, S. eds.), Kluwer Academic Publishers, 1999; Tomizuka, K. et al., Proc. Natl. Acad. Sci. USA (2000) 97, pp. 722-727, etc.).

[0065]

Such a human antibody-producing mouse can be generated, inter alia, by the following method. A genetically modified animal is created in which the endogenous immunoglobulin heavy and light chain gene loci have been severed and human immunoglobulin heavy and light chain gene loci have been inserted instead through a vector representing an artificial yeast chromosome (YAC) or the like, by obtaining a knockout -animal and transgenic animal and mating of these animals.

In addition, according to the recombinant DNA method, by using cDNAs encoding each of such a heavy chain and a light chain of a human antibody, and preferably a vector including such cDNAs, eukaryotic cells are transformed, and the transformed cell that produces the recombinant human monoclonal antibody is cultured, whereby the antibody can also be obtained from the culture supernatant.

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

Regarding the preparation of a human antibody, a detailed description is provided in International Publication No. WO 2007/077028. The contents of International Publication No. WO 2007/077028 are incorporated into this application by reference.

[0066]

In addition, a method for producing a human antibody using a phage display that is selected from a library of human antibodies is also known (see Wormstone, I. M. et al., Investigative Ophthalmology & Visual Science. (2002) 43 (7), pp. 2301-2308; Carmen, S. et al., Briefings in Functional Genomics and Proteomics (2002), 1 (2), pp. 189-203 Siriwardena, D. et al., Ophthalmology (2002) 109 (3), pp. 427- 431, etc.).

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

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

If the DNA sequence of an antigen-binding scFv is determined, a human antibody can be generated by obtaining an expression vector comprising this sequence and introducing the vector into a suitable host to express it (International Publication No. WO 92/01047, WO 92/20791, WO 93/06213 , WO 93/11236, WO 93/19172, WO 95/01438, WO 95/15388 Annu Rev Immunol (1994) 12 pp 433-455 Nature Biotechnology (2005) 23(9) pp. 1105-1116).

[0067]

One aspect of the present invention relates to an isolated protein that binds to HER3. In one embodiment of the present invention, the isolated HER3 binding protein of the present invention comprises a heavy chain variable region amino acid sequence comprising: (a) a CDRH1 contained in the amino acid sequence shown in SEQ ID NOs: 2, 6, 10, 14, 18, 22, 26, 30, 34, 36, 40, 42, 46, 50, 54, 60, 62, 66, 70, 74, 78, 80, 84, 88, 92, 96, 100, 104, 108, 112, 116, 120, 122, 126, 130, 134, 138, 142, 146, 150, 154, 158, 162, 166, 170, 174, 178, 182, 186, 190, 194, 198, 202, 206, 2 10, 214, 218, 222, 226 or 230, (b) CDRH2 contained in the amino acid sequence shown in SEQ ID NO: 2, 6, 10, 14, 18, 22, 26, 30, 34, 36, 40, 42, 46, 50, 54, 60, 62, 66, 70, 74, 78, 80, 84, 88, 92, 96, 100, 104, 108, 112, 116, 120, 122, 126, 130, 134, 138, 142, 146, 150, 154, 158, 162, 166, 170, 174, 178, 182, 186, 190, 194, 198, 202, 206, 210, 214, 218, 222, 226 or 230, and CDRH3 contained in the amino acid sequence shown in SEQ ID NOs: 2, 6, 10, 14, 18, 22, 26, 30, 34, 36, 40, 42, 46, 50, 54, 60, 62, 66, 70 , 74, 78, 80, 84, 88, 92, 96, 100, 104, 108, 112, 116, 120, 122, 126, 130, 134, 138, 142, 146, 150, 154, 158, 162, 166 , 170, 174, 178, 182, 186, 190, 194, 198, 202, 206, 210, 214, 218, 222, 226, or 230, and a light chain variable region amino acid sequence comprising: the amino acid sequence shown in SEQ ID NO: 4, 8, 12, 16, 20, 24, 28, 32, 38, 44, 48, 52, 56, 58, 64, 68, 72, 76, 82, 86, 90 , 94, 98, 102, 106, 110, 114, 118, 124, 128, 132, 136, 140, 144, 148, 152, 156, 160, 164, 168, 172, 176, 180, 184, 188, 1 92 , 196, 200, 204, 208, 212, 216, 220, 224, 228 or 232, (e) CDRL2 contained in the amino acid sequence shown in SEQ ID NOs: 4, 8, 12, 16, 20, 24, 28 , 32, 38, 44, 48, 52, 56, 58, 64, 68, 72, 76, 82, 86, 90, 94, 98, 102, 106, 110, 114, 118, 124, 128, 132, 136 , 140, 144, 148, 152, 156, 160, 164, 168, 172, 176, 180, 184, 188, 192, 196, 200, 204, 208, 212, 216, 220, 224, 228 or 232, and (f) CDRL3 contained in the amino acid sequence shown in SEQ ID NOs: 4, 8, 12, 16, 20, 24, 28, 32, 38, 44, 48, 52, 56, 58, 64, 68, 72, 76, 82, 86, 90, 94, 98, 102, 106, 110, 114, 118, 124, 128, 132, 136, 140, 144, 148, 152, 156, 160, 164, 168, 172, 176, 180, 184, 188, 192, 196, 200, 204, 208, 212, 216, 220, 224, 228 or 232.

[0068]

The isolated HER3 binding protein of the present invention preferably comprises a heavy chain amino acid sequence comprising (a) a CDRH1 comprising an amino acid sequence represented by a sequence selected from the group consisting of SEQ ID NOs: 236, 251, 252, and 256; (b) a CDRH2 comprising an amino acid sequence represented by a sequence selected from the group consisting of SEQ ID NOs: 258, 278, 280 and 282; and (c) a CDRH3 comprising an amino acid sequence represented by a sequence selected from the group consisting of SEQ ID NOs: 283, 285, 309, 313 and 315 and a light chain amino acid sequence comprising (d) a CDRL1 comprising the amino acid sequence represented by a sequence selected from the group consisting of SEQ ID NOs: 320, 334, 337 and 340; (e) CDRL2 comprising an amino acid sequence represented by a sequence selected from the group consisting of SEQ ID NOs: 343, 356, 351 and 344; and (f) a CDRL3 comprising an amino acid sequence represented by a sequence selected from the group consisting of SEQ ID NOs: 360, 381, 385 and 387.

[0069]

In another embodiment of the present invention, the isolated binding protein of the present invention comprises a heavy chain variable region amino acid sequence selected from the group consisting of SEQ ID Nos: 2, 6, 10, 14, 18, 22, 26, 30, 34, 36 , 40, 42, 46, 50, 54, 60, 62, 66, 70, 74, 78, 80, 84, 88, 92, 96, 100, 104, 108, 112, 116, 120, 122, 126, 130 , 134, 138, 142, 146, 150, 154, 158, 162, 166, 170, 174, 178, 182, 186, 190, 194, 198, 202, 206, 210, 214, 218, 222, 226 and 230 , and/or a light chain variable region amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 8, 12, 16, 20, 24, 28, 32, 38, 44, 48, 52, 56, 58, 64 , 68, 72, 76, 82, 86, 90, 94, 98, 102, 106, 110, 114, 118, 124, 128, 132, 136, 140, 144, 148, 152, 156, 160, 164, 168 , 172, 176, 180, 184, 188, 192, 196, 200, 204, 208, 212, 216, 220, 224, 228 and 232.

[0070]

In yet another embodiment of the present invention, the isolated binding protein of the present invention comprises the heavy chain variable region amino acid sequence and the light chain variable region amino acid sequence shown in SEQ ID NOs: 2 and 4, 6 and 8, 10 and 12, 14 and 16, 18&20, 22&24, 26&28, 30&32, 36&38, 42&44, 46&48, 50&52, 54&56, 60&58, 62&64, 66&68, 70& 72, 74 and 76, 78 and 82, 80 and 82, 84 and 86, 88 and 90, 92 and 94, 96 and 98, 100 and 102, 104 and 106, 108 and 110, 112 and 114, 116 and 118, 122&124, 126&128, 130&132, 134&136, 138&140, 142&144, 146&148, 150&152, 154&156, 158&160, 162&164, 166&168, 1 70 and 172, 174 and 176, 178 and 180, 182 and 184, 186 and 188, 190 and 192, 194 and 196, 198 and 200, 202 and 204, 206 and 208, 210 and 212, 214 and 216, 218 and 2 20, 222 and 224, 226 and 228, or 230 and 232, or the heavy chain variable region amino acid sequence set forth in SEQ ID NO: 34, 40, 60, 62, or 120 and the light chain variable region amino acid sequence set forth in SEQ ID NO: 58 or 64, respectively.

The isolated HER3 binding protein of the present invention more preferably comprises the heavy chain variable region amino acid sequence set forth in SEQ ID NO: 42, 54, 70, 92, or 96 and the light chain variable region amino acid sequence set forth in SEQ ID NO: 44, 56, 72, 94 or 98.

[0071]

An antibody comprising a heavy chain variable region amino acid sequence and a light chain variable region amino acid sequence set forth in SEQ ID NOs: 2 and 4 is referred to as "U1-39", an antibody comprising a heavy chain variable region amino acid sequence and a light chain variable region amino acid sequence chains shown in SEQ ID NOs: 6 and 8 are referred to as "U1-40", an antibody comprising the heavy chain variable region amino acid sequence and the light chain variable region amino acid sequence shown in SEQ ID NOs: 10 and 12 is referred to as " U1-38", an antibody comprising a heavy chain variable region amino acid sequence and a light chain variable region amino acid sequence shown in SEQ ID NOs: 14 and 16 is referred to as "U1-41", an antibody comprising a heavy chain variable region amino acid sequence and the light chain variable region amino acid sequence set forth in SEQ ID NOs: 18 and 20 is referred to as "U1-42", an antibody comprising the heavy chain variable region amino acid sequence and the light chain variable region amino acid sequence set forth in SEQ ID NOs: 22 and 24, referred to as "U1-43", an antibody comprising the amino acid sequence of the heavy chain variable region and the amino acid sequence of the variable light chain shown in SEQ ID NOs: 26 and 28, referred to as "U1-44", an antibody comprising the amino acid sequence heavy chain variable region and light chain variable region amino acid sequence set forth in SEQ ID NOs: 30 and 32, referred to as "U1-45", an antibody comprising the heavy chain variable region amino acid sequence and the light chain variable region amino acid sequence set forth in SEQ ID NOs: 36 and 38, referred to as "U1-47", an antibody comprising the amino acid sequence of the variable region of the heavy chain and the amino acid sequence of the variable region of the light chain shown in SEQ ID NOs: 42 and 44, referred to as "U1-49", an antibody comprising a heavy chain variable region amino acid sequence and a light chain variable region amino acid sequence set forth in SEQ ID NOs: 46 and 48 is referred to as "U1-50", an antibody comprising a heavy chain variable region amino acid sequence and a light chain variable region amino acid sequence chains shown in SEQ ID NOs: 50 and 52 are referred to as "U1-51", an antibody comprising the heavy chain variable region amino acid sequence and the light chain variable region amino acid sequence shown in SEQ ID NOs: 54 and 56 is referred to as " U1-53", an antibody comprising the amino acid sequence of the heavy chain variable region and the amino acid sequence of the variable region of the light chain shown in SEQ ID NOs: 60 and 58 is referred to as "U1-55", an antibody comprising the amino acid sequence of the heavy chain variable region and the light chain variable region amino acid sequence set forth in SEQ ID NOs: 62 and 64 is referred to as "U1-57", an antibody comprising the heavy chain variable region amino acid sequence and the light chain variable region amino acid sequence set forth in SEQ ID NOs: 66 and 68, referred to as "U1-58", an antibody comprising the amino acid sequence of the heavy chain variable region and the amino acid sequence of the variable region of the light chain shown in SEQ ID NOs: 70 and 72, referred to as "U1-59", an antibody comprising the amino acid sequence heavy chain variable region and light chain variable region amino acid sequence set forth in SEQ ID NOs: 74 and 76, referred to as "U1-52", an antibody comprising the heavy chain variable region amino acid sequence and the light chain variable region amino acid sequence set forth in SEQ ID NOs: 78 and 82, referred to as "U1-61", an antibody comprising the amino acid sequence of the variable region of the heavy chain and the amino acid sequence of the variable region of the light chain shown in SEQ ID NOs: 80 and 82, referred to as "U1-61.1", an antibody comprising a heavy chain variable region amino acid sequence and a light chain variable region amino acid sequence set forth in SEQ ID NOs: 84 and 86 is referred to as "U1-62", an antibody comprising a heavy chain variable region amino acid sequence and a light chain variable region amino acid sequence chains shown in SEQ ID NOs: 88 and 90 are referred to as "U1-2", an antibody comprising the heavy chain variable region amino acid sequence and the light chain variable region amino acid sequence shown in SEQ ID NOs: 92 and 94 is referred to as " U1-7", an antibody comprising the amino acid sequence of a heavy chain variable region and an amino acid sequence of a light chain variable region shown in SEQ ID NOs: 96 and 98, is referred to as "U1-9", an antibody comprising an amino acid sequence of a heavy chain variable region and the light chain variable region amino acid sequence set forth in SEQ ID NOs: 100 and 102 is referred to as "U1-10", an antibody comprising the heavy chain variable region amino acid sequence and the light chain variable region amino acid sequence set forth in SEQ ID NOs: 104 and 106, referred to as "U1-12", an antibody comprising the amino acid sequence of the heavy chain variable region and the amino acid sequence of the variable light chain shown in SEQ ID NOs: 108 and 110, referred to as "U1-13", an antibody comprising the amino acid sequence heavy chain variable region and light chain variable region amino acid sequence set forth in SEQ ID NOs: 112 and 114, referred to as "U1-14", an antibody comprising the heavy chain variable region amino acid sequence and the light chain variable region amino acid sequence set forth in SEQ ID NOs: 116 and 118, referred to as "U1-15", an antibody comprising the amino acid sequence of the variable region of the heavy chain and the amino acid sequence of the variable region of the light chain shown in SEQ ID NOs: 122 and 124, referred to as "U1-20", an antibody comprising a heavy chain variable region amino acid sequence and a light chain variable region amino acid sequence set forth in SEQ ID NOs: 126 and 128 is referred to as "U1-21", an antibody comprising a heavy chain variable region amino acid sequence and a light chain variable region amino acid sequence chains shown in SEQ ID NOs: 130 and 132 are referred to as "U1-22", an antibody comprising the heavy chain variable region amino acid sequence and the light chain variable region amino acid sequence shown in SEQ ID NOs: 134 and 136 is referred to as " U1-23", an antibody comprising a heavy chain variable region amino acid sequence and a light chain variable region amino acid sequence set forth in SEQ ID NOs: 138 and 140 is referred to as "U1-24", an antibody comprising a heavy chain variable region amino acid sequence and the light chain variable region amino acid sequence set forth in SEQ ID NOs: 142 and 144 is referred to as "U1-25", an antibody comprising the heavy chain variable region amino acid sequence and the light chain variable region amino acid sequence set forth in SEQ ID NOs: 146 and 148, referred to as "U1-26", an antibody comprising the amino acid sequence of the heavy chain variable region and the amino acid sequence of the variable light chain region shown in SEQ ID NOs: 150 and 152, referred to as "U1-27", an antibody comprising the amino acid sequence heavy chain variable region and light chain variable region amino acid sequence set forth in SEQ ID NOs: 154 and 156, referred to as "U1-28", an antibody comprising the heavy chain variable region amino acid sequence and the light chain variable region amino acid sequence set forth in SEQ ID NOs: 158 and 160, referred to as "U1-31", an antibody comprising the amino acid sequence of the variable region of the heavy chain and the amino acid sequence of the variable region of the light chain shown in SEQ ID NOs: 162 and 164, referred to as "U1-32", an antibody comprising a heavy chain variable region amino acid sequence and a light chain variable region amino acid sequence as set forth in SEQ ID NOs: 166 and 168, referred to as "U1-35", an antibody comprising a heavy chain variable region amino acid sequence and a light chain variable region amino acid sequence chains shown in SEQ ID NOs: 170 and 172 are referred to as "U1-36", an antibody comprising the heavy chain variable region amino acid sequence and the light chain variable region amino acid sequence shown in SEQ ID NOs: 174 and 176 is referred to as " U1-37", an antibody comprising a heavy chain variable region amino acid sequence and a light chain variable region amino acid sequence set forth in SEQ ID NOs: 178 and 180 is referred to as "U1-34", an antibody comprising a heavy chain variable region amino acid sequence and the light chain variable region amino acid sequence set forth in SEQ ID NOs: 182 and 184 is referred to as "U1-1", an antibody comprising the heavy chain variable region amino acid sequence and the light chain variable region amino acid sequence set forth in SEQ ID NOs: 186 and 188, referred to as "U1-3", an antibody comprising the amino acid sequence of the heavy chain variable region and the amino acid sequence of the variable light chain shown in SEQ ID NOs: 190 and 192, referred to as "U1-4", an antibody comprising the amino acid sequence heavy chain variable region and light chain variable region amino acid sequence set forth in SEQ ID NOs: 194 and 196, referred to as "U1-5", an antibody comprising the heavy chain variable region amino acid sequence and the light chain variable region amino acid sequence set forth in SEQ ID NOs: 198 and 200, referred to as "U1-6", an antibody comprising the amino acid sequence of the variable region of the heavy chain and the amino acid sequence of the variable region of the light chain shown in SEQ ID NOs: 202 and 204, referred to as "U1-8", an antibody comprising a heavy chain variable region amino acid sequence and a light chain variable region amino acid sequence as set forth in SEQ ID NOs: 206 and 208, referred to as "U1-11", an antibody comprising a heavy chain variable region amino acid sequence and a light chain variable region amino acid sequence chains set forth in SEQ ID NOs: 210 and 212 are referred to as "U1-16", an antibody comprising the heavy chain variable region amino acid sequence and the light chain variable region amino acid sequence shown in SEQ ID NOs: 214 and 216 is referred to as " U1-17", an antibody comprising the amino acid sequence of a heavy chain variable region and an amino acid sequence of a light chain variable region shown in SEQ ID NOs: 218 and 220 is referred to as "U1-18", an antibody comprising an amino acid sequence of a heavy chain variable region and the light chain variable region amino acid sequence set forth in SEQ ID NOs: 222 and 224 is referred to as "U1-33", an antibody comprising the heavy chain variable region amino acid sequence and the light chain variable region amino acid sequence set forth in SEQ ID NOs: 226 and 228, referred to as "U1-29", an antibody comprising the amino acid sequence of the heavy chain variable region and the amino acid sequence of the variable light chain shown in SEQ ID NOs: 230 and 232, referred to as "U1-30", an antibody comprising the amino acid sequence heavy chain variable region set forth in SEQ ID NO: 34 is referred to as "U1-46", an antibody comprising the amino acid sequence of the heavy chain variable region set forth in SEQ ID NO: 40 is referred to as "U1-48", an antibody comprising the amino acid sequence of the heavy chain variable region and the amino acid sequence of the light chain variable region shown in SEQ ID NOs: 60 and 58 are referred to as "U1-55.1", an antibody comprising the heavy chain variable region amino acid sequence shown in SEQ ID NOs: 120, is referred to as "U1-19", and an antibody comprising the heavy chain variable region amino acid sequence and the light chain variable region amino acid sequence shown in SEQ ID NOs: 62 and 64 is referred to as "U1-57.1". These antibodies are described in detail in the Examples.

The isolated HER3 binding protein of the present invention further preferably comprises the heavy chain variable region amino acid sequence and the light chain variable region amino acid sequence shown in SEQ ID NOs: 42 and 44, respectively, the heavy chain variable region amino acid sequence and the light chain variable region amino acid sequence. chains shown in SEQ ID NOs: 54 and 56, respectively, the amino acid sequence of the heavy chain variable region and the amino acid sequence of the light chain variable region, shown in SEQ ID NOs: 70 and 72, respectively, the amino acid sequence of the heavy chain variable region and the amino acid sequence of the variable region light chain regions set forth in SEQ ID NOs: 92 and 94, respectively, or the heavy chain variable region amino acid sequence and the light chain variable region amino acid sequence set forth in SEQ ID NOs: 96 and 98, respectively, and even more preferred HER3 binding the proteins are U1-49, U1-53, U1-59, U1-7 or U1-9 which is an anti-HER3 antibody.

[0072]

[Chemical formula 14]

[0073]

[Chemical Formula 15]

[0074]

When a newly formed monoclonal antibody binds to a partial peptide or partial tertiary structure to which a U1-49, U1-53, U1-59, U1-7, or U1-9 antibody binds, it can be determined that the antibody binds to the same epitope, as U1-49, U1-53, U1-59, U1-7 or U1-9 antibody. In addition, by confirming that the antibody competes with the U1-49, U1-53, U1-59, U1-7, or U1-9 antibody for binding to HER3 (i.e., the antibody inhibits binding between U1-49, U1- 53, U1-59, U1-7 or U1-9 antibody and HER3), it can be determined that the antibody binds to the same epitope as U1-49, U1-53, U1-59, U1-7 or U1- 9 antibody, even when the specific sequence or structure of the epitope has not been determined. Once confirmed that the epitope is the same, there is good reason to expect that the antibody has a biological activity equivalent to that of the U1-49, U1-53, U1-59, U1-7, or U1-9 antibody.

[0075]

In accordance with the present invention, the binding protein of the present invention interacts with at least one epitope in the extracellular portion of HER3. The epitopes are preferably located in the L1 domain (aa 19-184), which is the amino terminal domain, in the S1 domain (aa 185-327) and S2 (aa 500-632), which are two Cysteine-rich domains, in the domain L2 (328-499), which is flanked by two Cysteine-rich domains, or a combination of HER3 domains. Epitopes can also be located in combinations of domains, for example, but not limited to, an epitope consisting of parts of L1 and S1. In addition, the binding protein of the present invention is also characterized in that its binding to HER3 reduces HER3-mediated signal transduction. In accordance with the present invention, a decrease in HER3-mediated signal transduction can be caused, for example, by down-regulating HER3 leading to at least partial disappearance of the HER3 molecule from the cell surface, or by stabilizing HER3 on the cell surface in an essentially inactive form, i.e. .e. in a form that exhibits lower signal transduction compared to the unstabilized form. Alternatively, a decrease in HER3-mediated signal transduction can also be caused by, for example, reducing or inhibiting the binding of a ligand or other HER family member to HER3, GRB2 to HER-2, or GRB2 to SHC, inhibition of tyrosine receptor phosphorylation, AKT phosphorylation, PYK2 tyrosine phosphorylation or phosphorylation of ERK2 or a decrease in tumor invasiveness. Alternatively, the reduction in HER3-mediated signal transduction can also be caused by the influence of, for example, reducing or inhibiting the formation of HER3-containing dimers with other members of the HER family. One example, among others, would be to reduce or inhibit the formation of the HER3-EGFR protein complex.

[0076]

In addition, in accordance with the present invention, minor changes in the amino acid sequences shown in SEQ ID NOs: 1-232 are contemplated as being covered by the present invention, provided that even such changes in the amino acid sequence still retain at least 75%, more preferably at least 80%, 90%, 95% and most preferably 99% of the sequences shown in SEQ ID NOs: 1-232. Changes can take place in the framework regions (ie, outside the CDRs), in the CDRs, or in the framework regions and in the CDRs. Preferred changes in the amino acid sequences shown in SEQ ID NOs: 1-232, ie. deletions, insertions and/or substitutions of at least one amino acid occur near functional domain boundaries. Structural and functional domains can be identified by comparing nucleotide and/or amino acid sequence data with public or proprietary sequence databases. Computerized comparison methods can be used to identify sequence motifs or predicted protein conformational domains that occur in other binding proteins of known structure and/or function. Methods for identifying protein sequences that fit into a known three-dimensional structure are known. See, for example, Bowie et al, Science 253, 164 (1991); Proteins, Structures and Molecular Principles (Creighton, Ed., W. H. Freeman and Company, New York (1984)); Introduction to Protein Structure (C. Branden and J. Tooze, eds., Garland Publishing, New York, N.Y. (1991)); and Thornton et at., Nature 354, 105 (1991), all of which are incorporated herein by reference. Thus, one skilled in the art will be able to determine sequence motifs and structural conformations that can be used to define structural and functional domains in accordance with the invention. From antibodies produced by combining heavy and light chains having alterations in such amino acid sequences, one can select an antibody equivalent to the parent antibody (parent antibody) or even better than the parent antibody. As mentioned above, a HER3 binding protein, an anti-HER3 antibody, and the like. of the present invention retain HER3 binding activity even though they include changes in their amino acid sequences.

In the present invention, the term "homology" has the same meaning as "identity". Homology between two amino acid sequences can be determined using the default parameters of the Blast algorithm, version 2.2.2 (Altschul, Stephen F., Thomas L. Madden, Alejandro A. Schaeffer, Jinghui Zhang, Zheng Zhang, Webb Miller, and David J. Lipman (1997), "Gapped BLAST and PSI-BLAST: a new generation of protein database search programs", Nucleic Acids Res. 25: 3389-3402). The Blast algorithm can also be used via the Internet with access to the site www.ncbi.nlm.nih.gov/blast.

[0077]

A chimeric antibody, a humanized antibody, or a human antibody obtained by the above method can be subjected to a known method for evaluating antigen binding properties to select preferred antibodies.

Also included in the anti-HER3 antibody of the present invention are MEHD-7945A (or diligotuzumab), RG-7116, MM-111, MM-121 (or seribantumab, MM-141, LJM-716, huHER3-8, tri-specific anti- EGFR/ErbB3 zy-body, GSK-2849330, REGN-1400, KTN-3379, AV-203, monospecific surro-body (ErbB3), lumretuzumab, MP-EV-20, ZW-9, Dimercept™, anti-Erb3 surro -body (SL-175 or SL-176), SYM-013, variants thereof, active fragments, modified products, etc.

[0078]

As one example of another criterion for use in comparing antibody properties, antibody stability can be mentioned. Differential scanning calorimetry (DSC) is a tool for fast and accurate measurement of the mean thermal denaturation temperature (Tm), which can be used as a suitable indicator of the relative conformational stability of proteins. By measuring Tm values using DSC and comparing the values, the difference in thermal stability can be determined. It is known that the storage stability of antibodies shows some correlation with the thermal stability of antibodies (Lori Burton, et. al., Pharmaceutical Development and Technology (2007) 12, pp. 265-273), and the preferred antibody can be selected using thermal stability as a criterion for evaluation. . Examples of other criteria for selecting an antibody include the following: yield in a suitable host cell is high; and aggregability in aqueous solution is low. For example, the antibody that shows the highest yield does not always show the highest thermal stability, and therefore it is necessary to select the most suitable antibody for human administration by making a comprehensive evaluation based on the criteria described above.

[0079]

The antibody of the present invention encompasses a modified version of the antibody. A modified variant refers to a variant that is obtained by subjecting an antibody of the present invention to a chemical or biological modification. Examples of a chemically modified variant include those chemically modified by linking a chemical group to an amino acid backbone, variants chemically modified with an N-linked or O-linked carbohydrate chain, and so on. Examples of a biologically modified variant include those obtained by post-translational modification (such as N-linked or O-linked glycosylation, N- or C-terminal processing, deamidation, aspartic acid isomerization, or methionine oxidation), and variants in which the methionine residue was added to the N terminus by expression in a prokaryotic host cell. In addition, an antibody labeled in such a way as to allow detection or isolation of an antibody or antigen of the present invention, such as an enzyme-labeled antibody, a fluorescently labeled antibody, and an affinity-labeled antibody, is also included within the meaning of the modified variant. Such a modified variant of an antibody of the present invention is useful for improving the stability and retention in circulation of an antibody, reducing its antigenicity, detecting or isolating an antibody or antigen, and so on.

[0080]

In addition, by regulating the modification of the glycan that binds to the antibody of the present invention (glycosylation, defucosylation, etc.), antibody-dependent cellular cytotoxic activity can be increased. A method for regulating antibody glycan modification is known from International Publication No. WO 1999/54342, WO 2000/61739, WO 2002/31140, etc. However, the method is not limited to this. The antibody of the present invention also includes an antibody in which glycan modification is regulated.

In the case where an antibody is obtained by first isolating the antibody gene and then introducing the gene into a suitable host, a combination of a suitable host and a suitable expression vector can be used. Specific examples of an antibody gene include a combination of a gene encoding an antibody heavy chain sequence and a gene encoding its light chain sequence described herein. When a host cell is transformed, the heavy chain gene sequence and the light chain gene sequence can be inserted into the same expression vector, as well as into different expression vectors separately.

In the case where eukaryotic cells are used as the host, animal cells, plant cells and eukaryotic microorganisms can be used. As an example of animal cells, mammalian cells can be mentioned, for example, monkey COS cells (Gluzman, Y., Cell, (1981) 23, pp. 175-182, ATCC CRL-1650), NIH3T3 mouse fibroblasts (ATCC No. CRL-1658 ) and dihydrofolate reductase-deficient strains (Urlaub, G. and Chasin, L. A., Proc. Natl. Acad. Sci. USA (1980) 77, pp. 4126-4220) of Chinese hamster ovary cells (CHO cells; ATCC: CCL-61) .

In the case where prokaryotic cells are used, for example, Escherichia coli and Bacillus subtilis can be mentioned as an example.

By introducing the gene of the desired antibody into these cells through transformation and culturing the thus transformed cells in vitro, an antibody can be obtained. In the culture method described above, the yield may sometimes vary depending on the sequence of the antibody, and therefore, from antibodies having an equivalent binding activity, using the yield as a judgment criterion, an antibody that can be easily obtained as a pharmaceutical agent can be selected. Therefore, in the antibody of the present invention, also included is an antibody obtained by a method for producing an antibody, characterized in that it includes the step of culturing the transformed host cell and the step of collecting the desired antibody from the cultured product obtained in the culturing step.

[0081]

It is known that the lysine residue at the carboxyl end of the heavy chain of an antibody that is produced in a cultured mammalian cell can be deleted/eliminated (Journal of Chromatography A, 705: 129-134 (1995)), and it is also known that two amino acid residues (glycine and lysine) at the carboxyl end of the heavy chain of an antibody that is produced in a cultured mammalian cell can be deleted/eliminated and a new proline residue at the carboxyl end can be amidated (Analytical Biochemistry, 360: 75-83 (2007)). However, such deletion/elimination and heavy chain sequence modification do not affect the antigen binding affinity and effector function (complement activation, antibody-dependent cellular cytotoxicity, etc.) of the antibody. Therefore, the antibody of the present invention also includes an antibody and a functional antibody fragment subject to such modification, and also covers a deletion variant in which one or two amino acids are deleted at the carboxyl terminus of a heavy chain, a variant obtained by amidating a deletion variant (e.g., a heavy chain , in which the carboxy-terminal proline residue is amidated), and the like. The type of deletion variant including deletion at the carboxyl end of the heavy chain of an antibody according to the invention is not limited to the above-described variants, as long as the antigen binding affinity and effector function are maintained. The two heavy chains of an antibody of the invention may be of the same type selected from the group consisting of the full length heavy chain and the deletion variant described above, or may be of two types in a combination selected from them. The ratio of the amount of each deletion variant may be influenced by the type of cultured mammalian cells that produce the antibody of the invention and culture conditions, however, as an example, one can point to the case where one amino acid residue at the carboxyl terminus is deleted in each of the two heavy chains contained as essential components in an antibody according to the invention. The scope of the whole antibody (also simply referred to as "antibody" in the present invention) of the present invention also includes deletion variants thereof, mixtures containing one or two or more deletion variants thereof, etc. An "antibody" of the present invention includes an antibody comprising a heavy or light chain in which the N-terminal glutamate is present in the form of pyroglutamate as a result of cyclization, and/or a heavy or light chain in which part of the cysteine residues are in the form of cysteinyl.

[0082]

In a preferred embodiment of the present invention, the anti-HER3 antibody of the present invention is an IgA, IgD-, IgE ₁ IgG- or IgM-type, preferably IgG- or IgM-type, including but not limited to IgGI-, IgG2 -, IgG3-, IgG4-, IgMI- and IgM2-type. In most preferred embodiments, the antibody is an IgGI-, IgG2-, or IgG4-type antibody.

[0083]

As the biological activity of an antibody, generally, an antigen-binding activity, an activity of internalizing an antigen in antigen-expressing cells by binding to an antigen, an action to neutralize antigen activity, an activity to enhance antigen activity, an antibody-dependent activity can be exemplified. cellular cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), and antibody-dependent cell-mediated phagocytosis (ADCP). The function of an antibody according to the invention is HER3 binding activity, preferably HER3 internalization activity in HER3 expressing cells by binding to HER3. In addition, the antibody of the present invention may have ADCC activity, CDC activity and/or ADCP activity in addition to internalization activity in cells.

[0084]

In some aspects, for example, in connection with the generation of antibodies as therapeutic candidates against HER3, it may be desirable that the anti-HER3 antibody of the present invention be capable of fixing complement and participating in complement-dependent cytotoxicity (CDC). There are a number of antibody isotypes that have this ability, including, without limitation, the following: mouse IgM, mouse IgG2a, mouse IgG2b, mouse IgG3, human IgM, human IgGI, human IgG3, and human IgA. It should be understood that the antibodies that are generated need not be of such an isotype originally, rather the generated antibody can be of any isotype and the isotype of the antibody can be switched by adding molecularly cloned genes or V region cDNA to molecularly cloned genes or constant region cDNA in the appropriate expression vectors using conventional molecular biological methods that are well known in the art, and then expressing the antibodies in host cells using methods known in the art. An isotype-switched antibody may also contain an Fc region that is molecularly engineered to have a CDC higher than natural variants (Idusogie et al., J Immunol., 166, 2571-2575) and recombinantly expressed in host cells. using methods known in the art. Such methods include the use of direct recombinant methods (see, for example, US Pat. No. 4,816,397), cell fusion methods (see, for example, US Pat. Nos. 5,916,771 and 6,207,418), among others. In the cell fusion method, a myeloma or other cell line, such as CHO, which has a heavy chain with any desired isotype is obtained, and another myeloma or other cell line, such as CHO, which has a light chain is obtained. These cells can then be fused, and a cell line expressing the intact antibody can be isolated. As an example, it is easy to switch the isotype of a human anti-HER3 IgG4 antibody that has the desired binding to the HER3 antigen to generate a human IgM, human IgGI, or human IgG3 isotype, yet it will have the same variable region (which determines the specificity antibodies and some of its affinity). Such a molecule may then be able to fix complement and participate in CDC.

[0085]

In addition, it may also be desirable that the anti-HER3 antibody of the present invention has the ability to bind to Fc receptors on effector cells such as monocytes and natural killer (NK) cells and participate in antibody-dependent cellular cytotoxicity (ADCC). There are a number of antibody isotypes that have this ability, including, without limitation, the following: mouse IgG2a, mouse IgG2b, mouse IgG3, human IgGI, and human IgG3. It should be understood that the antibodies that are generated need not be of such an isotype originally, rather the generated antibody can be of any isotype and the isotype of the antibody can be switched by adding molecularly cloned genes or V region cDNA to molecularly cloned genes or constant region cDNA in the appropriate expression vectors using conventional molecular biological methods that are well known in the art, and then expressing the antibodies in host cells using methods known in the art. An isotype-switched antibody may also contain an Fc region that is molecularly engineered to have an ADCC higher than natural variants (Shields et al. J Biol Chem., 276, 6591-6604) and recombinantly expressed in host cells. using methods known in the art. Such methods include the use of direct recombinant methods (see, for example, US Pat. No. 4,816,397), cell fusion methods (see, for example, US Pat. Nos. 5,916,771 and 6,207,418), among others. In the cell fusion method, a myeloma or other cell line, such as CHO, which has a heavy chain with any desired isotype is obtained, and another myeloma or other cell line, such as CHO, which has a light chain is obtained. These cells can then be fused, and a cell line expressing the intact antibody can be isolated. As an example, it is easy to switch the isotype of a human anti-HER3 IgG4 antibody that has the desired binding to the HER3 antigen to generate a human human IgGI or human IgG3 isotype, but it will have the same variable region (which determines the specificity of the antibody and some of its affinity). Such a molecule may then be able to bind to FcyR on effector cells and participate in ADCC.

[0086]

The resulting antibody can be purified to homogeneity. Isolation and purification of the antibody can be carried out using the traditional method of separating and purifying proteins. For example, an antibody can be isolated and purified by the appropriate choice and combination of column chromatography, filtration, ultrafiltration, precipitation of salts, dialysis, preparative polyacrylamide gel electrophoresis, isoelectric focusing electrophoresis, and the like. (Strategies for Protein Purification and Characterization: A Laboratory Course Manual, Daniel R. Marshak et al. eds., Cold Spring Harbor Laboratory Press (1996); Antibodies: A Laboratory Manual. Ed Harlow and David Lane, Cold Spring Harbor Laboratory (1988 )), but the method is not limited to this.

Examples of such chromatography include affinity chromatography, ion exchange chromatography, hydrophobic chromatography, size exclusion chromatography, reverse phase chromatography, and adsorption chromatography.

Such chromatography can be carried out using liquid chromatography such as high performance liquid chromatography (HPLC) or fast resolution liquid chromatography.

As an example of a column to be used in affinity chromatography, a Protein A column and a Protein G column can be mentioned. can be given as an example.

In addition, using a carrier containing an antigen immobilized thereon, the antibody can also be purified using the antibody's antigen-binding property.

[0087]

[Antineoplastic Compound]

Disclosed is an antineoplastic compound that is used in an anti-HER3 antibody-drug conjugate of the present invention. The antitumor compound used in the present invention is not particularly limited as long as it is a compound having an antitumor effect and containing a substituent group or a partial structure capable of being bound to a linker structure. When part or all of the antitumor compound linker is cleaved in the tumor cells, the antitumor compound is released to exhibit an antitumor effect. Since the linker is cleaved off at the drug-binding position, the antitumor compound is released in its unmodified structure to exhibit its inherent antitumor effect.

As the antitumor compound used in the present invention, exatecan, a camptothecin derivative ((1S,9S)-1-amino-9-ethyl-5-fluoro-2,3-dihydro-9-hydroxy-4-methyl- 1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-10,13(9H,15H)-dione shown in the following formula).

[0088]

[Chemical Formula 16]

[0089]

Although it has an excellent antitumor effect, exatecan is not a commercial product marketed as an antitumor drug. The compound can be easily prepared in a known manner, and the amino group at the 1-position can preferably be used as the binding position to the linker structure. In addition, exatecan can also be released in tumor cells with part of the linker still attached to it. However, it is an excellent compound showing excellent antitumor effect even in this structure.

Because exatecan has a camptothecin structure, the equilibrium is known to shift to a closed lactone ring structure (closed ring) in an acidic aqueous environment (e.g. pH 3 or similar), but it shifts to an open lactone ring structure (ring open) in an aqueous alkaline environment (for example, pH 10 or similar). A drug conjugate with an introduced exatecan residue corresponding to the closed ring structure and the ring open structure is also expected to have the same antitumor effect, and it goes without saying that any such structure is within the scope of the present invention.

[0090]

Other examples of an antitumor compound may include doxorubicin, daunorubicin, mitomycin C, bleomycin, cyclocytidine, vincristine, vinblastine, methotrexate, a platinum-based antitumor agent (cisplatin or derivatives thereof), taxol or derivatives thereof, and other camptothecin or derivatives thereof (antitumor agent described in Japanese Laid-Open Patent Application No. 6-87746).

[0091]

With respect to the antibody-drug conjugate, the number of conjugated drug molecules per antibody molecule is a key factor influencing efficacy and safety. The preparation of an antibody-drug conjugate is carried out by determining the reaction conditions, including the amount of raw materials and reagents used for the reaction, in order to have a constant number of conjugated drug molecules, and, as a rule, a mixture is obtained containing different amounts of conjugated drug molecules, as opposed to chemical reactions of low molecular weight compounds. The number of drug molecules conjugated in an antibody molecule is expressed or defined as the mean, ie the average number of conjugated drug molecules. Unless otherwise specified in principle, the number of conjugated drug molecules means the average value, except in the case of an antibody-drug conjugate having a certain number of conjugated drug molecules, which is included in a mixture of antibody-drug conjugates having a different number of conjugated drug molecules. The number of exatecan molecules conjugated to an antibody molecule is controlled, and as an average number of conjugated drug molecules per antibody, about 1 to 10 exatecan molecules can be bound. Preferably 2 to 8, and more preferably 3 to 8 molecules. At the same time, a person skilled in the art will be able to calculate the reaction for conjugating the required number of drug molecules to an antibody molecule based on the description of the Examples of the present application, and will be able to obtain an antibody-drug conjugate with a controlled amount of conjugated exatecan molecules.

With regard to the antibody-drug conjugate of the present invention, aggregation, insolubility, fragmentation or the like are unlikely to occur even when the number of conjugated drug molecules per antibody molecule is increased.

[0092]

{Linker structure}

With regard to the anti-HER3 antibody-drug conjugate of the present invention, a linker structure for conjugating an anti-tumor compound to an anti-HER3 antibody is explained below. The linker has the following structure:

-L ¹ -L ² -L ^P -NH-(CH ₂ )n ¹ -L ^a -(CH ₂ )n ² -C(=O)- or -L ¹ -L ² -L ^P -,

the antibody is linked to the L ¹ end (opposite the end to which the L ² is linked), and the antitumor compound is linked to the carbonyl group of the -L ^a -(CH ₂ )n ² -C(=O)- moiety or the C end of the L ^P .

n ¹ is an integer having a value from 0 to 6, preferably an integer having a value from 1 to 5, and more preferably from 1 to 3.

[0093]

^1.L1

L ¹ is represented by the structure shown below:

-(Succinimide-3-yl-N)-(CH ₂ )n ³ -C(=O)-

In the above structure, n ³ is an integer having a value of 2 to 8, and "-(Succinimide-3-yl-N)-" has the structure represented by the following formula:

[0094]

[Chemical Formula 17]

[0095]

Position 3 of the partial structure shown above is the binding position for the anti-HER3 antibody. Binding to the antibody at position 3 is characterized by the formation of a thioether bond. The nitrogen atom in position 1 of this structural fragment is connected to the carbon atom of methylene, which is present in the linker, including this structure. for example, -(succinimide-3-yl-N)-(CH ₂ )n ³ -C(=O)-L ² - is the structure represented by the following formula (here, "antibody -S-" is derived from an antibody).

[0096]

[Chemical Formula 18]

[0097]

In the formula, n ³ is an integer having a value of 2 to 8, and preferably 2 to 5.

[0098]

Specific examples of L ¹ include the following.

-(Succinimide-3-yl-N)-CH ₂ CH ₂ -C(=O)-,

-(Succinimide-3-yl-N)-CH ₂ CH ₂ CH ₂ -C(=O)-,

-(Succinimide-3-yl-N)-CH ₂ CH ₂ CH ₂ CH ₂ -C (= O) -,

-(Succinimide-3-yl-N) -CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ -C (= O) -

[0099]

^2.L2

L ² has the structure represented by the following formula:

-NH-(CH ₂ CH ₂ -O)n ⁴ -CH ₂ CH ₂ -C (= O) -

L ² may not be present, in which case L ² is a single bond. In the drug-linker structure of the present invention, in particular, L ^P is directly linked to the drug, in which case L ² is particularly preferably a single bond. n ⁴ is an integer having a value of 1 to 6, and preferably 2 to 4. L ² is linked to L ¹ at its terminal amino group and is linked to L ^P at the opposite end carbonyl group.

[0100]

Specific examples of L ² include the following.

-NH-CH ₂ CH ₂ -O-CH ₂ CH ₂ -C (= O) -,

-NH-CH ₂ CH ₂ -O-CH ₂ CH ₂ -O-CH ₂ CH ₂ -C (= O) -,

-NH-CH ₂ CH ₂ -O-CH ₂ CH ₂ -O-CH ₂ CH ₂ -O-CH ₂ CH ₂ -C (= O) -,

-NH-CH ₂ CH ₂ -O-CH ₂ CH ₂ -O-CH ₂ CH ₂ -O-CH ₂ CH ₂ -O-CH ₂ CH ₂ -C (= O) -,

-NH-CH ₂ CH ₂ -O-CH ₂ CH ₂ -O-CH ₂ CH ₂ -O-CH ₂ CH ₂ -O-CH ₂ CH ₂ -O-CH ₂ CH ₂ -C (= O) -,

-NH-CH ₂ CH ₂ -O-CH ₂ CH ₂ -O-CH ₂ CH ₂ -O-CH ₂ CH ₂ -O-CH ₂ CH ₂ -O-CH ₂ CH ₂ -O-CH ₂ CH ₂ - C(=O)-

[0101]

3. L ^P

L ^P is a peptide residue consisting of 2-7 amino acids. For example, it consists of an oligopeptide residue in which 2-7 amino acids are linked by a peptide bond. L ^P is linked to L ² at the N terminus and is linked to the amino group of the -NH-(CH ₂ )n ¹ -L ^a -(CH ₂ )n ² -C(=O)- fragment of the linker at the C terminus.

[0102]

The amino acid constituting L ^P is not particularly limited, and examples include L- or D-amino acid, L-amino acid is preferred. In addition, it may be an amino acid having a structure such as beta-alanine, epsilon-aminocaproic acid or gamma-aminobutyric acid, in addition to an alpha-amino acid, it may also be a non-natural type amino acid such as an N-methylated amino acid.

The amino acid sequence of L ^P is not particularly limited, but examples of its constituent amino acids include phenylalanine (Phe; F), tyrosine (Tyr; Y), leucine (Leu; L), glycine (Gly; G), alanine (Ala; A), valine (Val; V), lysine (Lys; K), citrulline (Cit), serine (Ser; S), glutamic acid (Glu; E), and aspartic acid (Asp; D). Of these, preferred examples include phenylalanine, glycine, valine, lysine, citrulline, serine, glutamic acid, and aspartic acid. Depending on the type of amino acid, the drug release pattern can be controlled. The number of amino acids can be from 2 to 7.

[0103]

Specific examples of L ^P include the following:

-GGF-,

-DGGF-,

-(D-)D-GGF-,

-EGGF-,

-GGFG-,

-SGGF-,

-KGGF-,

-DGGFG-,

-GGFGG-,

-DDGGFG-,

-KDGGFG-,

-GGFGGGF-

"(D-)D" as described above means D-aspartic acid. Examples of a particularly preferred L ^P antibody-drug conjugate of the present invention include a -GGFG- and -DGGFG- peptide residue. In addition, in the drug-linker structure of the present invention, L ^P is directly linked to the drug, and for such a case, preferred examples of L ^P include the pentapeptide residue -DGGFG-.

[0104]

4. L ^a -(CH ₂ )n ² -C(=O)-

L ^a in L ^a -(CH ₂ )n ² -C(=O)- represents an -O- structure or a single bond. n ² is an integer having a value of 0 to 5, preferably 0 to 3, and more preferably 0 or 1.

Examples of L ^a -(CH ₂ )n ² -C(=O)- include the following:

-O-CH ₂ -C(=O)-,

-O-CH ₂ CH ₂ -C (= O) -,

-O-CH ₂ CH ₂ CH ₂ -C (= O) -,

-O-CH ₂ CH ₂ CH ₂ CH ₂ -C (= O) -,

-O-CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ -C (= O) -,

-CH ₂ -C(=O)-,

-CH ₂ CH ₂ -C(=O)-,

-CH ₂ CH ₂ CH ₂ -C(=O)-,

-CH ₂ CH ₂ CH ₂ CH ₂ -C (= O) -,

-CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ -C(=O)-.

Of these, structures with

-O-CH ₂ -C(=O)-,

-O-CH ₂ CH ₂ -C (= O) -

or those in which L ^a is a single bond and n ² is 0 are preferred.

[0105]

Specific examples of the linker structure represented as -NH-(CH ₂ )n ¹ -L ^a -(CH ₂ )n ² -C(=O)- include the following:

-NH-CH ₂ -C(=O)-,

-NH-CH ₂ CH ₂ -C (= O) -,

-NH-CH ₂ -O-CH ₂ -C(=O)-,

-NH-CH ₂ CH ₂ -OC(=O)-,

-NH-CH ₂ CH ₂ -O-CH ₂ -C (= O) -,

-NH-CH ₂ CH ₂ CH ₂ -C (= O) -,

-NH-CH ₂ CH ₂ CH ₂ CH ₂ -C (= O) -,

-NH-CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ -C (= O) -

[0106]

Of these, the following are preferred:

-NH-CH ₂ CH ₂ CH ₂ -C (= O) -,

-NH-CH ₂ -O-CH ₂ -C(=O)-,

-NH-CH ₂ CH ₂ -OC(=O)-

[0107]

As for the linker -NH-(CH ₂ )n ¹ -L ^a -(CH ₂ )n ² -C(=O)-, a chain length of 4-7 atoms is preferred, and those with a chain length of 5 or 6 atoms.

[0108]

As for the anti-HER3 antibody-drug conjugate of the present invention, when it enters the interior of tumor cells, the linker group is considered to be cleaved off, and the drug derivative having the structure represented as NH ₂ -(CH ₂ )n ¹ -L ^a - (CH ₂ )n ² -C(=O)-(NH-DX), is released with the manifestation of antitumor activity. Examples of an antitumor derivative exhibiting an antitumor effect when released from an antibody-drug conjugate of the present invention include an antitumor derivative containing a structural moiety in which the terminal portion of the structure represented by -NH-(CH ₂ )n ¹ -L ^a -(CH ₂ )n ² -C(=O)- linker is an amino group, and particularly preferred include the following:

NH ₂ -CH ₂ CH ₂ -C (= O) - (NH-DX),

NH ₂ -CH ₂ CH ₂ CH ₂ -C (= O) - (NH-DX),

NH ₂ -CH ₂ -O-CH ₂ -C (= O) - (NH-DX),

NH ₂ -CH ₂ CH ₂ -O-CH ₂ -C (= O) - (NH-DX).

Meanwhile, in the case of NH ₂ -CH ₂ -O-CH ₂ -C(=O)-(NH-DX), it was confirmed that since the aminal structure in the molecule is unstable, it again undergoes self-decomposition to release the next HO- CH ₂ -C(=O)-(NH-DX). Such compounds can also preferably be used as an intermediate in the process for preparing an antibody-drug conjugate of the present invention.

In addition, with regard to the structure of the drug-linker of the present invention, there is a case where L ^P is directly linked to the drug. In such a case where the C terminus of L ^P is glycine, the anticancer drug to be released is exatecan as such or a compound where the glycine is linked to the amino group of exatecan.

[0109]

For the antibody-drug conjugate of the present invention in which exatecan is used as the drug, a drug-linker structural fragment having the following structure

-L ¹ -L ² -L ^P -NH-(CH ₂ )n ¹ -L ^a -(CH ₂ )n ² -C(=O)-(NH-DX) or

-L ¹ -L ² -L ^P -(NH-DX),

to which the antibody binds is preferred. having the following structure, was associated with the antibody. The number of conjugable said drug-linker structural fragments can be from 1 to 10 as the average conjugated amount per antibody, preferably from 2 to 8, and more preferably from 3 to 8.

-(Succinimide-3-yl-N)-CH ₂ CH ₂ -C(=O)-GGFG-NH-CH ₂ CH ₂ -C(=O)-(NH-DX),

-(Succinimide-3-yl-N)-CH ₂ CH ₂ -C (= O) -GGFG-NH-CH ₂ CH ₂ CH ₂ -C (= O) - (NH-DX),

-(Succinimide-3-yl-N)-CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ -C (= O) -GGFG-NH-CH ₂ CH ₂ -C (= O) - (NH-DX),

-(Succinimide-3-yl-N)-CH ₂ CH ₂ CH _{2 CH 2 CH 2} -C (= O) -GGFG-NH-CH ₂ CH ₂ CH ₂ -C (= O) - (NH-DX) ,

-(Succinimide-3-yl-N)-CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ -C (= O) -GGFG-NH-CH ₂ CH ₂ CH 2 CH _{2 CH 2 -C} (= O) - ( NH-DX),

-(Succinimid-3-yl-N)-CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ -C (= O) -DGGFG-NH-CH ₂ CH ₂ -C (= O) - (NH-DX),

-(Succinimide-3-yl-N)-CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ -C (= O) -DGGFG-NH-CH ₂ CH ₂ CH ₂ -C (= O) - (NH-DX) ,

-(Succinimide-3-yl-N ₎ -CH ₂ CH ₂ CH 2 CH ₂ CH ₂ -C (= O) -DGGFG-NH-CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ -C (= O) - ( NH-DX),

-(Succinimide-3-yl-N)-CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ -C (= O) -GGFG-NH-CH ₂ -O-CH ₂ -C (= O) - (NH-DX ),

-(Succinimide-3-yl-N)-CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ -C (= O) -GGFG-NH-CH ₂ CH ₂ -O-CH ₂ -C (= O) - (NH -DX),

-(Succinimide-3-yl-N)-CH ₂ CH ₂ -C(=O)-NH-CH ₂ CH ₂ -O-CH ₂ CH ₂ -O-CH ₂ CH ₂ -C(=O)-GGFG -NH-CH ₂ CH ₂ -C (= O) - (NH-DX),

-(Succinimide-3-yl-N)-CH ₂ CH ₂ -C(=O)-NH-CH ₂ CH ₂ -O-CH ₂ CH ₂ -O-CH ₂ CH ₂ -C(=O)-GGFG -NH-CH ₂ CH ₂ CH ₂ -C (= O) - (NH-DX),

-(Succinimide-3-yl-N) -CH ₂ CH ₂ -C (= O) -NH-CH ₂ CH ₂ -O-CH ₂ CH ₂ -O-CH ₂ CH ₂ -O-CH ₂ CH ₂ - O-CH ₂ CH ₂ -C (= O) -GGFG-NH-CH ₂ CH ₂ -C (= O) - (NH-DX),

-(Succinimide-3-yl-N)-CH ₂ CH ₂ -C (= O) -NH-CH ₂ CH ₂ -O-CH ₂ CH ₂ -O-CH ₂ CH ₂ O-CH ₂ CH ₂ -O -CH ₂ CH ₂ -C (= O) -GGFG-NH-CH ₂ CH ₂ CH ₂ -C (= O) - (NH-DX),

-(Succinimide-3-yl-N)-CH ₂ CH ₂ -C(=O)-GGFG-(NH-DX),

-(Succinimide-3-yl-N)-CH ₂ CH ₂ -C(=O)-DGGFG-(NH-DX),

-(Succinimide-3-yl-N)-CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ -C (= O) -GGFG-(NH-DX),

-(Succinimide-3-yl-N)-CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ -C(=O)-DGGFG-(NH-DX).

Of these, the following are more preferable.

-(Succinimide-3-yl-N)-CH ₂ CH ₂ -C (= O) -GGFG-NH-CH ₂ CH ₂ CH ₂ -C (= O) - (NH-DX),

-(Succinimide-3-yl-N)-CH ₂ CH ₂ CH _{2 CH 2 CH 2} -C (= O) -GGFG-NH-CH ₂ CH ₂ CH ₂ -C (= O) - (NH-DX) ,

-(Succinimide-3-yl-N)-CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ -C (= O) -DGGFG-NH-CH ₂ CH ₂ CH ₂ -C (= O) - (NH-DX) ,

-(Succinimide-3-yl-N)-CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ -C (= O) -GGFG-NH-CH ₂ -O-CH ₂ -C (= O) - (NH-DX ),

-(Succinimide-3-yl-N)-CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ -C (= O) -GGFG-NH-CH ₂ CH ₂ -O-CH ₂ -C (= O) - (NH -DX),

-(Succinimide-3-yl-N)-CH ₂ CH ₂ -C(=O)-NH-CH ₂ CH ₂ -O-CH ₂ CH ₂ -O-CH ₂ CH ₂ -C(=O)-GGFG -NH-CH ₂ CH ₂ CH ₂ -C (= O) - (NH-DX),

-(Succinimide-3-yl-N) -CH ₂ CH ₂ -C (= O) -NH-CH ₂ CH ₂ -O-CH ₂ CH ₂ -O-CH ₂ CH ₂ -O-CH ₂ CH ₂ - O-CH ₂ CH ₂ -C (= O) -GGFG-NH-CH ₂ CH ₂ CH ₂ -C (= O) - (NH-DX),

-(Succinimide-3-yl-N)-CH ₂ CH ₂ -C(=O)-DGGFG-(NH-DX),

-(Succinimide-3-yl-N)-CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ -C(=O)-DGGFG-(NH-DX).

Even more preferred are the following.

-(Succinimide-3-yl-N)-CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ -C (= O) -GGFG-NH-CH ₂ -O-CH ₂ -C (= O) - (NH-DX ),

-(Succinimide-3-yl-N)-CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ -C (= O) -GGFG-NH-CH ₂ CH ₂ -O-CH ₂ -C (= O) - (NH -DX),

-(Succinimide-3-yl-N)-CH ₂ CH ₂ -C(=O)-NH-CH ₂ CH ₂ -O-CH ₂ CH ₂ -O-CH ₂ CH ₂ -C(=O)-GGFG -NH-CH ₂ CH ₂ CH ₂ -C (= O) - (NH-DX),

-(Succinimide-3-yl-N)-CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ -C(=O)-DGGFG-(NH-DX).

The following are especially preferred.

-(Succinimide-3-yl-N)-CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ -C (= O) -GGFG-NH-CH ₂ -O-CH ₂ -C (= O) - (NH-DX ),

-(Succinimide-3-yl-N)-CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ -C (= O) -GGFG-NH-CH ₂ CH ₂ -O-CH ₂ -C (= O) - (NH -DX),

-(Succinimide-3-yl-N)-CH ₂ CH ₂ -C(=O)-NH-CH ₂ CH ₂ -O-CH ₂ CH ₂ -O-CH ₂ CH ₂ -C(=O)-GGFG -NH-CH ₂ CH ₂ CH ₂ -C(=O)-(NH-DX).

[0110]

With regard to the linker structure used to conjugate an anti-HER3 antibody and a drug to obtain the antibody-drug conjugate of the present invention, a preferred linker can be designed by linking the preferred structures shown for each linker part described above. With regard to the structure of the linker, linkers with the following structure are preferably used. In this case, the left end of the structure is the position of binding to the antibody, and the right end is the position of binding to the drug.

-(Succinimide-3-yl-N)-CH ₂ CH ₂ -C(=O)-GGFG-NH-CH ₂ CH ₂ -C(=O)-,

-(Succinimide-3-yl-N)-CH ₂ CH ₂ -C(=O)-GGFG-NH-CH ₂ CH ₂ CH ₂ -C(=O)-,

-(Succinimide-3-yl-N)-CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ -C (= O) -GGFG-NH-CH ₂ CH ₂ -C (= O) -,

-(Succinimide-3-yl-N)-CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ -C (= O) -GGFG-NH-CH ₂ CH ₂ CH ₂ -C (= O) -,

-(Succinimide-3-yl-N)-CH ₂ CH ₂ CH 2 CH _{2 CH 2} -C (= O) -GGFG-NH-CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ -C (= O) -,

-(Succinimid-3-yl-N)-CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ -C (= O) -DGGFG-NH-CH ₂ CH ₂ -C (= O) -,

-(Succinimide-3-yl-N)-CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ -C (= O) -DGGFG-NH-CH ₂ CH ₂ CH ₂ -C (= O) -,

-(Succinimide-3-yl-N)-CH ₂ CH ₂ CH 2 CH _{2 CH 2} -C (= O) -DGGFG-NH-CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ -C (= O) -,

-(Succinimide-3-yl-N)-CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ -C(=O)-GGFG-NH-CH ₂ -O-CH ₂ -C(=O)-,

-(Succinimide-3-yl-N)-CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ -C (= O) -GGFG-NH-CH ₂ CH ₂ -O-CH ₂ -C (= O) -,

-(Succinimide-3-yl-N)-CH ₂ CH ₂ -C(=O)-NH-CH ₂ CH ₂ -O-CH ₂ CH ₂ -O-CH ₂ CH ₂ -C(=O)-GGFG -NH-CH ₂ CH ₂ -C (= O) -,

-(Succinimide-3-yl-N)-CH ₂ CH ₂ -C(=O)-NH-CH ₂ CH ₂ -O-CH ₂ CH ₂ -O-CH ₂ CH ₂ -C(=O)-GGFG -NH-CH ₂ CH ₂ CH ₂ -C (= O) -,

-(Succinimide-3-yl-N) -CH ₂ CH ₂ -C (= O) -NH-CH ₂ CH ₂ -O-CH ₂ CH ₂ -O-CH ₂ CH ₂ -O-CH ₂ CH ₂ - O-CH ₂ CH ₂ -C (= O) -GGFG-NH-CH ₂ CH ₂ -C (= O) -,

-(Succinimide-3-yl-N) -CH ₂ CH ₂ -C (= O) -NH-CH ₂ CH ₂ -O-CH ₂ CH ₂ -O-CH ₂ CH ₂ -O-CH ₂ CH ₂ - O-CH ₂ CH ₂ -C (= O) -GGFG-NH-CH ₂ CH ₂ CH ₂ -C (= O) -,

-(Succinimide-3-yl-N)-CH ₂ CH ₂ -C (= O) -GGFG-,

-(Succinimide-3-yl-N)-CH ₂ CH ₂ -C(=O)-DGGFG-,

-(Succinimide-3-yl-N)-CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ -C (= O) -GGFG-,

-(Succinimide-3-yl-N)-CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ -C(=O)-DGGFG-.

Of these, the following are more preferable.

-(Succinimide-3-yl-N)-CH ₂ CH ₂ -C(=O)-GGFG-NH-CH ₂ CH ₂ CH ₂ -C(=O)-,

-(Succinimide-3-yl-N)-CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ -C (= O) -GGFG-NH-CH ₂ CH ₂ CH ₂ -C (= O) -,

-(Succinimide-3-yl-N)-CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ -C (= O) -DGGFG-NH-CH ₂ CH ₂ CH ₂ -C (= O) -,

-(Succinimide-3-yl-N)-CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ -C(=O)-GGFG-NH-CH ₂ -O-CH ₂ -C(=O)-,

-(Succinimide-3-yl-N)-CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ -C (= O) -GGFG-NH-CH ₂ CH ₂ -O-CH ₂ -C (= O) -,

-(Succinimide-3-yl-N)-CH ₂ CH ₂ -C(=O)-NH-CH ₂ CH ₂ -O-CH ₂ CH ₂ -O-CH ₂ CH ₂ -C(=O)-GGFG -NH-CH ₂ CH ₂ CH ₂ -C (= O) -,

-(Succinimide-3-yl-N) -CH ₂ CH ₂ -C (= O) -NH-CH ₂ CH ₂ -O-CH ₂ CH ₂ -O-CH ₂ CH ₂ -O-CH ₂ CH ₂ - O-CH ₂ CH ₂ -C (= O) -GGFG-NH-CH ₂ CH ₂ CH ₂ -C (= O) -,

-(Succinimide-3-yl-N)-CH ₂ CH ₂ -C(=O)-DGGFG-,

-(Succinimide-3-yl-N)-CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ -C(=O)-DGGFG-.

Even more preferred are the following.

-(Succinimide-3-yl-N)-CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ -C(=O)-GGFG-NH-CH ₂ -O-CH ₂ -C(=O)-,

-(Succinimide-3-yl-N)-CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ -C (= O) -GGFG-NH-CH ₂ CH ₂ -O-CH ₂ -C (= O) -,

-(Succinimide-3-yl-N)-CH ₂ CH ₂ -C(=O)-NH-CH ₂ CH ₂ -O-CH ₂ CH ₂ -O-CH ₂ CH ₂ -C(=O)-GGFG -NH-CH ₂ CH ₂ CH ₂ -C (= O) -,

-(Succinimide-3-yl-N)-CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ -C(=O)-DGGFG-.

The following are especially preferred.

-(Succinimide-3-yl-N)-CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ -C(=O)-GGFG-NH-CH ₂ -O-CH ₂ -C(=O)-,

-(Succinimide-3-yl-N)-CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ -C (= O) -GGFG-NH-CH ₂ CH ₂ -O-CH ₂ -C (= O) -,

-(Succinimide-3-yl-N)-CH ₂ CH ₂ -C(=O)-NH-CH ₂ CH ₂ -O-CH ₂ CH ₂ -O-CH ₂ CH ₂ -C(=O)-GGFG -NH-CH ₂ CH ₂ CH ₂ -C(=O)-.

[0111]

[Obtaining method]

The following are explanations regarding a representative method for preparing an antibody-drug conjugate of the present invention or preparing an intermediate thereof. That being said, the compounds are described below with the compound number shown in each reaction formula. In particular, they are referred to as "compound of formula (1)", "compound (1)", or the like. Connections to other numbers are also described in a similar way.

[0112]

1. Production method 1

An antibody-drug conjugate represented by formula (1), in which an antibody is conjugated to a drug-linker structure via a thioether, can be obtained, for example, by the following method.

[0113]

[Chemical Formula 19]

[0114]

[in the formula, AB is an antibody with a sulfhydryl group, and L ^1' corresponds to L ¹ having a structure in which the end group of the linker is converted to a maleimidyl group (the formula is shown below).

[0115]

[Chemical Formula 20]

[0116]

(in the formula, the nitrogen atom represents the binding position)

For example, it is a linker having a structure which, in the L ¹ structure, is represented as -(succinimide-3-yl-N)-(CH ₂ )n ² -C(=O)-, indicated by -(succinimide-3 -yl-N)- group converted to maleimidyl group. In addition, -(NH-DX) is a structure represented by the following formula:

[0117]

[Chemical Formula 21]

[0118]

and it is a group which is formed by removing one hydrogen atom of the amino group at position 1 of exatecan.]

[0119]

Further, the compound of formula (1) in the above reaction formula can be interpreted as a structure in which one structural fragment from the drug to the end of the linker is associated with one antibody. However, this is only a description provided for convenience, and in fact there are many cases in which several of these structural fragments are associated with a single antibody molecule. The same applies to the explanation of the production method described below.

[0120]

For example, the antibody-drug conjugate (1) can be obtained by reacting the compound (2), which is obtained by the method described below, with an antibody (3a) containing a sulfhydryl group.

An antibody (3a) containing a sulfhydryl group can be prepared by a method well known in the art (Hermanson, G.T, Bioconjugate Techniques, pp. 56-136, pp. 456-493, Academic Press (1996)). Examples include: the Troth reagent is reacted with the amino group of the antibody; N-succinimidyl S-acetylthioalkanoates are reacted with the amino group of the antibody, followed by reaction with hydroxylamine; after interaction with N-succinimidyl 3-(pyridyldithio)propionate, the antibody is subjected to interaction with a reducing agent; the antibody is reacted with a reducing agent such as dithiothreitol, 2-mercaptoethanol, and tris(2-carboxyethyl)phosphine hydrochloride (TCEP) to reduce the hinge disulfide bond in the antibody to form a sulfhydryl group, but is not limited to this. In particular, by using 0.3-3 molar equivalents of TCEP as a reducing agent for hinge disulfide bonds in an antibody and reacting with the antibody in a buffer solution containing a chelating agent, an antibody can be obtained with partially or completely reduced hinge disulfide bonds in antibody. Examples of the chelating agent include ethylenediaminetetraacetic acid (EDTA) and diethylenetriaminepentaacetic acid (DTPA). It can be used at a concentration of 1 mM to 20 mM. Examples of a buffer solution that can be used include a solution of sodium phosphate, sodium borate or sodium acetate. In particular, by reacting the antibody with TCEP at 4°C - 37°C for 1-4 hours, an antibody (3a) containing partially or completely reduced sulfhydryl groups can be obtained.

Meanwhile, by carrying out the addition reaction of a sulfhydryl group to a drug-linker moiety, the drug-linker moiety can be conjugated via a thioether bond.

Using 2 to 20 molar equivalents of compound (2) per antibody (3a) containing a sulfhydryl group, an antibody-drug conjugate (1) can be prepared in which 2 to 8 drug molecules per antibody are conjugated. Specifically, it is sufficient to add the solution containing the compound (2) dissolved therein to the buffer solution containing the sulfhydryl group-containing antibody (3a) for reaction. Here, examples of a buffer solution that can be used include a solution of sodium acetate, sodium phosphate, and sodium borate. The pH of the reaction is 5-9, and more preferably the reaction is carried out at about pH 7. Examples of the solvent for dissolving the compound (2) include an organic solvent such as dimethyl sulfoxide (DMSO), dimethylformamide (DMF), dimethylacetamide (DMA) and N- methyl-2-pyridone (NMP).

The reaction can be carried out by adding an organic solvent containing compound (2) dissolved therein at 1-20% v/v to a buffer solution containing an antibody (3a) containing a sulfhydryl group. The reaction temperature is 0-37°C, more preferably 10-25°C, and the reaction time is 0.5 to 2 hours. The reaction can be stopped by deactivating the reactivity of the unreacted compound (2) with a thiol-containing reagent. Examples of the thiol-containing reagent include cysteine and N-acetyl-L-cysteine (NAC). More specifically, 1-2 molar equivalents of NAC are added to the compound (2) used, and the reaction can be stopped by incubation at room temperature for 10-30 minutes.

The resulting antibody-drug conjugate (1) can be subjected, after concentration, to buffer exchange, purification and measurement of antibody concentration and the average number of conjugated drug molecules per antibody molecule in accordance with the general procedures described below, to identify the antibody-drug conjugate ( 1).

[0121]

General Procedure A: Concentration of an Aqueous Solution of an Antibody or Antibody-Drug Conjugate

In an Amicon Ultra (50,000 MWCO, Millipore Corporation) container, the antibody or antibody drug conjugate solution was added and the antibody or antibody drug conjugate solution was concentrated by centrifugation (centrifugation for 5-20 minutes at 2000 G - 3800 G) using a centrifuge (Allegra X-15R, Beckman Coulter, Inc.).

[0122]

General Procedure B: Measurement of Antibody Concentration

Using a UV detector (Nanodrop 1000, Thermo Fisher Scientific Inc.), the antibody concentration was measured according to the manufacturer's method. Here, the absorbance at 280 nm can be determined from the amino acid sequence of the antibody using a known calculation method (Protein Science, 1995, vol. 4, 2411-2423), and the absorbance at 280 nm is used different for each antibody (from 1.3 mlmg ^-1 cm ^-1 to 1.8 mlmg ^-1 cm ^-1 ). In the case of U1-59, a 280 nm absorption coefficient of 1.768 mlmg ^-1 cm ^-1 was used as a calculated value according to its amino acid sequence.

[0123]

General Procedure C-1: Buffer Exchange for Antibody

NAP-25 column (Cat. No. 17-0852-02, GE Healthcare Japan Corporation) using Sephadex G-25 vehicle was equilibrated with phosphate buffer (10 mM, pH 6.0; referred to as PBS6.0/EDTA in the specification) containing sodium chloride (137 mm) and ethylenediaminetetraacetic acid (EDTA, 5 mm), in accordance with the method specified by the manufacturer. An aqueous solution of the antibody was injected in an amount of 2.5 ml into one NAP-25 column, and then a fraction (3.5 ml), which was eluted with 3.5 ml of PBS6.0/EDTA, was collected. The resulting fraction was concentrated using General Procedure A. After measuring the antibody concentration using General Procedure B, the antibody concentration was adjusted to 10 mg/mL using PBS6.0/EDTA.

[0124]

General Procedure D: Purification of the Antibody-Drug Conjugate

The NAP-25 column was equilibrated with an acetate buffer containing sorbitol (5%) (10 mM, pH 5.5; referred to as ABS in the specification). An aqueous solution of the antibody-drug conjugate (about 2.5 ml) was injected into the NAP-25 column and then eluted with the buffer in the amount specified by the manufacturer to collect the antibody fraction. The collected fraction was again applied to the NAP-25 column, and by repeating the gel filtration purification process with elution buffer 2 to 3 times in total, an antibody-drug conjugate was obtained that did not include the unconjugated linker drug and a small molecular weight compound (tris(2 -carboxyethyl)phosphine hydrochloride (TCEP), N-acetyl-L-cysteine (NAC) and dimethyl sulfoxide).

[0125]

General procedure E: Measurement of the concentration of antibody in the antibody-drug conjugate and the average number of conjugated drug molecules per antibody molecule (1).

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 at two wavelengths of 280 nm and 370 nm, and then performing the calculation as shown below.

Since the total absorbance at any wavelength is equal to the sum of the absorbances of each light-absorbing chemical that are present in the system (absorbance additivity), when the molar absorbances of the antibody and drug remain the same before and after conjugation between the antibody and the drug, the concentration of the antibody and the concentration drug in the antibody-drug conjugate is expressed by the following equations.

A ₂₈₀ =A _D.280+ A _A.280 =E _D.280 C _D+ E _A.280 C _A Equation (I)

A ₃₇₀ =A _D.370 + A _A.370 =E _D.370 C _D+ E _A.370 C _A Equation (II)

In the above equations, A ₂₈₀ is the absorbance of an aqueous solution of the antibody-drug conjugate at 280 nm, A ₃₇₀ is the absorbance of an aqueous solution of the antibody-drug conjugate at 370 nm, A _A.280 is the absorbance of the antibody at 280 nm, A _{A .370} is the absorbance of the antibody at 370 nm, A _D.280 is the absorbance of the precursor conjugate at 280 nm, A _D.370 is the absorbance of the precursor conjugate at 370 nm, E _A.280 is the molar absorbance of the antibody at 280 nm, E _A.370 is the molar absorbance of the antibody at 370 nm, E _D.280 is the molar absorbance of the conjugate precursor at 280 nm, E _D.370 is the molar absorbance of the conjugate precursor at 370 nm, C _A is the concentration of the antibody in the antibody-drug conjugate, and _CD is the drug concentration in the antibody-drug conjugate.

As for E _A.280 , E _A.370 , E _D.280 and E _D.370 in the above equations, use pre-determined values (calculated value based on the calculation or measurement of the value obtained by UV measurement of the compound). For example, the E _{of A.280} can be calculated from the amino acid sequence of an antibody using a known calculation method (Protein Science, 1995, vol. 4, 2411-2423). E _A.370 is generally zero. In the case of U1-59, an E _{of A.280} of 259400 was used as a calculated value according to its amino acid sequence. E _D.280 and E _D.370 can be obtained based on the Bouguer-Lambert-Beer law (Absorption = molar concentration × molar absorption coefficient × length of the absorbing layer) by measuring the absorption of a solution in which a conjugate precursor suitable for use is dissolved at a certain molar concentration. By measuring A ₂₈₀ and A ₃₇₀ in an aqueous solution of the antibody-drug conjugate and solving parallel equations (I) and (II), and using the obtained values, C _A and _CD can be obtained. Further, by dividing C _D by C _A , you can get the average amount of drug that binds to the antibody.

In the present invention, the method for determining the average number of conjugated drug molecules per antibody described above is referred to as "UV method".

[0126]

General procedure F: Measurement of the average number of conjugated drug molecules per antibody molecule in the antibody-drug conjugate - (2).

The average number of conjugated drug molecules per antibody molecule in the antibody-drug conjugate can also be determined by high performance liquid chromatography (HPLC) using the method described below, in addition to General Procedure E described above.

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

The antibody-drug conjugate solution (about 1 mg/ml, 60 µl) is mixed with dithiothreitol (DTT) aqueous solution (100 mM, 15 µl). The mixture is incubated at 37°C for 30 minutes, the disulfide bond between the L chain and H chain of the antibody-drug conjugate is cleaved. The resulting sample is used in HPLC analysis.

[F-2. HPLC analysis]

HPLC analysis is carried out under the following measurement conditions:

HPLC system: Agilent 1290 HPLC system (Agilent Technologies, Inc.)

Detector: UV absorption spectrometer (wavelength measurement: 280 nm)

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

Column temperature: 80°C

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

Mobile phase B: acetonitrile solution containing 0.04% TFA

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

Injection volume: 15 µl

[F-3. Data analysis]

[F-3-1] Compared to the L chain (L ₀ ) and H chain (H ₀ ) of the unconjugated drug-conjugated antibody L (L chain linked to one drug molecule: L ₁ ) and H (H chain , associated with one drug molecule: H ₁ , H chain associated with two drug molecules: H ₂ , H chain associated with three drug molecules: H ₃ ) chains show higher hydrophobicity in proportion to the number of conjugated drug molecules and, thus have a longer retention time. Therefore, these chains are eluted in the order L ₀ and L ₁ or H ₀ , H ₁ , H ₂ and H ₃ . The detected peaks can be assigned to any of L ₀ , L ₁ , H ₀ , H ₁ , H ₂ and H ₃ by comparing retention times with L ₀ and H ₀ .

[F-3-2] Since the linker drug has UV absorption, the peak area values are corrected in response to the number of conjugated drug-linker molecules according to the following formula using the molar absorption coefficients of L chain, H chain and drug- linker.

[0127]

[Math Formula 1]

[0128]

[Math Formula 2]

[0129]

Here, as the molar absorbance (280 nm) of the L chain or H chain of each antibody, a value calculated from the amino acid sequence of the L chain or H chain of each antibody by a known calculation method can be used (Protein Science, 1995, vol. 4, 2411-2423 ). In the case of U1-59, the molar absorption coefficient of 34690 and the molar absorption coefficient of 95000 were used as the calculated value for the L chain or H chain, respectively, according to its amino acid sequence. The actual measured molar absorbance (280 nm) of the compound in which the maleimide group was converted to a succinimide thioester by reacting each linker drug with mercaptoethanol or N-acetylcysteine was used as the molar absorbance (280 nm) of the linker drug.

[F-3-3] The peak area ratio (%) of each chain is calculated for all corrected peak areas according to the following formula.

[0130]

[Math Formula 3]

Corrected values of the corresponding peak areas A _Li , A _Hi :L _i , H _i

[0131]

[F-3-4] The average number of conjugated drug molecules per antibody molecule in the antibody-drug conjugate is calculated according to the following formula.

Average number of conjugated drug molecules=(L ₀ peak area ratio × 0+L ₀ peak area ratio × 1+H ₀ peak area ratio × 0+H ₁ peak area ratio × 1+H ₂ peak area ratio × 2+H ₃ peak area ratio × 3)/100 × 2

[0132]

The following describes the intermediates used in Production Method 1. The compound represented by formula (2) in Production Method 1 is a compound represented by the following formula:

[0133]

(maleimide-N-yl)-(CH ₂ )n ³ -C(=O)-L ² -L ^P -NH-(CH ₂ )n ¹ -L ^a -(CH ₂ )n ² -C(=O )-(NH-DX) or

(maleimide-N-yl)-(CH ₂ )n ³ -C(=O)-L ² -L ^P -(NH-DX).

In this formula,

n ³ is an integer having a value from 2 to 8,

L ² is -NH-(CH ₂ CH ₂ -O)n ⁴ -CH ₂ CH ₂ -C(=O)- or a single bond,

where n ⁴ is an integer having a value from 1 to 6,

L ^P is a peptide residue consisting of 2-7 amino acids selected from phenylalanine, glycine, valine, lysine, citrulline, serine, glutamic acid and aspartic acid,

n ¹ is an integer having a value from 0 to 6,

n ² is an integer having a value from 0 to 5,

L ^a is -O- or a single bond,

(maleimid-N-yl)- is a maleimidyl group (2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl group) represented by the following formula:

[Chemical Formula 22]

[0134]

where the nitrogen atom represents the binding position, and

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

[Chemical Formula 23]

[0135]

where the nitrogen atom of the amino group in position 1 represents the binding position.

[0136]

With respect to the peptide residue L ^P , those comprising an amino acid selected from phenylalanine, glycine, valine, lysine, citrulline, serine, glutamic acid and aspartic acid are preferred as a process intermediate. Of the L ^P peptide residues, those consisting of 4 or 5 amino acids are preferred as an intermediate. More specifically, those in which L ^P is a tetrapeptide residue -GGFG- or a pentapeptide -DGGFG- are preferred as an intermediate in the preparation process, more preferred is -GGFG-.

[0137]

In addition, with regard to -NH-(CH ₂ )n ¹ -L ^a -(CH ₂ )n ² -, those containing -NH-CH ₂ CH ₂ -, -NH-CH ₂ CH ₂ CH ₂ -, -NH-CH ₂ CH ₂ CH ₂ CH ₂ -, -NH-CH ₂ CH _{2 CH 2} CH _{2 CH 2 -} , -NH-CH ₂ -O-CH ₂ - or -NH-CH ₂ CH ₂ -O- CH ₂ - are preferred as an intermediate in the preparation process. The connection -NH-CH ₂ CH ₂ CH ₂ -, -NH-CH ₂ -O-CH ₂ - or -NH-CH ₂ CH ₂ -O-CH ₂ is more preferred.

As for n ³ , compounds in which n ³ is an integer having a value of 2 to 8 are preferred as an intermediate in the production method.

As for L ² , compounds in which L ² is a single bond or -NH-(CH ₂ CH ₂ -O)n ⁴ -CH ₂ CH ₂ -C(=O)- and n ⁴ is an integer, having a value of 2 to 4 are preferred as an intermediate in the production process.

[0138]

In addition, compounds in which n ³ is an integer having a value of 2 to 5, L ² is a single bond, and -NH-(CH ₂ )n ¹ -L ^a -(CH ₂ )n ² - is -NH-CH ₂ CH ₂ -, -NH-CH ₂ CH ₂ CH ₂ -, -NH-CH ₂ CH ₂ CH ₂ CH ₂ -, -NH-CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ -, -NH -CH ₂ -O-CH ₂ - or -NH-CH ₂ CH ₂ -O-CH ₂ - are preferred as an intermediate in the production process. In addition, more preferred of these are those in which -NH-(CH ₂ )n ¹ -L ^a -(CH ₂ )n ² - is -NH-CH ₂ CH ₂ -, -NH-CH ₂ CH ₂ CH ₂ -, -NH-CH ₂ -O-CH ₂ - or -NH-CH ₂ CH ₂ -O-CH ₂ -. In addition, those in which n ³ is an integer having a value of 2 or 5 are preferred.

[0139]

In addition, those in which n ³ is an integer having a value of 2 to 5, L ² is -NH-(CH ₂ CH ₂ -O)n ⁴ -CH ₂ CH ₂ -C(=O)- , n ⁴ is an integer having a value of 2 to 4, and -NH-(CH ₂ )n ¹ -L ^a -(CH ₂ )n ² - is -NH-CH ₂ CH ₂ -, -NH- CH ₂ CH ₂ CH ₂ -, -NH-CH ₂ CH ₂ CH ₂ CH ₂ -, -NH-CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ -, -NH-CH ₂ -O-CH ₂ - or -NH -CH ₂ CH ₂ -O-CH ₂ - are preferred as an intermediate in the production process. More preferred of these are those in which n ⁴ is an integer having the value of 2 or 4. In addition, those in which -NH-(CH ₂ )n ¹ -L ^a - is -NH-CH ₂ CH ₂ CH ₂ -, -NH-CH ₂ -O-CH ₂ - or -NH-CH ₂ CH ₂ -O-CH ₂ - are preferred.

[0140]

Preferred examples of intermediates that are useful in preparing the compound of the present invention include those exemplified below:

(maleimid-N-yl)-CH ₂ CH ₂ -C (= O) -GGFG-NH-CH ₂ CH ₂ -C (= O) - (NH-DX),

(maleimid-N-yl)-CH ₂ CH ₂ CH ₂ -C (= O) -GGFG-NH-CH ₂ CH ₂ -C (= O) - (NH-DX),

(maleimid-N-yl)-CH ₂ CH ₂ CH ₂ CH ₂ -C (= O) -GGFG-NH-CH ₂ CH ₂ -C (= O) - (NH-DX),

(maleimid-N-yl)-CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ -C (= O) -GGFG-NH-CH ₂ CH ₂ -C (= O) - (NH-DX),

(maleimid-N-yl)-CH ₂ CH ₂ -C (= O) -GGFG-NH-CH ₂ CH ₂ CH ₂ -C (= O) - (NH-DX),

(maleimid-N-yl)-CH ₂ CH ₂ CH ₂ -C (= O) -GGFG-NH-CH ₂ CH ₂ CH ₂ -C (= O) - (NH-DX),

(maleimid-N-yl)-CH ₂ CH ₂ CH ₂ CH ₂ -C (= O) -GGFG-NH-CH ₂ CH ₂ CH ₂ -C (= O) - (NH-DX),

(maleimid-N-yl)-CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ -C (= O) -GGFG-NH-CH ₂ CH ₂ CH ₂ -C (= O) - (NH-DX),

(maleimid-N-yl)-CH ₂ CH ₂ -C (= O) -GGFG-NH-CH ₂ CH ₂ CH ₂ CH ₂ -C (= O) - (NH-DX),

(maleimid-N-yl)-CH ₂ CH ₂ CH ₂ -C (= O) -GGFG-NH-CH ₂ CH ₂ CH ₂ CH ₂ -C (= O) - (NH-DX),

(maleimid-N-yl)-CH ₂ CH ₂ CH ₂ CH ₂ -C (= O) -GGFG-NH-CH ₂ CH ₂ CH ₂ CH ₂ -C (= O) - (NH-DX),

(maleimid-N-yl)-CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ -C (= O) -GGFG-NH-CH ₂ CH ₂ CH ₂ CH ₂ -C (= O) - (NH-DX),

(maleimid-N-yl)-CH ₂ CH ₂ -C (= O) -GGFG-NH-CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ -C (= O) - (NH-DX),

(maleimid-N-yl)-CH ₂ CH ₂ CH ₂ -C (= O) -GGFG-NH-CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ -C (= O) - (NH-DX),

(maleimid-N-yl)-CH ₂ CH ₂ CH ₂ CH ₂ -C (= O) -GGFG-NH-CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ -C (= O) - (NH-DX),

(maleimid-N-yl) -CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ -C (= O) -GGFG-NH-CH ₂ CH ₂ CH 2 CH ₂ CH _{2 -C} (= O) - (NH-DX ),

(maleimid-N-yl)-CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ -C (= O) -DGGFG-NH-CH ₂ CH ₂ CH ₂ -C (= O) - (NH-DX),

(maleimid-N-yl)-CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ -C (= O) -DGGFG-NH-CH ₂ CH ₂ CH ₂ CH ₂ -C (= O) - (NH-DX),

(maleimid-N-yl)-CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ -C (= O) -DGGFG-NH-CH ₂ CH 2 CH ₂ CH ₂ CH _{2 -C} (= O) - (NH-DX ),

(maleimid-N-yl)-CH ₂ CH ₂ -C(=O)-GGFG-NH-CH ₂ -O-CH ₂ -C(=O)-(NH-DX),

(maleimid-N-yl)-CH ₂ CH ₂ CH ₂ -C(=O)-GGFG-NH-CH ₂ -O-CH ₂ -C(=O)-(NH-DX),

(maleimid-N-yl)-CH ₂ CH ₂ CH ₂ CH ₂ -C (= O) -GGFG-NH-CH ₂ -O-CH ₂ -C (= O) - (NH-DX),

(maleimid-N-yl)-CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ -C (= O) -GGFG-NH-CH ₂ -O-CH ₂ -C (= O) - (NH-DX),

(maleimid-N-yl)-CH ₂ CH ₂ -C (= O) -GGFG-NH-CH ₂ CH ₂ -O-CH ₂ -C (= O) - (NH-DX),

(maleimid-N-yl)-CH ₂ CH ₂ CH ₂ -C (= O) -GGFG-NH-CH ₂ CH ₂ -O-CH ₂ -C (= O) - (NH-DX),

(maleimid-N-yl)-CH ₂ CH ₂ CH ₂ CH ₂ -C (= O) -GGFG-NH-CH ₂ CH ₂ -O-CH ₂ -C (= O) - (NH-DX),

(maleimid-N-yl)-CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ -C (= O) -GGFG-NH-CH ₂ CH ₂ -O-CH ₂ -C (= O) - (NH-DX) ,

(maleimid-N-yl) -CH ₂ CH ₂ -C (= O) -NH-CH ₂ CH ₂ -O-CH ₂ CH ₂ -O-CH ₂ CH ₂ -C (= O) -GGFG-NH- CH ₂ CH ₂ CH ₂ -C (= O) - (NH-DX),

(maleimid-N-yl)-CH ₂ CH ₂ -C (= O) -NH-CH ₂ CH ₂ -O-CH ₂ CH ₂ -O-CH ₂ CH ₂ -O-CH ₂ CH ₂ -C (= O)-GGFG-NH-CH ₂ CH ₂ CH ₂ -C (= O) - (NH-DX),

(maleimid-N-yl)-CH ₂ CH ₂ -C (= O) -NH-CH ₂ CH ₂ -O-CH ₂ CH ₂ -O-CH ₂ CH ₂ -O-CH ₂ CH ₂ -O-CH ₂ CH ₂ -C (= O) -GGFG-NH-CH ₂ CH ₂ CH ₂ -C (= O) - (NH-DX),

(maleimide-N-yl)-CH ₂ CH ₂ -C (= O) -GGFG-(NH-DX),

(maleimid-N-yl)-CH ₂ CH ₂ CH ₂ -C (= O) -GGFG- (NH-DX),

(maleimid-N-yl)-CH ₂ CH ₂ CH ₂ CH ₂ -C (= O) -GGFG- (NH-DX),

(maleimid-N-yl)-CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ -C (= O) -GGFG-(NH-DX),

(maleimide-N-yl)-CH ₂ CH ₂ -C (= O) -DGGFG-(NH-DX),

(maleimid-N-yl)-CH ₂ CH ₂ CH ₂ -C (= O) -DGGFG- (NH-DX),

(maleimid-N-yl)-CH ₂ CH ₂ CH ₂ CH ₂ -C(=O)-DGGFG-(NH-DX) or

(maleimid-N-yl)-CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ -C(=O)-DGGFG-(NH-DX).

[0141]

By reacting a drug-linker compound selected from the above group of intermediates with an anti-HER3 antibody or a reactive derivative thereof, a thioether bond can be formed at the disulfide bond site present in the hinge portion of the anti-HER3 antibody, and as a result, an anti-HER3 antibody-drug conjugate of the present invention can be prepared. In this case, it is preferable to use a reactive derivative of the anti-HER3 antibody. A reactive derivative obtained by reconstitution of an anti-HER3 antibody is particularly preferred.

[0142]

The following are compounds that are more preferred as an intermediate:

(maleimid-N-yl)-CH ₂ CH ₂ -C (= O) -GGFG-NH-CH ₂ CH ₂ -C (= O) - (NH-DX),

(maleimid-N-yl)-CH ₂ CH ₂ -C (= O) -GGFG-NH-CH ₂ CH ₂ CH ₂ -C (= O) - (NH-DX),

(maleimid-N-yl)-CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ -C (= O) -GGFG-NH-CH ₂ CH ₂ -C (= O) - (NH-DX),

(maleimid-N-yl)-CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ -C (= O) -GGFG-NH-CH ₂ CH ₂ CH ₂ -C (= O) - (NH-DX),

(maleimid-N-yl) -CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ -C (= O) -GGFG-NH-CH ₂ CH ₂ CH 2 CH ₂ CH _{2 -C} (= O) - (NH-DX ),

(maleimid-N-yl)-CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ -C (= O) -DGGFG-NH-CH ₂ CH ₂ CH ₂ -C (= O) - (NH-DX),

(maleimid-N-yl)-CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ -C (= O) -GGFG-NH-CH ₂ -O-CH ₂ -C (= O) - (NH-DX),

(maleimid-N-yl)-CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ -C (= O) -GGFG-NH-CH ₂ CH ₂ -O-CH ₂ -C (= O) - (NH-DX) ,

(maleimid-N-yl) -CH ₂ CH ₂ -C (= O) -NH-CH ₂ CH ₂ -O-CH ₂ CH ₂ -O-CH ₂ CH ₂ -C (= O) -GGFG-NH- CH ₂ CH ₂ -C (= O) - (NH-DX),

(maleimid-N-yl) -CH ₂ CH ₂ -C (= O) -NH-CH ₂ CH ₂ -O-CH ₂ CH ₂ -O-CH ₂ CH ₂ -C (= O) -GGFG-NH- CH ₂ CH ₂ CH ₂ -C (= O) - (NH-DX),

(maleimid-N-yl)-CH ₂ CH ₂ -C (= O) -NH-CH ₂ CH ₂ -O-CH ₂ CH ₂ -O-CH ₂ CH ₂ -O-CH ₂ CH ₂ -O-CH ₂ CH ₂ -C (= O) -GGFG-NH-CH ₂ CH ₂ CH ₂ -C (= O) - (NH-DX),

(maleimide-N-yl)-CH ₂ CH ₂ -C (= O) -DGGFG-(NH-DX),

(maleimid-N-yl)-CH ₂ CH ₂ CH ₂ -C (= O) -DGGFG- (NH-DX),

(maleimid-N-yl)-CH ₂ CH ₂ CH ₂ CH ₂ -C (= O) -DGGFG- (NH-DX),

(maleimid-N-yl)-CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ -C(=O)-DGGFG-(NH-DX).

[0143]

In addition, from the above group of intermediates, the intermediates represented by the following formulas are more preferred:

(maleimid-N-yl) -CH ₂ CH ₂ -C (= O) -NH-CH ₂ CH ₂ -O-CH ₂ CH ₂ -O-CH ₂ CH ₂ -C (= O) -GGFG-NH- CH ₂ CH ₂ CH ₂ -C (= O) - (NH-DX),

(maleimid-N-yl)-CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ -C (= O) -GGFG-NH-CH ₂ -O-CH ₂ -C (= O) - (NH-DX),

(maleimid-N-yl)-CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ -C (= O) -GGFG-NH-CH ₂ CH ₂ -O-CH ₂ -C (= O) - (NH-DX) ,

(maleimid-N-yl)-CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ -C(=O)-DGGFG-NH-CH ₂ CH ₂ CH ₂ -C(=O)-(NH-DX) or

(maleimid-N-yl)-CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ -C(=O)-DGGFG-(NH-DX).

Particularly preferred are compounds which are represented by the following formula:

(maleimid-N-yl) -CH ₂ CH ₂ -C (= O) -NH-CH ₂ CH ₂ -O-CH ₂ CH ₂ -O-CH ₂ CH ₂ -C (= O) -GGFG-NH- CH ₂ CH ₂ CH ₂ -C (= O) - (NH-DX),

(maleimid-N-yl)-CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ -C(=O)-GGFG-NH-CH ₂ -O-CH ₂ -C(=O)-(NH-DX) or

(maleimid-N-yl)-CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ -C (= O) -GGFG-NH-CH ₂ CH ₂ -O-CH ₂ -C (= O) - (NH-DX) .

[0144]

2. Production method 2

The compound represented by formula (2) or a pharmacologically acceptable salt thereof used as an intermediate in the above production method can be obtained by the following method, for example.

[0145]

[Chemical Formula 24]

[0146]

[in the formula, L ^1' corresponds to L ¹ having a structure in which the terminal group is converted to a maleimidyl group, and P ¹ , P ² and P ³ represent a protecting group].

[0147]

Compound (6) can be obtained by derivatizing carboxylic acid (5) to an active ester, a mixed acid anhydride, an acid halide or the like. and, in the presence of a base, its interaction with NH ₂ -DX [meaning exatecan; chemical name: (1S,9S)-1-amino-9-ethyl-5-fluoro-2,3-dihydro-9-hydroxy-4-methyl-1H,12H-benzo[de]pyrano[3',4' :6,7]indolizino[1,2-b]quinoline-10,13(9H,15H)-dione] (4) or a pharmacologically acceptable salt thereof.

Reagents and reaction conditions that are traditionally used for peptide synthesis can be used for this reaction. There are various types of active ester, for example, it can be obtained by reacting phenols such as p-nitrophenol, N-hydroxybenzotriazole, N-hydroxysuccinimide or the like with carboxylic acid (5) using a condensing agent such as N, N'-dicyclohexylcarbodiimide or 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride; in addition, the active ester can also be obtained by reacting the carboxylic acid (5) with pentafluorophenyl trifluoroacetate or the like; interactions of carboxylic acid (5) with 1-benzotriazolyloxytripyrrolidinophosphonium hexafluorophosphite; interactions of carboxylic acid (5) with diethylcyanophosphonate (Shioiiri method); interactions of carboxylic acid (5) with triphenylphosphine and 2,2'-dipyridyl disulfide (Mukayama method); reacting the carboxylic acid (5) with a triazine derivative such as 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMTMM); or the like. Further, the reaction can also be carried out, for example, by an acid halide method in which the carboxylic acid (5) is treated with an acid halide such as thionyl chloride and oxalyl chloride in the presence of a base.

By reacting the active ester, mixed acid anhydride or carboxylic acid halide (5) obtained as described above with the compound (4) in the presence of a suitable base in an inert solvent at a reaction temperature ranging from -78C to 15C, the compound can be obtained (6). Here, "inert solvent" means a solvent that does not inhibit the desired reaction for which such a solvent is used.

[0148]

Specific examples of the base used for each step described above include an alkali metal or alkaline earth metal carbonate, alkoxide, hydroxide or hydride such as sodium carbonate, potassium carbonate, sodium ethoxide, potassium butoxide, sodium hydroxide, potassium hydroxide, sodium hydride or potassium hydride; an organometallic base represented by an alkyllithium such as n-butyllithium or a dialkylaminolithium such as lithium diisopropylamide; an organometallic base such as bissilylamine, including lithium bis(trimethylsilyl)amide; and an organic base such as pyridine, 2,6-lutidine, collidine, 4-dimethylaminopyridine, triethylamine, N-methylmorpholine, diisopropylethylamine, and diazabicyclo[5.4.0]undec-7-ene (DBU).

[0149]

Examples of the inert solvent that is used for the reaction of the present invention include a halogenated hydrocarbon solvent such as dichloromethane, chloroform and carbon tetrachloride; an ether solvent such as tetrahydrofuran, 1,2-dimethoxyethane and dioxane; aromatic hydrocarbon solvent such as benzene and toluene; and an amide solvent such as N,N-dimethylformamide, N,N-dimethylacetamide and N-methylpyrrolidin-2-one. In addition to these, a sulfoxide solvent such as dimethyl sulfoxide and sulfolane; a ketone solvent such as acetone and methyl ethyl ketone; and an alcohol solvent such as methanol and ethanol can be used in some cases. Alternatively, these solvents can be used as a mixed solvent.

[0150]

As for the protecting group P ¹ for the amino terminal group of compound (6), an amino protecting group which is generally used for peptide synthesis, such as t-butyloxycarbonyl group, 9-fluorenylmethyloxycarbonyl group and benzyloxycarbonyl group, can be used. Examples of another protecting group for an amino group include an alkanoyl group such as an acetyl group; an alkoxycarbonyl group such as a methoxycarbonyl group and an ethoxycarbonyl group; an arylmethoxycarbonyl group such as a para-methoxybenzyloxycarbonyl group and a para(or ortho)nitrobenzyloxycarbonyl group; an arylmethyl group such as a benzyl group and a triphenylmethyl group; an aroyl group such as a benzoyl group; and an arylsulfonyl group such as a 2,4-dinitrobenzenesulfonyl group and an ortho-nitrobenzenesulfonyl group. The protecting group P ¹ can be selected depending on, for example, the properties of the compound containing the amino group to be protected.

By removing the protecting group P ¹ for the terminal amino group of the resulting compound (6), compound (7) can be obtained. For this deprotection procedure, you can select reagents and conditions depending on the protecting group.

The compound (9) can be obtained by derivatizing the carboxylic acid of the peptide (8) with the N terminus protected by a P ² group into an active ester, a mixed acid anhydride or the like. and its interaction with the resulting compound (7). The reaction conditions, reagents, base and inert solvent used to form a peptide bond between the carboxylic acid of the peptide (8) and compound (7) can be suitably selected from those described for the synthesis of compound (6). The protecting group P ² can be suitably selected from those described for the protecting group of the compound (6), and the choice can be made based on, for example, the properties of the compound containing the amino group to be protected. As is commonly used for peptide synthesis, by repeating the reaction sequentially and deprotecting the amino acid or carboxylic acid constituting the peptide (8) for elongation, compound (9) can also be obtained.

By removing the protecting group P ² as the protecting group for the amino group of the obtained compound (9), compound (10) can be obtained. For this deprotection procedure, you can select reagents and conditions depending on the protecting group.

Compound (2) can be obtained by derivatizing carboxylic acid (11) to an active ester, a mixed acid anhydride, an acid halide or the like. and its interaction with the resulting compound (10). The reaction conditions, reagents, base and inert solvent used to form the peptide bond between the carboxylic acid (11) and compound (10) can be suitably selected from those described for the synthesis of compound (6).

[0151]

Compound (9) can also be obtained by the following method, for example.

Compound (13) can be obtained by derivatizing the carboxylic acid of peptide (8) with the ^P2 -protected N-terminus into an active ester, a mixed acid anhydride, or the like. and its interaction with an amine compound (12) containing a carboxy group protected by a P ³ group in the presence of a base. The reaction conditions, reagents, base and inert solvent used to form a peptide bond between the carboxylic acid of the peptide (8) and compound (12) can be suitably selected from those described for the synthesis of compound (6).

The protecting group P ² for the amino group of the compound (13) is not particularly limited, as long as it is a protecting group that is conventionally used. For example, examples of a protecting group for a hydroxyl group include an alkoxymethyl group such as a methoxymethyl group; an arylmethyl group such as a benzyl group, a 4-methoxybenzyl group and a triphenylmethyl group; an alkanoyl group such as an acetyl group; an aroyl group such as a benzoyl group; and a silyl group such as a tert-butyldiphenylsilyl group. The carboxy group may be protected by an ester with an alkyl group such as a methyl group, an ethyl group and a tert-butyl group, an allyl group, or an arylmethyl group such as a benzyl group. As for the amino group, an alkyloxycarbonyl group such as a t-butyloxycarbonyl group, a methoxycarbonyl group and an ethoxycarbonyl group can be mentioned; an arylmethoxy carbonyl group such as an allyloxycarbonyl group, a 9-fluorenylmethyloxy carbonyl group, a benzyloxycarbonyl group, a para-methoxybenzyloxycarbonyl group, and a para(or ortho)nitrobenzyloxycarbonyl group; an alkanoyl group such as an acetyl group; an arylmethyl group such as a benzyl group and a triphenylmethyl group; an aroyl group such as a benzoyl group; and an arylsulfonyl group such as a 2,4-dinitrobenzenesulfonyl group or an ortho-nitrobenzenesulfonyl group.

As for the protecting group P ³ for a carboxy group, a protecting group conventionally used as a protecting group for a carboxy group in the field of organic chemical synthesis, in particular peptide synthesis, can be used. The carboxyl group may be protected as an ester with an alkyl group such as a methyl group, an ethyl group, or t-butyl, an allyl group, and an arylmethyl group such as a benzyl group.

In such a case, it is preferable that the amino protecting group and the carboxy protecting group can be removed using different methods or different conditions. For example, a representative example includes a combination in which P ² is a tert-butyloxycarbonyl group and P ³ is a benzyl group. The protecting groups can be selected from the above groups depending on, for example, the properties of the compound containing the amino group and the carboxy group to be protected. To remove the protecting groups, you can select reagents and conditions depending on the protecting group.

By removing the protecting group P ³ for the carboxy group of the resulting compound (13), compound (14) can be obtained. For this deprotection procedure, the reagents and conditions are selected depending on the protecting group.

The compound (9) can be obtained by derivatizing the obtained compound (14) into an active ester, a mixed acid anhydride, an acid halide or the like. and interaction with compound (4) in the presence of a base. For this reaction, the reagents and reaction conditions that are commonly used for peptide synthesis can also be used, and the reaction conditions, reagents, base, and inert solvent for use in the reaction can be suitably selected from those described for the synthesis of compound (6).

[0152]

Compound (2) can also be obtained, for example, by the following method.

By removing the protecting group P ² for the amino group of compound (13), compound (15) can be obtained. For this deprotection procedure, you can select reagents and conditions depending on the protecting group.

Compound (16) can be obtained by derivatizing a carboxylic acid derivative (11) into an active ester, a mixed acid anhydride, an acid halide or the like. and its interaction with compound (15) obtained in the presence of a base. The reaction conditions, reagents, base and inert solvent used to form an amide bond between the carboxylic acid of the peptide (11) and compound (15) can be suitably selected from those described for the synthesis of compound (6).

By deprotecting the carboxy group of the resulting compound (16), compound (17) can be obtained. This deprotection procedure can be carried out in a manner analogous to the deprotection of the carboxy group to obtain compound (14).

Compound (2) can be obtained by derivatizing compound (17) into an active ester, a mixed acid anhydride, an acid halide or the like. and its interaction with compound (4) in the presence of a base. For this reaction, the reagents and reaction conditions that are commonly used for peptide synthesis can also be used, and the reaction conditions, reagents, base, and inert solvent for use in the reaction can be suitably selected from those described for the synthesis of compound (6).

[0153]

3. Production method 3

The compound represented by formula (2) used as an intermediate can also be obtained by the following method.

[0154]

[Chemical Formula 25]

[0155]

[in this formula, L ^1' corresponds to L ¹ having a structure in which the terminal group is converted to a maleimidyl group and P ⁴ is a protecting group].

[0156]

Compound (19) can be obtained by derivatizing compound (11) to an active ester, a mixed acid anhydride or the like. and its interaction with the carboxylic acid of the peptide (18) with the C-terminus protected by the group P ⁴ in the presence of a base. The reaction conditions, reagents, base and inert solvent used to form a peptide bond between the carboxylic acid of the peptide (18) and compound (11) can be suitably selected from those described for the synthesis of compound (6). The protecting group P ⁴ for the carboxy group of the compound (18) can be suitably selected from the above protecting groups.

By deprotecting the carboxy group of the obtained compound (19), compound (20) can be obtained. This deprotection procedure can be carried out in a manner analogous to the deprotection of the carboxy group to obtain compound (14).

Compound (2) can be obtained by derivatizing the resulting compound (20) into an active ester, a mixed acid anhydride or the like. and its interaction with compound (7). For this reaction, the reagents and reaction conditions that are commonly used for peptide synthesis can also be used, and the reaction conditions, reagents, base, and inert solvent for use in the reaction can be suitably selected from those described for the synthesis of compound (6).

[0157]

4. Production method 4

Below, in the context of the intermediate (10) described in the production method 2, the method for obtaining the compound (10b) having n ¹ =1 and L ^a =0 is described in detail. The compound represented by formula (10b), its salt or solvate can be obtained according to, for example, the following method.

[0158]

[Chemical Formula 26]

[0159]

[in the formula, L ^P has the meaning defined above, L represents an acyl group, including an alkanoyl group such as an acetyl group, or an aroyl group such as a benzoyl group, or represents a hydrogen atom or the like, X and Y are an oligopeptide consisting of 1-3 amino acids, P ⁵ and P ⁷ are a protecting group for an amino group, and P ⁶ is a protecting group for a carboxy group].

[0160]

The compound represented by formula (21) can be obtained using or using the method described in Japanese Patent Laid-Open No. 2002-60351 or literature (J. Org. Chem., Vol. 51, page 3196, 1986), and if necessary, by deprotection or modification of functional groups. Alternatively, it can also be obtained by treating an amino-terminated amino acid or acid amide of an amino-protected oligopeptide with an aldehyde or a ketone.

By reacting compound (21) with a compound (22) containing a hydroxyl group under a temperature condition from cooling to room temperature in an inert solvent in the presence of an acid or a base, compound (23) can be obtained.

Here, examples of the acid that can be used may include an inorganic acid such as hydrofluoric acid, hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, and boric acid; an organic acid such as acetic acid, citric acid, p-toluenesulfonic acid and methanesulfonic acid; and a Lewis acid such as tetrafluoroborate, zinc chloride, tin chloride, aluminum chloride and iron chloride. Of these, sulfonic acids are preferred, and p-toluenesulfonic acid is particularly preferred. As for the base, any one of the above bases can be appropriately selected and used. Preferred examples include an alkali metal alkoxide such as potassium t-butoxide, an alkali metal or alkaline earth metal hydroxide such as sodium hydroxide and potassium hydroxide; an alkali metal hydride such as sodium hydride and potassium hydride; an organometallic base represented by a dialkylaminolithium such as lithium diisopropylamide; and an organometallic base of bissilylamine such as lithium bis(trimethylsilyl)amide.

Examples of a solvent that can be used for the reaction include an ether solvent such as tetrahydrofuran and 1,4-dioxane; and an aromatic hydrocarbon solvent such as benzene and toluene. Such solvents may be prepared as a mixture with water.

In addition, the protecting group for the amino group represented as P ⁵ is not particularly limited, as long as it is a group commonly used to protect the amino group. Representative examples may include amino protecting groups as described in Production Method 2. However, the amino protecting group represented as P ⁵ may be cleaved off during the reaction. In such a case, the protecting group can be reintroduced by suitably reacting with a suitable amino group protecting reagent, if necessary.

Compound (24) can be obtained by removing the protecting group P ⁶ of compound (23). Here, although representative examples of the protecting group for the carboxy group represented by P ⁶ are described in Production Method 2, it can be suitably selected from these examples. In the compound (23), it is desirable that the amino protecting group P ⁵ and the carboxy protecting group P ⁶ are protective groups that can be removed using different methods or different conditions. For example, a representative example may include a combination in which P ⁵ is a 9-fluorenylmethyloxycarbonyl group and P ⁶ is a benzyl group. The protecting groups can be selected depending on, for example, the properties of the compound containing the amino group and the carboxy group to be protected. For deprotection, reagents and conditions are chosen depending on the protecting group.

Compound (26) can be obtained by derivatizing compound (24) into an active ester, a mixed acid anhydride, an acid halide or the like. and its interaction with the compound (4) or its pharmacologically acceptable salt in the presence of a base to obtain the compound (25), followed by removal of the protective group P ⁵ of the resulting compound (25). For the reaction between compound (4) and carboxylic acid (24) and the reaction to remove the protecting group P ⁶ , the same reagents and reaction conditions as described for Production Method 2 can be used.

Compound (10b) can be obtained by reacting compound (26) with amino-terminal protected amino acid or amino-protected oligopeptide (27) to obtain compound (9b) and deprotection P ⁷ of the obtained compound (9b). The amino protecting group represented as P ⁷ is not particularly limited, as long as it is commonly used to protect the amino group. Representative examples include protecting groups for the amino group, which are described in Production Method 2. To remove the protecting group, the reagents and conditions are selected depending on the protecting group. For the reaction between compound (26) and compound (27), reagents and reaction conditions that are traditionally used for peptide synthesis can be used. The compound (10b) obtained by the above method can be converted into the compound (1) of the present invention according to the method described above.

[0161]

5. Production method 5

The compound represented by formula (2) as an intermediate can also be obtained with the method shown below.

[0162]

[Chemical Formula 27]

[0163]

[in the formula, L ^1' corresponds to L ¹ having a structure in which the terminal group is converted to a maleimidyl group, L ^P is a structure consisting of -L ^p1 -L ^p2 -, and P ³ , P ⁸ , P ⁹ , P ¹⁰ , P ¹¹ and P ¹² represent a protective group].

[0164]

Since L ^P is formed by linking L ^p1 to L ^p2 , the hydrophilic amino acid at the N-terminus of L ^P is derived from L ^p1 , and thus those containing the hydrophilic amino acid at the N-terminus are suitably used as L ^p1 . In this case, several hydrophilic amino acids may be present. In addition, when L ^p2 with a hydrophilic amino acid is used, an L ^p containing several hydrophilic amino acids at the N-terminus of the L ^P or at the N-terminus and other positions can be obtained depending on the position of the hydrophilic amino acid.

[0165]

Compound (29) can be obtained by derivatizing the P ² -protected peptide or amino acid (28) into an active ester, a mixed acid anhydride, or the like. and its interaction with the resulting compound (7). The reaction conditions, reagents, base and solvent used to form an amide bond between the peptide or amino acid (28) and compound (7) can be suitably selected from those described for the synthesis of compound (6). The protecting group P ⁸ for the amino group may be suitably selected from those described for the protecting group of the compound (6), and the selection may be made based on a property of the compound or the like. As is commonly used for peptide synthesis, compound (29) can also be obtained by repeating the reaction sequentially and deprotecting the amino acid or peptide constituent amino acid (28) for elongation.

By removing P ⁸ as a protecting group for the amino group of the resulting compound (29), compound (23) can be obtained. For this deprotection procedure, you can select reagents and conditions depending on the protecting group.

Compound (32) can be obtained by derivatizing an amino acid or peptide (31) with an N-terminus protected by a P ⁸ group and protected by a carboxy group, a hydroxy group or an amino group in the side chain into an active ester, a mixed acid anhydride or the like. and its interaction with the resulting compound (30). The reaction conditions, reagents, base and inert solvent used to form a peptide bond between the amino acid or peptide (31) and compound (30) can be suitably selected from those described for the synthesis of compound (6). With regard to the protecting groups P ⁸ and P ⁹ , the protecting groups can be suitably selected from those described as protecting groups for the amino group, carboxy group or hydroxy group of the compound (6). However, in such a case, it is necessary that the protecting group P ⁹ for the amino group and the protecting group P ¹⁰ for the functional group in the side chain can be removed using different methods or different conditions. For example, a representative example includes a combination where P ⁹ is a 9-fluorenylmethyloxycarbonyl group and P ¹⁰ is a t-butyl group or the like. as a protecting group for a carboxy group, a methoxymethyl group, or the like. as a protecting group for a hydroxy group, or a t-butyloxycarbonyl group, or the like. as a protecting group for the amino group. The protecting group P ¹⁰ for a functional group in the side chain is preferably a protecting group that can be removed by treatment under acidic conditions. However, it is not limited to this, and may be selected from the above groups depending on, for example, the properties of the amino group, carboxy group or hydroxy group of the compound to be protected. For deprotection, reagents and conditions are selected depending on the protecting group. As is commonly used for peptide synthesis, compound (32) can also be obtained by repeating the reaction sequentially and deprotecting the constituent amino acid or peptide to be elongated.

By removing P ⁹ as the protecting group of the terminal amino group of the resulting compound (32), compound (33) can be obtained. For this deprotection procedure, you can select reagents and conditions depending on the protecting group.

The compound can be obtained (34) by derivatizing the carboxylic acid derivative (11) to an active ester, a mixed acid anhydride, an acid halide or the like. and its interaction with the resulting compound (33). Here, the carboxylic acid derivative (11) is a compound with a structure in which the terminal portion of the L ^1' linker contains a maleimidyl group.

The reaction conditions, reagents, base and solvent used to form a peptide bond between the carboxylic acid derivative (11) and compound (33) can be suitably selected from those described for the synthesis of compound (6).

By removing the protecting group P ¹⁰ for the carboxy group, hydroxy group or amino group in the amino acid side chain of the peptide portion of the obtained compound (34), compound (2) can be obtained. You can select reagents and conditions depending on the protecting group.

[0166]

Compound (29) can also be obtained, for example, by the following method.

Compound (35) can be obtained by derivatizing a ^P8 -protected peptide or amino acid (28) into an active ester, a mixed acid anhydride, or the like. and its interaction with an amine compound (12) containing a terminal carboxy group protected by a P ³ group in the presence of a base. The reaction conditions, reagents, base and solvent used to form a peptide bond between the peptide or amino acid (28) and compound (12) can be suitably selected from those described for the synthesis of compound (6). The protecting group P ⁸ for the amino group of the compound (35) can be suitably selected from those described as the protecting group for the compound (6) and used. As for the protecting group P ³ for the carboxy group, a protecting group conventionally used as a protecting group for the carboxy group in the field of organic chemical synthesis, in particular peptide synthesis, can be used. Specific examples include an alkyl ester such as a methyl group, an ethyl group and t-butyl, an allyl ester and a benzyl ester, and can be suitably selected from the protecting groups which are described for the compound (6) and used. In such a case, it is necessary that the amino protecting group P ⁸ and the carboxy protecting group P ³ can be removed using different methods or different conditions. For example, a representative example includes a combination in which P ⁸ is a tert-butyloxycarbonyl group and P ³ is a benzyl group. The protecting groups can be selected from the above groups depending on, for example, the properties of the compound containing the amino group and the carboxy group to be protected. For deprotection, reagents and conditions are selected depending on the protecting group.

By removing the protecting group P ³ for the carboxy group of the resulting compound (35), compound (36) can be obtained. For this deprotection procedure, the reagents and conditions are selected depending on the protecting group.

Compound (29) can be obtained by derivatizing the resulting compound (36) into an active ester, a mixed acid anhydride, an acid halide or the like. and its interaction with compound (4) in the presence of a base. The reagents and reaction conditions that are commonly used for peptide synthesis can also be used for this reaction, and the reaction conditions, reagents, base and solvent used for the reaction can be suitably selected from those described for the synthesis of compound (6).

[0167]

Compound (32) can also be obtained, for example, by the following method.

By removing the protecting group P ⁸ for the amino group of compound (35), compound (37) can be obtained. For this deprotection procedure, you can select reagents and conditions depending on the protecting group.

Compound (38) can be obtained by derivatizing an amino acid or peptide (31) into an active ester, a mixed acid anhydride, an acid halide or the like. and its interaction with the resulting compound (37) in the presence of a base. The reaction conditions, reagents, base and solvent used to form an amide bond between the amino acid or peptide (31) and compound (37) can be suitably selected from those described for the synthesis of compound (6). In such a case, it is necessary that the protecting group P ⁹ and P ¹⁰ for the amino acid or peptide (31) and the protecting group P ³ for the compound (37) can be removed using different methods or different conditions. For example, a representative example includes a combination in which P ⁹ is a 9-fluorenylmethyloxycarbonyl group, P ¹⁰ is a t-butyloxycarbonyl group, a t-butyl group, or a methoxymethyl group, and P ³ is a benzyl group. In addition, the protecting group P ¹⁰ for a functional group in the side chain is preferably a protecting group that can be removed by treatment under acidic conditions as described above. However, the protecting group is not limited to this, and may be selected from the above groups depending on, for example, the properties of the amino group, carboxy group, or hydroxy group of the compound to be protected. For deprotection, reagents and conditions are selected depending on the protecting group.

By removing the protecting group P ³ for the carboxy group of the resulting compound (38), compound (39) can be obtained. For this deprotection procedure, you can select reagents and conditions depending on the protecting group.

Compound (32) can be obtained by derivatizing compound (39) into an active ester, a mixed acid anhydride, an acid halide or the like. and its interaction with compound (4) in the presence of a base. For this reaction, the reagents and reaction conditions that are commonly used for peptide synthesis can also be used, and the reaction conditions, reagents, base, and solvent for use in the reaction can be suitably selected from those described for the synthesis of compound (6).

[0168]

Compound (34) can also be obtained, for example, by the following method.

By removing the protecting group P ⁹ for the amino group of compound (38), compound (40) can be obtained. For this deprotection procedure, you can select reagents and conditions depending on the protecting group.

Compound (41) can be obtained by derivatizing a carboxylic acid derivative (11) into an active ester, a mixed acid anhydride, an acid halide or the like. and its interaction with the resulting compound (40) in the presence of a base. The reaction conditions, reagents, base and solvent used to form an amide bond between the carboxylic acid derivative (11) and compound (40) can be suitably selected from those described for the synthesis of compound (6).

By removing the protecting group P ³ for the carboxy group of the obtained compound (41), compound (42) can be obtained. For this deprotection procedure, you can select reagents and conditions depending on the protecting group.

Compound (34) can be obtained by derivatizing compound (42) into an active ester, a mixed acid anhydride, an acid halide or the like. and its interaction with compound (4) in the presence of a base. For this reaction, the reagents and reaction conditions that are commonly used for peptide synthesis can also be used, and the reaction conditions, reagents, base, and solvent for use in the reaction can be suitably selected from those described for the synthesis of compound (6).

[0169]

Compound (34) can also be obtained, for example, by the following method.

Compound (44) can be obtained by derivatizing a carboxylic acid derivative (11) into an active ester, a mixed acid anhydride, an acid halide or the like. and its interaction with an amino acid or peptide (43) containing a carboxy group protected by a P ¹¹ group and a carboxy group, a hydroxy group or an amino group in the side chain protected by a P ¹⁰ group, in the presence of a base. The reaction conditions, reagents, base and solvent used to form an amide bond between the carboxylic acid derivative (11) and compound (43) can be suitably selected from those described for the synthesis of compound (6). With respect to the protecting groups P ¹⁰ and P ¹¹ of the compound (44), the protecting groups can be suitably selected from those described as the protecting group for the carboxy group, hydroxy group or amino group of the compound (6). Here, in such a case, it is necessary that the carboxy protecting group P ¹¹ and the side chain functional group P ¹⁰ can be removed using different methods or different conditions. For example, a representative example includes a combination in which P ¹¹ is a benzyl group and P ¹⁰ is a t-butyl group or the like. as a protecting group for a carboxy group, a methoxymethyl group, or the like. as a protecting group for a hydroxy group, or a t-butyloxycarbonyl group, or the like. as a protecting group for the amino group. The protecting group P ¹⁰ for a functional group in the side chain is preferably a protecting group that can be removed by treatment under acidic conditions. However, it is not limited to this, and may be selected from the above groups depending on, for example, the properties of the amino group, carboxy group or hydroxy group of the compound to be protected. To remove the protecting group, you can select reagents and conditions depending on the protecting group.

By removing the protecting group P ¹¹ for the carboxy group of the resulting compound (44), compound (45) can be obtained. For this deprotection procedure, you can select reagents and conditions depending on the protecting group.

Compound (34) can be obtained by derivatizing compound (45) into an active ester, a mixed acid anhydride, an acid halide or the like. and its interaction with compound (30) in the presence of a base. For this reaction, the reagents and reaction conditions that are commonly used for peptide synthesis can also be used, and the reaction conditions, reagents, base, and solvent for use in the reaction can be suitably selected from those described for the synthesis of compound (6).

Compound (47) can be obtained by derivatizing compound (45) into an active ester, a mixed acid anhydride, an acid halide or the like. and its interaction with an amino acid or peptide (46) containing a carboxy group protected by a P ¹² group in the presence of a base. For this reaction, the reagents and reaction conditions commonly used for peptide synthesis can be used, and the reaction conditions, reagents, base and solvent can be suitably selected from those described for the synthesis of compound (6). With respect to the protecting groups P ¹⁰ and P ¹² of the compound (47), the protecting groups can be suitably selected and used from those described as protecting groups for the carboxy group, hydroxy group or amino group of the compound (6). Here, in such a case, it is necessary that the P ¹² protecting group for the carboxy group and the P ¹⁰ protecting group for the functional group in the side chain can be removed using different methods or different conditions. For example, a representative example includes a combination in which P ¹² is a benzyl group and P ¹⁰ is a t-butyl group or the like. as a protecting group for a carboxy group, a methoxymethyl group, or the like. as a protecting group for a hydroxy group, or a t-butyloxycarbonyl group, or the like. as a protecting group for the amino group. The protecting group P ¹⁰ for a functional group in the side chain is preferably a protecting group that can be removed by treatment under acidic conditions. However, it is not limited to this, and may be selected from the above groups depending on, for example, the properties of the amino group, carboxy group or hydroxy group of the compound to be protected. To remove the protecting group, you can select reagents and conditions depending on the protecting group. In addition, the compound (47) can also be obtained by sequentially repeating the reaction and removing the protection of the constituent amino acid or peptide for elongation.

By removing the protecting group P ¹² for the carboxy group of the resulting compound (47), compound (48) can be obtained. You can select reagents and conditions depending on the protecting group.

Compound (34) can be obtained by derivatizing compound (48) into an active ester, a mixed acid anhydride, an acid halide or the like. and its interaction with compound (7) in the presence of a base. For this reaction, the reagents and reaction conditions that are commonly used for peptide synthesis can also be used, and the reaction conditions, reagents, base, and solvent for use in the reaction can be suitably selected from those described for the synthesis of compound (6).

Compound (47) can also be obtained, for example, by the following method.

The peptide (49) can be obtained by derivatizing the amino acid or peptide (46) into an active ester, a mixed acid anhydride, an acid halide, or the like. and its interaction with an amino acid or peptide (31) with an N-terminus protected by a P ⁹ group and a carboxy group, a hydroxy group or an amino group in the side chain protected by a P ¹⁰ group, in the presence of a base. The reaction conditions, reagents, base and solvent used to form a peptide bond between the amino acid or peptide (46) and the amino acid or peptide (31) can be suitably selected from those described for the synthesis of compound (6). However, in this case, it is necessary that the protecting group P ¹² for the carboxy group of the amino acid or peptide (46) and the protecting group P ⁹ and P ¹⁰ for the amino acid or peptide (31) can be removed in the same way as described above, but using different ways or conditions. For example, a representative example includes a combination in which P ⁹ is a 9-fluorenylmethyloxycarbonyl group, P ¹⁰ is a t-butyl group, or the like. as a protecting group for a carboxy group, a methoxymethyl group, or the like. as a protecting group for a hydroxy group, or a t-butyloxycarbonyl group as a protecting group, or the like. for an amino group, and P ¹² represents a benzyl group. The protecting group P ¹⁰ for a functional group in the side chain is preferably a protecting group that can be removed by treatment under acidic conditions. However, it is not limited to this, and may be selected from the above groups depending on, for example, the properties of the amino group, carboxy groups or hydroxy group of the compound to be protected. To remove the protecting group, you can select reagents and conditions depending on the protecting group.

By removing the P ⁹ protecting group from the N-terminal portion of the resulting peptide (49), compound (50) can be obtained. You can select reagents and conditions depending on the protecting group.

Compound (47) can be obtained by derivatizing a carboxylic acid derivative (11) into an active ester, a mixed acid anhydride, an acid halide or the like. and its interaction with the resulting peptide (50) in the presence of a base. The reaction conditions, reagents, base and solvent used to form the amide bond between the carboxylic acid derivative (11) and the peptide (50) can be suitably selected from those described for the synthesis of compound (6).

[0170]

6. Production method 6

Within intermediate (2), compounds in which the linker has the structure represented as -L ¹ -L ² -L ^P - and said L ^P is a peptide residue containing a hydrophilic amino acid at the N-terminus and said hydrophilic amino acid located at the N-terminus, other than glycine, can also be obtained in the following way.

[0171]

[Chemical Formula 28]

[0172]

[in this formula, L ^1' corresponds to L ¹ having a structure in which the terminal group is modified to form a maleimidyl group, L ^P is a structure consisting of -L ^p1 -L ^p2 -, and P ⁸ , P ⁹ , P ¹⁰ and P ¹² are a protecting group].

[0173]

Since L ^P is formed by linking L ^p1 to L ^p2 , the hydrophilic amino acid at the N-terminus of L ^P is derived from L ^p1 , and thus those containing the hydrophilic amino acid at the N-terminus are suitably used as L ^p1 . In this case, several hydrophilic amino acids may be present. In addition, when L ^p2 with a hydrophilic amino acid is used, an L ^p containing several hydrophilic amino acids at the N-terminus of the L ^P or at the N-terminus and other positions can be obtained depending on the position of the hydrophilic amino acid.

Compound (51) can be obtained by derivatizing a peptide or amino acid (28) described in Production Method 5 with an N-terminal group protected by a P ⁸ group into an active ester, a mixed acid anhydride or the like. and interactions with compound (4) and its salt. The reaction conditions, reagents, base and solvent used to form a peptide bond between the peptide or amino acid (28) and compound (4) can be suitably selected from those described for the synthesis of compound (6). The protecting group P ⁸ can be suitably selected and used from those described as the protecting group for the compound (6), and can be selected depending on, for example, the property of the compound containing the amino group to be protected. In addition, as is commonly used for peptide synthesis, by repeating the reaction sequentially and deprotecting the amino acid or peptide constituting the peptide or amino acid (28) for elongation, compound (51) can also be obtained.

By removing the protecting group P ⁸ for the amino group of the resulting compound (51), compound (52) can be obtained. For this deprotection procedure, you can select reagents and conditions depending on the protecting group.

Compound (53) can be obtained by derivatizing an amino acid or peptide (31) with an N-terminus protected by a P ⁹ group and a carboxy group, a hydroxy group or an amino group in the side chain protected by a P ¹⁰ group, as described in Production Method 4, in an active ester, a mixed acid anhydride, or the like. and its interaction with the resulting compound (52). The reaction conditions, reagents, base and solvent used to form a peptide bond between the amino acid or peptide (31) and compound (52) can be suitably selected from those described for the synthesis of compound (6). Protecting groups P ⁹ and P ¹⁰ are the same as those described in Preparation Method 5. In addition, as is commonly used for peptide synthesis, compound (53) can also be obtained by repeating the reaction and deprotecting the amino acid constituent or peptide for elongation in sequence.

By removing the P ⁹ group as a protecting group for the amino group of the resulting compound (53), compound (54) can be obtained. For this deprotection procedure, you can select reagents and conditions depending on the protecting group.

The compound can be obtained (55) by derivatizing the carboxylic acid derivative (11) to an active ester, a mixed acid anhydride, an acid halide or the like. and its interaction with the resulting compound (54). The reaction conditions, reagents, base and solvent used to form a peptide bond between the carboxylic acid derivative (11) and compound (54) can be suitably selected from those described for the synthesis of compound (6).

By removing the protecting group P ¹⁰ for the carboxy group, hydroxy group or amino group of the obtained compound (55), compound (2) can be obtained. For this deprotection procedure, you can select reagents and conditions depending on the protecting group.

[0174]

Compound (53) can also be obtained, for example, by the following method.

By removing the protecting group P ¹² for the carboxy group of compound (49) described in Production Method 5, peptide (56) can be obtained. For this deprotection procedure, you can select reagents and conditions depending on the protecting group.

Compound (53) can be obtained by derivatizing the resulting peptide (56) into an active ester, a mixed acid anhydride, an acid halide or the like. and its interaction with the compound (4) or its salt. The reaction conditions, reagents, base and solvent used to form a peptide bond between compound (56) and compound (4) can be suitably selected from those described for the synthesis of compound (6).

[0175]

Compound (55) can also be obtained, for example, by the following method.

Compound (55) can be produced by derivatizing compound (48) described in Production Method 5 to an active ester, mixed acid anhydride, or the like. and reacting it with compound (4) in the presence of a base, or by derivatizing the amino acid or peptide (45) described in Production Method 5 into an active ester, a mixed acid anhydride, or the like. and its interaction with compound (52) in the presence of a base. The reaction conditions, reagents, base and solvent used to form each peptide bond can be suitably selected from those described for the synthesis of compound (6).

[0176]

7. Production method 7

Within the intermediate compound represented by formula (2), compounds containing the linker structure -L ¹ -L ² -L ^P -NH-(CH ₂ )n ¹ -L ^a -NH-(CH ₂ )n ² -C(= O)-, where the specified L ^P is a peptide residue containing a hydrophilic amino acid at the N-terminus, and the specified hydrophilic amino acid located at the N-terminus other than glycine, can also be obtained by the following method.

[0177]

[Chemical Formula 29]

[0178]

[in this formula, L ^1' corresponds to L ¹ having a structure in which the terminal group is modified to form a maleimidyl group, L ^P is a structure consisting of -L ^p1 -L ^p2 -, and P ⁹ and P ¹³ are protective group].

[0179]

The intermediate compound represented by formula (2) includes the following two variants, that is, the structure in which the linker is represented as -L ¹ -L ² -L ^P -NH-(CH ₂ )n ¹ -L ^a -NH-(CH ₂ )n ² -C(=O)-, and a structure in which the linker is represented as -L ¹ -L ² -L ^P -.

Compound (2) with a structure in which the linker is represented as -L ¹ -L ² -L ^P -NH-(CH ₂ )n ¹ -L ^a -NH-(CH ₂ )n ² -C(=O)-, can be obtained in the following way.

Compound (57) can be synthesized in the same manner as compound (32) described in Production Method 5. However, unlike compound (32), it is not necessary that in order to remove the P ⁹ protecting group for the amino group and the P ¹³ protecting group for functional group in the side chain were used in different ways or different conditions. The functional group in the side chain is a carboxy group or a hydroxy group, and the P ⁹ protecting group for an amino group and the P ¹³ protecting group for a carboxy group or a hydroxy group in the side chain can be removed at the same time. For example, a representative example includes a combination in which P ⁹ is a t-butyloxycarbonyl group and P ¹³ is a t-butyl group or a trityl group, or P ³ is a benzyloxy carbonyl group and P ¹³ is a benzyl group. The protecting groups can be suitably selected from the above groups relating to the protecting groups for the compound (6) depending on, for example, the properties of the amino group, carboxy group or hydroxy group of the compound to be protected. For deprotection, reagents and conditions are selected depending on the protecting group. Using a protected amino acid or peptide that satisfies the above properties, compound (57) can be synthesized in the same manner as Production Method 5.

By successively or simultaneously removing the protecting groups P ⁹ and P ¹³ of compound (57), compound (51) can be obtained. You can select reagents and conditions depending on the protecting group.

The functional group in the hydrophilic side chain L ^P in compound (58) is not particularly protected, however, by reacting with compound (11) derivatized to an active ester, mixed acid anhydride or the like in the presence of a base, compound (2 ). The reaction conditions, reagents, base and solvent used to form each peptide bond can be suitably selected from those described for the synthesis of compound (6).

Compound (2) with a structure in which the linker is represented as -L ¹ -L ² -L ^P - can be obtained by the following method.

Compound (59) can also be synthesized in the same manner as compound (53) described in Production Method 6. However, unlike compound (53), it is not necessary that P³ for amino group and protecting group P⁸ different methods or different conditions were used for the functional group in the side chain. The functional group in the side chain is a carboxy group or a hydroxy group, and the protecting group P⁹ for the amino group and the protecting group P¹³ for the carboxy group or the hydroxy group in the side chain can be removed at the same time. For example, a representative example includes a combination in which P⁹is a tert-butyloxycarbonyl group and P¹³ is a tert-butyl group or a trityl group, or P³represents benzyloxy carbonyl group and P¹³ is a benzyl group. The protecting groups can be suitably selected from the above groups relating to the protecting groups for the compound (6) depending on, for example, the properties of the amino group, carboxy group or hydroxy group of the compound to be protected. To remove the protecting groups, the reagents and conditions are selected depending on the protecting group. Using a protected amino acid or peptide that satisfies the above properties, compound (59) can be synthesized in the same manner as Production Method 6.

By sequential or simultaneous removal of the protecting groups P ⁹ and P ¹³ of compound (59), compound (53) can be obtained. You can select reagents and conditions depending on the protecting group.

The functional group in the hydrophilic side chain L ^P in compound (60) is not particularly protected. However, by reacting with compound (11) derivatized to an active ester, mixed acid anhydride or the like in the presence of a base, a compound (2 Reaction conditions, reagents, base and solvent used to form each peptide bond can be suitably choose from those described for the synthesis of compound (6).

[0180]

8. Production method 8

Compound (43) shown in Production Method 5, in which the -L ^P - linker has the structure -L ^p1 -Gly-Gly-Phe-Gly-, can also be produced by the following method.

[0181]

[Chemical Formula 30]

[0182]

[in this formula, P ⁹ and P ¹⁰ represent a protecting group].

[0183]

Compound (62) can be produced by derivatizing the amino acid or peptide (31) described in Production Method 5 into an active ester, a mixed acid anhydride, an acid halide or the like. and its interactions with glycylglycyl-L-phenylalanyl-N-[(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13 ,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl]glycinamide (that is, the free form of the pharmaceutical compound disclosed in International Publication No. WO 1997/46260) (61) or a salt thereof in the presence of a base. The reaction conditions, reagents, base and solvent used to form a peptide bond between the amino acid or peptide (31) and compound (61) can be suitably selected from those described for the synthesis of compound (6). The protecting group P ³ for the N-terminal group and the protecting group P ¹⁰ for the functional group in the side chain are the same as those described in Production Method 5. However, the protecting group P ¹⁰ for the functional group in the side chain may not be present, and by carrying out the reaction using the amino acid or peptide (31) with only the N-terminal group protected, compound (62) can be obtained.

[0184]

9. Production method 9

Of the compounds represented by formula (2), a compound in which the linker has the structure represented as -L ¹ -L ² -L ^P - and said L ^P is an oligopeptide in which the C-terminus is 2 or 3 or more glycines and is linked to a drug, and the N-terminus of said peptide residue is glycine in the case where a hydrophilic amino acid is present at the N-terminus, can also be obtained according to the following method.

[0185]

[Chemical Formula 31]

[0186]

[in this formula, L ^1' corresponds to L ¹ having a structure in which the terminal group is converted to a maleimidyl group, L ^P is a structure consisting of L ^p1 -L ^p2 , and P ¹² and P ¹⁴ represent a protecting group].

[0187]

Since L ^P is formed by linking L ^p1 with L ^p2 , the amount of C-terminal glycine of L ^P contained therein can be calculated taking into account the amount of glycine at the C-terminus of L ^P and the amount of reuse during the reaction.

Peptide (63) is an oligopeptide in which the C-terminus is 2 or 3 or more glycines and the N-terminus is glycine when the N-terminus of said peptide residue is a hydrophilic amino acid and, in addition, said N the end is protected by the P ¹⁴ group. As is commonly used for peptide synthesis, peptide (63) can be synthesized by successively repeating the condensation reaction of a constituent amino acid or peptide and deprotection.

Compound (64) can be obtained by derivatizing peptide (63) into an active ester, a mixed acid anhydride or the like. and its interaction with the compound (4) or its salt. The reaction conditions, reagents, base and solvent used to form a peptide bond between the peptide (63) and compound (4) can be suitably selected from those described for the synthesis of compound (6). The protecting group P ¹⁴ can be suitably selected and used from those described for the synthesis of compound (6).

Compound (64) can also be obtained by derivatizing an amino acid or peptide (65) with an N-terminal group protected by a P ¹⁴ group into an active ester, a mixed acid anhydride or the like. and its interaction with compound (52) described in Preparation Method 6. The reaction conditions, reagents, base, and solvent used to form a peptide bond between an amino acid or peptide (65) and compound (52) may be suitably selected from those described for the synthesis of compound (6). The protecting group P ¹⁴ can be suitably selected and used from those described for the synthesis of compound (6).

By removing the protecting group P ¹⁴ for the amino group of the obtained compound (64), compound (66) can be obtained. You can select reagents and conditions depending on the protecting group.

The compound (2) can be obtained by derivatizing the carboxylic acid derivative (11) into an active ester, a mixed acid anhydride, an acid halide or the like. and its interaction with the resulting compound (66). The reaction conditions, reagents, base and solvent used to form an amide bond between the carboxylic acid derivative (11) and compound (66) can be suitably selected from those described for the synthesis of compound (6).

Compound (2) can also be obtained by the following method.

In compound (67), glycine at the N-terminus of L ^p1 is linked to L ² , and can be obtained in the same manner as compound (45) described in Production Method 5. Compound (68) can be obtained by derivatization of amino acid or peptide (46) described in Production Method 5 into an active ester, a mixed acid anhydride, an acid halide or the like. and interactions with compound (67). Here, the amino acid or peptide (46) is an oligopeptide consisting of glycine or containing a C-terminus consisting of 2 or 3 or more glycines, in which the C-terminus is protected by a P ¹² group. The reaction conditions, reagents, base and solvent used to form an amide bond between the amino acid or peptide (46) and compound (67) can be suitably selected from those described for the synthesis of compound (6).

Compound (68) can also be obtained by derivatizing compound (11) into an active ester, a mixed acid anhydride or the like. and its interaction with a peptide (69) containing a C-terminus protected by a P ¹² group. Here, peptide (69) is an oligopeptide in which the C-terminus is composed of 2 or 3 or more glycines, and the N-terminus is glycine in the case where the N-terminus of said peptide residue is a hydrophilic amino acid. As is commonly used for peptide synthesis, peptide (69) can be obtained by successively repeating the condensation reaction of a constituent amino acid or peptide and deprotection. The reaction conditions, reagents, base and solvent used to form a peptide bond between the peptide (69) and compound (11) can be suitably selected from those described for the synthesis of compound (6). The protecting group P ¹² is preferably a protecting group that can be removed under acidic conditions, but is not limited to, and can be suitably selected and used from those described for the synthesis of compound (6).

By removing the protecting group P ¹² for the carboxy group of the resulting compound (68), compound (70) can be obtained. For this deprotection procedure, you can select reagents and conditions depending on the protecting group.

Compound (2) can be obtained by derivatizing compound (70) to an active ester, a mixed acid anhydride or the like. and its interaction with the compound (4) or its salt. The reaction conditions, reagents, base and solvent used to form a peptide bond between compound (70) and compound (4) can be suitably selected from those described for the synthesis of compound (6).

In addition to the above, the compound (2) can also be obtained according to the following method.

Compound (2) can be produced by derivatizing compound (52) described in Production Method 6 to an active ester, a mixed acid anhydride or the like. and its interaction with compound (67) in the presence of a base. The reaction conditions, reagents, base and solvent used to form a peptide bond between compound (67) and compound (52) can be suitably selected from those described for the synthesis of compound (6).

[0188]

However, all of the intermediates of Methods 1-9 may be present in salt and/or hydrate form.

[0189]

The antibody-drug conjugate of the present invention, when exposed to air or subjected to purification procedures such as recrystallization, may, for example, absorb moisture and contain adsorbed water or hydrate. Such a compound and a salt containing water are also included in the present invention.

A compound labeled with various radioactive or non-radioactive isotopes is also included in the present invention. One or more of the atoms contained in the antibody-drug conjugate of the present invention may contain an atomic isotope in a ratio that does not exist in nature. Examples of an atomic isotope include deuterium ( ² H), tritium ( ³ H), iodine-125 ( ¹²⁵ I) and carbon-14 ( ¹⁴ C). In addition, the compound of the present invention can be labeled with a radioactive isotope such as tritium ( ³ H), iodine-125 ( ¹²⁵ I), carbon-14 ( ¹⁴ C), copper-64 ( ⁶⁴ Cu), zirconium-89 ( ⁸⁹ Zr), iodine-124 ( ¹²⁴ I), fluorine-18 ( ¹⁸ F), indium-111 ( ¹¹¹ I), carbon-11 ( ¹¹ C) and iodine-131 ( ¹³¹ I). A radioactively labeled compound is useful as a therapeutic or prophylactic agent, a research reagent such as an assay reagent, and a diagnostic agent such as an in vivo diagnostic imaging agent. Without being associated with radioactivity, any isotopic variant of the antibody-drug conjugate of the present invention is within the scope of the present invention.

[0190]

{Pharmaceutical/drugs}

The anti-HER3 antibody-drug conjugate of the present invention exhibits cytotoxic activity against cancer cells, and thus can be used as a drug, especially as a therapeutic and/or prophylactic for cancer.

Namely, the anti-HER3 antibody-drug conjugate of the present invention can be selectively used as a chemotherapy drug, which is the main method for treating cancer, and as a result, it is possible to delay the development of cancer cells, inhibit their growth, and then destroy cancer cells. This may provide cancer patients with the ability to not have symptoms caused by cancer, or the ability to achieve an improvement in the QOL of cancer patients and achieve a therapeutic effect while maintaining the life of cancer patients. Even if the anti-HER3 antibody-drug conjugate of the present invention does not reach the level of destruction of cancer cells, it is able to achieve a higher QOL of cancer patients to achieve their longer survival period by inhibiting or controlling the growth of cancer cells.

In such drug therapy, the anti-HER3 antibody-drug conjugate of the present invention can be used as a drug alone. In addition, the anti-HER3 antibody-drug conjugate of the present invention can be used as a drug in combination with additional therapy in adjuvant therapy, and can be used in combination with surgery, radiation therapy, hormonal therapy or the like. In addition, the anti-HER3 antibody-drug conjugate of the present invention can also be used as a neoadjuvant drug therapy drug.

In addition to the therapeutic use described above, the effect of suppressing the growth of a minor amount of metastatic cancer cells and then killing them can also be expected. In particular, when HER3 expression is confirmed in primary cancer cells, cancer metastasis inhibition or a prophylactic effect can be expected when the anti-HER3 antibody drug conjugate of the present invention is administered. For example, an effect of inhibiting and killing cancer cells in body fluid upon metastasis formation, or an effect such as inhibiting and killing minor amounts of cancer cells immediately after implantation in any tissue can be expected. In addition, inhibition of cancer metastases or a prophylactic effect, in particular after surgical removal of the cancer, can be expected.

The anti-HER3 antibody-drug conjugate of the present invention can be expected to exhibit a therapeutic effect when administered to patients as a systemic therapy, as well as when administered topically to cancerous tissues.

The antibody drug conjugate (1) was excellent in antitumor activity, safety and physical properties, and showed anticancer effect against breast cancer, lung cancer and melanoma in vitro.

The antibody-drug conjugate (2) had excellent antitumor activity, safety and physical properties, and showed anticancer effect against breast cancer, lung cancer, colorectal cancer and melanoma in vitro and stronger anticancer effect against breast cancer and melanoma in vivo compared to U1-59.

The antibody-drug conjugate (3) had excellent antitumor activity, safety and physical properties, and showed anticancer effect against breast cancer, lung cancer, ovarian cancer, colorectal cancer and melanoma in vitro and stronger anticancer effect against breast cancer, lung cancer , gastric cancer and melanoma in vivo compared to U1-59.

The antibody-drug conjugate (4) was excellent in antitumor activity, safety and physical properties, and showed an anticancer effect against breast cancer in vitro.

The antibody-drug conjugate (5) was excellent in antitumor activity, safety and physical properties, and showed anticancer effect against breast cancer, lung cancer and melanoma in vitro.

The antibody-drug conjugate (6) had excellent antitumor activity, safety and physical properties, and showed an anticancer effect against breast cancer, lung cancer and melanoma in vitro and a stronger anticancer effect against breast cancer in vivo compared to U1-59.

The antibody-drug conjugate (7) had excellent antitumor activity, safety and physical properties, and showed an anticancer effect against breast cancer, lung cancer and melanoma in vitro.

The antibody-drug conjugate (8) had excellent antitumor activity, safety and physical properties, and showed an anticancer effect against breast cancer, lung cancer and melanoma in vitro and a stronger anticancer effect against breast cancer in vivo compared to U1-59.

The antibody drug conjugate (9) was excellent in antitumor activity, safety and physical properties, and showed anticancer effect against breast cancer, lung cancer, ovarian cancer, colorectal cancer and melanoma in vitro.

The antibody-drug conjugate (10) had excellent antitumor activity, safety and physical properties, and showed anticancer effect against breast cancer, lung cancer, colorectal cancer and melanoma in vitro and stronger anticancer effect against breast cancer, lung cancer, colorectal cancer , gastric cancer and melanoma in vivo compared with U1-59.

The antibody-drug conjugate (11) was excellent in antitumor activity, safety and physical properties, and showed an anticancer effect against breast cancer in vitro.

The antibody-drug conjugate (12) had excellent antitumor activity, safety and physical properties, and showed anticancer effect against breast cancer, lung cancer, ovarian cancer, colorectal cancer and melanoma in vitro.

The antibody-drug conjugate (13) had excellent antitumor activity, safety and physical properties, and showed anticancer effect against breast cancer, lung cancer, colorectal cancer and melanoma in vitro and stronger anticancer effect against breast cancer (including triple negative breast cancer). gland), lung cancer, gastric cancer, pancreatic cancer and melanoma in vivo compared to U1-59.

The antibody-drug conjugate (14) had excellent antitumor activity, safety and physical properties, and showed an anticancer effect against breast cancer in vitro .

The antibody-drug conjugate (16a) had excellent antitumor activity, safety and physical properties, and showed anticancer effect against breast cancer (including luminal and triple negative), melanoma, ovarian cancer, bladder cancer, lung cancer, head and neck cancer and gastric cancer in vivo when administered alone or in combination with trastuzumab, gefinitib, cetuximab, panitumumab, or pertuzumab.

[0191]

Examples of the type of cancer to which the anti-HER3 antibody-drug conjugate of the present invention is applied include lung cancer, kidney cancer, urothelial cancer, colorectal cancer, prostate cancer, glioblastoma multiforme, ovarian cancer, pancreatic cancer, breast cancer, metastatic breast cancer, luminal breast cancer, melanoma, liver cancer, bladder cancer, gastric cancer (stomach cancer), gastrointestinal stromal tumor, cervical cancer, head and neck cancer, esophageal cancer, epidermoid cancer, peritoneal cancer, glioblastoma multiforme adult, hepatic cancer, hepatocellular carcinoma, colon cancer, rectal cancer, colon and rectal cancer, endometrial cancer, uterine cancer, salivary gland cancer, renal cancer, vulvar cancer, thyroid cancer, hepatic carcinoma, anal carcinoma, cancer penis. Chemotherapy is the only existing treatment indicated especially for triple negative breast cancer (which lacks the expression of HER2, estrogen and progesterone receptors) of breast cancer types considered to have a poor prognosis. There have been almost no reports regarding HER3 expression in triple negative breast cancer. However, if HER3 expression is observed in triple negative breast cancer patients, the anti-HER3 antibody drug conjugate of the present invention can be used as a therapeutic agent and/or a prophylactic agent. However, the cancer is not limited to these types, as long as it is a cancer cell expressing, in the cancer cell as an object of treatment, a protein that the antibody from the antibody-drug conjugate can recognize.

Treatment using the anti-HER3 antibody-drug conjugate of the present invention can be targeted to a cancer cell expressing, in the cancer cell being treated, a HER3 protein that the antibody from the antibody-drug conjugate can recognize. In the description of the present invention, "a cancer expressing HER3 protein" means a cancer including cells containing HER3 protein on their surface, or a cancer that secretes HER3 protein into the blood. The HER3 protein is overexpressed in various human tumors and can be determined using a method that is commonly used, such as the immunohistochemical staining (IHC) method for determining the overexpression of HER3 protein in tumor samples (primary, metastatic), the fluorescent in situ hybridization (FISH) method. ) to determine the amplification of the HER3 gene or enzyme-linked immunosorbent assay (ELISA) to determine the overexpression of the HER3 protein in blood samples.

The anti-HER3 antibody-drug conjugate of the present invention exhibits an antitumor effect by recognizing and then internalizing the HER3 protein expressed on the surface of a cancer cell. Thus, the object of treatment of the anti-HER3 antibody-drug conjugate of the present invention is not limited to "cancer expressing HER3 protein", and it may also be, for example, leukemia, malignant lymphoma, plasmacytoma, myeloma or sarcoma.

[0192]

The anti-HER3 antibody-drug conjugate of the present invention may preferably be administered to a mammal, but more preferably administered to a human.

[0193]

Substances used in the pharmaceutical composition containing the anti-HER3 antibody-drug conjugate of the present invention can be appropriately selected and used from formulation additives or the like that are commonly used in the art, considering dose or concentration used to administer.

[0194]

The anti-HER3 antibody drug conjugate of the present invention may be administered as a pharmaceutical composition containing at least one pharmaceutically acceptable ingredient. For example, the pharmaceutical composition typically contains at least one pharmaceutical carrier (eg, a sterilized liquid). As described in this application, examples of the liquid include water and oil (mineral oil and oil of animal origin, vegetable origin or synthetic origin). The oil may be, for example, peanut oil, soybean oil, mineral oil, sesame oil or the like. Water is the more typical carrier when the pharmaceutical composition is administered intravenously. Physiological saline solution, aqueous dextrose solution and aqueous glycerol solution can also be used as a liquid carrier, in particular in an injection solution. A suitable pharmaceutical carrier may suitably be selected from those known in the art. If desired, the composition may also contain trace amounts of a humectant, emulsifier or pH buffering agent. Examples of a suitable pharmaceutical carrier are disclosed in "Remington's Pharmacetical Sciences" by E. W. Martin. The compositions correspond to the route of administration.

[0195]

Various delivery systems are known and can be used to administer the anti-HER3 antibody drug conjugate of the present invention. Examples of routes of administration include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, and subcutaneous routes. Administration can be by injection or bolus injection, for example. According to a particular preferred embodiment, administration of the antibody-drug conjugate is by injection. Parenteral administration is the preferred route of administration.

[0196]

According to a representative embodiment, the pharmaceutical composition is intended 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 medicinal product may contain a solubilizing agent and local anesthetics to relieve pain at the injection site (eg, lignocaine). Typically, the ingredient above is provided individually either as a lyophilized powder or an anhydrous concentrate contained in a container, which is obtained as a hermetically sealed ampoule or sachet containing a certain amount of active substance, or as a mixture in a unit dosage form. When the drug is in a form for administration by injection, it can be administered from an injection vial that contains sterile pharmaceutical grade water or saline. When the drug is administered by injection, an ampoule of sterile water or saline for injection can be provided so that the above ingredients are mixed together before administration.

[0197]

The pharmaceutical composition of the present invention may only include an anti-HER3 antibody-drug conjugate disclosed herein as an active ingredient, or may include an anti-HER3 antibody-drug conjugate and at least one drug (e.g., an agent for cancer treatment) in addition to the conjugate. The anti-HER3 antibody-drug conjugate of the present invention can be administered with another cancer treatment agent, and the anti-cancer effect can be enhanced accordingly. For example, another drug, such as an anti-cancer agent used for this purpose, can be administered before administration of a pharmaceutical composition comprising an antibody-drug conjugate of the present invention as an active ingredient, or after administration of a pharmaceutical composition comprising an antibody-drug conjugate in as an active ingredient, or can be administered simultaneously with the anti-HER3 antibody-drug conjugate, separately (individually) or after the administration of the antibody-drug conjugate, and the administration can be carried out by varying the interval between injections for each. In the present invention, the case where the anti-HER3 antibody-drug conjugate of the present invention is administered simultaneously with another drug in the form of a single preparation containing the antibody-drug conjugate and this drug, and the case where the anti-HER3 antibody-drug conjugate is the drug of the present invention and the other drug are administered simultaneously or sequentially as different preparations, or administered by varying the administration interval for each, both are included in the scope of "a pharmaceutical composition comprising an antibody-drug conjugate and another drug". Examples of a cancer treatment agent include 5-FU, trastuzumab, trastuzumab emtansine (T-DM1), cetuximab, gefitinib, panitumumab, pertuzumab, abraxan, erlotinib, carboplatin, cisplatin, gemcitabine, capecitabine, irinotecan (CPT-11), paclitaxel, docetaxel , pemetrexed, sorafenib, vinblastine, vinorelbine, vermurafenib, drugs described in International Publication No. WO 2003/038043, LH-RH analogs (leuprorelin, goserelin or the like), estramustine phosphate, estrogen antagonist (tamoxifen, raloxifene or t .p.) and an aromatase inhibitor (anastrozole, letrozole, exemestane or the like), but are not limited to this, provided that it is a drug that has antitumor activity. These cancer treatments can be classified, according to their targets, into: anti-FGER agents such as cetuximab, gefitinib, and panitumumab; anti-HER2 agents such as trastuzumab, T-DM1 and pertuzumab; anti-HER3 agents such as patritumab, MM-121 and MM-111; anti-VEGF agents such as infliximab and adalimumab; etc. In addition, they can be classified into: anti-EGFR antibodies such as cetuximab and panitumumab; anti-HER2 antibodies such as trastuzumab and pertuzumab; anti-HER3 antibodies such as patritumab, MM-121 and MM-111; anti-VEGF antibodies such as infliximab and adalimumab; etc. The anti-HER3 antibody-drug conjugate of the present invention exhibits excellent therapeutic effect when administered in combination with an anti-HER2 agent or an anti-HER2 antibody in i) treatment of gastric cancer, breast cancer, triple negative breast cancer or the like. or when administered in combination with an anti-EGFR agent or an anti-EGFR antibody in ii) the treatment of lung cancer, head and neck cancer, gastric cancer, breast cancer, triple negative breast cancer, or the like. One or two or more drugs may be used in addition to the conjugate, and these drugs may be anti-cancer agents or may be drugs to alleviate side effects caused by additional drugs.

[0198]

In the present invention, "a pharmaceutical composition comprising an anti-HER3 antibody-drug conjugate and another drug" has the same meaning as "a pharmaceutical composition in which the anti-HER3 antibody-drug conjugate is to be administered in combination with another drug." means." In the present invention, the phrase "administered in combination" used for the anti-HER3 antibody-drug-other drug conjugate means that the anti-HER3 antibody-drug-other drug conjugate is administered to the recipient within a certain period of time. A single preparation containing the anti-HER3 antibody-drug conjugate and another drug may be administered, or the anti-HER3 antibody-drug conjugate and another drug may be separately formulated and may be administered as separate preparations. In the case of individual preparations, the time of their administration is not particularly limited, and these preparations may be administered at the same time or may be administered at different times or on different days alternately. In the case where the anti-HER3 antibody-drug conjugate and the other drug are administered separately at different times or on different days, the order of their administration is not particularly limited. Since individual drugs are usually administered according to their respective routes of administration, the frequency of their administration may be the same or may be different. In addition, such separate preparations can be administered by the same route of administration (administration route) or by different routes of administration (administration routes). It is not necessary that the anti-HER3 antibody-drug conjugate and the other drug exist in the body at the same time, and it is sufficient that the anti-HER3 antibody-drug conjugate and the other drug be administered to the body within a certain period (e.g. , 1 month, preferably 1 week, more preferably several days, even more preferably 1 day). Alternatively, when one of the active ingredients is administered, the other active ingredient may have already disappeared from the body.

Examples of a dosage form of a "pharmaceutical composition in which an anti-HER3 antibody-drug conjugate is administered in combination with another drug" may include: 1) administering a single formulation comprising an anti-HER3 antibody-drug conjugate and another drug, 2 ) simultaneous administration by the same route of administration of two drugs obtained by separately formulating the anti-HER3 antibody-drug conjugate and another drug, 3) administration alternately by the same route of administration of two drugs obtained by separately formulating the anti-HER3 conjugate an antibody-drug and another drug, 4) simultaneous administration by different routes of administration of two drugs obtained by separately formulating an anti-HER3 conjugate of an antibody-drug and another drug, and 5) administration alternately by different routes of administration of two drugs obtained by separately formulating an anti-HER3 antibody-drug conjugate and another drug. Dose, dosing interval, dosage form, preparation, etc., of the "pharmaceutical composition in which the anti-HER3 antibody-drug conjugate is administered in combination with another drug" corresponds to the pharmaceutical composition containing the anti-HER3 antibody-drug conjugate according to the present invention, but are not limited to this.

Such a pharmaceutical composition, formulated as two different preparations, may be in the form of a kit containing them.

In the present invention, "combination" of an anti-HER3 antibody-drug conjugate and another drug means that the anti-HER3 antibody-drug-drug conjugate is "administered in combination".

[0199]

The pharmaceutical composition can be formulated as a lyophilized preparation or liquid preparation, in the form of a preparation containing the desired composition and having the required purity. When the composition is formulated as a lyophilized preparation, it may be a preparation containing additives suitable for formulating, which are used in this field. Also, with regard to the liquid composition, it can be formulated as a liquid preparation containing various additives for formulation, which are used in this field.

[0200]

The composition and concentration of the pharmaceutical composition may vary depending on the route of administration. However, the anti-HER3 antibody drug conjugate contained in the pharmaceutical composition of the present invention can exhibit a pharmaceutical effect even at a low dose when the antibody drug conjugate has a higher antigen affinity, i.e. higher affinity (=lower Kd value) expressed as the dissociation constant (i.e. Kd value) for the antigen. Thus, with regard to determining the dose of the antibody-drug conjugate, the dose can be determined in light of the situation regarding the affinity between the antibody-drug conjugate and the antigen. When the antibody-drug conjugate of the present invention is administered to a human, for example, about 0.001-100 mg/kg can be administered once or administered several times at an interval of once within 1-180 days.

Examples

[0201]

The present invention is more specifically described in light of the examples below. However, the present invention is not limited to them. In addition, this should not be considered as a limitation in any way. In addition, unless specifically stated otherwise, the reagent, solvent, and starting material described in the specification are readily available from a commercial supplier.

[0202]

Reference Example 1 Obtaining U1-59

U1-59 was obtained in accordance with the method described in International Publication No. WO 2007/077028.

[0203]

Example 1 Antibody drug conjugate (1)

[Chemical Formula 32]

[0204]

Stage 1: tert-Butyl (4-{[(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15 -hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl]amino}-4-oxobutyl)carbamate

4-(tert-Butoxycarbonylamino)butanoic acid (0.237 g, 1.13 mmol) was dissolved in dichloromethane (10 mL), N-hydroxysuccinimide (0.130 g, 1.13 mmol) and 1-ethyl-3-(3-dimethylaminopropyl) were charged )carbodiimide hydrochloride (0.216 g, 1.13 mmol) and stirred for 1 hour. The reaction solution was added dropwise to a solution of N,N-dimethylformamide (10 ml) containing exatecan mesylate (0.500 g, 0.94 mmol) and triethylamine (0.157 ml, 1.13 mmol) and stirred at room temperature for 1 day . The solvent was removed under reduced pressure, and the resulting residues were purified by silica gel column chromatography [chloroform-chloroform:methanol=8:2 (v/v)] to give the title compound (0.595 g, quantitative yield).

¹ H-NMR (400 MHz, DMSO-d ₆ ) delta: 0.87 (3H, t, J=7.2 Hz), 1.31 (9H, s), 1.58 (1H, t, J= 7.2 Hz), 1.66 (2H, t, J=7.2 Hz), 1.89-1.82 (2H, m), 2.12-2.21 (3H, m), 2, 39 (3H, s), 2.92 (2H, t, J=6.5 Hz), 3.17 (2H, s), 5.16 (1H, d, J=19.2 Hz), 5, 24 (1H, d, J=18.8 Hz), 5.42 (2H, s), 5.59-5.55 (1H, m), 6.53 (1H, s), 6.78 (1H , t, J=6.3 Hz), 7.30 (1H, s), 7.79 (1H, d, J=11.0 Hz), 8.40 (1H, d, J=8.6 Hz ).

MS (APCI) m/z: 621 (M+H) ⁺ .

[0205]

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

The compound (0.388 g, 0.61 mmol) obtained in Step 1 above was dissolved in dichloromethane (9 ml). After adding triperoxonane acid (9 ml) the mixture was stirred for 4 hours. The solvent was removed under reduced pressure, and the resulting residues were purified by silica gel column chromatography [chloroform - separated organic layer chloroform:methanol:water=7:3:1 (v/v/v)] to give the title compound (0.343 g, quantitative yield ).

¹ H-NMR (400 MHz, DMSO-d ₆ ) 0.87 (3H, t, J=7.2 Hz), 1.79-1.92 (4H, m), 2.10-2.17 ( 2H, m), 2.27 (2H, t, J=7.0 Hz), 2.40 (3H, s), 2.80-2.86 (2H, m), 3.15-3.20 (2H, m), 5.15 (1H, d, J=18.8 Hz), 5.26 (1H, d, J=18.8 Hz), 5.42 (2H, s), 5.54 -5.61(1H, m), 6.55(1H, s), 7.32(1H, s), 7.72(3H, br s), 7.82(1H, d, J=11 .0 Hz), 8.54 (1H, d, J=8.6 Hz).

MS (APCI) m/z: 521 (M+H) ⁺ .

[0206]

Step 3: N-(t-butoxycarbonyl)glycylglycyl-L-phenylalanyl-N-(4-{[(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo -2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl]amino }-4-oxobutyl)glycinamide

N-(tert-butoxycarbonyl)glycylglycyl-L-phenylalanylglycine (0.081 g, 0.19 mmol) was dissolved in dichloromethane (3 ml), N-hydroxysuccinimide (0.021 g, 0.19 mmol) and 1-ethyl-3-( 3-dimethylaminopropyl)carbodiimide hydrochloride (0.036 g, 0.19 mmol) and stirred for 3.5 hours. The reaction solution was added dropwise to a solution of N,N-dimethylformamide (1.5 ml) containing the compound (0.080 g, 0.15 mmol) obtained in Step 2 above, and stirred at room temperature for 4 hours. The solvent was removed under reduced pressure, and the resulting residues were purified by silica gel column chromatography [chloroform-chloroform:methanol=8:2 (v/v)] to give the title compound (0.106 g, 73%).

¹ H-NMR (400 MHz, DMSO-d ₆ ) delta: 0.87 (3H, t, J=7.4 Hz), 1.36 (9H, s), 1.71 (2H, m), 1 .86 (2H, t, J=7.8 Hz), 2.15-2.19 (4H, m), 2.40 (3H, s), 2.77 (1H, dd, J=12.7 , 8.8 Hz), 3.02 (1H, dd, J=14.1, 4.7 Hz), 3.08-3.11 (2H, m), 3.16-3.19 (2H, m), 3.54 (2H, d, J=5.9 Hz), 3.57-3.77 (4H, m), 4.46-4.48 (1H, m), 5.16 (1H , d, J=19.2 Hz), 5.25 (1H, d, J=18.8 Hz), 5.42 (2H, s), 5.55-5.60 (1H, m), 6 .53 (1H, s), 7.00 (1H, t, J=6.3 Hz), 7.17-7.26 (5H, m), 7.31 (1H, s), 7.71 ( 1H, t, J=5.7 Hz), 7.80(1H, d, J=11.0 Hz), 7.92(1H, t, J=5.7 Hz), 8.15(1H, d, J=8.2 Hz), 8.27 (1H, t, J=5.5 Hz), 8.46 (1H, d, J=8.2 Hz).

MS (APCI) m/z: 939 (M+H) ⁺ .

[0207]

Step 4: Glycylglycyl-L-phenylalanyl-N-(4-{[(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9, 10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl]amino}-4-oxobutyl)glycinamide trifluoroacetate

The compound (1.97 g, 2.10 mmol) obtained in Step 3 above was dissolved in dichloromethane (7 ml). After adding trifluoroacetic acid (7 ml), the mixture was stirred for 1 hour. The solvent was removed under reduced pressure and toluene was charged for azeotropic distillation. The resulting residues were purified by silica gel column chromatography [chloroform - separated organic layer chloroform:methanol:water=7:3:1 (v/v/v)] to obtain the title compound (1.97 g, 99%).

¹ H-NMR (400 MHz, DMSO-d ₆ ) delta: 0.87 (3H, t, J=7.4 Hz), 1.71-1.73 (2H, m), 1.82-1, 90 (2H, m), 2.12-2.20 (4H, m), 2.40 (3H, s), 2.75 (1H, dd, J=13.7, 9.4 Hz), 3 .03-3.09(3H, m), 3.18-3.19(2H, m), 3.58-3.60(2H, m), 3.64(1H, d, J=5, 9 Hz), 3.69 (1H, d, J=5.9 Hz), 3.72 (1H, d, J=5.5 Hz), 3.87 (1H, dd, J=16.8, 5.9 Hz), 4.50-4.56 (1H, m), 5.16 (1H, d, J=19.2 Hz), 5.25 (1H, d, J=18.8 Hz) , 5.42(2H, s), 5.55-5.60(1H, m), 7.17-7.27(5H, m), 7.32(1H, s), 7.78-7 .81(2H, m), 7.95-7.97(3H, m), 8.33-8.35(2H, m), 8.48-8.51(2H, m).

MS (APCI) m/z: 839 (M+H) ⁺ .

[0208]

Step 5: N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]glycylglycyl-L-phenylalanyl-N-(4-{[(1S,9S)- 9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4 ':6,7]indolizino[1,2-b]quinolin-1-yl]amino}-4-oxobutyl)glycinamide

To an N,N-dimethylformamide (1.2 mL) solution of the compound (337 mg, 0.353 mmol) obtained in Step 4 above were added triethylamine (44.3 mL, 0.318 mmol) and N-succinimidyl 6-maleimide hexanoate (119 .7 mg, 0.388 mmol) and stirred at room temperature for 1 hour. The solvent was removed under reduced pressure, and the resulting residues were purified by silica gel column chromatography [chloroform-chloroform:methanol=5:1 (v/v)] to give the title compound as a pale yellow solid (278.0 mg, 76% ).

¹ H-NMR (400 MHz, DMSO-d ₆ ) delta: 0.87 (3H, t, J=7.3 Hz), 1.12-1.22 (2H, m), 1.40-1, 51 (4H, m), 1.66-1.76 (2H, m), 1.80-1.91 (2H, m), 2.05-2.21 (6H, m), 2.39 ( 3H, s), 2.79 (1H, dd, J=14.0, 9.8 Hz), 2.98-3.21 (5H, m), 3.55-3.77 (8H, m) , 4.41-4.48 (1H, m), 5.15 (1H, d, J=18.9 Hz), 5.24 (1H, d, J=18.9 Hz), 5.40 ( 1H, d, J=17.1 Hz), 5.44 (1H, d, J=17.1 Hz), 5.54-5.60 (1H, m), 6.53 (1H, s), 6.99 (2H, s), 7.20-7.27 (5H, m), 7.30 (1H, s), 7.70 (1H, t, J=5.5 Hz), 7.80 (1H, d, J=11.0 Hz), 8.03 (1H, t, J=5.8 Hz), 8.08 (1H, t, J=5.5 Hz), 8.14 (1H , d, J=7.9 Hz), 8.25 (1H, t, J=6.1 Hz), 8.46 (1H, d, J=8.5 Hz).

MS (APCI) m/z: 1032 (M+H) ⁺ .

[0209]

Step 6: Antibody Drug Conjugate (1)

Antibody Recovery: U1-59 prepared in Reference Example 1 was prepared at an antibody concentration of 10 mg/ml by changing the medium to PBS6.0/EDTA using General Procedure B and General Procedure C described in Preparation Method 1. Solution (1, 00 ml) was added to a 2.0 ml polypropylene tube and loaded with an aqueous solution of 10 mM TCEP (Tokyo Chemical Industry Co., Ltd.) (0.0307 ml; 4.6 equivalents per antibody molecule) and 1 M aqueous solution dipotassium hydrogen phosphate (Nacalai Tesque, Inc.; 0.050 ml). After confirming that the solution had a pH of 7.4 +/- 0.1, the disulfide bond in the hinge portion of the antibody was restored by incubation at 37° C. for 1 hour.

Conjugation between antibody and drug-linker component: After incubating the solution in a water bath at 22°C for 10 minutes, dimethyl sulfoxide (Sigma-Aldrich Co. LLC; 0.0586 ml) and dimethyl sulfoxide solution (0.0615 ml; 9.2 equivalents) were added per antibody molecule) containing 10 mM of the compound obtained in Step 5 above and incubated in a water bath at 22° C. for 40 minutes to conjugate the drug-linker component to the antibody. An aqueous solution (0.0123 ml) of 100 mM NAC (Sigma-Aldrich Co. LLC) was then added and mixed using a tube rotator (MTR-103, manufactured by AS ONE Corporation) at room temperature for 20 minutes to stop the drug- linker.

Purification: The above solution was purified using General Procedure D (ABS used as buffer solution) described in Preparation Method 1 to obtain 6 ml of a solution containing the title antibody drug conjugate.

Physico-chemical characterization: Using General procedure E described in Production Method 1 (E _D.280 =7280 and E _D.370 =23400 were used as the molar absorption coefficient of the linker drug), the following characteristic values were obtained.

Antibody concentration: 1.29 mg/mL, antibody yield: 7.74 mg (77%) and average number of conjugated drug molecules (n) per antibody molecule measured using General Procedure E: 4.9.

[0210]

Example 2 Antibody drug conjugate (2)

[Chemical Formula 33]

[0211]

Step 1: Antibody Drug Conjugate (2)

Using U1-59 obtained in Reference Example 1 and the compound obtained in Step 5 of Example 1 above, the title antibody drug conjugate was prepared in the same manner as Step 6 of Example 1.

Antibody concentration: 12.0 mg/mL, antibody yield: 226.8 mg (91%) and average number of conjugated drug molecules (n) per antibody molecule measured using General Procedure E: 4.9.

[0212]

Example 3 Antibody Drug Conjugate (3)

[Chemical Formula 34]

[0213]

Step 1: Antibody Drug Conjugate (3)

Using U1-59 prepared in Reference Example 1 and the compound obtained in Step 5 of Example 1, the title antibody drug conjugate was prepared in the same manner as Step 6 of Example 1.

Antibody concentration: 16.9 mg/mL, antibody yield: 219.7 mg (88%) and average number of conjugated drug molecules (n) per antibody molecule measured using General Procedure E: 4.9.

[0214]

Example 4 Antibody Drug Conjugate (4)

[Chemical Formula 35]

[0215]

Step 1: Antibody Drug Conjugate (4)

Antibody Recovery: U1-59 prepared in Reference Example 1 was prepared at an antibody concentration of 10 mg/ml by changing the medium to PBS6.0/EDTA using General Procedure B and General Procedure C described in Preparation Method 1. Solution (1, 00 ml) was added to a 1.5 ml polypropylene tube and loaded with an aqueous solution of 10 mM TCEP (Tokyo Chemical Industry Co., Ltd.) (0.0187 ml; 2.8 equivalents per antibody molecule) and 1 M aqueous solution of dipotassium hydrogen phosphate (Nacalai Tesque, Inc.; 0.0170 ml). After confirming that the solution had a pH of 7.0 +/- 0.1, the disulfide bond in the hinge portion of the antibody was restored by incubation at 37° C. for 1 hour.

Conjugation between antibody and drug-linker component: incubated at 15° C. for 1 hour to conjugate the drug-linker component to the antibody. An aqueous solution (0.0123 ml; 18.4 equivalents per antibody molecule) of 100 mM NAC (Sigma-Aldrich Co. LLC) was then added and incubated at room temperature for an additional 20 minutes to stop the linker drug reaction.

Purification: The above solution was purified using General Procedure D (ABS used as buffer solution) described in Preparation Method 1 to obtain 6 ml of a solution containing the title antibody drug conjugate. Thereafter, the solution was concentrated using General Procedure A.

Physico-chemical characterization: Using General procedure E described in Production Method 1 (E _D.280 =5000 and E _D.370 =19000 were used as the molar absorption coefficient of the linker drug), the following characteristic values were obtained.

Antibody concentration: 1.02 mg/ml, antibody yield: 6.1 mg (61%) and average number of conjugated drug molecules (n) per antibody molecule measured using General Procedure E: 2.9; and the average number of conjugated drug molecules (n) per antibody molecule measured using General Procedure F (E _D.280 =5000 was used as the molar absorption coefficient of the linker drug): 3.2.

[0216]

Example 5 Antibody drug conjugate (5)

[Chemical Formula 36]

[0217]

Stage 1: tert-Butyl (5S,14S)-5-benzyl-1-{[(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2, 3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl]amino}-14 -{[(9H-fluoren-9-ylmethoxy)carbonyl]amino}-1,4,7,10,13-pentaoxo-3,6,9,12-tetraazahexadecane-16-oat

Under ice-cooling, to a solution in N,N-dimethylformamide (10.0 ml) of glycylglycyl-L-phenylalanyl-N-[(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10 ,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-1 -yl]glycinamide (free form of the pharmaceutical compound described in International Publication No. WO 1997/46260; 0.250 g, 0.332 mmol), N-hydroxysuccinimide (57.2 mg, 0.497 mmol) and 4-tert-butyl N-[(9H -fluoren-9-ylmethoxy)carbonyl]-L-aspartic acid (0.205 g, 0.497 mmol) N,N'-dicyclohexylcarbodiimide (0.123 g, 0.497 mmol) was added and stirred at room temperature for 2 days. The solvent was removed under reduced pressure, and the resulting residues were purified by silica gel column chromatography [chloroform-chloroform:methanol=9:1 (v/v)] to give the title compound as a pale yellow solid (0.278 g, 73%).

¹ H-NMR (400 MHz, DMSO-d ₆ ) delta: 0.86 (3H, t, J=7.1 Hz), 1.35 (9H, s), 1.79-1.90 (2H, m), 2.03-2.25(2H, m), 2.40(3H, s), 2.40-2.51(2H, m), 2.64-2.82(2H, m) , 2.98 (1H, dd, J=13.7, 4.6 Hz), 3.16 (2H, br.s), 3.55 (1H, dd, J=16.7, 5.7 Hz ), 3.63-3.80(4H, m), 4.16-4.34(3H, m), 4.36-4.50(2H, m), 5.23(2H, s), 5.37 (1H, d, J=16.5 Hz), 5.43 (1H, d, J=16.5 Hz), 5.51-5.62 (1H, m), 6.52 (1H , s), 7.10-7.25 (5H, m), 7.26-7.33 (3H, m), 7.39 (2H, t, J=7.3 Hz), 7.65- 7.72(3H, m), 7.80(1H, d, J=11.0 Hz), 7.86(2H, d, J=7.3 Hz), 7.98(1H, t, J =5.5 Hz), 8.07 (1H, d, J=7.8 Hz), 8.15 (1H, t, J=5.5 Hz), 8.31 (1H, t, J=5 .5 Hz), 8.41 (1H, d, J=8.7 Hz).

MS (ESI) m/z: 1147 (M+H) ⁺ .

[0218]

Stage 2: tert-Butyl (5S,14S)-14-amino-5-benzyl-1-{[(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13- dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl] amino}-1,4,7,10,13-pentaoxo-3,6,9,12-tetraazahexadecane-16-oate

To a solution of the compound (0.279 g, 0.242 mmol) obtained in Step 1 above in N,N-dimethylformamide (2.00 ml) was added piperidine (0.240 ml, 2.42 mmol) and stirred at room temperature for 1 hour . The solvent was removed under reduced pressure, and the resulting residues were purified by silica gel column chromatography [chloroform-chloroform:methanol=2:1 (v/v)] to give the title compound as a pale yellow solid (0.265 g, quantitative yield).

¹ H-NMR (400 MHz, DMSO-d ₆ ) delta: 0.88 (3H, t, J=7.2 Hz), 1.39 (9H, s), 1.81-1.94 (1H, m), 2.07-2.28 (2H, m), 2.37 (1H, dd, J=15.8, 8.0 Hz), 2.43 (3H, s), 2.60 (1H , dd, J=15.8, 4.9 Hz), 2.75-2.82 (1H, m), 3.00 (1H, dd, J=13.9, 4.5 Hz), 3, 16-3.25(2H, m), 3.50-3.61(2H, m), 3.65-3.81(5H, m), 4.40-4.51(1H, m), 5.27 (2H, dd, J=24.1, 19.0 Hz), 5.43 (2H, dd, J=21.3, 16.2 Hz), 5.56-5.65 (1H, m), 6.55 (1H, s), 7.15-7.28 (5H, m), 7.33 (1H, s), 7.83 (1H, d, J=11.0 Hz), 8.04 (1H, t, J=5.7 Hz), 8.09 (1H, d, J=8.2 Hz), 8.26-8.39 (2H, m), 8.44 (1H , d, J=8.2 Hz).

[0219]

Stage 3: tert-Butyl (5S,14S)-5-benzyl-14-{[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]amino}-1- {[(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[ de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl]amino}-1,4,7,10,13-pentaoxo-3,6,9,12 -tetraazahexadecane-16-oate

To a solution of the compound (0.100 g, 0.108 mmol) obtained in Step 2 above in N,N-dimethylformamide (2.00 mL) was added N-succinimidyl 6-maleimide hexanoate (40.0 mg, 0.130 mmol) and stirred at room temperature for 2 days. The solvent was removed under reduced pressure, and the resulting residues were purified by silica gel column chromatography [chloroform-chloroform:methanol=9:1 (v/v)] to give the title compound as a pale yellow solid (80.0 mg, 66% ).

¹ H-NMR (400 MHz, DMSO-d ₆ ) delta: 0.88 (3H, t, J=7.2 Hz), 1.13-1.23 (2H, m), 1.37 (9H, s), 1.42-1.54(4H, m), 1.80-1.96(2H, m), 2.08-2.25(4H, m), 2.35-3.76( 15H, m), 2.43(3H, s), 4.39-4.49(1H, m), 4.55-4.67(1H, m), 5.21-5.34(2H, m), 5.43 (2H, dd, J=21.1, 16.4 Hz), 5.56-5.64 (1H, m), 6.55 (1H, s), 7.01 (2H , d, J=0.8 Hz), 7.16-7.26 (5H, m), 7.33 (1H, s), 7.83 (1H, d, J=11.3 Hz), 8 .04-8.18 (3H, m), 8.30-8.37 (1H, m), 8.43 (1H, d, J=8.6 Hz).

MS (ESI) m/z: 1118 (M+H) ⁺ .

[0220]

Step 4: N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-alpha-aspartylglycylglycyl-L-phenylalanyl-N-[(1S,9S) -9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3', 4':6,7]indolizino[1,2-b]quinolin-1-yl]glycinamide

Under ice-cooling, trifluoroacetic acid (4.00 ml) was added to the compound (70.0 mg, 62.6 μmol) obtained in Step 3 above, and stirred at room temperature for 1 hour. The solvent was removed under reduced pressure to give the title compound as a pale yellow solid (55.0 mg, 83%).

¹ H-NMR (400 MHz, DMSO-d ₆ ) delta: 0.88 (3H, t, J=7.4 Hz), 1.14-1.24 (2H, m), 1.41-1, 53(4H, m), 1.79-1.95(2H, m), 2.08-2.28(4H, m), 2.37-2.60(2H, m), 2.42( 3H, s), 2.63-2.82 (2H, m), 2.99 (1H, dd, J=14.1, 5.1 Hz), 3.12-3.25 (2H, m) , 3.29-3.44(1H, m), 3.52-3.80(6H, m), 4.38-4.48(1H, m), 4.56(1H, dd, J= 13.7, 7.4 Hz), 5.27 (2H, dd, J=24.3, 18.8 Hz), 5.43 (2H, dd, J=21.5, 16.4 Hz), 5.57-5.62(1H, m), 6.55(1H, s), 7.01(2H, s), 7.15-7.26(5H, m), 7.33(1H, s), 7.82 (1H, d, J=11.0 Hz), 7.98 (1H, br s), 8.08 (1H, d, J=6.7 Hz), 8.15 ( 1H, d, J=7.8 Hz), 8.34(1H, br s), 8.44(1H, d, J=8.6 Hz), 12.26(1H, br s).

MS (ESI) m/z: 1062 (M+H) ⁺ .

[0221]

Step 5: Antibody Drug Conjugate (5)

Using U1-59 prepared in Reference Example 1 and the compound obtained in Step 4 above, the title antibody drug conjugate was prepared in the same manner as Step 6 of Example 1.

Antibody concentration: 1.36 mg/mL, antibody yield: 8.16 mg (82%) and the average number of conjugated drug molecules (n) per antibody molecule measured using General Procedure E (as molar drug uptake coefficient linker means used E _D.280 =7620, E _D.370 =23700): 5.0.

[0222]

Example 6 Antibody Drug Conjugate (6)

[Chemical Formula 37]

[0223]

Step 1: Antibody Drug Conjugate (6)

Using U1-59 prepared in Reference Example 1 and the compound obtained in Step 4 of Example 5 above, the title antibody drug conjugate was prepared in the same manner as Step 6 of Example 1.

Antibody concentration: 11.5 mg/mL, antibody yield: 224.2 mg (90%), and the average number of conjugated drug molecules (n) per antibody molecule measured using General Procedure E (as molar drug absorption coefficient linker means used E _D.280 =7620, E _D.370 =23700): 4.6.

[0224]

Example 7 Antibody Drug Conjugate (7)

[Chemical Formula 38]

[0225]

Stage 1: tert-Butyl (3S,12S)-12-benzyl-21-{[(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2, 3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl]amino}-3 -{[(9H-fluoren-9-ylmethoxy)carbonyl]amino}-4,7,10,13,16,21-hexaoxo-5,8,11,14,17-pentaazagenicosan-1-oate

(2S)-4-tert-Butoxy-2-{[(9H-fluoren-9-ylmethoxy)carbonyl]amino}-4-oxobutanoic acid (0.625 g, 1.52 mmol) was dissolved in dichloromethane (10.0 ml) , loaded N-hydroxysuccinimide (0.175 g, 1.52 mol) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (0.291 g, 1.52 mmol) and stirred for 1 hour. The reaction solution was added dropwise to a solution of N,N-dimethylformamide (10.0 ml) containing the compound (1.00 g, 1.01 mmol) obtained in the above Step 4 of Example 1, and stirred at room temperature for 20 hours. The solvent was removed under reduced pressure, and the resulting residues were purified by silica gel column chromatography [chloroform-chloroform:methanol=8:2 (v/v)] to give the title compound as a pale yellow solid (0.873 g, 70%).

¹ H-NMR (400 MHz, DMSO-d ₆ ) delta: 0.88 (3H, t, J=7.4 Hz), 1.37 (9H, s), 1.68-1.78 (2H, m), 1.81-1.93(2H, m), 2.10-2.23(4H, m), 2.41(3H, s), 2.68-2.85(3H, m) , 2.99-3.22(5H, m), 3.58-3.81(6H, m), 4.19-4.36(3H, m), 4.38-4.52(2H, m), 5.17 (1H, d, J=19.2 Hz), 5.25 (1H, d, J=19.2 Hz), 5.43 (2H, s), 5.54-5, 62 (1H, m), 6.55 (1H, s), 7.15-7.34 (8H, m), 7.41 (2H, t, J=7.2 Hz), 7.66-7 .75 (4H, m), 7.81 (1H, d, J=11.0 Hz), 7.88 (2H, d, J=7.4 Hz), 8.01-8.06 (1H, m), 8.14 (1H, d, J=8.2 Hz), 8.17-8.22 (1H, m), 8.25-8.30 (1H, m), 8.47 (1H , d, J=8.6 Hz).

MS (APCI) m/z: 1232 (M+H) ⁺ .

[0226]

Stage 2: tert-Butyl (3S,12S)-12-benzyl-3-{[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]amino}-21- {[(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[ de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl]amino}-4,7,10,13,16,21-hexaoxo-5,8,11 ,14,17-pentaazagenicosan-1-oate

The compound (0.800 g, 0.649 mmol) obtained in Step 1 above was dissolved in N,N-dimethylformamide (3.00 ml), piperidine (0.643 ml, 6.49 mmol) was charged and stirred for 1 hour. The solvent was removed to dryness under reduced pressure and the resulting residues were dissolved in N,N-dimethylformamide (10 ml). After N-succinimidyl 6-maleimide hexanoate (0.300 g, 0.974 mmol) was added, the mixture was stirred for 20 hours. The solvent was removed under reduced pressure, and the resulting residues were purified by silica gel column chromatography [chloroform-chloroform:methanol=8:2 (v/v)] to give the title compound as a pale yellow solid (0.224 g, 29%).

¹ H-NMR (400 MHz, DMSO-d ₆ ) delta: 0.87 (3H, t, J=7.6 Hz), 1.15-1.22 (2H, m), 1.35 (9H, s), 1.44-1.47(4H, m), 1.71-1.73(2H, m), 1.80-1.91(2H, m), 2.08(2H, t, J=7.6Hz), 2.13-2.20(4H, m), 2.40(3H, s), 2.67(1H, dt, J=11.1, 4.8Hz), 2.78 (1H, dd, J=13.6, 9.4 Hz), 2.99-3.17 (6H, m), 3.31-3.36 (2H, m), 3.57- 3.76(6H, m), 4.45-4.47(1H, m), 4.57-4.60(1H, m), 5.16(1H, d, J=18.7 Hz) , 5.25 (1H, d, J=18.7 Hz), 5.42 (2H, s), 5.55-5.60 (1H, m), 6.53 (1H, s), 6, 99 (2H, s), 7.15-7.27 (5H, m), 7.31 (1H, s), 7.70 (1H, t, J=5.4 Hz), 7.80 (1H , d, J=10.9 Hz), 7.99 (1H, t, J=5.7 Hz), 8.09-8.12 (3H, m), 8.25 (1H, t, J= 6.0 Hz), 8.45 (1H, d, J=9.1 Hz).

MS (APCI) m/z: 1203 (M+H) ⁺ .

[0227]

Step 3: N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-alpha-aspartylglycylglycyl-L-phenylalanyl-N-(4-{[( 1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano [3',4':6,7]indolizino[1,2-b]quinolin-1-yl]amino}-4-oxobutyl)glycinamide

The compound (0.224 g, 0.186 mmol) obtained in Step 2 above was reacted in the same manner as Step 2 of Example 1 to give the title compound as a pale yellow solid (21.2 mg, 10%) .

¹ H-NMR (400 MHz, DMSO-d ₆ ) delta: 0.87 (3H, t, J=7.2 Hz), 1.13-1.21 (2H, m), 1.42-1, 45(6H, m), 1.70-1.72(2H, m), 1.85-1.88(2H, m), 2.06-2.20(6H, m), 2.39( 3H, s), 2.63-2.67(1H, m), 2.78-2.81(1H, m), 3.04-3.12(6H, m), 3.63-3, 70(6H, m), 4.46-4.52(2H, m), 5.16(1H, d, J=19.2Hz), 5.25(1H, d, J=18.8Hz) ), 5.42(2H, s), 5.55-5.58(1H, m), 6.53(1H, s), 6.99(2H, s), 7.18-7.23( 6H, m), 7.30 (1H, s), 7.71 (1H, t, J=5.5 Hz), 7.79 (1H, d, J=10.9 Hz), 7.99- 8.02(1H, m), 8.10-8.11(3H, m), 8.27-8.30(1H, m), 8.47-8.50(1H, m).

MS (APCI) m/z: 1147 (M+H) ⁺ .

[0228]

Step 4: Antibody Drug Conjugate (7)

Using U1-59 prepared in Reference Example 1 and the compound obtained in Step 3 above, the title antibody drug conjugate was prepared in the same manner as Step 6 of Example 1.

Antibody concentration: 1.39 mg/mL, antibody yield: 8.34 mg (83%) and the average number of conjugated drug molecules (n) per antibody molecule measured using General Procedure E (as molar drug uptake coefficient linker means used E _D.280 =7670, E _D.370 =24800): 4.7.

[0229]

Example 8 Antibody Drug Conjugate (8)

[Chemical Formula 39]

[0230]

Stage 1: Antibody drug conjugate (8)

Using U1-59 prepared in Reference Example 1 and the compound obtained in Step 3 of Example 7 above, the title antibody drug conjugate was prepared in the same manner as Step 6 of Example 1.

Antibody concentration: 11.2 mg/mL, antibody yield: 228.5 mg (91%) and the average number of conjugated drug molecules (n) per antibody molecule measured using General Procedure E (as molar drug absorption coefficient linker means used E _D.280 =7670, E _D.370 =24800): 4.7.

[0231]

Example 9 Antibody Drug Conjugate (9)

[0232]

[Chemical Formula 40]

[0233]

Step 1: N-{3-[2-(2-{[3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanoyl]amino}ethoxy)ethoxy]propanoyl}glycylglycyl -L-phenylalanyl-N-(4-{[(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13, 15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl]amino}-4-oxobutyl)glycinamide

The compound (100 mg, 0.119 mmol) obtained in the above Step 4 of Example 1 was reacted in the same manner as Step 5 of Example 1 using N-succinimidyl 3-(2-(2-(3-maleimidepropanamide)ethoxy) ethoxy)propanoate (50.7 mg, 0.119 mmol) in place of N-succinimidyl 6-maleimide hexanoate to give the title compound as a pale yellow solid (66.5 mg, 48%).

¹ H-NMR (400 MHz, DMSO-d ₆ ) delta: 0.85 (3H, t, J=7.4 Hz), 1.65-1.74 (2H, m), 1.77-1, 90 (2H, m), 2.07-2.19 (4H, m), 2.30 (2H, t, J=7.2 Hz), 2.33-2.36 (2H, m), 2 .38 (3H, s), 2.76 (1H, dd, J=13.7, 9.8 Hz), 2.96-3.18 (9H, m), 3.42-3.44 (4H , m), 3.53-3.76 (10H, m), 4.43 (1H, td, J=8.6, 4.7 Hz), 5.14 (1H, d, J=18.8 Hz), 5.23 (1H, d, J=18.8 Hz), 5.38 (1H, d, J=17.2 Hz), 5.42 (1H, d, J=17.2 Hz) , 5.52-5.58(1H, m), 6.52(1H, s), 6.98(2H, s), 7.12-7.17(1H, m), 7.18-7 .25 (4H, m), 7.29 (1H, s), 7.69 (1H, t, J=5.5 Hz), 7.78 (1H, d, J=11.3 Hz), 7 .98-8.03(2H, m), 8.11(1H, d, J=7.8Hz), 8.16(1H, t, J=5.7Hz), 8.23(1H, t, J=5.9 Hz), 8.44 (1H, d, J=9.0 Hz).

MS (APCI) m/z: 1149 (M+H) ⁺ .

[0234]

Step 2: Antibody Drug Conjugate (9)

Using U1-59 prepared in Reference Example 1 and the compound obtained in Step 1 above, the title antibody drug conjugate was prepared in the same manner as Step 6 of Example 1.

Antibody concentration: 2.08 mg/mL, antibody yield: 18.7 mg (94%) and the average number of conjugated drug molecules (n) per antibody molecule measured using General Procedure E (as molar drug uptake coefficient linker means used E _D.280 =4964, E _D.370 =18982): 5.6.

[0235]

Example 10 Antibody Drug Conjugate (10)

[Chemical Formula 41]

[0236]

Step 1: Antibody Drug Conjugate (10)

Using U1-59 obtained in Reference Example 1 and the compound obtained in Step 1 of Example 9 above, the title antibody drug conjugate was prepared in the same manner as Step 6 of Example 1.

Antibody concentration: 19.7 mg/mL, antibody yield: 236.4 mg (95%), and the average number of conjugated drug molecules (n) per antibody molecule measured using General Procedure E (as molar drug absorption coefficient linker means used E _D.280 =4964, E _D.370 =18982): 6.2; and the average number of conjugated drug molecules (n) per antibody molecule measured using General Procedure F (E _D.280 =4964 was used as the molar absorption coefficient of the linker drug): 6.4.

[0237]

Example 11 Antibody Drug Conjugate (11)

[0238]

[Chemical Formula 42]

[0239]

Stage 1: Antibody drug conjugate (11)

Using U1-59 obtained in Reference Example 1 and the compound obtained in Step 1 of Example 9 above, the title antibody drug conjugate was prepared in the same manner as Step 1 of Example 4.

Antibody concentration: 0.88 mg/mL, antibody yield: 5.28 mg (53%) and the average number of conjugated drug molecules (n) per antibody molecule measured using General Procedure E (as molar drug uptake coefficient linker means used E _D.280 =4964, E _D.370 =18982): 3.0; and the average number of conjugated drug molecules (n) per antibody molecule, measured using General Procedure F (E _D.280 =4964 was used as the molar absorption coefficient of the linker drug): 3.3.

[0240]

Example 12 Antibody Drug Conjugate (12)

[Chemical Formula 43]

[0241]

Stage 1: ({N-[(9H-fluoren-9-ylmethoxy)carbonyl]glycyl}amino)methyl acetate

Pyridine (1.16 ml, 14.7 mmol) and tetraacetate lead (6.84 g, 14.7 mmol) and heated at reflux temperature for 5 hours. After the reaction solution was cooled to room temperature, the insoluble matter was removed by filtration through Celite, and the filtrate was concentrated under reduced pressure. The obtained residues were dissolved in ethyl acetate, washed with water and brine, and then the organic layer was dried over anhydrous magnesium sulfate. After the solvent was removed under reduced pressure, the resulting residues were purified by silica gel column chromatography [hexane:ethyl acetate=9:1 (v/v)-ethyl acetate] to give the title compound as a colorless solid (3.00 g, 67%).

¹ H-NMR (400 MHz, CDCl ₃ ) delta: 2.07 (3H, s), 3.90 (2H, d, J=5.1 Hz), 4.23 (1H, t, J=7, 0 Hz), 4.46 (2H, d, J=6.6 Hz), 5.26 (2H, d, J=7.0 Hz), 5.32 (1H, br s), 6.96 (1H, br s), 7.32 (2H, t, J=7.3 Hz), 7.41 (2H, t, J=7.3 Hz), 7.59 (2H, d, J= 7.3 Hz), 7.77 (2H, d, J=7.3 Hz).

[0242]

Stage 2: Benzyl [({N-[(9H-fluoren-9-ylmethoxy)carbonyl]glycyl}amino)methoxy]acetate

To a tetrahydrofuran (40.0 mL) solution of the compound (3.68 g, 10.0 mmol) obtained in Step 1 above and benzyl glycolate (4.99 g, 30.0 mmol) was added potassium tert-butoxide (2 .24 g, 20.0 mmol) at °C and stirred at room temperature for 15 minutes. The reaction solution was charged with ethyl acetate and water at °C and extracted with ethyl acetate and chloroform. The resulting organic layer was dried over sodium sulfate and filtered. The solvent was removed under reduced pressure. The resulting residues were dissolved in dioxane (40.0 ml) and water (10.0 ml), sodium bicarbonate (1.01 g, 12.0 mmol) and 9-fluorenylmethyl chloroformate (2.59 g, 10.0 mmol) were charged and stirred at room temperature for 2 hours. The reaction solution was charged with water and extracted with ethyl acetate. The resulting organic layer was dried over sodium sulfate and filtered. The solvent was removed under reduced pressure, and the resulting residues were purified by silica gel column chromatography [hexane:ethyl acetate=100:0 (v/v) - 0:100] to give the title compound as a colorless oily substance (1.88 g, 40%) .

¹ H-NMR (400 MHz, CDCl ₃ ) delta: 3.84 (2H, d, J=5.5 Hz), 4.24 (3H, t, J=6.5 Hz), 4.49 (2H , d, J=6.7 Hz), 4.88 (2H, d, J=6.7 Hz), 5.15-5.27 (1H, m), 5.19 (2H, s), 6 .74 (1H, br s), 7.31-7.39 (7H, m), 7.43 (2H, t, J=7.4 Hz), 7.61 (2H, d, J=7 .4 Hz), 7.79 (2H, d, J=7.4 Hz).

[0243]

Stage 3: [({N-[(9H-fluoren-9-ylmethoxy)carbonyl]glycyl}amino)methoxy]acetic acid

The compound (1.88 g, 3.96 mmol) obtained in Step 2 above was dissolved in ethanol (40.0 ml) and ethyl acetate (20.0 ml). After adding the palladium-carbon catalyst (376 mg), the mixture was stirred at room temperature under a hydrogen atmosphere for 2 hours. The insoluble matter was removed by filtration through Celite, and the solvent of the filtrate was removed under reduced pressure to give the title compound as a colorless solid (1.52 g, quant.).

¹ H-NMR (400 MHz, DMSO-d ₆ ) delta: 3.62 (2H, d, J=6.3 Hz), 3.97 (2H, s), 4.18-4.32 (3H, m), 4.60 (2H, d, J=6.7 Hz), 7.29-7.46 (4H, m), 7.58 (1H, t, J=5.9 Hz), 7, 72 (2H, d, J=7.4 Hz), 7.90 (2H, d, J=7.4 Hz), 8.71 (1H, t, J=6.5 Hz).

[0244]

Step 4: 9H-Fluoren-9-ylmethyl(2-{[(2-{[(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2, 3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl]amino}-2 -oxoethoxy)methyl]amino}-2-oxoethyl)carbamate

Under ice-cooling, to a solution in N,N-dimethylformamide (10.0 ml) of exatecan mesylate (0.283 g, 0.533 mmol), N-hydroxysuccinimide (61.4 mg, 0.533 mmol) and the compound (0.205 g, 0.533 mmol) obtained in Step 3 above, N,N-diisopropylethylamine (92.9 µl, 0.533 mmol) and N,N'-dicyclohexylcarbodiimide (0.143 g, 0.693 mmol) were added and stirred at room temperature for 3 days. The solvent was removed under reduced pressure, and the resulting residues were purified by silica gel column chromatography [chloroform - separated organic layer chloroform:methanol:water=7:3:1 (v/v/v)] to give the title compound as a pale brown solid substances (0.352 g, 82%).

¹ H-NMR (400 MHz, DMSO-d ₆ ) delta: 0.81 (3H, t, J=7.4 Hz), 1.73-1.87 (2H, m), 2.06-2, 20(2H, m), 2.34(3H, s), 3.01-3.23(2H, m), 3.58(2H, d, J=6.7Hz), 3.98(2H , s), 4.13-4.25 (3H, m), 4.60 (2H, d, J=6.7 Hz), 5.09-5.22 (2H, m), 5.32- 5.42(2H, m), 5.50-5.59(1H, m), 6.49(1H, s), 7.24-7.30(3H, m), 7.36(2H, t, J=7.4 Hz), 7.53(1H, t, J=6.3 Hz), 7.66(2H, d, J=7.4 Hz), 7.75(1H, d, J=11.0 Hz), 7.84(2H, d, J=7.4 Hz), 8.47(1H, d, J=8.6 Hz), 8.77(1H, t, J= 6.7 Hz).

MS (ESI) m/z: 802 (M+H) ⁺ .

[0245]

Step 5: N-[(2-{[(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15 -hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl]amino}-2-oxoethoxy)methyl]glycinamide

To a solution of the compound (0.881 g, 1.10 mmol) obtained in the above Step 4 in N,N-dimethylformamide (11.0 ml) was added piperidine (1.1 ml) and stirred at room temperature for 2 hours. The solvent was removed under reduced pressure to give a mixture containing the title compound. The mixture was used for the next reaction without further purification.

[0246]

Step 6: N-[(9H-fluoren-9-ylmethoxy)carbonyl]glycylglycyl-L-phenylalanyl-N-[(2-{[(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4 -methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b ]quinolin-1-yl]amino}-2-oxoethoxy)methyl]glycinamide

Under ice-cooling, to a solution in N,N-dimethylformamide (50.0 mL) of the mixture (0.439 mmol) obtained in Step 5 above, of N-hydroxysuccinimide (0.101 g, 0.878 mmol) and N-[(9H-fluoren- 9-ylmethoxy)carbonyl]glycylglycyl-L-phenylalanine (compound described in Japanese Patent Laid-Open No. 2002-60351; 0.440 g, 0.878 mmol) was added N,N'-dicyclohexylcarbodiimide (0.181 g, 0.878 mmol) and stirred at room temperature in within 4 days. The solvent was removed under reduced pressure, and the resulting residues were purified by silica gel column chromatography [chloroform-chloroform:methanol=9:1 (v/v)] to give the title compound as a pale orange solid (0.269 g, 58%).

MS (ESI) m/z: 1063 (M+H) ⁺ .

[0247]

Stage 7: Glycylglycyl-L-phenylalanyl-N-[(2-{[(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9 ,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl]amino}-2-oxoethoxy) methyl]glycinamide

To a solution of the compound (0.269 g, 0.253 mmol) obtained in Step 6 above in N,N-dimethylformamide (4.00 ml) was added piperidine (0.251 ml, 2.53 mmol) and stirred at room temperature for 2 hours . The solvent was removed under reduced pressure to give a mixture containing the title compound. The mixture was used for the next reaction without further purification.

[0248]

Step 8: N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]glycylglycyl-L-phenylalanyl-N-[(2-{[(1S,9S) -9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3', 4':6,7]indolizino[1,2-b]quinolin-1-yl]amino}-2-oxoethoxy)methyl]glycinamide

To a solution of the compound (0.253 mmol) obtained in Step 7 above in N,N-dimethylformamide (10.0 mL) was added N-succinimidyl 6-maleimide hexanoate (0.156 g, 0.506 mmol) and stirred at room temperature for 3 days. The solvent was removed under reduced pressure, and the resulting residues were purified by silica gel column chromatography [chloroform-chloroform:methanol=9:1 (v/v)] to give the title compound as a pale yellow solid (0.100 g, 38%).

¹ H-NMR (400 MHz, DMSO-d ₆ ) delta: 0.83 (3H, t, J=7.2 Hz), 1.09-1.21 (2H, m), 1.33-1, 47(4H, m), 1.75-1.90(2H, m), 2.00-2.23(4H, m), 2.36(3H, s), 2.69-2.81( 1H, m), 2.94-3.03(1H, m), 3.06-3.22(2H, m), 3.23-3.74(6H, m), 3.98(2H, s), 4.39-4.50(1H, m), 4.60(2H, d, J=6.7 Hz), 5.17(2H, s), 5.39(2H, s), 5.53-5.61(1H, m), 6.50(1H, s), 6.96(2H, s), 7.11-7.24(5H, m), 7.28(1H, s), 7.75 (1H, d, J=11.0 Hz), 7.97 (1H, t, J=5.7 Hz), 8.03 (1H, t, J=5.9 Hz) , 8.09 (1H, d, J=7.8 Hz), 8.27 (1H, t, J=6.5 Hz), 8.48 (1H, d, J=9.0 Hz), 8 .60 (1H, t, J=6.5 Hz).

MS (ESI) m/z: 1034 (M+H) ⁺ .

[0249]

Step 9: Antibody Drug Conjugate (12)

Using U1-59 obtained in Reference Example 1 and the compound obtained in Step 8 above, the title antibody drug conjugate was prepared in the same manner as Step 6 of Example 1.

Antibody concentration: 2.11 mg/mL, antibody yield: 19.0 mg (95%), and the average number of conjugated drug molecules (n) per antibody molecule measured using General Procedure E (as molar drug absorption coefficient linker means used E _D.280 =5178, E _D.370 =20217): 4.9.

[0250]

Example 13 Antibody Drug Conjugate (13)

[Chemical Formula 44]

[0251]

Stage 1: Antibody drug conjugate (13)

Using U1-59 prepared in Reference Example 1 and the compound obtained in Step 8 of Example 12 above, the title antibody drug conjugate was prepared in the same manner as Step 6 of Example 1.

Antibody concentration: 22.2 mg/mL, antibody yield: 244.2 mg (98%) and the average number of conjugated drug molecules (n) per antibody molecule measured using General Procedure E (as molar drug uptake linker means used E _D.280 =5178, E _D.370 =20217): 6.2; and the average number of conjugated drug molecules (n) per antibody molecule, measured using General Procedure F (E _D.280 =5178 was used as the molar absorption coefficient of the linker drug): 7.0.

[0252]

Example 14 Antibody Drug Conjugate (14)

[Chemical Formula 45]

[0253]

Stage 1: Antibody drug conjugate (14)

Antibody Recovery: U1-59 prepared in Reference Example 1 was prepared at an antibody concentration of 10 mg/ml by changing the medium to PBS6.0/EDTA using General Procedure B and General Procedure C described in Preparation Method 1. Solution (1, 00 ml) was added to a 2.0 ml polypropylene tube and loaded with an aqueous solution of 10 mM TCEP (Tokyo Chemical Industry Co., Ltd.) (0.0160 ml; 2.4 equivalents per antibody molecule) and 1 M aqueous solution dipotassium hydrogen phosphate (Nacalai Tesque, Inc.; 0.0150 ml). After confirming that the solution had a pH of 7.0 +/- 0.1, the disulfide bond in the hinge portion of the antibody was restored by incubation at 37C for 1 hour.

Conjugation between antibody and drug-linker component: After incubating the solution in a water bath at 15°C for 10 minutes, dimethyl sulfoxide (Sigma-Aldrich Co. LLC; 0.0209 ml) and dimethyl sulfoxide solution (0.0315 ml; 5.0 equivalents) were added per antibody molecule) containing 10 mM of the compound obtained in Step 8 of Example 12 above and incubated in a water bath at 15° C. for 60 minutes to conjugate the drug-linker component to the antibody. An aqueous solution (0.0050 ml) of 100 mM NAC (Sigma-Aldrich Co. LLC) was then added and mixed using a tube rotator (MTR-103, manufactured by AS ONE Corporation) at room temperature for an additional 20 minutes to stop the drug reaction. -linker.

Following the same purification procedures and physico-chemical characterization as in Step 6 of Example 1, the following characteristic values were obtained.

Antibody concentration: 1.46 mg/ml, antibody yield: 8.76 mg (88%) and the average number of conjugated drug molecules (n) per antibody molecule measured using General Procedure E (as molar drug uptake coefficient linker means used E _D.280 =5178, E _D.370 =20217): 2.5; and the average number of conjugated drug molecules (n) per antibody molecule, measured using General Procedure F (E _D.280 =5178 was used as the molar absorption coefficient of the linker drug): 2.9.

[0254]

Example 15 Antibody Drug Conjugate (15)

[Chemical Formula 46]

[0255]

Stage 1: Antibody drug conjugate (15)

Antibody recovery: U1-59 prepared in Reference Example 1 was prepared at 10 mg/mL antibody concentration by medium change to PBS6.0/EDTA using General Procedure B and General Procedure C described in Preparation Method 1. Solution (100 ml ) was added to a 250 ml polycarbonate Erlenmeyer flask and charged with 1 M dipotassium hydrogen phosphate aqueous solution (1.70 ml) followed by 10 mM TCEP aqueous solution (4.010 ml; 6.0 equivalents per antibody molecule) at room temperature while stirring with using a magnetic stirrer. After confirming that the solution had a pH of 7.0 +/- 0.1, stirring was stopped and the hinge disulfide bond in the antibody was restored by incubation at 37° C. for 1 hour.

Conjugation between antibody and drug-linker component: After this solution was cooled to 15° C., a DMSO solution (6.684 ml; 10.0 equivalents per antibody molecule) containing 10 mM of the compound obtained in Step 8 of Example 12 above was gradually added thereto. , while stirring. The mixture was stirred at 15°C for the first 30 minutes, then the stirring was stopped and incubated for another 1 hour to conjugate the drug-linker component to the antibody. An aqueous solution (0.862 ml; 12.9 equivalents per antibody molecule) of 100 mM NAC was then added with stirring and incubated at room temperature for 20 minutes to stop the reaction of unreacted linker drug.

Purification: 20% aqueous acetic acid solution (about 0.6 ml) and ABS (100 ml) were gradually added to the solution with stirring to bring the pH of the solution to 5.5 +/- 0.1. This solution was subjected to microfiltration (Millipore Corp. Millex-HV filter, 0.45 µm, PVDF membrane) to remove whitish matter. This solution was subjected to ultrafiltration purification using an ultrafiltration device consisting of an ultrafiltration membrane (Merck Japan, Ltd., Pellicon XL Cassette, Biomax 50 kDa), a hose pump (Cole-Parmer International, USA, MasterFlex pump model 77521-40, pump head model 7518-00) and hose (Cole-Parmer International, USA, MasterFlex hose L/S16). For example, by adding ABS dropwise (1600 ml total) as purification buffer to the reaction solution during ultrafiltration purification, unconjugated drug linkers and other small molecular weight reagents were removed while the buffer solution was replaced with ABS and then the solution concentrated. The resulting purified solution was subjected to microfiltration (0.22 µm (Millipore Corp. Millex-GV filter, PVDF membrane) to obtain 37.5 ml of a solution containing the title antibody drug conjugate.

Antibody concentration: 26.5 mg/mL, antibody yield: 993.0 mg (90%), average number of conjugated drug molecules (n) per antibody molecule measured using General Procedure E (as molar drug absorption coefficient linker agents used E _D.280 =5178 and E _D.370 =20217): 6.3, and the average number of conjugated drug molecules (n) per antibody molecule measured using General Procedure F (as molar factor linker drug uptake used E _D.280 =5178): 7.3.

[0256]

Example 16a Antibody Drug Conjugate (16a)

[Chemical Formula 47]

[0257]

Step 1: Antibody Drug Conjugate (16a)

Antibody recovery: U1-59 prepared in Reference Example 1 was prepared with an antibody concentration of 10 mg/mL by changing the medium to PBS6.0/EDTA using General Procedure B and General Procedure C described in Preparation Method 1. Solution (15 ml ) was added to a 50 ml polyethylene terephthalate container and loaded with 1 M dipotassium hydrogen phosphate aqueous solution (0.255 ml) followed by 10 mM TCEP aqueous solution (0.601 ml; 6.0 equivalents per antibody molecule) at room temperature with stirring using magnetic mixers. After confirming that the solution had a pH of 7.0 +/- 0.1, the disulfide bond in the hinge portion of the antibody was restored by incubation at 37° C. for 2 hours.

Conjugation between antibody and drug-linker component: After this solution was cooled to 15° C., a DMSO solution (1.002 ml; 10.0 equivalents per antibody molecule) containing 10 mM of the compound obtained in Step 8 of Example 12 above was gradually added with stirring The mixture was stirred at 15° C. for 30 minutes to conjugate the drug-linker component to the antibody. An aqueous solution (0.129 ml; 12.9 equivalents per antibody molecule) of 100 mM NAC was then added to the mixture with stirring and incubated at room temperature for 20 minutes to stop the reaction of the unreacted linker drug. Following the same purification procedures and physicochemical characterization methods as described for Step 6 of Example 1, the following characterization values were obtained.

Antibody concentration: 2.36 mg/ml, antibody yield: 140 mg (59.5 ml) (94%), average number of conjugated drug molecules (n) per antibody molecule measured using General Procedure E (as molar absorption coefficient of the drug-linker used E _D.280 =5178 and E _D.370 =20217): 6.4, and the average number of conjugated drug molecules (n) per antibody molecule, measured using the General procedure F ( E _D.280 =5178 was used as the molar absorption coefficient of the linker drug: 7.7.

[0258]

Example 16b Antibody Drug Conjugate (16b)

[Chemical Formula 48]

[0259]

Antibody Recovery: U1-59 prepared in Reference Example 1 was prepared at an antibody concentration of 10 mg/mL by medium change to PBS6.0/EDTA using General Procedure B and General Procedure C described in Preparation Method 1. Solution (900 ml ) was added to a 2000 ml polycarbonate Erlenmeyer flask and charged with 1 M dipotassium hydrogen phosphate aqueous solution (15.3 ml) followed by 10 mM TCEP aqueous solution (36.1 ml; 6.0 equivalents per antibody molecule) at room temperature at stirring using a magnetic stirrer. After confirming that the solution had a pH of 7.0 +/- 0.1, stirring was stopped and the hinge disulfide bond in the antibody was restored by incubation at 37° C. for 2 hours.

Conjugation between antibody and drug-linker component: After this solution was cooled to 15° C., a DMSO solution (60.16 ml; 10.0 equivalents per antibody molecule) containing 10 mM of the compound prepared in Step 8 of Example 12 was gradually added with stirring. The mixture was stirred at 15° C. for 30 minutes to conjugate the drug-linker component to the antibody. An aqueous solution (7.76 ml; 12.9 equivalents per antibody molecule) of 100 mM NAC was then added to the mixture with stirring and incubated at room temperature for 20 minutes to stop the reaction of unreacted linker drug.

Purification: 20% aqueous acetic acid solution (about 5 ml) and ABS (1000 ml) were gradually added to the solution with stirring to bring the pH of the solution to 5.5 +/- 0.1. This solution was subjected to microfiltration (Millipore Corp. Stericup, 0.45 μm, PVDF membrane) to remove whitish matter. This solution was subjected to ultrafiltration purification using an ultrafiltration device consisting of an ultrafiltration membrane (Merck Japan, Ltd., Pellicon 2 minicassette, Ultracel 30 KDa, 0.1 m ² ), a peristaltic pump (Cole-Parmer International, USA, MasterFlex pump model 7528 -20, pump head model 77800-62) and hose (Cole-Parmer International, USA, MasterFlex L/S24 and 25 hoses). For example, by adding ABS dropwise (16 L in total) as a purification buffer to the reaction solution during ultrafiltration purification, unconjugated drug-linker components and other small molecular weight reagents were removed during buffer exchange with ABS, and then the solution was concentrated. to obtain about 500 ml of a solution containing the title antibody-drug conjugate.

Antibody concentration: 19.66 mg/mL, antibody yield: 9830 mg (109%), and the average number of conjugated drug molecules (n) per antibody molecule measured using General Procedure E (as molar drug uptake coefficient -linker used E _D.280 =5178 and E _D.370 =20217): 6.5.

[0260]

Example 16c Antibody Drug Conjugate (16c)

[Chemical Formula 49]

[0261]

Using U1-59 obtained in Reference Example 1 and the compound obtained in the above Step 8 of Example 12, the title antibody-drug conjugate was prepared in the same manner as in Example 16b.

Antibody concentration: 16.21 mg/ml, antibody yield: 9726 mg (600 ml, 108%), and the average number of conjugated drug molecules (n) per antibody molecule measured using General Procedure E (as molar factor linker drug uptake used E _D.280 =5178 and E _D.370 =20217): 6.5.

[0262]

Example 16d Antibody drug conjugate (16d)

[Chemical Formula 50]

[0263]

The antibody-drug conjugates (16a), (16b) and (16c) obtained in Examples 16a, 16b and 16c, respectively, were mixed (about 18 g total) and then ultrafiltered in the same manner as in Example 16b (used 11 l ABS). The resulting purified solution was subjected to microfiltration (Millipore Corp. Stericup, 0.45 µm and 0.22 µm, PVDF membrane) to obtain 745 ml of a solution containing the title antibody drug conjugate. Then, 110 ml of ABS was added to obtain 855 ml of a solution containing the title antibody-drug conjugate.

Antibody concentration: 20.0 mg/mL, antibody yield: 17.1 g (94%), average number of conjugated drug molecules (n) per antibody molecule, measured using General Procedure E (as molar drug uptake coefficient linker agents used E _D.280 =5178 and E _D.370 =20217): 6.5, and the average number of conjugated drug molecules (n) per antibody molecule measured using General Procedure F (as molar factor linker drug uptake used E _D.280 =5178): 7.8.

[0264]

Test Example 1 HER3 Binding Affinity of Antibody-Drug Conjugate Compared to U1-59

Method:

The human breast cancer cell line HCC1569 (CRL-2330) from ATCC was cultured in RPMI1640 medium (purchased from Invitrogen Corp. containing 10% bovine serum albumin (manufactured by Invitrogen Corp.) and 2 mM L-glutamine (manufactured by Invitrogen Corp.)) . Cells were detached from the culture plate using ACCUTASE(R) SOLUTION (Millipore Corp., SCR005) or EDTA (5 mM, phosphate buffered saline (PBS, 137 mM sodium chloride, 2.7 mM potassium chloride, 1.47 mM dihydrogen phosphate potassium and 10.5 mm disodium hydrogen phosphate)) and the number of living cells was measured by treatment with trypan blue. The same number of cells suspended in fluorescently activated buffer (PBS containing 3% FBS and 0.004% sodium azide) for cell sorting (FACS) was used to inoculate 96-well U-bottom plates and the cells were pelleted by centrifugation and suspended in 100 µl ice-cold dilution of antibody or antibody-drug conjugate or FACS buffer.

Serial dilutions of the antibody or each antibody-drug conjugate were made at a ratio of 1/3 using FACS buffer and adjusted to 30 μg/ml - 5 ng/ml (200 nM - 0.03 nM). Cells treated with FACS buffer without addition of the primary antibody were used as controls.

U1-59, antibody-drug conjugate (3), antibody-drug conjugate (10), or antibody-drug conjugate (13) were evaluated as antibody or antibody-drug conjugates.

Cells from each group were reacted with the primary antibody dilution for 45 minutes on ice and then washed with FACS buffer. Then, 100 μl of FACS buffer or secondary antibody reaction solution diluted 1/100 (phycoerythrin (PE)-linked anti-human antibody, Dianova GmbH #709-116-149) was added to the mixture. Cells were treated for 45 minutes on ice in the dark and then washed with FACS buffer and dead cells were discarded using FACS buffer or FACS buffer supplemented with 7-aminoactinomycin D (7AAD, Sigma-Aldrich Co. LLC, #A9400, 1.1 µg/ml).

Live cell fluorescence signals were assessed using an Accuri C6 flow cytometer (BD Biosciences/Accuri(R) Cytometers Inc., serial number 1424) and the CFlow program (CFlow sampler Version 1.0.264.13).

To correct for PE- and 7-AAD-produced signals, the fluorescence signals of U1-59 (30 μg/ml) and cells labeled with PE-labeled secondary antibody or 7-AAD were evaluated.

To quantify U1-59 specific fluorescence signals in cells, values obtained by subtracting FL-2 signals from cells treated with secondary antibody or 7-AAD alone were used. The balance of binding affinity (KD) and maximum binding strength (Bmax) was calculated using the GraphPad Prism program (version 5.04 for Windows(R) (single-site-specific binding)).

[0265]

The results are shown in FIG. 3 and in Table 1. FIG. 3 and Table 1 show the mean fluorescence intensity of HCC1569 treated with serial dilutions of U1-59 or each antibody drug conjugate. The balance of binding affinity KD and maximum binding strength Bmax was calculated using the GraphPad Prism program.

[0266]

Table 1 U1-59 conjugate
Antibody-
medicinal
remedy (3) conjugate
Antibody-
medicinal
remedy (10) conjugate
Antibody-
medicinal
remedy (13) _Bmax 31830 28981 28841 28415 Kd (nM) 1.672 1.653 1.554 2.757

[0267]

The antibody-drug conjugate (3) or antibody-drug conjugate (10) showed an average KD binding affinity for HCC-1569 equivalent to the KD of the unconjugated anti-HER3 antibody U1-59. The antibody-drug conjugate (13) also showed a mean KD binding affinity equivalent to the KD of the unconjugated anti-HER3 antibody U1-59 (2.7 nM vs 1.6 nM). The KD values of the various antibody-drug conjugates indicate that antibody-drug conjugation processes do not impair U1-59 binding affinity in any meaningful way.

[0268]

Test Example 2 Inhibition of HER3 Signal by Anti-HER3 Antibody-Drug Conjugate

Method:

The human lung cancer cell line A549 (CRS-300114) from Cell Lines Service was dissociated by trypsinization and 50,000 live cells were inoculated in 3 ml DMEM/F12 (Invitrogen Corp., #21331-020) + 10% FBS (Invitrogen Corp., # 10270-106) in each of the 6 wells. After culturing the cells for 3 days, the medium was replaced with 2 ml of fresh medium.

The antibody or each antibody-drug conjugate was added directly to 2 ml of medium in each of 6 wells so that the final concentration was 10 μg/ml (20 μl of a stock solution of 1 μg/μl of antibody or antibody-drug conjugate was added).

U1-59, an antibody-drug conjugate (3), an antibody-drug conjugate (10), or an antibody-drug conjugate (13) was used as an antibody or an antibody-drug conjugate. The untreated group was used as a control.

Cells were cultured for 2 days, washed once with PBS and treated with 100 µl of ice-cold buffer (50 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), pH 7.5, 150 mM sodium chloride , 1 mM ethylenediaminetetraacetic acid (EDTA), 12.5% glycerol, 1% Triton X-100, and 10 mM tetrabasic sodium pyrophosphate supplemented with proteinase inhibitors (Roche Diagnostics, Inc., #11697 498 001), 10 mM sodium fluoride, 1 mM sodium vanadate, 1 mM phenylmethanesulfonyl fluoride (PMSF) and 10 μg/ml aprotinin (Sigma-Aldrich Co. LLC, A1153)) for 30 minutes at 4°C for lysis. The lysate was washed for 20 minutes at 13,000 rpm at 4°C and the supernatant was used in protein concentration measurements using the Bradford assay (Sigma-Aldrich Co. LLC, #B6916, BSA standard from Thermo Fisher Scientific Inc., #23209). A 4-fold concentration of LDS buffer (Invitrogen Corp. containing DTT (final concentration: 166.67 mM)) was added to each sample (amount of proteins: 120 μg) and its volume was finally adjusted to 40 μl with water. The sample was boiled for 10 minutes at 7° C. and added to wells with NuPage Mini Bis-Tris gel (4%-12%, 1.5 mm thick, 10 slot/gel, Invitrogen Corp.). 7.5 μl Novex(R) sharp ladder (Invitrogen Corp., P/N 57318) was added as protein standards. The sample was electrophoresed for 70 minutes at 175 V with 1 x MOPS of Running Buffer (Invitrogen Corp.) containing NuPage antioxidant (Invitrogen Corp., NP0005, Lot 1356629) added to the internal volume. Proteins separated by gel electrophoresis were transferred to nitrocellulose membrane (GE Healthcare Life Sciences) having a pore size of 0.45 µm using NuPage transfer buffer (Invitrogen Corp.) containing 10% methanol and NuPage antioxidant (Invitrogen Corp., NP0005, Lot 1356629, 1:1000 dilution) The proteins were transferred for 80 minutes at a constant volume of 30 V.

The blotting membrane was cut, separated into fractions of 100 kDa or more and 30-100 kDa, washed twice with PBS containing 0.1% Tween-20, and blocked by shaking for 1 hour at room temperature using Odyssey blocking solution (LI -COR, Inc., #927-40000). The blotting membrane thus blocked was treated overnight at 4° C. with a diluted primary antibody solution (mixture of Odyssey blocking solution and PBS in equal amounts).

Anti-HER3 antibody (Santa Cruz Biotechnology, Inc., SC-81455, 1:500 dilution) and anti-phosphorylated HER antibody (Cell Signaling Technology, Inc., #4791, 1:1000) were used as the primary antibody, and anti α-actin antibody (Neomarkers, #MS1295, dilution 1:3333) was used as a control for electrophoresis.

The blotting membrane was washed three times (each time for 5 minutes) with PBS containing 0.1% Tween-20 and reacted with a dilution containing a secondary antibody (a mixture of Odyssey blocking solution and PBS in equal amounts) for 1 hour at room temperature. temperature in a dark room.

Goat Anti-Mouse IRDye 680RD (LI-COR, Inc., #926-68070, diluted 1:25000) or Goat Anti-Rabbit IR Dye 800CW (LI-COR, Inc., #926-32211, diluted 1:10000) used as a secondary antibody. The blotting membrane was washed three times (6 minutes each time) with PBS containing 0.1% Tween-20, followed by signal detection using an Odyssey IR video signal conditioner (LI-COR, Inc.).

[0269]

The results are shown in FIG. 4. A549 cells were cultured for 2 days with U1-59 or different antibody-drug conjugates. HER3 or phosphorylated HER3 was analyzed by Western blotting. pan-Actin was determined as a control for electrophoresis.

[0270]

As a result of culturing A549 for 2 days with 10 μg/ml U1-59 or antibody-drug conjugate, HER3 phosphorylation was reduced compared to untreated cells. This decrease in HER3 phosphorylation was equivalent to U1-59 and the antibody-drug conjugate, suggesting that a variety of drug conjugation processes did not interfere with U1-59's HER3 signal inhibition function.

When A549 was treated with U1-59 or each antibody-drug conjugate for 2 days, a decrease in HER3 expression was also observed compared to untreated cells. The extent of this reduction in expression was equivalent to U1-59 and the antibody-drug conjugate. This suggests that a variety of drug conjugation processes did not disrupt U1-59 mediated internalization (see Internalization Test Example) and U1-59 induced HER down-regulation (see Signal Inhibition Test Example).

[0271]

Test Example 3 Reducing Cell Surface Expression of HER3 with U1-59 and Antibody Drug Conjugate

Method:

HER3 internalization by U1-59 and each antibody-drug conjugate was assessed by flow cytometry. 70,000 live HCC1569 cells (from ATCC) were suspended in 0.5 ml RPMI1640 (Invitrogen Corp., #31870-025) (containing 10% FBS (Invitrogen Corp., #10270-106 or PAN Biotech GmbH, #1505-P131304) and 2 mM glutamine (Invitrogen Corp., #25030-024)) and inoculated into each well of a 24-well plate. The cells were cultured for 4 days and the medium was changed to 0.5 ml of fresh medium prior to the start of the internalization test. 5 μg of antibody or each antibody-drug conjugate was added to 0.5 ml in each well of a 24-well plate so that the final concentration was 10 μg/ml. Cells were cultured for 1 hour at 37° C. in the presence of an antibody or an antibody-drug conjugate. U1-59, an antibody-drug conjugate (3), an antibody-drug conjugate (10), or an antibody-drug conjugate (13) was used as an antibody or an antibody-drug conjugate. Cells as positive control or negative control were untreated in some procedures.

For flow cytometric analysis, cells were washed once with PBS and separated from the plate using 5 mM EDTA (100 μl/well) dissolved in ACCUTASE(R) SOLUTION (Millipore Corp., SCR005) or PBS. Cells were suspended in 200 μl of ice-cold FACS buffer (PBS containing 3% FBS and 0.004% sodium azide), then added to each well of a 96-well U-bottom plate and left on ice. Cells were washed once using FACS buffer. To each sample was added 100 μl U1-59 (10 μg/ml) diluted with FACS buffer or FACS buffer alone. Cells were treated for 45 minutes with shaking on ice and then washed with FACS buffer and 100 µl of PE-labeled anti-human secondary antibody (Dianova GmbH, 709-116-149) diluted at a ratio of 1:100 was added to each well in FACS buffer, or FACS buffer alone. Cells were processed for 45 minutes in a dark room with shaking on ice. Cells were washed with FACS buffer and treated with FACS buffer or FACS buffer containing 7AAD (Sigma-Aldrich Co. LLC, A9400, 1.1 μg - 1.25 μg/ml) to stain dead cells. Fluorescence signals from live cells were measured using an Accuri C6 flow cytometer. U1-59 (10 μg/ml) and cells stained with PE-labeled secondary antibody or 7-AAD alone were used to correct for PE and 7-AAD signals.

To quantify HER3-specific signals, the values obtained for cells stained with secondary antibody or 7-AAD alone were subtracted from the FL-2 values for cells stained with primary antibody and secondary antibody and then with 7-AAD.

When FL-2 signals from cells that were not treated with U1-59 or antibody drug conjugate (no internalization) were determined as the maximum value, the decrease in HER3 (internalization) on the surface of cells treated with U1-59 or antibody drug conjugate was calculated at 37C.

The average calculated for 2-3 wells was used for the positive control (no 37° C. treatment) and negative control (no primary antibody added), and well internalization was quantified for each treatment group.

[0272]

The results are shown in FIG. 5. This graph shows the mean reduction in HER3 expression on the surface of HCC1569 cells treated with U1-59 or each antibody drug conjugate (37° C., 1 hour). HER3 expression in the group without addition of U1-59 or antibody-drug conjugate was defined as the maximum value of HER3 expression in cells. The secondary antibody or 7-AAD treatment group values were used as background values. Groups treated with U1-59 or each antibody-drug conjugate for 1 hour were used as treatment groups. FL-2 values were almost the same for U1-59 and the antibody-drug conjugate.

[0273]

The decrease in generated fluorescence caused by HCC-1569 treatment of U1-59 cells or each antibody-drug conjugate means a decrease in HER3 expression. Compared to the approximately 50% reduction in HER3 expression induced by U1-59, each antibody-drug conjugate showed a reduction in HER3 expression of the same or greater value, suggesting that drug-antibody conjugation did not impair HER3 internalization function. antibodies.

[0274]

Test Example 4 In Vitro Inhibition of Mitogenic or Survival Signal by HER3 Antibody-Drug Conjugate in a Human Cancer Cell Line

Method:

The inhibitory activity of each HER3 antibody-drug conjugate against mitogenic or survival signals was measured in the presence of 10% FBS. Cell growth and development was assessed by measuring adenosine triphosphate (ATP) activity in untreated and antibody drug conjugate treatment groups. Adherent cancer cell lines (human breast cancer cell line HCC1569 (CRL-2330) from ATCC, human breast cancer cell line MDA-MB-453 (CLB-22) from ATCC, and human colorectal cancer cell line HT-29 (CPQ- 57) from ProQinase GmbH) were cultured in 2D culture systems and pop-up (non-adherent) cancer cell lines (human melanoma cell line A375 (CRL-1619) from ATCC and human lung cancer cell line A549 (CRS-300114) from Cell Lines Service) were cultured in 3D culture systems.

Processing of adherent cells

Each cancer cell line was suspended in 100 µl of each medium at low density (500 cells/well for HT-29, 800 cells/well for MDA-MB-453 and 1000 cells/well for HCC-1569) and inoculated into 96-MicroWell Optical Bottom plate (Thermo Fisher Scientific Inc./Nunc, #165306, with white walls and transparent bottom). For HCC-1569 and MDA-MB-453, cells were cultured in RPMI1640 medium (Invitrogen Corp., 31870-025) containing 10% FBS (Invitrogen Corp., 10270-106) and 2 mM glutamine (Invitrogen Corp., 25030 -024). For HT-29, cells were cultured in McCoy's 5A medium (Invitrogen Corp., 26600-023) containing 10% FBS (Invitrogen Corp., 10270-106) and 2 mM glutamine (Invitrogen Corp., 25030-024). The rim of the well of each plate was filled with 100 μl of medium.

The cells were cultured for 3 days and the medium was changed to 95 μl of fresh medium before treatment with the antibody-drug conjugate.

An antibody-drug conjugate (3), an antibody-drug conjugate (10), and an antibody-drug conjugate (13) were used. The untreated group was used as a control to measure normal cell growth.

By adding 5 μl of each antibody-drug conjugate, concentrated to 20-fold concentration, to 95 μl of culture medium (10% FBS) contained in each well of a 96-well plate, the final concentration was established. Only 5 µl of medium was added to each control well. The test was carried out in three repetitions for each sample.

To calculate the antibody-drug concentration at which cell growth or survival was reduced by 50%, the antibody-drug conjugate concentrations were obtained by 4-fold dilutions (10 μg/mL to 0.15 ng/mL or 40 μg/mL to 0.15 ng/mL) and cells were treated with these concentrations of antibody-drug conjugate and compared with the untreated group for ATP activity. Evaluation was performed by 5 day culture of HT-29 and 7 day culture of HCC-1569 and MDA-MB-453 thus supplemented with the antibody-drug conjugate. The CellTiter-Glo(R) luminescent cell survival assay was used to assess the activity of the antibody-drug conjugate. This method involves the measurement of living cells with metabolic activity based on ATP activity and finally the determination of the number of living cells, and used CellTiter-Glo(R) Luminescent Cell (Promega K.K., G7573) as a kit.

100 µl of CellTiter-Glo(R) reagent was added to each well of a 96-well plate and the plate was stored for 25 minutes - 65 minutes at room temperature in a dark room before measurement using a Wallac Victor2 1420 Multilabel Counter (luminescence measurement program, measurement time : 0.5 sec). Wells containing only culture solution without cell inoculation were used for analysis as controls. To measure the reduction in ATP activity, the average luminescence from 3 wells was calculated under each of the conditions (Microsoft Excel 2010). To remove cell-independent signals, the average luminescence from the control wells was subtracted from the average luminescence of the cells treated with the antibody-drug conjugate (Microsoft Excel 2010). The percentage reduction (%) of luminescence was calculated by comparison with cells from the untreated group (Microsoft Excel 2010). This value was interpreted as the percentage inhibition (%) of cell growth or survival.

Pop-up cell processing

Since A375 and A549 have a faster growth rate compared to other cell lines, growth measurements were performed in non-adherent 3D culture systems.

Each cancer cell line was suspended in 75 μl of each medium at low density (500 cells/well for A375 and 1500 cells/well for A549) and inoculated into a 96-well round-bottom non-adhesive 3D culture plate (Prime Surface 96U; Sumitomo Bakelite Co, Ltd.; order no. MS-9096U). For A375, cells were cultured in DMEM medium (Invitrogen Corp., 41965-039) containing 10% FBS (Invitrogen Corp., 10270-106) and 2 mM glutamine (Invitrogen Corp., 25030-024). For A549, cells were cultured in a DMEM/F12 medium (Invitrogen Corp., 21331-020) containing 10% FBS (Invitrogen Corp., 10270-106) and 2 mM glutamine (Invitrogen Corp., 25030-024). The outer wells of each plate were filled with 150 µl of medium. Cells were cultured for 3 days and the final dose was adjusted to 142.5 µl or 150 µl by adding 67.5 or 75 µl of fresh medium before adding the antibody-drug conjugate. An antibody-drug conjugate (3), an antibody-drug conjugate (10), and an antibody-drug conjugate (13) were used. The untreated group was used as a control to measure normal cell growth.

By adding 7.5 or 8 µl of each antibody-drug conjugate, concentrated to 20-fold concentration, to 142.5 or 150 µl of culture medium (10% FBS) contained in each well of a 96-well plate, the final concentration was established. . The final dose was set at 150 μl or 158 μl. Only 7.5 or 8 µl of medium was added to each control well. The test was carried out in three repetitions for each sample.

To calculate the antibody-drug concentration at which cell growth or survival was reduced by 50%, the antibody-drug conjugate concentrations were obtained by 4-fold dilutions (10 μg/mL to 0.15 ng/mL or 40 μg/mL to 0.15 ng/mL) and cells were treated with these concentrations of antibody-drug conjugate and compared with the untreated group for ATP activity. The evaluation was carried out by 7-day culturing of the cells thus supplemented with the antibody-drug conjugate. The CellTiter-Glo(R) luminescent cell survival assay was used to assess the activity of the antibody-drug conjugate. This method involves the measurement of living cells with metabolic activity based on ATP activity and finally the determination of the number of living cells, and used CellTiter-Glo(R) Luminescent Cell (Promega K.K., G7573) as a kit.

Before measurement, 50 µl of medium was removed from each well and 100 µl of CellTiter-Glo(R) reagent was added to each well of a 96-well plate and the plate was stored for 30 minutes to 55 minutes at room temperature in a dark room before measurement using a counter Wallac Victor2 1420 Multilabel Counter (luminescence measurement program, measurement time: 0.5 sec). Prior to measurement, 180 μl was collected from each well and transferred to a measurable 96-MicroWell Optical Bottom white plate. Wells containing only culture solution without cell inoculation were used for analysis as controls. A method for calculating the concentration of an antibody-drug conjugate at which a 50% inhibition of cell growth or survival is obtained is described in the evaluation method for adherent cells.

[0275]

The results for the human breast cancer lines HCC1569 and MDA-MB453 are shown in FIG. 6 and 7, respectively. The results for the A375 human melanoma line are shown in FIG. 8. Results for the HT29 human colorectal cancer line are shown in FIG. 9. Results for the A549 human lung cancer line are shown in FIG. 10. A in each FIG. shows cell growth or survival data obtained for an antibody-drug conjugate in the presence of 10% FBS. The y-axis represents the luminescence value, indicative of ATP activity, for each sample. The abscissa axis represents the concentration of each antibody-drug conjugate. Data are presented as mean +/- standard deviation of three repetitions. B in each Fig. indicates the percentage reduction in luminescence caused by treatment with the antibody-drug conjugate when the luminescence of the untreated group is determined to be 100%.

[0276]

Three types of antibody-drug conjugates were added to various human cancer cell lines in the presence of 10% FBS and evaluated for in vitro growth in 2D or 3D systems. The percentage inhibition of cell growth or development in the untreated group or upon treatment with each antibody-drug conjugate was calculated from ATP activity data obtained in the CellTiter-Glo(R) assay. In an ATP activity assay, these antibody drug conjugates strongly inhibited cell growth or survival of two types of breast cancer cell lines (HCC-1569 and MDA-MB-453) and one type of human melanoma line (A375).

Addition of each antibody drug conjugate and culture (in the presence of 10% FBS) for 7 days reduced ATP activity by 55-75% in HCC1569, by 60-83% in MDA-MB-453 and by 60-70% in A375 . The inhibitory activity of the antibody drug conjugate against cell growth or survival was not strong in the HT-29 human colorectal cancer line and the A549 human lung cancer line compared to the human breast cancer line and the human melanoma line. In HCC-1569 and MDA-MB-453, the antibody-drug conjugate (10) showed strong inhibitory activity, which was not significantly different from that of the antibody-drug conjugate (3) in vitro . In contrast, in both human breast cancer lines, the antibody-drug conjugate (13) showed low activity, and a concentration of 15 nM was required to achieve 50% inhibition of cell growth or survival, while with the antibody-drug conjugate ( 3) or an antibody-drug conjugate (10), such inhibition was achieved at 1 nM or less. In human melanoma lines, compared to human breast cancer lines, the activity of the antibody-drug conjugate (13) was equivalent to that of the antibody-drug conjugate (3) or the antibody-drug conjugate (10).

All antibody-drug conjugates showed a maximum percentage of inhibition of the order of 61-68%. The antibody-drug conjugates achieved 50% inhibition of ATP activity at a concentration of 1 to 4 nM.

In addition to the assay described above, the inhibitory activity of the antibody-drug conjugate (13) against cell growth or survival of the human ovarian cancer cell line OVCAR-8 was also confirmed in vitro (data not shown).

[0277]

Test Example 5 Comparison of Percentage In Vitro Cell Growth Inhibition or Survival of a Human Cancer Cell Line Depending on the Number of Drug Molecules (High or Medium) Loaded on the Antibody-Drug Conjugate

Method:

Antibody-drug conjugates with varying amounts of loaded drug molecules were evaluated for in vitro inhibitory activity against cell growth or survival. High drug loading is a state where 5-7 drug molecules are conjugated to an antibody, and medium drug loading is a state where about 3 drug molecules are conjugated to an antibody. The average number of drug molecules conjugated to a single antibody was measured using the UV method (described elsewhere in the present invention).

[0278]

Average number of drug molecules conjugated to one antibody:

High drug load <HDL>

Antibody drug conjugate (3): 4.9

Antibody drug conjugate (10): 6.2

Antibody drug conjugate (13): 6.2

Average drug load <MDL>

Antibody drug conjugate (4): 2.9

Antibody drug conjugate (11): 3.0

Antibody drug conjugate (14): 2.5

[0279]

The inhibitory activity of each HER3 antibody-drug conjugate against mitogenic or survival signals was measured in the presence of 10% FBS. Cell growth and development was assessed by measuring adenosine triphosphate (ATP) activity in untreated and antibody drug conjugate treatment groups. The cancer cell line was suspended in 100 µl of each medium at low density (750 cells/well for human breast cancer cell line MDA-MB-453 (CLB-22)) from ATCC) and inoculated into a 96-MicroWell Optical Bottom white plate (Thermo Fisher Scientific Inc./Nunc, #165306). Cells were cultured in RPMI1640 medium (Invitrogen Corp., 31870-025) containing 10% FBS (Invitrogen Corp., 10270-106) and 2 mM glutamine (Invitrogen Corp., 25030-024). The outer wells of each plate were filled with 100 µl of medium.

The cells were cultured for 3 days and the medium was changed to 95 μl of fresh medium before the addition of the antibody-drug conjugate. Antibody-drug conjugate (3), antibody-drug conjugate (10), antibody-drug conjugate (13), antibody-drug conjugate (4), antibody-drug conjugate (11) and antibody-drug conjugate were used. agent (14). The untreated group was used as a control to measure normal cell growth.

By adding 5 μl of each antibody-drug conjugate, concentrated to 20-fold concentration, to 95 μl of culture medium (10% FBS) contained in each well of a 96-well plate, the final concentration was established. Only 5 µl of medium was added to each control well. The test was carried out in three repetitions for each sample. To calculate the antibody-drug concentration at which cell growth or survival was reduced by 50%, the antibody-drug conjugate concentrations were obtained by 4-fold dilutions (10 μg/mL to 0.15 ng/mL) and cells were treated with these concentrations. conjugate antibody-drug and compared with the untreated group for ATP activity. The evaluation was carried out by 7-day culturing of the cells thus supplemented with the antibody-drug conjugate. The CellTiter-Glo(R) luminescent cell survival assay was used to assess the activity of the antibody-drug conjugate. This method involves measuring live cells with metabolic activity based on ATP activity and finally quantifying live cells, and used CellTiter-Glo(R) Luminescent Cell (Promega K.K., G7573) as a kit. 100 µl CellTiter-Glo(R) reagent was added to each well of a 96-well plate and the plate was stored for 25 minutes to 55 minutes at room temperature in a dark room before measurement using a Wallac Victor2 1420 Multilabel Counter (luminescence measurement program, measurement time: 0.5 sec). Wells containing only culture solution without cell inoculation were used for analysis as controls. To measure the reduction in ATP activity, the average luminescence from 3 wells was calculated under each of the conditions (Microsoft Excel 2010). To remove cell-independent signals, the average luminescence from the control wells was subtracted from the average luminescence of the cells treated with the antibody-drug conjugate (Microsoft Excel 2010). Percent reduction (%) in luminescence was calculated by comparison with cells from the untreated group (Microsoft Excel 2010). This value was interpreted as the percentage inhibition (%) of cell growth or survival.

[0280]

The results are shown in FIG. 11-13. Fig. 11 shows the results of comparing the antibody-drug conjugate (3) with the antibody-drug conjugate (4). Fig. 12 shows the results of comparing the antibody-drug conjugate (10) with the antibody-drug conjugate (11). Fig. 13 shows the results of comparing the antibody-drug conjugate (13) with the antibody-drug conjugate (14). On each Fig. the left chart shows the percentage of cell growth inhibition or survival obtained for the antibody-drug conjugate in the presence of 10% FBS in one of the three replicate tests. The y-axis represents the luminescence showing the ATP activity of each sample. The abscissa axis represents the concentration of each antibody-drug conjugate. The right chart shows a comparison of the decrease in luminescence induced by antibody drug conjugate treatment between high drug load (HDL) and medium drug load (MDL) when the luminescence of the untreated group was determined to be 100%.

[0281]

High drug loading and medium drug loading antibody-drug conjugates inhibited cell growth or survival by treatment and 7 day culture with MDA-MB-453. As already shown in the results for high drug loading, the medium drug loading antibody-drug conjugate (11) showed high activity at the same level as the antibody-drug conjugate (4). When compared between the amounts of loaded drug molecules, antibody-drug conjugates having a high number of loaded drug molecules showed a greater decrease in ATP compared to conjugates with a medium drug load. The antibody-drug conjugate (3), the antibody-drug conjugate (10) and the antibody-drug conjugate (13), having a large number of loaded drug molecules, showed inhibition percentages of 68%, 76%, and 56%, respectively, at concentration of 10 μg/ml, while the antibody-drug conjugate (4), the antibody-drug conjugate (11) and the antibody-drug conjugate (14), having an average number of loaded drug molecules, showed an inhibition of only 44%, 47% and 27%, respectively, at this concentration. Antibody-drug conjugates having a high number of loaded drug molecules showed a better concentration that causes 50% inhibition of cancer cell growth or survival compared to antibody-drug conjugates having an average number of loaded drug molecules. For an antibody-drug conjugate (3) or an antibody-drug conjugate (10) having a large number of loaded drug molecules, a concentration of 15 ng/ml (1 nM) of the antibody-drug conjugate is needed to reduce ATP activity by 50%. The antibody-drug conjugate (13) reduced ATP activity by 50% at least at the highest concentration tested. In contrast, a decrease in ATP activity corresponding to this was not observed at a concentration of 1000 ng/ml (67 nM) or lower in the concentration range of the evaluated antibody-drug conjugates having an average number of loaded drug molecules. An in vitro comparison between conjugates with a high number of loaded drug molecules and those with an average number of loaded drug molecules suggests that an antibody-drug conjugate having a high number of loaded drug molecules also has a better in vivo inhibitory activity against cancer cell growth. .

[0282]

Test Example 6 Antibody drug conjugates (3), (10) and (13) showed antitumor effect in in vivo antitumor test using human breast cancer cell line

Used five-week-old female BALB/C "naked" mice having a body weight of 15 to 20 g after acclimatization. Mice were housed in individually ventilated cages (IVC, maximum 4 mice per cage) maintained at room temperature and constant humidity. After randomization, the body weight of the mice was measured continuously every other day and the behavior of the animals was recorded every day. 5×10 ⁶ cells of the human breast cancer cell line HCC1569 (CRL-2330) from ATCC were suspended in a solution prepared from 50 μl of PBS and Matrigel (PBS: PAA #H21-002, Matrigel: BD #354230) mixed at a ratio of 1 :1 and transplanted subcutaneously into the right flank of each BALB/C nude mouse using a 29 G needle.

The body weight of the animals was measured using a balance (Mettler Toledo PB602-L). The major axis and minor axis of the tumor were measured twice a week using an electronic digital caliper (hand compass, OMC Fontana) and the tumor volume (mm ³ ) was calculated. The calculation was carried out in accordance with the following formula (the same applies to the Test Examples described below).

Tumor volume (mm ³ )=1/2 × Major axis (mm) × [Minor axis (mm)] ²

On Day 19, when the tumor size reached about 150 mm ³ , 70 animals were randomized into 7 groups based on their tumor sizes. On the same day, the antibody-drug conjugate (3), (10) or (13) or PBS for the control group was injected into the tail vein of each animal at the following doses.

[0283]

Administration Group: PBS was administered by intravenous injection once a week at the same dose as the antibody drug conjugate was administered.

Administration group: The antibody-drug conjugate (3) was administered by intravenous injection once a week at 3 or 10 mg/kg.

Administration group: The antibody-drug conjugate (10) was administered by intravenous injection once a week at 3 or 10 mg/kg.

Administration group: The antibody-drug conjugate (13) was administered by intravenous injection once a week at 3 or 10 mg/kg.

[0284]

All data were presented as mean +/- SEM. The size of the tumors and the body weight of the animals were assessed by the mean +/- SEM. All data was analyzed using Microsoft Excel 2009 (the same applies to the Test Examples described below).

[0285]

The results are shown in FIG. 14. The PBS administration group was euthanized on Day 53 post-transplant because the tumors exceeded the acceptable maximum size. Inhibition of tumor growth of the human breast cancer cell line was observed in all groups of administration of the antibody-drug conjugate compared with the control group. No body weight loss was observed in mice in the treated groups.

[0286]

Test Example 7 Antibody drug conjugates (3), (10) and (13) showed anti-tumor effect in anti-tumor test using human melanoma

Used 5-6-week-old female NRMI nude mice having a body weight of 22 to 26 g after acclimatization. Mice were housed in individually ventilated cages (IVC, maximum 4 mice per cage) maintained at room temperature and constant humidity. After randomization, the body weight of the mice was measured continuously every other day and the behavior of the animals was recorded every day.

5×10 ⁶ human melanoma cell line HT-144 (HTB-63) from ATCC were suspended in a solution prepared from 50 µl of PBS and Matrigel (PBS: PAA #H21-002, Matrigel: BD #354230) mixed at a ratio of 1 :1, and transplanted subcutaneously into the right flank of each NRMI nude mouse using a 29 G needle.

Measurement of body weight and size of tumors and measurement and calculation of tumor volume were carried out in the same manner as in Test Example 6.

On Day 22, when the tumor size reached about 150 mm ³ , 80 animals were randomly divided into 8 groups based on their tumor sizes. On the same day, U1-59 or the antibody-drug conjugate (3), (10) or (13) or PBS for the control group was injected into the tail vein of each animal at the following doses.

[0287]

Administration Group: PBS was administered by intravenous injection once a week at the same dose as the antibody drug conjugate was administered.

Administration group: U1-59 was administered by subcutaneous injection twice a week at 25 mg/kg.

Administration group: The antibody-drug conjugate (3) was administered by intravenous injection once a week at 3 or 10 mg/kg.

Administration group: The antibody-drug conjugate (10) was administered by intravenous injection once a week at 3 or 10 mg/kg.

Administration group: The antibody-drug conjugate (13) was administered by intravenous injection once a week at 3 or 10 mg/kg.

[0288]

The results are shown in FIG. 15. The PBS and U1-59 administration groups were sacrificed on Days 48 and 52, respectively, post-transplant because the tumor sizes exceeded the acceptable maximum. Tumor growth inhibition of the human melanoma cell line was observed in all antibody drug conjugate administration groups compared to the control group and the U1-59 administration group.

[0289]

Test Example 8 Antibody-Drug Conjugates (3), (10) and (13) Demonstrated Antitumor Effect in Antitumor Test Using Human Breast Cancer Cell Line

Sixteen-week-old female SCID nude mice having a body weight of 17 to 25.5 g after acclimatization were used. Mice were placed in individually ventilated cages maintained at room temperature and constant humidity.

With respect to solid tumors derived from the human breast cancer cell line MDA-MB-453 (CLB-22) from ATCC, MDA-MB-453 in the first passage (Batch 1089) was transplanted into 3 mice (two sites per mouse: right and left side of the body). After 13-17 weeks, tumor sections were isolated and frozen. For the second passage, a section of the tumor (2 x 2 x 2 mm) from the first passage was then subcutaneously transplanted (10 mice, two sites per mouse: right and left side of the body) and allowed to grow to form a tumor for 7 weeks. The thus formed tumor was prepared into tumor sections (2 x 2 x 2 mm, second passage) and transplanted into the right flank of each SCID nude mouse.

The body weight of the animals was measured using a balance. The major axis (length) and diameter of the tumor were measured using an electronic digital caliper (Pro-Max 150 mm hand compasses, Fred V. Fowler Co., Inc.) and the tumor volume (mm ³ ) was calculated. The calculation was carried out in accordance with the following formula.

Tumor volume (mm ³ )=pi/6 × Major axis (mm) × [Diameter (mm)] ²

On Day 40, when the tumor size reached about 143 mm ³ , 72 animals were randomized into 8 groups based on their tumor sizes. On the same day, U1-59 or the antibody-drug conjugate (3), (10) or (13) or PBS for the control group was injected into the tail vein of each animal at the following doses.

[0290]

Administration Group: PBS was administered by intravenous injection once a week at the same dose as the antibody drug conjugate was administered.

Administration group: U1-59 was administered by subcutaneous injection twice a week at 25 mg/kg.

Administration group: The antibody-drug conjugate (3) was administered by intravenous injection once a week at 3 or 10 mg/kg.

Administration group: The antibody-drug conjugate (10) was administered by intravenous injection once a week at 3 or 10 mg/kg.

Administration group: The antibody-drug conjugate (13) was administered by intravenous injection once a week at 3 or 10 mg/kg.

[0291]

The results are shown in FIG. 16. Tumor growth inhibition of the human breast cancer cell line was observed in all antibody drug conjugate administration groups compared to the control group and the U1-59 group. No body weight loss was observed in mice in the treated groups. In addition, in another antitumor trial using the human breast cancer cell line HCC1954 or JIMT1-PR10 (trastuzumab-, pertuzumab- and T-DM1-resistant), tumor growth inhibition was also observed in the antibody-drug conjugate (16a) group by compared with the control group.

[0292]

Test Example 9 Antibody-Drug Conjugates (3), (10) and (13) Demonstrated Antitumor Effect in Antitumor Test Using Human Colorectal Cancer Line

Used 5-6-week-old female NRMI nude mice having a body weight of 15 to 20 g after acclimatization. Mice were housed in individually ventilated cages (IVC, maximum 4 mice per cage) maintained at room temperature and constant humidity. After randomization, the body weight of the mice was measured continuously every other day and the behavior of the animals was recorded every day.

4×10 ⁶ human colorectal cancer cell line HT-29 (CPQ-57) from ProQinase GmbH were suspended in a solution prepared from PBS and Matrigel (PBS: PAA #H21-002, Matrigel: BD #354230) mixed at a ratio of 1 :1, and transplanted subcutaneously into the right flank of each NRMI nude mouse using a 29 G needle.

Measurement of body weight and size of tumors and measurement and calculation of tumor volume were carried out in the same manner as in Test Example 6.

On Day 8, when the tumor size reached about 150 mm ³ , 70 animals were randomized into 7 groups based on their tumor sizes. On the same day, the antibody-drug conjugate (3), (10) or (13) or PBS for the control group was injected into the tail vein of each animal at the following doses.

[0293]

Administration Group: PBS was administered by intravenous injection once a week at the same dose as the antibody drug conjugate was administered.

Administration group: The antibody-drug conjugate (3) was administered by intravenous injection once a week at 3 or 10 mg/kg.

Administration group: The antibody-drug conjugate (10) was administered by intravenous injection once a week at 3 or 10 mg/kg.

Administration group: The antibody-drug conjugate (13) was administered by intravenous injection once a week at 3 or 10 mg/kg.

[0294]

The results are shown in FIG. 17. PBS administration group (6 of 10 mice), groups administered with antibody drug conjugate (3) at 3 mg/kg (3 of 10 mice) and at 10 mg/kg (4 of 10 mice), groups treated with the antibody-drug conjugate (10) at 3 mg/kg (2 of 10 mice) and at 10 mg/kg (2 of 10 mice), and the group that received the antibody-drug conjugate (13) at 3 mg /kg (2 out of 10 mice) were euthanized on Day 50 post-transplant because tumors exceeded acceptable maximum levels or an ulcer formed. Tumor growth inhibition of the human colorectal cancer cell line was observed in all antibody drug conjugate treatment groups compared to the control group. The antibody-drug conjugate (13) showed stronger antitumor activity compared to the antibody-drug conjugate (3) or the antibody-drug conjugate (10). No body weight loss was observed in mice in the treated groups.

[0295]

Test Example 10 Antibody-Drug Conjugates (3), (10) and (13) Demonstrated Antitumor Effect in Antitumor Test Using Human Lung Cancer Line

Used 5-6-week-old female CD1 "naked" mice having a body weight of 24 to 28 g after acclimatization. Mice were housed in individually ventilated cages (IVC, maximum 4 mice per cage) maintained at room temperature and constant humidity. After randomization, the body weight of the mice was measured continuously every other day and the behavior of the animals was recorded every day.

5×10 ⁶ cells of the human lung cancer cell line A549 (CRS-300114) from Cell Lines Service were suspended in a solution prepared from 200 μl of PBS and Matrigel (PBS: PAA #H21-002, Matrigel: BD #354230) mixed at a ratio of 1:1, and transplanted subcutaneously into the right flank of each CD1 nude mouse using a 29 G needle.

Measurement of body weight and size of tumors and measurement and calculation of tumor volume were carried out in the same manner as in Test Example 6.

On Day 38, when the tumor size reached about 200 mm ³ , 70 animals were randomly divided into 7 groups based on their tumor sizes. On the same day, the antibody-drug conjugate (3), (10) or (13) or PBS for the control group was injected into the tail vein of each animal at the following doses.

[0296]

Administration Group: PBS was administered by intravenous injection once a week at the same dose as the antibody drug conjugate was administered.

Administration group: The antibody-drug conjugate (3) was administered by intravenous injection once a week at 3 or 10 mg/kg.

Administration group: The antibody-drug conjugate (10) was administered by intravenous injection once a week at 3 or 10 mg/kg.

Administration group: The antibody-drug conjugate (13) was administered by intravenous injection once a week at 3 or 10 mg/kg.

[0297]

The results are shown in FIG. 18. Tumor growth inhibition of the human lung cancer cell line was observed in all antibody drug conjugate treatment groups compared to the control group. No body weight loss was observed in mice in the treated groups.

[0298]

Test Example 11 Antibody-Drug Conjugate (13) Demonstrated Antitumor Effect in an In Vivo Antitumor Test Using Human Triple Negative Breast Cancer Cell Line

Triple negative breast cancer refers to breast cancer that does not express either hormone receptors (estrogen receptor and progesterone receptor) or HER2. Because these receptors are not expressed, hormonal treatments (tamoxifen, etc.) or anti-HER2 treatments (trastuzumab, trastuzumab emtansine, or pertuzumab) cannot be used to treat this cancer. This breast cancer therefore results in a low survival rate and many therapeutics are still in clinical trials. Since HER3 expression was confirmed in the human triple negative breast cancer cell line MDA-MB-468 (data not shown), the antitumor activity of the antibody-drug conjugate was evaluated.

Used 5-6 week old females naked mice (Charles River Laboratories Japan, Inc.) having a body weight of 15 to 20 g after acclimatization. Mice were housed in individually ventilated cages (IVC, maximum 4 mice per cage) maintained at room temperature and constant humidity. After randomization, the body weight of the mice was measured continuously every other day and the behavior of the animals was recorded every day.

5×10 ⁶ cells of the human triple negative breast cancer cell line MDA-MB-468 (CRL-2322) from ATCC were suspended in a solution prepared from 200 μl of PBS and Matrigel (PBS: PAA #10010-023, Matrigel: BD # 354234) mixed at a 1:1 ratio and transplanted subcutaneously into the right flank of each nude mouse using a 29 G needle. The body weight of the animals was measured using a balance (Mettler Toledo PB602-L).

The major axis and minor axis of the tumor were measured using an electronic digital caliper (CD-15CX, Mitsutoyo Corp), and the tumor volume (mm ³ ) was calculated. The calculation was carried out in accordance with the following formula.

Tumor volume (mm ³ )=1/2 × Major axis (mm) × [Minor axis (mm)] ²

On Day 20, when the tumor size reached about 170 mm ³ , 18 animals were randomly divided into 3 groups based on their tumor sizes. On the same day, U1-59 or the antibody-drug conjugate (13) or PBS for the control group was injected into the tail vein of each animal at the following doses.

[0299]

Administration Group: PBS was administered by intravenous injection once a week at the same dose as the antibody drug conjugate was administered.

Administration group: U1-59 was injected intravenously once a week at 10 mg/kg.

Administration group: The antibody-drug conjugate (13) was administered by intravenous injection once a week at 10 mg/kg.

[0300]

The results are shown in FIG. 19. Tumor growth inhibition of the human triple negative breast cancer cell line was observed in the antibody drug conjugate administration group compared to the control group and the U1-59 group. No body weight loss was observed in mice in the treated groups.

[0301]

Test Example 12 Antibody-Drug Conjugate (16a) Demonstrated Antitumor Effect in an In Vivo Antitumor Test Using a Human Luminal Breast Cancer Cell Line

Human luminal breast cancer refers to breast cancer that expresses hormone receptors (estrogen receptor) but does not express HER2. Because these receptors are not expressed, anti-HER2 treatments (trastuzumab, trastuzumab emtansine, pertuzumab) cannot be used to treat this cancer. This breast cancer therefore results in a low survival rate and many therapeutics are still in clinical trials. Since HER3 expression was confirmed in the human luminal breast cancer cell line MCF-7 (data not shown), the antitumor activity of the antibody-drug conjugate was evaluated.

Used 5-6-week-old female Nude-Foxn1 ^nu athymic nude mice (ProQinase GmbH) having a body weight of 15 to 20 g after acclimatization. Mice were housed in individually ventilated cages (IVC, maximum 4 mice per cage) maintained at room temperature and constant humidity. After randomization, the body weight of the mice was measured continuously every other day and the behavior of the animals was recorded every day.

5×10 ⁶ cells of the human luminal breast cancer cell line MCF-7 (CRQ-#327) were suspended in a solution prepared from 200 μl of PBS and Matrigel (PBS: PAA #10010-023, Matrigel: BD #354234) mixed at 1:1 ratio and transplanted subcutaneously into the right flank of each nude mouse using a 29 G needle. The body weight of the animals was measured using a balance (Mettler Toledo PB602-L).

The major axis and minor axis of the tumor were measured using an electronic digital caliper (CD-15CX, Mitsutoyo Corp), and the tumor volume (mm ³ ) was calculated. The calculation was carried out in accordance with the following formula.

Tumor volume (mm ³ )=1/2 × Major axis (mm) × [Minor axis (mm)] ²

On Day 11, when the tumor size reached about 250 mm ³ , 20 animals were randomly divided into 2 groups based on their tumor sizes. On the same day, the antibody-drug conjugate (16a) or PBS for the control group was administered at the following doses.

[0302]

Administration group: PBS was administered by intravenous injection at the same dose as the antibody-drug conjugate was administered.

Administration group: The antibody-drug conjugate (16a) was administered by intravenous injection at a single dose of 10 mg/kg.

[0303]

The results are shown in FIG. 20. Tumor growth inhibition of the human luminal breast cancer cell line was observed in the antibody drug conjugate administration group compared to the control group. No body weight loss was observed in mice in the treated groups.

[0304]

Test Example 13 Antibody drug conjugate (16a) showed antitumor effect in in vivo antitumor test using human melanoma cell line

Used 5-6 week old females naked mice (Charles River Laboratories Japan, Inc.) having a body weight of 15 to 20 g after acclimatization. Mice were housed in individually ventilated cages (IVC, maximum 4 mice per cage) maintained at room temperature and constant humidity. After randomization, the body weight of the mice was measured continuously every other day and the behavior of the animals was recorded every day.

3×10 ⁶ human melanoma cell line WM-266-4 (CRL-1676) from ATCC were mixed and suspended in Matrigel (BD #354234) and transplanted subcutaneously into the right flank of each nude mouse using a 29 G needle. The body weight of the animals was measured using a balance (Mettler Toledo PB602-L).

The major axis and minor axis of the tumor were measured using an electronic digital caliper (CD-15CX, Mitsutoyo Corp), and the tumor volume (mm ³ ) was calculated. The calculation was carried out in accordance with the following formula.

Tumor volume (mm ³ )=1/2 × Major axis (mm) × [Minor axis (mm)] ²

On Day 19, when the tumor size reached about 220 mm ³ , 8 animals were randomly divided into 2 groups based on their tumor sizes. On the same day, the antibody-drug conjugate (16a) or PBS for the control group was administered at the following doses.

[0305]

Administration group: PBS was administered by intravenous injection at the same dose as the antibody-drug conjugate was administered.

Administration group: The antibody-drug conjugate (16a) was administered by intravenous injection at a single dose of 10 mg/kg.

[0306]

The results are shown in FIG. 21. Inhibition of tumor growth of the human melanoma cell line was observed in the antibody drug conjugate administration group compared to the control group. No body weight loss was observed in mice in the treated groups. In another C32 human melanoma model, tumor growth inhibition was also observed in the antibody drug conjugate (16a) administration group compared to the control group.

[0307]

Test Example 14 Antibody drug conjugate (16a) showed antitumor effect in in vivo antitumor test using human ovarian cancer cell line

Used 5-6 week old females naked mice (Charles River Laboratories Japan, Inc.) having a body weight of 15 to 20 g after acclimatization. Mice were housed in individually ventilated cages (IVC, maximum 4 mice per cage) maintained at room temperature and constant humidity. After randomization, the body weight of the mice was measured continuously every other day and the behavior of the animals was recorded every day.

5×10 ⁶ human ovarian cancer cell line OVCAR-8 (HTB-161) from ATCC were mixed and suspended in Matrigel (BD #354234) and transplanted subcutaneously into the right flank of each nude mouse using a 29 G needle. The body weight of the animals was measured using a balance (Mettler Toledo PB602-L).

The major axis and minor axis of the tumor were measured using an electronic digital caliper (CD-15CX, Mitsutoyo Corp), and the tumor volume (mm ³ ) was calculated. The calculation was carried out in accordance with the following formula.

Tumor volume (mm ³ )=1/2 × Major axis (mm) × [Minor axis (mm)] ²

On Day 21, when the tumor size reached about 140 mm ³ , 8 animals were randomly divided into 2 groups based on their tumor sizes. On the same day, the antibody-drug conjugate (16a) or PBS for the control group was administered at the following doses.

[0308]

Administration group: PBS was administered by intravenous injection at the same dose as the antibody-drug conjugate was administered.

Administration group: The antibody-drug conjugate (16a) was administered by intravenous injection at a single dose of 10 mg/kg.

[0309]

The results are shown in FIG. 22. Tumor growth inhibition of the human ovarian cancer cell line was observed in the antibody-drug conjugate administration group compared to the control group. No body weight loss was observed in mice in the treated groups.

[0310]

Test Example 15 Antibody drug conjugate (16a) showed antitumor effect in in vivo antitumor test using human bladder cancer cell line

Used 5-6 week old females naked mice (Charles River Laboratories Japan, Inc.) having a body weight of 15 to 20 g after acclimatization. Mice were housed in individually ventilated cages (IVC, maximum 4 mice per cage) maintained at room temperature and constant humidity. After randomization, the body weight of the mice was measured continuously every other day and the behavior of the animals was recorded every day.

8×10 ⁶ human bladder cancer cell line SW-780 (CRL-2169) from ATCC were mixed and suspended in Matrigel (BD #354234) and transplanted subcutaneously into the right flank of each nude mouse using a 29 G needle. The body weight of the animals was measured using a balance (Mettler Toledo PB602-L).

The major axis and minor axis of the tumor were measured using an electronic digital caliper (CD-15CX, Mitsutoyo Corp), and the tumor volume (mm ³ ) was calculated. The calculation was carried out in accordance with the following formula.

Tumor volume (mm ³ )=1/2 × Major axis (mm) × [Minor axis (mm)] ²

On Day 7, when the tumor size reached about 190 mm ³ , 10 animals were randomly divided into 2 groups based on their tumor sizes. On the same day, the antibody-drug conjugate (16a) or PBS for the control group was administered at the following doses.

[0311]

Administration group: PBS was administered by intravenous injection at the same dose as the antibody-drug conjugate was administered.

Administration group: The antibody-drug conjugate (16a) was administered by intravenous injection at a single dose of 10 mg/kg.

[0312]

The results are shown in FIG. 23. Tumor growth inhibition of the human bladder cancer cell line was observed in the antibody drug conjugate administration group compared to the control group. No body weight loss was observed in mice in the treated groups.

[0313]

Test Example 16 Antibody drug conjugate (16a) demonstrated HER3-dependent antitumor effect in an in vivo antitumor test using a human breast cancer cell line. The human breast cancer line MDA-MB-453 expresses HER3 and is responsive to the antibody-drug conjugate (13) as described in Test Example 8. However, since this pharmaceutical effect has not yet been demonstrated to be mediated by HER3, HER3 masked by introducing U1-59 in advance and assessed whether or not the pharmaceutical effect was reduced.

Used 5-6 week old females naked mice (Charles River Laboratories Japan, Inc.) having a body weight of 15 to 20 g after acclimatization. Mice were housed in individually ventilated cages (IVC, maximum 4 mice per cage) maintained at room temperature and constant humidity. After randomization, the body weight of the mice was measured continuously every other day and the behavior of the animals was recorded every day.

1×10 ⁷ cells of the human breast cancer cell line MDA-MB-453 (CLB-22) from ATCC were mixed and suspended in Matrigel (BD #354234) and transplanted subcutaneously into the right flank of each nude mouse using a 29 G needle. The body weight of the animals was measured using a balance (Mettler Toledo PB602-L).

The major axis and minor axis of the tumor were measured using an electronic digital caliper (CD-15CX, Mitsutoyo Corp), and the tumor volume (mm ³ ) was calculated. The calculation was carried out in accordance with the following formula.

Tumor volume (mm ³ )=1/2 × Major axis (mm) × [Minor axis (mm)] ²

On Day 11, when the tumor size reached about 130 mm ³ , 16 animals were randomized into 4 groups based on their tumor sizes. On the same day, the antibody-drug conjugate (16a) and/or U1-59 or PBS for the control group was administered at the following doses.

[0314]

Administration group: PBS was administered by intravenous injection at the same dose as the antibody-drug conjugate was administered.

Administration group: U1-59 was administered by intravenous injection at a single dose of 30 mg/kg.

Administration group: The antibody-drug conjugate (16a) was administered by intravenous injection at a single dose of 3 mg/kg.

Administration group: 30 minutes after administration (intravenous injection at a single dose) of U1-59, the antibody-drug conjugate (16a) was injected intravenously at a single dose of 3 mg/kg.

[0315]

The results are shown in FIG. 24. Inhibition of tumor growth of the human breast cancer cell line was observed in the antibody-drug conjugate administration group compared to the control group, while this tumor-inhibiting effect was attenuated by pre-administration of U1-59. These results demonstrated that the tumor-inhibiting effect of the antibody-drug conjugate is a pharmaceutical effect mediated by HER3. No body weight loss was observed in mice in the treated groups.

[0316]

Test Example 17 Antibody drug conjugate (16a) showed antitumor effect in combination with trastuzumab in an in vivo antitumor test using a human breast cancer cell line

Trastuzumab is approved as a therapeutic agent for human HER2-positive breast cancer. However, resistance to trastuzumab is known and a mutation in PIK3CA <H1047R or H420R> has been reported to be involved in one of the mechanisms underlying such resistance. This trial assessed whether or not the combined use of trastuzumab and an antibody-drug conjugate is effective in a trastuzumab-resistant breast cancer cell line.

Used 5-6 week old females naked mice (Charles River Laboratories Japan, Inc.) having a body weight of 15 to 20 g after acclimatization. Mice were housed in individually ventilated cages (IVC, maximum 4 mice per cage) maintained at room temperature and constant humidity. After randomization, the body weight of the mice was measured continuously every other day and the behavior of the animals was recorded every day.

1×10 ⁷ human breast cancer cell line MDA-MB-453 (CLB-22, H1047R mutation in PIK3CA) from ATCC were mixed and suspended in Matrigel (PBS: PAA #10010-023, Matrigel: BD #354234) and subcutaneously transplanted into the right flank of each nude mouse using a 29 G needle. The body weight of the animals was measured using a balance (Mettler Toledo PB602-L).

The major axis and minor axis of the tumor were measured using an electronic digital caliper (CD-15CX, Mitsutoyo Corp), and the tumor volume (mm ³ ) was calculated. The calculation was carried out in accordance with the following formula.

Tumor volume (mm ³ )=1/2 × Major axis (mm) × [Minor axis (mm)] ²

On Day 11, when the tumor size reached about 130 mm ³ , 16 animals were randomized into 4 groups based on their tumor sizes. On the same day, the antibody-drug conjugate (16a), trastuzumab used in the combination of the conjugate and trastuzumab or PBS for the control group was administered at the following doses.

[0317]

Administration group: PBS was administered by intravenous injection at the same dose as the antibody-drug conjugate was administered.

Administration group: Trastuzumab (Roche Diagnostics, Inc.) was administered by intravenous injection at a single dose of 1 mg/kg.

Administration group: The antibody-drug conjugate (16a) was administered by intravenous injection at a single dose of 3 mg/kg.

Administration group: 30 minutes after administration (single dose intravenous injection) of trastuzumab (Roche Diagnostics, Inc.), the antibody-drug conjugate (16a) was administered by intravenous injection at a single dose of 3 mg/kg.

[0318]

The results are shown in FIG. 25. The antitumor effect achieved by combined use on the human breast cancer cell line (PIK3CA H1047R) was observed in the trastuzumab and antibody drug conjugate group compared with the administration of each drug separately. These results demonstrate that the pharmaceutical effect of the antibody-drug conjugate is enhanced when it is used in combination with trastuzumab. No body weight loss was observed in mice in the treated groups. In another antitumor trial using the human breast cancer cell line HCC1954(PIK3CA H1047R), a combined antitumor effect was also observed in the trastuzumab and antibody drug conjugate (16a) administration group compared to each drug alone.

[0319]

Test Example 18 Antibody drug conjugate (15) showed antitumor effect in combination with trastuzumab in an in vivo antitumor trial using a human breast cancer cell line

Trastuzumab is approved as a therapeutic agent for human HER2-positive breast cancer. However, resistance to trastuzumab is known and a mutation in PIK3CA <H1047R or H420R> has been reported to be involved in one of the mechanisms underlying such resistance. This trial assessed whether or not the combined use of trastuzumab and an antibody-drug conjugate is effective in a trastuzumab-resistant breast cancer cell line.

Used 5-6 week old females naked mice (Charles River Laboratories Japan, Inc.) having a body weight of 15 to 20 g after acclimatization. Mice were housed in individually ventilated cages (IVC, maximum 4 mice per cage) maintained at room temperature and constant humidity. After randomization, the body weight of the mice was measured continuously every other day and the behavior of the animals was recorded every day.

5×10 ⁶ cells of the human breast cancer cell line JIMT-1 (ACC-589, H420R mutation in PIK3CA) from ATCC were suspended in PBS (PAA #10010-023) and transplanted subcutaneously into the right flank of each nude mouse using 29 G needles. The body weight of the animals was measured using a balance (Mettler Toledo PB602-L).

The major axis and minor axis of the tumor were measured using an electronic digital caliper (CD-15CX, Mitsutoyo Corp), and the tumor volume (mm ³ ) was calculated. The calculation was carried out in accordance with the following formula.

Tumor volume (mm ³ )=1/2 × Major axis (mm) × [Minor axis (mm)] ²

On Day 10, when the tumor size reached about 200 mm ³ , 24 animals were randomly divided into 4 groups based on their tumor sizes. On the same day, the antibody-drug conjugate (15), trastuzumab used in combination of the conjugate and trastuzumab or PBS for the control group was administered at the following doses.

[0320]

Administration Group: PBS was administered by intravenous injection once a week at the same dose as the antibody drug conjugate was administered.

Administration group: Trastuzumab (Roche Diagnostics, Inc.) was administered by intravenous injection once a week at 10 mg/kg.

Administration group: The antibody-drug conjugate (15) was administered by intravenous injection once a week at 10 mg/kg.

Administration group: 30 minutes after administration (IV injection once a week) of trastuzumab (Roche Diagnostics, Inc.), the antibody-drug conjugate (15) was injected intravenously once a week at 10 mg/kg.

[0321]

The results are shown in FIG. 26. The antitumor effect achieved by combined use on the human breast cancer cell line (PIK3CA H420R) was observed in the trastuzumab and antibody drug conjugate group compared with the administration of each drug alone. These results demonstrate that the pharmaceutical effect of the antibody-drug conjugate is enhanced when it is used in combination with trastuzumab. No body weight loss was observed in mice in the treated groups.

[0322]

Test Example 19 Antibody drug conjugate (16a) showed antitumor effect in combination with gefitinib in an in vivo antitumor trial using a human lung cancer line

Gefitinib is approved as a therapeutic agent for human lung cancer. This trial evaluated whether or not the combined use of gefitinib and an antibody drug conjugate is effective.

Used 5-6 week old females naked mice (Charles River Laboratories Japan, Inc.) having a body weight of 15 to 20 g after acclimatization. Mice were housed in individually ventilated cages (IVC, maximum 4 mice per cage) maintained at room temperature and constant humidity. After randomization, the body weight of the mice was measured continuously every other day and the behavior of the animals was recorded every day.

3×10 ⁶ cells of the human lung cancer cell line PC-9 (RCB0446) from ATCC were suspended in PBS (PAA #10010-023) and transplanted subcutaneously into the right flank of each nude mouse using a 29 G needle. The body weight of the animals was measured using a balance (Mettler Toledo PB602-L).

The major axis and minor axis of the tumor were measured using an electronic digital caliper (CD-15CX, Mitsutoyo Corp), and the tumor volume (mm ³ ) was calculated. The calculation was carried out in accordance with the following formula.

Tumor volume (mm ³ )=1/2 × Major axis (mm) × [Minor axis (mm)] ²

On Day 14, when the tumor size reached about 270 mm ³ , 16 animals were randomized into 4 groups based on their tumor sizes. On the same day, the antibody-drug conjugate (16a), gefitinib used in the combination of the conjugate and gefitinib or PBS for the control group was administered at the following doses.

[0323]

Administration Group: PBS was administered by intravenous injection once a week at the same dose as the antibody drug conjugate was administered.

Administration Group: Gefitinib (AstraZeneca) was administered orally once daily at 6 mg/kg.

Administration group: The antibody-drug conjugate (16a) was administered by intravenous injection once a week at 10 mg/kg.

Administration group: 30 minutes after administration (oral administration once a day) of gefitinib (AstraZeneca), the antibody-drug conjugate (16a) was administered by intravenous injection once a week at 10 mg/kg.

[0324]

The results are shown in FIG. 27. The antitumor effect achieved by combined use on the human lung cancer cell line was observed in the gefitinib and antibody-drug conjugate group compared with the administration of each drug separately. These results demonstrate that the pharmaceutical effect of the antibody-drug conjugate is enhanced when it is used in combination with gefitinib. No body weight loss was observed in mice in the treated groups.

[0325]

Test Example 20 Antibody drug conjugate (16a) demonstrated antitumor effect in combination with cetuximab or panitumumab in an in vivo antitumor trial using a triple negative human breast cancer cell line

The anti-EGFR antibody cetuximab or panitumumab is in clinical trials against human triple negative breast cancer. This trial evaluated whether or not the combined use of cetuximab or panitumumab and an antibody drug conjugate is effective.

Used 5-6 week old females naked mice (Charles River Laboratories Japan, Inc.) having a body weight of 15 to 20 g after acclimatization. Mice were housed in individually ventilated cages (IVC, maximum 4 mice per cage) maintained at room temperature and constant humidity. After randomization, the body weight of the mice was measured continuously every other day and the behavior of the animals was recorded every day.

5×10 ⁶ cells of the human triple negative breast cancer cell line MDA-MB-468 (CRL-2322) from ATCC were suspended in a solution prepared from 200 μl of PBS and Matrigel (PBS: PAA #10010-023, Matrigel: BD #354234 ) mixed at a 1:1 ratio and transplanted subcutaneously into the right flank of each nude mouse using a 29 G needle. The body weight of the animals was measured using a balance (Mettler Toledo PB602-L).

The major axis and minor axis of the tumor were measured using an electronic digital caliper (CD-15CX, Mitsutoyo Corp), and the tumor volume (mm ³ ) was calculated. The calculation was carried out in accordance with the following formula.

Tumor volume (mm ³ )=1/2 × Major axis (mm) × [Minor axis (mm)] ²

On Day 21, when the tumor size reached about 160 mm ³ , 30 animals were randomly divided into 6 groups based on their tumor sizes. On the same day, the antibody-drug conjugate (16a), cetuximab or panitumumab used in combination of the conjugate and cetuximab or panitumumab or PBS for the control group was administered at the following doses.

[0326]

Administration group: PBS was administered at the same dose as the antibody-drug conjugate was administered.

Administration group: Cetuximab (Bristol-Myers Squibb Company) was administered at a single dose of 10 mg/kg.

Administration group: Panitumumab (Amgen Inc.) was administered at a single dose of 10 mg/kg.

Administration group: The antibody-drug conjugate (16a) was administered at a single dose of 10 mg/kg.

Administration group: 30 minutes after administration (single dose administration) of cetuximab (Bristol-Myers Squibb Company), the antibody-drug conjugate (16a) was administered at a single dose of 10 mg/kg.

Administration group: 30 minutes after administration (single dose administration) of panitumumab (Amgen Inc.), the antibody-drug conjugate (16a) was administered at a single dose of 10 mg/kg.

[0327]

The results are shown in FIG. 28. The antitumor effect achieved by combined use on the human triple negative breast cancer cell line was observed in the group of administration of cetuximab (Fig. 28A) or panitumumab (Fig. 28B) and antibody-drug conjugate compared with the administration of each drug separately. . These results demonstrate that the pharmaceutical effect of the antibody-drug conjugate is enhanced when it is used in combination with cetuximab or panitumumab. No body weight loss was observed in mice in the treated groups. Combination antitumor effect on another human triple-negative breast cancer cell line, MDA-MB-231, was also observed in the cetuximab or panitumumab and antibody drug conjugate (16a) group compared to each drug alone.

[0328]

Test Example 21 Antibody drug conjugate (16a) showed antitumor effect in combination with cetuximab or panitumumab in an in vivo antitumor trial using a human head and neck cancer cell line

The anti-EGFR antibody cetuximab is approved against human head and neck cancer, while panitumumab is in clinical trials against this cancer. This trial evaluated whether or not the combined use of cetuximab or panitumumab and an antibody drug conjugate is effective.

Used 5-6 week old females naked mice (Charles River Laboratories Japan, Inc.) having a body weight of 15 to 20 g after acclimatization. Mice were housed in individually ventilated cages (IVC, maximum 4 mice per cage) maintained at room temperature and constant humidity. After randomization, the body weight of the mice was measured continuously every other day and the behavior of the animals was recorded every day.

4×10 ⁶ cells of the Fadu human head and neck cancer cell line (HTB-43) from ATCC were suspended in 200 μl of PBS (PAA #10010-023) and transplanted subcutaneously into the right flank of each nude mouse using a 29 G needle. The body weight of the animals was measured using a balance (Mettler Toledo PB602-L).

The major axis and minor axis of the tumor were measured using an electronic digital caliper (CD-15CX, Mitsutoyo Corp), and the tumor volume (mm ³ ) was calculated. The calculation was carried out in accordance with the following formula.

Tumor volume (mm ³ )=1/2 × Major axis (mm) × [Minor axis (mm)] ²

On Day 6, when the tumor size reached about 330 mm ³ , 30 animals were randomized into 6 groups based on their tumor sizes. On the same day, the antibody-drug conjugate (16a), cetuximab or panitumumab used in combination of the conjugate and cetuximab or panitumumab or PBS for the control group was administered at the following doses.

[0329]

Administration group: PBS was administered at the same dose as the antibody-drug conjugate was administered.

Administration group: Cetuximab (Bristol-Myers Squibb Company) was administered at a single dose of 5 mg/kg.

Administration group: Panitumumab (Amgen Inc.) was administered at a single dose of 5 mg/kg.

Administration group: The antibody-drug conjugate (16a) was administered at a single dose of 10 mg/kg.

Administration group: 30 minutes after administration (single dose administration) of cetuximab (Bristol-Myers Squibb Company), the antibody-drug conjugate (16a) was administered at a single dose of 10 mg/kg.

Administration group: 30 minutes after administration (single dose administration) of panitumumab (Amgen Inc.), the antibody-drug conjugate (16a) was administered at a single dose of 10 mg/kg.

[0330]

The results are shown in FIG. 29. The antitumor effect achieved by the combined application on the human head and neck cancer cell line was observed in the cetuximab (Fig. 29A) or panitumumab (Fig. 29B) and antibody-drug conjugate group compared with the administration of each drug alone. These results demonstrate that the pharmaceutical effect of the antibody-drug conjugate is enhanced when it is used in combination with cetuximab or panitumumab. No body weight loss was observed in mice in the treated groups.

[0331]

Test Example 22 Antibody drug conjugate (16a) demonstrated antitumor effect in combination with cetuximab or pertuzumab in an in vivo antitumor trial by tumor transplantation from a patient with gastric cancer

The anti-EGFR antibody cetuximab and the anti-HER2 antibody pertuzumab are in clinical trials against human gastric cancer. This trial assessed whether or not the combined use of cetuximab or pertuzumab and an antibody-drug conjugate is effective. Evaluation was performed by conducting an anti-tumor test close to clinical conditions using the patient's stroma, using patient-derived tumors transplanted into mice, instead of the general evaluation system using a human cancer cell line.

Used 5-6 week old females naked mice (Charles River Laboratories Japan, Inc.) having a body weight of 15 to 20 g after acclimatization. Mice were housed in individually ventilated cages (IVC, maximum 4 mice per cage) maintained at room temperature and constant humidity. After randomization, the body weight of the mice was measured continuously every other day and the behavior of the animals was recorded every day.

Tumor from a patient with gastric cancer NIBIO-G016 from the National Institute of Biomedical Innovation was transplanted subcutaneously into the right flank of each nude mouse. The body weight of the animals was measured using a balance (Mettler Toledo PB602-L).

The major axis and minor axis of the tumor were measured using an electronic digital caliper (CD-15CX, Mitsutoyo Corp), and the tumor volume (mm ³ ) was calculated. The calculation was carried out in accordance with the following formula.

Tumor volume (mm ³ )=1/2 × Major axis (mm) × [Minor axis (mm)] ²

On Day 56, when the tumor size reached about 220 mm ³ , 30 animals were randomly divided into 6 groups based on their tumor sizes. On the same day, the antibody-drug conjugate (16a), cetuximab or pertuzumab used in the combination of the conjugate and cetuximab or pertuzumab or PBS for the control group was administered at the following doses.

[0332]

Administration group: PBS was administered at the same dose as the antibody-drug conjugate was administered.

Administration group: Cetuximab (Bristol-Myers Squibb Company) was administered at a single dose of 10 mg/kg.

Administration group: Pertuzumab (Roche Diagnostics, Inc.) was administered at a single dose of 10 mg/kg.

Administration group: The antibody-drug conjugate (16a) was administered at a single dose of 10 mg/kg.

Administration group: 30 minutes after administration (single dose administration) of cetuximab (Bristol-Myers Squibb Company), the antibody-drug conjugate (16a) was administered at a single dose of 10 mg/kg.

Administration group: 30 minutes after administration (single dose administration) of pertuzumab (Roche Diagnostics, Inc.), the antibody-drug conjugate (16a) was administered at a single dose of 10 mg/kg.

[0333]

The results are shown in FIG. 30. The antitumor effect achieved by combined use on a patient-isolated gastric cancer tumor model was observed in the cetuximab (Fig. 30A) or pertuzumab (Fig. 30B) and antibody-drug conjugate group compared to each drug alone. These results demonstrate that the pharmaceutical effect of the antibody-drug conjugate is enhanced when it is used in combination with cetuximab or panitumumab in a patient-isolated tumor model. No body weight loss was observed in mice in the treated groups. In addition, in an antitumor test using the human pancreatic cancer cell line BxPC3, the antibody drug conjugate (13) showed a stronger antitumor effect compared to the PBS administration group or the U1-59 administration group. The antibody-drug conjugate also showed a strong antitumor effect in vivo. an antitumor model using the JIMT1 HER2-positive breast cancer cell line with acquired resistance to the combined use of trastuzumab and pertuzumab or to trastuzumab emtansine.

[0334]

Test Example 23 Safety of Antibody-Drug Conjugate in Non-Human Animals

The anti-HER3 antibody-drug conjugate of the present invention and the pharmaceutical composition containing such anti-HER3 antibody-drug conjugate are excellent in safety as a therapeutic or prophylactic for a certain disease. For example, when the antibody-drug conjugate (5), (10) or (13) was administered at a dose of up to 30 mg/kg to a hybrid rat, a total of twice once a week apart, no signs of serious toxicity were observed for either which of the antibody-drug conjugates as a result of observations up to Day 7 after the final administration. In addition, when the antibody-drug conjugate (5), (10) or (13) was administered at a dose of up to 30 mg/kg to the hybrid monkey, no noticeable signs of toxicity were observed for any of the antibody-drug conjugates as a result of observations in within 7 days.

In addition, when the antibody-drug conjugate (5), (10) or (13) was administered to a monkey using multiple doses (at 3-week intervals), no significant signs of toxicity were observed for any of the antibody-drug conjugates as a result observations. Therefore, the antibody-drug conjugate of the present invention has excellent safety as a pharmaceutical composition for the treatment or prevention of a disease.

[Free text sequence listing]

[0335]

SEQ ID NO: 1 - Nucleotide sequence encoding the amino acid sequence of the heavy chain variable region of human anti-HER3 antibody U1-39

SEQ ID NO: 2 - Amino acid sequence of the heavy chain variable region of human anti-HER3 antibody U1-39

SEQ ID NO: 3 - Nucleotide sequence encoding the amino acid sequence of the light chain variable region of the human anti-HER3 antibody U1-39

SEQ ID NO: 4 - Amino acid sequence of the light chain variable region of human anti-HER3 antibody U1-39

SEQ ID NO: 5 - Nucleotide sequence encoding the amino acid sequence of the heavy chain variable region of the human anti-HER3 antibody U1-40

SEQ ID NO: 6 - Amino acid sequence of the heavy chain variable region of human anti-HER3 antibody U1-40

SEQ ID NO: 7 - Nucleotide sequence encoding the amino acid sequence of the light chain variable region of the human anti-HER3 antibody U1-40

SEQ ID NO: 8 - Amino acid sequence of the light chain variable region of human anti-HER3 antibody U1-40

SEQ ID NO: 9 - Nucleotide sequence encoding the amino acid sequence of the heavy chain variable region of human anti-HER3 antibody U1-38

SEQ ID NO: 10 - Amino acid sequence of the heavy chain variable region of human anti-HER3 antibody U1-38

SEQ ID NO: 11 - Nucleotide sequence encoding the amino acid sequence of the light chain variable region of human anti-HER3 antibody U1-38

SEQ ID NO: 12 - Amino acid sequence of the light chain variable region of human anti-HER3 antibody U1-38

SEQ ID NO: 13 - Nucleotide sequence encoding the amino acid sequence of the heavy chain variable region of human anti-HER3 antibody U1-41

SEQ ID NO: 14 - Amino acid sequence of the heavy chain variable region of human anti-HER3 antibody U1-41

SEQ ID NO: 15 - Nucleotide sequence encoding the amino acid sequence of the light chain variable region of human anti-HER3 antibody U1-41

SEQ ID NO: 16 - Amino acid sequence of the light chain variable region of human anti-HER3 antibody U1-41

SEQ ID NO: 17 - Nucleotide sequence encoding the amino acid sequence of the heavy chain variable region of human anti-HER3 antibody U1-42

SEQ ID NO: 18 - Amino acid sequence of the heavy chain variable region of human anti-HER3 antibody U1-42

SEQ ID NO: 19 - Nucleotide sequence encoding the amino acid sequence of the light chain variable region of human anti-HER3 antibody U1-42

SEQ ID NO: 20 - Amino acid sequence of the light chain variable region of human anti-HER3 antibody U1-42

SEQ ID NO: 21 - Nucleotide sequence encoding the amino acid sequence of the heavy chain variable region of human anti-HER3 antibody U1-43

SEQ ID NO: 22 - Amino acid sequence of the heavy chain variable region of human anti-HER3 antibody U1-43

SEQ ID NO: 23 - Nucleotide sequence encoding the amino acid sequence of the light chain variable region of human anti-HER3 antibody U1-43

SEQ ID NO: 24 - Amino acid sequence of the light chain variable region of human anti-HER3 antibody U1-43

SEQ ID NO: 25 - Nucleotide sequence encoding the amino acid sequence of the heavy chain variable region of human anti-HER3 antibody U1-44

SEQ ID NO: 26 - Amino acid sequence of heavy chain variable region of human anti-HER3 antibody U1-44

SEQ ID NO: 27 - Nucleotide sequence encoding the amino acid sequence of the light chain variable region of human anti-HER3 antibody U1-44

SEQ ID NO: 28 - Amino acid sequence of the light chain variable region of human anti-HER3 antibody U1-44

SEQ ID NO: 29 - Nucleotide sequence encoding the amino acid sequence of the heavy chain variable region of human anti-HER3 antibody U1-45

SEQ ID NO: 30 - Amino acid sequence of the heavy chain variable region of human anti-HER3 antibody U1-45

SEQ ID NO: 31 - Nucleotide sequence encoding the amino acid sequence of the light chain variable region of human anti-HER3 antibody U1-45

SEQ ID NO: 32 - Amino acid sequence of the light chain variable region of human anti-HER3 antibody U1-45

SEQ ID NO: 33 - Nucleotide sequence encoding the amino acid sequence of the heavy chain variable region of human anti-HER3 antibody U1-46

SEQ ID NO: 34 - Amino acid sequence of the heavy chain variable region of human anti-HER3 antibody U1-46

SEQ ID NO: 35 - Nucleotide sequence encoding the amino acid sequence of the heavy chain variable region of human anti-HER3 antibody U1-47

SEQ ID NO: 36 - Amino acid sequence of the heavy chain variable region of human anti-HER3 antibody U1-47

SEQ ID NO: 37 - Nucleotide sequence encoding the amino acid sequence of the light chain variable region of human anti-HER3 antibody U1-47

SEQ ID NO: 38 - Amino acid sequence of the light chain variable region of human anti-HER3 antibody U1-47

SEQ ID NO: 39 - Nucleotide sequence encoding the amino acid sequence of the heavy chain variable region of human anti-HER3 antibody U1-48

SEQ ID NO:40 - Amino acid sequence of heavy chain variable region of human anti-HER3 antibody U1-48

SEQ ID NO:41 - Nucleotide sequence encoding the amino acid sequence of the heavy chain variable region of human anti-HER3 antibody U1-49

SEQ ID NO:42 - Amino acid sequence of the heavy chain variable region of human anti-HER3 antibody U1-49

SEQ ID NO:43 - Nucleotide sequence encoding the amino acid sequence of the light chain variable region of human anti-HER3 antibody U1-49

SEQ ID NO:44 - Amino acid sequence of the light chain variable region of human anti-HER3 antibody U1-49

SEQ ID NO:45 - Nucleotide sequence encoding the amino acid sequence of the heavy chain variable region of human anti-HER3 antibody U1-50

SEQ ID NO:46 - Amino acid sequence of heavy chain variable region of human anti-HER3 antibody U1-50

SEQ ID NO:47 - Nucleotide sequence encoding the amino acid sequence of the light chain variable region of the human anti-HER3 antibody U1-50

SEQ ID NO:48 - Light chain variable region amino acid sequence of human anti-HER3 antibody U1-50

SEQ ID NO: 49 - Nucleotide sequence encoding the amino acid sequence of the heavy chain variable region of human anti-HER3 antibody U1-51

SEQ ID NO: 50 - Amino acid sequence of the heavy chain variable region of human anti-HER3 antibody U1-51

SEQ ID NO: 51 - Nucleotide sequence encoding the amino acid sequence of the light chain variable region of human anti-HER3 antibody U1-51

SEQ ID NO: 52 - Amino acid sequence of the light chain variable region of human anti-HER3 antibody U1-51

SEQ ID NO: 53 - Nucleotide sequence encoding the amino acid sequence of the heavy chain variable region of human anti-HER3 antibody U1-53

SEQ ID NO: 54 - Amino acid sequence of the heavy chain variable region of human anti-HER3 antibody U1-53

SEQ ID NO: 55 - Nucleotide sequence encoding the amino acid sequence of the light chain variable region of human anti-HER3 antibody U1-53

SEQ ID NO: 56 - Amino acid sequence of the light chain variable region of human anti-HER3 antibody U1-53

SEQ ID NO: 57 - Nucleotide sequence encoding the amino acid sequence of the light chain variable region of human anti-HER3 antibody U1-55

SEQ ID NO: 58 - Amino acid sequence of the light chain variable region of human anti-HER3 antibody U1-55

SEQ ID NO: 59 - Nucleotide sequence encoding the amino acid sequence of the heavy chain variable region of human anti-HER3 antibody U1-55.1

SEQ ID NO: 60 - Amino acid sequence of the heavy chain variable region of human anti-HER3 antibody U1-55.1

SEQ ID NO: 61 - Nucleotide sequence encoding the amino acid sequence of the heavy chain variable region of human anti-HER3 antibody U1-57

SEQ ID NO: 62 - Amino acid sequence of the heavy chain variable region of human anti-HER3 antibody U1-57

SEQ ID NO: 63 - Nucleotide sequence encoding the amino acid sequence of the light chain variable region of human anti-HER3 antibody U1-57.1

SEQ ID NO: 64 - Amino acid sequence of the light chain variable region of human anti-HER3 antibody U1-57.1

SEQ ID NO: 65 - Nucleotide sequence encoding the amino acid sequence of the heavy chain variable region of human anti-HER3 antibody U1-58

SEQ ID NO: 66 - Amino acid sequence of the heavy chain variable region of human anti-HER3 antibody U1-58

SEQ ID NO: 67 - Nucleotide sequence encoding the amino acid sequence of the light chain variable region of human anti-HER3 antibody U1-58

SEQ ID NO: 68 - Light chain variable region amino acid sequence of human anti-HER3 antibody U1-58

SEQ ID NO: 69 - Nucleotide sequence encoding the amino acid sequence of the heavy chain variable region of the human anti-HER3 antibody U1-59

SEQ ID NO: 70 - Amino acid sequence of the heavy chain variable region of human anti-HER3 antibody U1-59

SEQ ID NO: 71 - Nucleotide sequence encoding the amino acid sequence of the light chain variable region of human anti-HER3 antibody U1-59

SEQ ID NO: 72 - Amino acid sequence of the light chain variable region of human anti-HER3 antibody U1-59

SEQ ID NO: 73 - Nucleotide sequence encoding the amino acid sequence of the heavy chain variable region of human anti-HER3 antibody U1-52

SEQ ID NO: 74 - Amino acid sequence of the heavy chain variable region of human anti-HER3 antibody U1-52

SEQ ID NO: 75 - Nucleotide sequence encoding the amino acid sequence of the light chain variable region of human anti-HER3 antibody U1-52

SEQ ID NO: 76 - Amino acid sequence of the light chain variable region of human anti-HER3 antibody U1-52

SEQ ID NO: 77 - Nucleotide sequence encoding the amino acid sequence of the heavy chain variable region of human anti-HER3 antibody U1-61

SEQ ID NO: 78 - Amino acid sequence of the heavy chain variable region of human anti-HER3 antibody U1-61

SEQ ID NO: 79 - Nucleotide sequence encoding the amino acid sequence of the heavy chain variable region of human anti-HER3 antibody U1-61.1

SEQ ID NO: 80 - Amino acid sequence of the heavy chain variable region of human anti-HER3 antibody U1-61.1

SEQ ID NO: 81 - Nucleotide sequence encoding the amino acid sequence of the light chain variable region of human anti-HER3 antibody U1-61.1

SEQ ID NO: 82 - Light chain variable region amino acid sequence of human anti-HER3 antibody U1-61.1

SEQ ID NO: 83 - Nucleotide sequence encoding the amino acid sequence of the heavy chain variable region of human anti-HER3 antibody U1-62

SEQ ID NO: 84 - Amino acid sequence of the heavy chain variable region of human anti-HER3 antibody U1-62

SEQ ID NO: 85 - Nucleotide sequence encoding the amino acid sequence of the light chain variable region of the human anti-HER3 antibody U1-62

SEQ ID NO: 86 - Amino acid sequence of the light chain variable region of human anti-HER3 antibody U1-62

SEQ ID NO: 87 - Nucleotide sequence encoding the amino acid sequence of the heavy chain variable region of human anti-HER3 antibody U1-2

SEQ ID NO: 88 - Amino acid sequence of the heavy chain variable region of human anti-HER3 antibody U1-2

SEQ ID NO: 89 - Nucleotide sequence encoding the amino acid sequence of the light chain variable region of human anti-HER3 antibody U1-2

SEQ ID NO: 90 - Light chain variable region amino acid sequence of human anti-HER3 antibody U1-2

SEQ ID NO: 91 - Nucleotide sequence encoding the amino acid sequence of the heavy chain variable region of human anti-HER3 antibody U1-7

SEQ ID NO: 92 - Amino acid sequence of the heavy chain variable region of human anti-HER3 antibody U1-7

SEQ ID NO: 93 - Nucleotide sequence encoding the amino acid sequence of the light chain variable region of the human anti-HER3 antibody U1-7

SEQ ID NO: 94 - Amino acid sequence of the light chain variable region of human anti-HER3 antibody U1-7

SEQ ID NO: 95 - Nucleotide sequence encoding the amino acid sequence of the heavy chain variable region of human anti-HER3 antibody U1-9

SEQ ID NO: 96 - Amino acid sequence of heavy chain variable region of human anti-HER3 antibody U1-9

SEQ ID NO: 97 - Nucleotide sequence encoding the amino acid sequence of the light chain variable region of the human anti-HER3 antibody U1-9

SEQ ID NO: 98 - Light chain variable region amino acid sequence of human anti-HER3 antibody U1-9

SEQ ID NO: 99 - Nucleotide sequence encoding the amino acid sequence of the heavy chain variable region of human anti-HER3 antibody U1-10

SEQ ID NO: 100 - Amino acid sequence of the heavy chain variable region of human anti-HER3 antibody U1-10

SEQ ID NO: 101 - Nucleotide sequence encoding the amino acid sequence of the light chain variable region of human anti-HER3 antibody U1-10

SEQ ID NO: 102 - Amino acid sequence of the light chain variable region of human anti-HER3 antibody U1-10

SEQ ID NO: 103 - Nucleotide sequence encoding the amino acid sequence of the heavy chain variable region of human anti-HER3 antibody U1-12

SEQ ID NO: 104 - Amino acid sequence of the heavy chain variable region of human anti-HER3 antibody U1-12

SEQ ID NO: 105 - Nucleotide sequence encoding the amino acid sequence of the light chain variable region of human anti-HER3 antibody U1-12

SEQ ID NO: 106 - Amino acid sequence of the light chain variable region of human anti-HER3 antibody U1-12

SEQ ID NO: 107 - Nucleotide sequence encoding the amino acid sequence of the heavy chain variable region of human anti-HER3 antibody U1-13

SEQ ID NO: 108 - Amino acid sequence of the heavy chain variable region of human anti-HER3 antibody U1-13

SEQ ID NO: 109 - Nucleotide sequence encoding the amino acid sequence of the light chain variable region of human anti-HER3 antibody U1-13

SEQ ID NO: 110 - Amino acid sequence of the light chain variable region of human anti-HER3 antibody U1-13

SEQ ID NO: 111 - Nucleotide sequence encoding the amino acid sequence of the heavy chain variable region of human anti-HER3 antibody U1-14

SEQ ID NO: 112 - Amino acid sequence of the heavy chain variable region of human anti-HER3 antibody U1-14

SEQ ID NO: 113 - Nucleotide sequence encoding the amino acid sequence of the light chain variable region of human anti-HER3 antibody U1-14

SEQ ID NO: 114 - Light chain variable region amino acid sequence of human anti-HER3 antibody U1-14

SEQ ID NO: 115 - Nucleotide sequence encoding the amino acid sequence of the heavy chain variable region of human anti-HER3 antibody U1-15

SEQ ID NO: 116 - Amino acid sequence of heavy chain variable region of human anti-HER3 antibody U1-15

SEQ ID NO: 117 - Nucleotide sequence encoding the amino acid sequence of the light chain variable region of human anti-HER3 antibody U1-15

SEQ ID NO: 118 - Light chain variable region amino acid sequence of human anti-HER3 antibody U1-15

SEQ ID NO: 119 - Nucleotide sequence encoding the amino acid sequence of the heavy chain variable region of human anti-HER3 antibody U1-19

SEQ ID NO: 120 - Amino acid sequence of the heavy chain variable region of human anti-HER3 antibody U1-19

SEQ ID NO: 121 - Nucleotide sequence encoding the amino acid sequence of the heavy chain variable region of human anti-HER3 antibody U1-20

SEQ ID NO: 122 - Amino acid sequence of the heavy chain variable region of human anti-HER3 antibody U1-20

SEQ ID NO: 123 - Nucleotide sequence encoding the amino acid sequence of the light chain variable region of human anti-HER3 antibody U1-20

SEQ ID NO: 124 - Amino acid sequence of the light chain variable region of human anti-HER3 antibody U1-20

SEQ ID NO: 125 - Nucleotide sequence encoding the amino acid sequence of the heavy chain variable region of human anti-HER3 antibody U1-21

SEQ ID NO: 126 - Amino acid sequence of the heavy chain variable region of human anti-HER3 antibody U1-21

SEQ ID NO: 127 - Nucleotide sequence encoding the amino acid sequence of the light chain variable region of human anti-HER3 antibody U1-21

SEQ ID NO: 128 - Light chain variable region amino acid sequence of human anti-HER3 antibody U1-21

SEQ ID NO: 129 - Nucleotide sequence encoding the amino acid sequence of the heavy chain variable region of human anti-HER3 antibody U1-22

SEQ ID NO: 130 - Amino acid sequence of the heavy chain variable region of human anti-HER3 antibody U1-22

SEQ ID NO: 131 - Nucleotide sequence encoding the amino acid sequence of the light chain variable region of human anti-HER3 antibody U1-22

SEQ ID NO: 132 - Amino acid sequence of the light chain variable region of human anti-HER3 antibody U1-22

SEQ ID NO: 133 - Nucleotide sequence encoding the amino acid sequence of the heavy chain variable region of human anti-HER3 antibody U1-23

SEQ ID NO: 134 - Amino acid sequence of the heavy chain variable region of human anti-HER3 antibody U1-23

SEQ ID NO: 135 - Nucleotide sequence encoding the amino acid sequence of the light chain variable region of human anti-HER3 antibody U1-23

SEQ ID NO: 136 - Light chain variable region amino acid sequence of human anti-HER3 antibody U1-23

SEQ ID NO: 137 - Nucleotide sequence encoding the amino acid sequence of the heavy chain variable region of human anti-HER3 antibody U1-24

SEQ ID NO: 138 - Amino acid sequence of the heavy chain variable region of human anti-HER3 antibody U1-24

SEQ ID NO: 139 - Nucleotide sequence encoding the amino acid sequence of the light chain variable region of human anti-HER3 antibody U1-24

SEQ ID NO: 140 - Light chain variable region amino acid sequence of human anti-HER3 antibody U1-24

SEQ ID NO: 141 - Nucleotide sequence encoding the amino acid sequence of the heavy chain variable region of human anti-HER3 antibody U1-25

SEQ ID NO: 142 - Amino acid sequence of the heavy chain variable region of human anti-HER3 antibody U1-25

SEQ ID NO: 143 - Nucleotide sequence encoding the amino acid sequence of the light chain variable region of human anti-HER3 antibody U1-25

SEQ ID NO: 144 - Amino acid sequence of the light chain variable region of human anti-HER3 antibody U1-25

SEQ ID NO: 145 - Nucleotide sequence encoding the amino acid sequence of the heavy chain variable region of human anti-HER3 antibody U1-26

SEQ ID NO: 146 - Amino acid sequence of the heavy chain variable region of human anti-HER3 antibody U1-26

SEQ ID NO: 147 - Nucleotide sequence encoding the amino acid sequence of the light chain variable region of human anti-HER3 antibody U1-26

SEQ ID NO: 148 - Amino acid sequence of the light chain variable region of human anti-HER3 antibody U1-26

SEQ ID NO: 149 - Nucleotide sequence encoding the amino acid sequence of the heavy chain variable region of human anti-HER3 antibody U1-27

SEQ ID NO: 150 - Amino acid sequence of the heavy chain variable region of human anti-HER3 antibody U1-27

SEQ ID NO: 151 - Nucleotide sequence encoding the amino acid sequence of the light chain variable region of human anti-HER3 antibody U1-27

SEQ ID NO: 152 - Light chain variable region amino acid sequence of human anti-HER3 antibody U1-27

SEQ ID NO: 153 - Nucleotide sequence encoding the amino acid sequence of the heavy chain variable region of human anti-HER3 antibody U1-28

SEQ ID NO: 154 - Amino acid sequence of the heavy chain variable region of human anti-HER3 antibody U1-28

SEQ ID NO: 155 - Nucleotide sequence encoding the amino acid sequence of the light chain variable region of human anti-HER3 antibody U1-28

SEQ ID NO: 156 - Light chain variable region amino acid sequence of human anti-HER3 antibody U1-28

SEQ ID NO: 157 - Nucleotide sequence encoding the amino acid sequence of the heavy chain variable region of human anti-HER3 antibody U1-31

SEQ ID NO: 158 - Amino acid sequence of the heavy chain variable region of human anti-HER3 antibody U1-31

SEQ ID NO: 159 - Nucleotide sequence encoding the amino acid sequence of the light chain variable region of human anti-HER3 antibody U1-31

SEQ ID NO: 160 - Light chain variable region amino acid sequence of human anti-HER3 antibody U1-31

SEQ ID NO: 161 - Nucleotide sequence encoding the amino acid sequence of the heavy chain variable region of human anti-HER3 antibody U1-32

SEQ ID NO: 162 - Amino acid sequence of heavy chain variable region of human anti-HER3 antibody U1-32

SEQ ID NO: 163 - Nucleotide sequence encoding the amino acid sequence of the light chain variable region of human anti-HER3 antibody U1-32

SEQ ID NO: 164 - Amino acid sequence of the light chain variable region of human anti-HER3 antibody U1-32

SEQ ID NO: 165 - Nucleotide sequence encoding the amino acid sequence of the heavy chain variable region of human anti-HER3 antibody U1-35

SEQ ID NO: 166 - Amino acid sequence of the heavy chain variable region of human anti-HER3 antibody U1-35

SEQ ID NO: 167 - Nucleotide sequence encoding the amino acid sequence of the light chain variable region of human anti-HER3 antibody U1-35

SEQ ID NO: 168 - Light chain variable region amino acid sequence of human anti-HER3 antibody U1-35

SEQ ID NO: 169 - Nucleotide sequence encoding the amino acid sequence of the heavy chain variable region of human anti-HER3 antibody U1-36

SEQ ID NO: 170 - Amino acid sequence of the heavy chain variable region of human anti-HER3 antibody U1-36

SEQ ID NO: 171 - Nucleotide sequence encoding the amino acid sequence of the light chain variable region of human anti-HER3 antibody U1-36

SEQ ID NO: 172 - Light chain variable region amino acid sequence of human anti-HER3 antibody U1-36

SEQ ID NO: 173 - Nucleotide sequence encoding the amino acid sequence of the heavy chain variable region of human anti-HER3 antibody U1-37

SEQ ID NO: 174 - Amino acid sequence of the heavy chain variable region of human anti-HER3 antibody U1-37

SEQ ID NO: 175 - Nucleotide sequence encoding the amino acid sequence of the light chain variable region of human anti-HER3 antibody U1-37

SEQ ID NO: 176 - Light chain variable region amino acid sequence of human anti-HER3 antibody U1-37

SEQ ID NO: 177 - Nucleotide sequence encoding the amino acid sequence of the heavy chain variable region of human anti-HER3 antibody U1-34

SEQ ID NO: 178 - Amino acid sequence of the heavy chain variable region of human anti-HER3 antibody U1-34

SEQ ID NO: 179 - Nucleotide sequence encoding the amino acid sequence of the light chain variable region of human anti-HER3 antibody U1-34

SEQ ID NO: 180 - Light chain variable region amino acid sequence of human anti-HER3 antibody U1-34

SEQ ID NO: 181 - Nucleotide sequence encoding the amino acid sequence of the heavy chain variable region of human anti-HER3 antibody U1-1

SEQ ID NO: 182 - Amino acid sequence of the heavy chain variable region of human anti-HER3 antibody U1-1

SEQ ID NO: 183 - Nucleotide sequence encoding the amino acid sequence of the light chain variable region of human anti-HER3 antibody U1-1

SEQ ID NO: 184 - Amino acid sequence of human anti-HER3 antibody U1-1 light chain variable region

SEQ ID NO: 185 - Nucleotide sequence encoding the amino acid sequence of the heavy chain variable region of human anti-HER3 antibody U1-3

SEQ ID NO: 186 - Amino acid sequence of heavy chain variable region of human anti-HER3 antibody U1-3

SEQ ID NO: 187 - Nucleotide sequence encoding the amino acid sequence of the light chain variable region of human anti-HER3 antibody U1-3

SEQ ID NO: 188 - Light chain variable region amino acid sequence of human anti-HER3 antibody U1-3

SEQ ID NO: 189 - Nucleotide sequence encoding the amino acid sequence of the heavy chain variable region of human anti-HER3 antibody U1-4

SEQ ID NO: 190 - Amino acid sequence of the heavy chain variable region of human anti-HER3 antibody U1-4

SEQ ID NO: 191 - Nucleotide sequence encoding the amino acid sequence of the light chain variable region of human anti-HER3 antibody U1-4

SEQ ID NO: 192 - Light chain variable region amino acid sequence of human anti-HER3 antibody U1-4

SEQ ID NO: 193 - Nucleotide sequence encoding the amino acid sequence of the heavy chain variable region of human anti-HER3 antibody U1-5

SEQ ID NO: 194 - Amino acid sequence of heavy chain variable region of human anti-HER3 antibody U1-5

SEQ ID NO: 195 - Nucleotide sequence encoding the amino acid sequence of the light chain variable region of human anti-HER3 antibody U1-5

SEQ ID NO: 196 - Light chain variable region amino acid sequence of human anti-HER3 antibody U1-5

SEQ ID NO: 197 - Nucleotide sequence encoding the amino acid sequence of the heavy chain variable region of human anti-HER3 antibody U1-6

SEQ ID NO: 198 - Amino acid sequence of the heavy chain variable region of human anti-HER3 antibody U1-6

SEQ ID NO: 199 - Nucleotide sequence encoding the amino acid sequence of the light chain variable region of human anti-HER3 antibody U1-6

SEQ ID NO:200 - Amino acid sequence of human anti-HER3 antibody U1-6 light chain variable region

SEQ ID NO: 201 - Nucleotide sequence encoding the amino acid sequence of the heavy chain variable region of human anti-HER3 antibody U1-8

SEQ ID NO: 202 - Amino acid sequence of heavy chain variable region of human anti-HER3 antibody U1-8

SEQ ID NO: 203 - Nucleotide sequence encoding the amino acid sequence of the light chain variable region of human anti-HER3 antibody U1-8

SEQ ID NO: 204 - Amino acid sequence of the light chain variable region of human anti-HER3 antibody U1-8

SEQ ID NO: 205 - Nucleotide sequence encoding the amino acid sequence of the heavy chain variable region of human anti-HER3 antibody U1-11

SEQ ID NO: 206 - Amino acid sequence of the heavy chain variable region of human anti-HER3 antibody U1-11

SEQ ID NO: 207 - Nucleotide sequence encoding the amino acid sequence of the light chain variable region of human anti-HER3 antibody U1-11

SEQ ID NO: 208 - Light chain variable region amino acid sequence of human anti-HER3 antibody U1-11

SEQ ID NO: 209 - Nucleotide sequence encoding the amino acid sequence of the heavy chain variable region of human anti-HER3 antibody U1-16

SEQ ID NO: 210 - Amino acid sequence of heavy chain variable region of human anti-HER3 antibody U1-16

SEQ ID NO: 211 - Nucleotide sequence encoding the amino acid sequence of the light chain variable region of human anti-HER3 antibody U1-16

SEQ ID NO: 212 - Amino acid sequence of the light chain variable region of human anti-HER3 antibody U1-16

SEQ ID NO: 213 - Nucleotide sequence encoding the amino acid sequence of the heavy chain variable region of human anti-HER3 antibody U1-17

SEQ ID NO: 214 - Amino acid sequence of the heavy chain variable region of human anti-HER3 antibody U1-17

SEQ ID NO: 215 - Nucleotide sequence encoding the amino acid sequence of the light chain variable region of human anti-HER3 antibody U1-17

SEQ ID NO: 216 - Light chain variable region amino acid sequence of human anti-HER3 antibody U1-17

SEQ ID NO: 217 - Nucleotide sequence encoding the amino acid sequence of the heavy chain variable region of human anti-HER3 antibody U1-18

SEQ ID NO: 218 - Amino acid sequence of heavy chain variable region of human anti-HER3 antibody U1-18

SEQ ID NO: 219 - Nucleotide sequence encoding the amino acid sequence of the light chain variable region of human anti-HER3 antibody U1-18

SEQ ID NO: 220 - Amino acid sequence of the light chain variable region of human anti-HER3 antibody U1-18

SEQ ID NO: 221 - Nucleotide sequence encoding the amino acid sequence of the heavy chain variable region of human anti-HER3 antibody U1-33

SEQ ID NO: 222 - Amino acid sequence of the heavy chain variable region of human anti-HER3 antibody U1-33

SEQ ID NO: 223 - Nucleotide sequence encoding the amino acid sequence of the light chain variable region of human anti-HER3 antibody U1-33

SEQ ID NO: 224 - Light chain variable region amino acid sequence of human anti-HER3 antibody U1-33

SEQ ID NO: 225 - Nucleotide sequence encoding the amino acid sequence of the heavy chain variable region of human anti-HER3 antibody U1-29

SEQ ID NO: 226 - Amino acid sequence of the heavy chain variable region of human anti-HER3 antibody U1-29

SEQ ID NO: 227 - Nucleotide sequence encoding the amino acid sequence of the light chain variable region of human anti-HER3 antibody U1-29

SEQ ID NO: 228 - Light chain variable region amino acid sequence of human anti-HER3 antibody U1-29

SEQ ID NO: 229 - Nucleotide sequence encoding the amino acid sequence of the heavy chain variable region of human anti-HER3 antibody U1-30

SEQ ID NO: 230 - Amino acid sequence of the heavy chain variable region of human anti-HER3 antibody U1-30

SEQ ID NO: 231 - Nucleotide sequence encoding the amino acid sequence of the light chain variable region of human anti-HER3 antibody U1-30

SEQ ID NO: 232 - Light chain variable region amino acid sequence of human anti-HER3 antibody U1-30

SEQ ID NO: 233 - Primer

SEQ ID NO: 234 - Primer

SEQ ID NO: 235 - Amino acid sequence of heavy chain CDRH1 of human anti-HER3 antibody U1-1

SEQ ID NO: 236 - Amino acid sequence of heavy chain CDRH2 of human anti-HER3 antibody U1-1

SEQ ID NO: 237 - Amino acid sequence of heavy chain CDRH3 of human anti-HER3 antibody U1-1

SEQ ID NO: 238 - Light chain CDRL1 amino acid sequence of human anti-HER3 antibody U1-1

SEQ ID NO: 239 - Light chain CDRL2 amino acid sequence of human anti-HER3 antibody U1-1

SEQ ID NO: 240 - Light chain CDRL3 amino acid sequence of human anti-HER3 antibody U1-1

SEQ ID NO: 241 - Amino acid sequence of heavy chain CDRH1 of human anti-HER3 antibody U1-2

SEQ ID NO: 242 - Amino acid sequence of heavy chain CDRH2 of human anti-HER3 antibody U1-2

SEQ ID NO: 243 - Amino acid sequence of heavy chain CDRH3 of human anti-HER3 antibody U1-2

SEQ ID NO: 244 - Light chain CDRL1 amino acid sequence of human anti-HER3 antibody U1-2

SEQ ID NO: 245 - Light chain CDRL2 amino acid sequence of human anti-HER3 antibody U1-2

SEQ ID NO: 246 - Light chain CDRL3 amino acid sequence of human anti-HER3 antibody U1-2

SEQ ID NO: 247 - Amino acid sequence of heavy chain CDRH1 of human anti-HER3 antibody U1-3

SEQ ID NO: 248 - Amino acid sequence of heavy chain CDRH2 of human anti-HER3 antibody U1-3

SEQ ID NO: 249 - Amino acid sequence of heavy chain CDRH3 of human anti-HER3 antibody U1-3

SEQ ID NO: 250 - Light chain CDRL1 amino acid sequence of human anti-HER3 antibody U1-3

SEQ ID NO: 251 - Light chain CDRL2 amino acid sequence of human anti-HER3 antibody U1-3

SEQ ID NO: 252 - Light chain CDRL3 amino acid sequence of human anti-HER3 antibody U1-3

SEQ ID NO: 253 - Amino acid sequence of heavy chain CDRH1 of human anti-HER3 antibody U1-4

SEQ ID NO: 254 - Amino acid sequence of heavy chain CDRH2 of human anti-HER3 antibody U1-4

SEQ ID NO: 255 - Amino acid sequence of heavy chain CDRH3 of human anti-HER3 antibody U1-4

SEQ ID NO: 256 - Light chain amino acid sequence of human anti-HER3 antibody U1-4 CDRL1

SEQ ID NO: 257 - Amino acid sequence of CDRL2 light chain of human anti-HER3 antibody U1-4

SEQ ID NO: 258 - Light chain CDRL3 amino acid sequence of human anti-HER3 antibody U1-4

SEQ ID NO: 259 - Amino acid sequence of heavy chain CDRH1 of human anti-HER3 antibody U1-5

SEQ ID NO: 260 - Amino acid sequence of heavy chain CDRH2 of human anti-HER3 antibody U1-5

SEQ ID NO: 261 - Amino acid sequence of heavy chain CDRH3 of human anti-HER3 antibody U1-5

SEQ ID NO: 262 - Light chain CDRL1 amino acid sequence of human anti-HER3 antibody U1-5

SEQ ID NO: 263 - Light chain CDRL2 amino acid sequence of human anti-HER3 antibody U1-5

SEQ ID NO: 264 - Light chain CDRL3 amino acid sequence of human anti-HER3 antibody U1-5

SEQ ID NO: 265 - Amino acid sequence of heavy chain CDRH1 of human anti-HER3 antibody U1-6

SEQ ID NO: 266 - Amino acid sequence of heavy chain CDRH2 of human anti-HER3 antibody U1-6

SEQ ID NO: 267 - Amino acid sequence of heavy chain CDRH3 of human anti-HER3 antibody U1-6

SEQ ID NO: 268 - Light chain CDRL1 amino acid sequence of human anti-HER3 antibody U1-6

SEQ ID NO: 269 - Light chain CDRL2 amino acid sequence of human anti-HER3 antibody U1-6

SEQ ID NO: 270 - Light chain CDRL3 amino acid sequence of human anti-HER3 antibody U1-6

SEQ ID NO: 271 - Amino acid sequence of heavy chain CDRH1 of human anti-HER3 antibody U1-7

SEQ ID NO: 272 - Amino acid sequence of heavy chain CDRH2 of human anti-HER3 antibody U1-7

SEQ ID NO: 273 - Amino acid sequence of heavy chain CDRH3 of human anti-HER3 antibody U1-7

SEQ ID NO: 274 - Light chain CDRL1 amino acid sequence of human anti-HER3 antibody U1-7

SEQ ID NO: 275 - Light chain CDRL2 amino acid sequence of human anti-HER3 antibody U1-7

SEQ ID NO: 276 - Light chain CDRL3 amino acid sequence of human anti-HER3 antibody U1-7

SEQ ID NO: 277 - Amino acid sequence of heavy chain CDRH1 of human anti-HER3 antibody U1-8

SEQ ID NO: 278 - Amino acid sequence of heavy chain CDRH2 of human anti-HER3 antibody U1-8

SEQ ID NO: 279 - Amino acid sequence of heavy chain CDRH3 of human anti-HER3 antibody U1-8

SEQ ID NO: 280 - Light chain CDRL1 amino acid sequence of human anti-HER3 antibody U1-8

SEQ ID NO: 281 - Light chain CDRL2 amino acid sequence of human anti-HER3 antibody U1-8

SEQ ID NO: 282 - Light chain CDRL3 amino acid sequence of human anti-HER3 antibody U1-8

SEQ ID NO: 283 - Amino acid sequence of heavy chain CDRH1 of human anti-HER3 antibody U1-9

SEQ ID NO: 284 - Amino acid sequence of heavy chain CDRH2 of human anti-HER3 antibody U1-9

SEQ ID NO: 285 - Amino acid sequence of heavy chain CDRH3 of human anti-HER3 antibody U1-9

SEQ ID NO: 286 - Light chain CDRL1 amino acid sequence of human anti-HER3 antibody U1-9

SEQ ID NO: 287 - Light chain CDRL2 amino acid sequence of human anti-HER3 antibody U1-9

SEQ ID NO: 288 - Light chain CDRL3 amino acid sequence of human anti-HER3 antibody U1-9

SEQ ID NO: 289 - Amino acid sequence of heavy chain CDRH1 of human anti-HER3 antibody U1-10

SEQ ID NO: 290 - Amino acid sequence of heavy chain CDRH2 of human anti-HER3 antibody U1-10

SEQ ID NO: 291 - Amino acid sequence of heavy chain CDRH3 of human anti-HER3 antibody U1-10

SEQ ID NO: 292 - Light chain CDRL1 amino acid sequence of human anti-HER3 antibody U1-10

SEQ ID NO: 293 - Light chain CDRL2 amino acid sequence of human anti-HER3 antibody U1-10

SEQ ID NO: 294 - Light chain CDRL3 amino acid sequence of human anti-HER3 antibody U1-10

SEQ ID NO: 295 - Amino acid sequence of heavy chain CDRH1 of human anti-HER3 antibody U1-11

SEQ ID NO: 296 - Amino acid sequence of heavy chain CDRH2 of human anti-HER3 antibody U1-11

SEQ ID NO: 297 - Amino acid sequence of heavy chain CDRH3 of human anti-HER3 antibody U1-11

SEQ ID NO: 298 - Light chain CDRL1 amino acid sequence of human anti-HER3 antibody U1-11

SEQ ID NO: 299 - Light chain CDRL2 amino acid sequence of human anti-HER3 antibody U1-11

SEQ ID NO: 300 - Light chain CDRL3 amino acid sequence of human anti-HER3 antibody U1-11

SEQ ID NO: 301 - Amino acid sequence of heavy chain CDRH1 of human anti-HER3 antibody U1-12

SEQ ID NO: 302 - Amino acid sequence of heavy chain CDRH2 of human anti-HER3 antibody U1-12

SEQ ID NO: 303 - Amino acid sequence of heavy chain CDRH3 of human anti-HER3 antibody U1-12

SEQ ID NO: 304 - Light chain CDRL1 amino acid sequence of human anti-HER3 antibody U1-12

SEQ ID NO: 305 - Light chain CDRL2 amino acid sequence of human anti-HER3 antibody U1-12

SEQ ID NO: 306 - Light chain CDRL3 amino acid sequence of human anti-HER3 antibody U1-12

SEQ ID NO: 307 - Amino acid sequence of heavy chain CDRH1 of human anti-HER3 antibody U1-13

SEQ ID NO: 308 - Amino acid sequence of heavy chain CDRH2 of human anti-HER3 antibody U1-13

SEQ ID NO: 309 - Amino acid sequence of heavy chain CDRH3 of human anti-HER3 antibody U1-13

SEQ ID NO: 310 - Light chain CDRL1 amino acid sequence of human anti-HER3 antibody U1-13

SEQ ID NO: 311 - Light chain CDRL2 amino acid sequence of human anti-HER3 antibody U1-13

SEQ ID NO: 312 - Light chain CDRL3 amino acid sequence of human anti-HER3 antibody U1-13

SEQ ID NO: 313 - Amino acid sequence of heavy chain CDRH1 of human anti-HER3 antibody U1-14

SEQ ID NO: 314 - Amino acid sequence of heavy chain CDRH2 of human anti-HER3 antibody U1-14

SEQ ID NO: 315 - Amino acid sequence of heavy chain CDRH3 of human anti-HER3 antibody U1-14

SEQ ID NO: 316 - Light chain CDRL1 amino acid sequence of human anti-HER3 antibody U1-14

SEQ ID NO: 317 - Light chain CDRL2 amino acid sequence of human anti-HER3 antibody U1-14

SEQ ID NO: 318 - Light chain CDRL3 amino acid sequence of human anti-HER3 antibody U1-14

SEQ ID NO: 319 - Amino acid sequence of heavy chain CDRH1 of human anti-HER3 antibody U1-15

SEQ ID NO: 320 - Amino acid sequence of heavy chain CDRH2 of human anti-HER3 antibody U1-15

SEQ ID NO: 321 - Amino acid sequence of heavy chain CDRH3 of human anti-HER3 antibody U1-15

SEQ ID NO: 322 - Light chain CDRL1 amino acid sequence of human anti-HER3 antibody U1-15

SEQ ID NO: 323 - Light chain CDRL2 amino acid sequence of human anti-HER3 antibody U1-15

SEQ ID NO: 324 - Light chain CDRL3 amino acid sequence of human anti-HER3 antibody U1-15

SEQ ID NO: 325 - Amino acid sequence of heavy chain CDRH1 of human anti-HER3 antibody U1-16

SEQ ID NO: 326 - Amino acid sequence of heavy chain CDRH2 of human anti-HER3 antibody U1-16

SEQ ID NO: 327 - Amino acid sequence of heavy chain CDRH3 of human anti-HER3 antibody U1-16

SEQ ID NO: 328 - Light chain CDRL1 amino acid sequence of human anti-HER3 antibody U1-16

SEQ ID NO: 329 - Light chain CDRL2 amino acid sequence of human anti-HER3 antibody U1-16

SEQ ID NO: 330 - Light chain CDRL3 amino acid sequence of human anti-HER3 antibody U1-16

SEQ ID NO: 331 - Amino acid sequence of heavy chain CDRH1 of human anti-HER3 antibody U1-17

SEQ ID NO: 332 - Amino acid sequence of heavy chain CDRH2 of human anti-HER3 antibody U1-17

SEQ ID NO: 333 - Amino acid sequence of heavy chain CDRH3 of human anti-HER3 antibody U1-17

SEQ ID NO: 334 - Light chain CDRL1 amino acid sequence of human anti-HER3 antibody U1-17

SEQ ID NO: 335 - Light chain CDRL2 amino acid sequence of human anti-HER3 antibody U1-17

SEQ ID NO: 336 - Light chain CDRL3 amino acid sequence of human anti-HER3 antibody U1-17

SEQ ID NO: 337 - Amino acid sequence of heavy chain CDRH1 of human anti-HER3 antibody U1-18

SEQ ID NO: 338 - Amino acid sequence of heavy chain CDRH2 of human anti-HER3 antibody U1-18

SEQ ID NO: 339 - Amino acid sequence of heavy chain CDRH3 of human anti-HER3 antibody U1-18

SEQ ID NO: 340 - Light chain amino acid sequence of human anti-HER3 antibody U1-18 CDRL1

SEQ ID NO: 341 - Light chain CDRL2 amino acid sequence of human anti-HER3 antibody U1-18

SEQ ID NO: 342 - Light chain CDRL3 amino acid sequence of human anti-HER3 antibody U1-18

SEQ ID NO: 343 - Amino acid sequence of heavy chain CDRH1 of human anti-HER3 antibody U1-19

SEQ ID NO: 344 - Amino acid sequence of heavy chain CDRH2 of human anti-HER3 antibody U1-19

SEQ ID NO: 345 - Amino acid sequence of heavy chain CDRH3 of human anti-HER3 antibody U1-19

SEQ ID NO: 346 - Amino acid sequence of heavy chain CDRH1 of human anti-HER3 antibody U1-20

SEQ ID NO: 347 - Amino acid sequence of heavy chain CDRH2 of human anti-HER3 antibody U1-20

SEQ ID NO: 348 - Amino acid sequence of heavy chain CDRH3 of human anti-HER3 antibody U1-20

SEQ ID NO: 349 - Light chain CDRL1 amino acid sequence of human anti-HER3 antibody U1-20

SEQ ID NO: 350 - Light chain CDRL2 amino acid sequence of human anti-HER3 antibody U1-20

SEQ ID NO: 351 - Light chain CDRL3 amino acid sequence of human anti-HER3 antibody U1-20

SEQ ID NO: 352 - Amino acid sequence of heavy chain CDRH1 of human anti-HER3 antibody U1-21

SEQ ID NO: 353 - Amino acid sequence of heavy chain CDRH2 of human anti-HER3 antibody U1-21

SEQ ID NO: 354 - Amino acid sequence of heavy chain CDRH3 of human anti-HER3 antibody U1-21

SEQ ID NO: 355 - Light chain CDRL1 amino acid sequence of human anti-HER3 antibody U1-21

SEQ ID NO: 356 - Light chain CDRL2 amino acid sequence of human anti-HER3 antibody U1-21

SEQ ID NO: 357 - Light chain CDRL3 amino acid sequence of human anti-HER3 antibody U1-21

SEQ ID NO: 358 - Amino acid sequence of heavy chain CDRH1 of human anti-HER3 antibody U1-22

SEQ ID NO: 359 - Amino acid sequence of heavy chain CDRH2 of human anti-HER3 antibody U1-22

SEQ ID NO: 360 - Amino acid sequence of heavy chain CDRH3 of human anti-HER3 antibody U1-22

SEQ ID NO: 361 - Light chain CDRL1 amino acid sequence of human anti-HER3 antibody U1-22

SEQ ID NO: 362 - Light chain CDRL2 amino acid sequence of human anti-HER3 antibody U1-22

SEQ ID NO: 363 - Light chain CDRL3 amino acid sequence of human anti-HER3 antibody U1-22

SEQ ID NO: 364 - Amino acid sequence of heavy chain CDRH1 of human anti-HER3 antibody U1-23

SEQ ID NO: 365 - Amino acid sequence of heavy chain CDRH2 of human anti-HER3 antibody U1-23

SEQ ID NO: 366 - Amino acid sequence of heavy chain CDRH3 of human anti-HER3 antibody U1-23

SEQ ID NO: 367 - Light chain CDRL1 amino acid sequence of human anti-HER3 antibody U1-23

SEQ ID NO: 368 - Light chain CDRL2 amino acid sequence of human anti-HER3 antibody U1-23

SEQ ID NO: 369 - Light chain CDRL3 amino acid sequence of human anti-HER3 antibody U1-23

SEQ ID NO: 370 - Amino acid sequence of heavy chain CDRH1 of human anti-HER3 antibody U1-24

SEQ ID NO: 371 - Amino acid sequence of heavy chain CDRH2 of human anti-HER3 antibody U1-24

SEQ ID NO: 372 - Amino acid sequence of heavy chain CDRH3 of human anti-HER3 antibody U1-24

SEQ ID NO: 373 - Light chain CDRL1 amino acid sequence of human anti-HER3 antibody U1-24

SEQ ID NO: 374 - Light chain CDRL2 amino acid sequence of human anti-HER3 antibody U1-24

SEQ ID NO: 375 - Light chain CDRL3 amino acid sequence of human anti-HER3 antibody U1-24

SEQ ID NO: 376 - Amino acid sequence of heavy chain CDRH1 of human anti-HER3 antibody U1-25

SEQ ID NO: 377 - Amino acid sequence of heavy chain CDRH2 of human anti-HER3 antibody U1-25

SEQ ID NO: 378 - Amino acid sequence of heavy chain CDRH3 of human anti-HER3 antibody U1-25

SEQ ID NO: 379 - Light chain CDRL1 amino acid sequence of human anti-HER3 antibody U1-25

SEQ ID NO: 380 - Light chain amino acid sequence of human anti-HER3 antibody U1-25 CDRL2

SEQ ID NO: 381 - Light chain CDRL3 amino acid sequence of human anti-HER3 antibody U1-25

SEQ ID NO: 382 - Amino acid sequence of heavy chain CDRH1 of human anti-HER3 antibody U1-26

SEQ ID NO: 383 - Amino acid sequence of heavy chain CDRH2 of human anti-HER3 antibody U1-26

SEQ ID NO: 384 - Amino acid sequence of heavy chain CDRH3 of human anti-HER3 antibody U1-26

SEQ ID NO: 385 - Light chain CDRL1 amino acid sequence of human anti-HER3 antibody U1-26

SEQ ID NO: 386 - Light chain CDRL2 amino acid sequence of human anti-HER3 antibody U1-26

SEQ ID NO: 387 - Light chain CDRL3 amino acid sequence of human anti-HER3 antibody U1-26

SEQ ID NO: 388 - Amino acid sequence of heavy chain CDRH1 of human anti-HER3 antibody U1-27

SEQ ID NO: 389 - Amino acid sequence of heavy chain CDRH2 of human anti-HER3 antibody U1-27

SEQ ID NO: 390 - Amino acid sequence of heavy chain CDRH3 of human anti-HER3 antibody U1-27

SEQ ID NO: 391 - Light chain CDRL1 amino acid sequence of human anti-HER3 antibody U1-27

SEQ ID NO: 392 - Light chain CDRL2 amino acid sequence of human anti-HER3 antibody U1-27

SEQ ID NO: 393 - Light chain CDRL3 amino acid sequence of human anti-HER3 antibody U1-27

SEQ ID NO: 394 - Amino acid sequence of heavy chain CDRH1 of human anti-HER3 antibody U1-28

SEQ ID NO: 395 - Amino acid sequence of heavy chain CDRH2 of human anti-HER3 antibody U1-28

SEQ ID NO: 396 - Amino acid sequence of heavy chain CDRH3 of human anti-HER3 antibody U1-28

SEQ ID NO: 397 - Light chain CDRL1 amino acid sequence of human anti-HER3 antibody U1-28

SEQ ID NO: 398 - Light chain CDRL2 amino acid sequence of human anti-HER3 antibody U1-28

SEQ ID NO: 399 - Light chain CDRL3 amino acid sequence of human anti-HER3 antibody U1-28

SEQ ID NO: 400 - Amino acid sequence of heavy chain CDRH1 of human anti-HER3 antibody U1-29

SEQ ID NO: 401 - Amino acid sequence of heavy chain CDRH2 of human anti-HER3 antibody U1-29

SEQ ID NO: 402 - Amino acid sequence of heavy chain CDRH3 of human anti-HER3 antibody U1-29

SEQ ID NO: 403 - Light chain CDRL1 amino acid sequence of human anti-HER3 antibody U1-29

SEQ ID NO: 404 - Light chain CDRL2 amino acid sequence of human anti-HER3 antibody U1-29

SEQ ID NO: 405 - Light chain CDRL3 amino acid sequence of human anti-HER3 antibody U1-29

SEQ ID NO: 406 - Amino acid sequence of heavy chain CDRH1 of human anti-HER3 antibody U1-30

SEQ ID NO: 407 - Amino acid sequence of heavy chain CDRH2 of human anti-HER3 antibody U1-30

SEQ ID NO: 408 - Amino acid sequence of heavy chain CDRH3 of human anti-HER3 antibody U1-30

SEQ ID NO: 409 - Light chain CDRL1 amino acid sequence of human anti-HER3 antibody U1-30

SEQ ID NO: 410 - Amino acid sequence of CDRL2 light chain of human anti-HER3 antibody U1-30

SEQ ID NO: 411 - Light chain CDRL3 amino acid sequence of human anti-HER3 antibody U1-30

SEQ ID NO: 412 - Amino acid sequence of heavy chain CDRH1 of human anti-HER3 antibody U1-31

SEQ ID NO: 413 - Amino acid sequence of heavy chain CDRH2 of human anti-HER3 antibody U1-31

SEQ ID NO: 414 - Amino acid sequence of heavy chain CDRH3 of human anti-HER3 antibody U1-31

SEQ ID NO: 415 - Light chain CDRL1 amino acid sequence of human anti-HER3 antibody U1-31

SEQ ID NO: 416 - Light chain CDRL2 amino acid sequence of human anti-HER3 antibody U1-31

SEQ ID NO: 417 - Light chain CDRL3 amino acid sequence of human anti-HER3 antibody U1-31

SEQ ID NO: 418 - Amino acid sequence of heavy chain CDRH1 of human anti-HER3 antibody U1-32

SEQ ID NO: 419 - Amino acid sequence of heavy chain CDRH2 of human anti-HER3 antibody U1-32

SEQ ID NO: 420 - Amino acid sequence of heavy chain CDRH3 of human anti-HER3 antibody U1-32

SEQ ID NO: 421 - Light chain CDRL1 amino acid sequence of human anti-HER3 antibody U1-32

SEQ ID NO: 422 - Light chain CDRL2 amino acid sequence of human anti-HER3 antibody U1-32

SEQ ID NO: 423 - Light chain CDRL3 amino acid sequence of human anti-HER3 antibody U1-32

SEQ ID NO: 424 - Amino acid sequence of heavy chain CDRH1 of human anti-HER3 antibody U1-33

SEQ ID NO: 425 - Amino acid sequence of heavy chain CDRH2 of human anti-HER3 antibody U1-33

SEQ ID NO: 426 - Amino acid sequence of heavy chain CDRH3 of human anti-HER3 antibody U1-33

SEQ ID NO: 427 - Light chain CDRL1 amino acid sequence of human anti-HER3 antibody U1-33

SEQ ID NO: 428 - Light chain CDRL2 amino acid sequence of human anti-HER3 antibody U1-33

SEQ ID NO: 429 - Light chain CDRL3 amino acid sequence of human anti-HER3 antibody U1-33

SEQ ID NO: 430 - Amino acid sequence of heavy chain CDRH1 of human anti-HER3 antibody U1-34

SEQ ID NO: 431 - Amino acid sequence of heavy chain CDRH2 of human anti-HER3 antibody U1-34

SEQ ID NO: 432 - Amino acid sequence of heavy chain CDRH3 of human anti-HER3 antibody U1-34

SEQ ID NO:433 - Light chain CDRL1 amino acid sequence of human anti-HER3 antibody U1-34

SEQ ID NO: 434 - Light chain CDRL2 amino acid sequence of human anti-HER3 antibody U1-34

SEQ ID NO: 435 - Light chain CDRL3 amino acid sequence of human anti-HER3 antibody U1-34

SEQ ID NO: 436 - Amino acid sequence of heavy chain CDRH1 of human anti-HER3 antibody U1-35

SEQ ID NO: 437 - Amino acid sequence of heavy chain CDRH2 of human anti-HER3 antibody U1-35

SEQ ID NO: 438 - Amino acid sequence of heavy chain CDRH3 of human anti-HER3 antibody U1-35

SEQ ID NO: 439 - Light chain CDRL1 amino acid sequence of human anti-HER3 antibody U1-35

SEQ ID NO: 440 - Light chain amino acid sequence of human anti-HER3 antibody U1-35 CDRL2

SEQ ID NO: 441 - Light chain CDRL3 amino acid sequence of human anti-HER3 antibody U1-35

SEQ ID NO: 442 - Amino acid sequence of heavy chain CDRH1 of human anti-HER3 antibody U1-36

SEQ ID NO: 443 - Amino acid sequence of heavy chain CDRH2 of human anti-HER3 antibody U1-36

SEQ ID NO: 444 - Amino acid sequence of heavy chain CDRH3 of human anti-HER3 antibody U1-36

SEQ ID NO: 445 - Amino acid sequence of light chain CDRL1 of human anti-HER3 antibody U1-36

SEQ ID NO: 446 - Light chain CDRL2 amino acid sequence of human anti-HER3 antibody U1-36

SEQ ID NO: 447 - Light chain CDRL3 amino acid sequence of human anti-HER3 antibody U1-36

SEQ ID NO: 448 - Amino acid sequence of heavy chain CDRH1 of human anti-HER3 antibody U1-37

SEQ ID NO: 449 - Amino acid sequence of heavy chain CDRH2 of human anti-HER3 antibody U1-37

SEQ ID NO: 450 - Amino acid sequence of heavy chain CDRH3 of human anti-HER3 antibody U1-37

SEQ ID NO: 451 - Light chain CDRL1 amino acid sequence of human anti-HER3 antibody U1-37

SEQ ID NO: 452 - Light chain CDRL2 amino acid sequence of human anti-HER3 antibody U1-37

SEQ ID NO: 453 - Light chain CDRL3 amino acid sequence of human anti-HER3 antibody U1-37

SEQ ID NO: 454 - Amino acid sequence of heavy chain CDRH1 of human anti-HER3 antibody U1-38

SEQ ID NO: 455 - Amino acid sequence of heavy chain CDRH2 of human anti-HER3 antibody U1-38

SEQ ID NO: 456 - Amino acid sequence of heavy chain CDRH3 of human anti-HER3 antibody U1-38

SEQ ID NO: 457 - Light chain CDRL1 amino acid sequence of human anti-HER3 antibody U1-38

SEQ ID NO:458 - Amino acid sequence of CDRL2 light chain of human anti-HER3 antibody U1-38

SEQ ID NO: 459 - Light chain CDRL3 amino acid sequence of human anti-HER3 antibody U1-38

SEQ ID NO: 460 - Amino acid sequence of heavy chain CDRH1 of human anti-HER3 antibody U1-39

SEQ ID NO: 461 - Amino acid sequence of heavy chain CDRH2 of human anti-HER3 antibody U1-39

SEQ ID NO: 462 - Amino acid sequence of heavy chain CDRH3 of human anti-HER3 antibody U1-39

SEQ ID NO: 463 - Light chain CDRL1 amino acid sequence of human anti-HER3 antibody U1-39

SEQ ID NO: 464 - Light chain CDRL2 amino acid sequence of human anti-HER3 antibody U1-39

SEQ ID NO: 465 - Light chain CDRL3 amino acid sequence of human anti-HER3 antibody U1-39

SEQ ID NO: 466 - Amino acid sequence of heavy chain CDRH1 of human anti-HER3 antibody U1-40

SEQ ID NO: 467 - Amino acid sequence of heavy chain CDRH2 of human anti-HER3 antibody U1-40

SEQ ID NO: 468 - Amino acid sequence of heavy chain CDRH3 of human anti-HER3 antibody U1-40

SEQ ID NO: 469 - Light chain CDRL1 amino acid sequence of human anti-HER3 antibody U1-40

SEQ ID NO: 470 - Light chain CDRL2 amino acid sequence of human anti-HER3 antibody U1-40

SEQ ID NO: 471 - Light chain CDRL3 amino acid sequence of human anti-HER3 antibody U1-40

SEQ ID NO: 472 - Amino acid sequence of heavy chain CDRH1 of human anti-HER3 antibody U1-41

SEQ ID NO: 473 - Amino acid sequence of heavy chain CDRH2 of human anti-HER3 antibody U1-41

SEQ ID NO: 474 - Amino acid sequence of heavy chain CDRH3 of human anti-HER3 antibody U1-41

SEQ ID NO: 475 - Light chain CDRL1 amino acid sequence of human anti-HER3 antibody U1-41

SEQ ID NO: 476 - Light chain CDRL2 amino acid sequence of human anti-HER3 antibody U1-41

SEQ ID NO: 477 - Light chain CDRL3 amino acid sequence of human anti-HER3 antibody U1-41

SEQ ID NO: 478 - Amino acid sequence of heavy chain CDRH1 of human anti-HER3 antibody U1-42

SEQ ID NO: 479 - Amino acid sequence of heavy chain CDRH2 of human anti-HER3 antibody U1-42

SEQ ID NO: 480 - Amino acid sequence of heavy chain CDRH3 of human anti-HER3 antibody U1-42

SEQ ID NO: 481 - Light chain CDRL1 amino acid sequence of human anti-HER3 antibody U1-42

SEQ ID NO: 482 - Light chain CDRL2 amino acid sequence of human anti-HER3 antibody U1-42

SEQ ID NO: 483 - Light chain CDRL3 amino acid sequence of human anti-HER3 antibody U1-42

SEQ ID NO: 484 - Amino acid sequence of heavy chain CDRH1 of human anti-HER3 antibody U1-43

SEQ ID NO: 485 - Amino acid sequence of heavy chain CDRH2 of human anti-HER3 antibody U1-43

SEQ ID NO: 486 - Amino acid sequence of heavy chain CDRH3 of human anti-HER3 antibody U1-43

SEQ ID NO: 487 - Light chain CDRL1 amino acid sequence of human anti-HER3 antibody U1-43

SEQ ID NO: 488 - Light chain CDRL2 amino acid sequence of human anti-HER3 antibody U1-43

SEQ ID NO: 489 - Light chain CDRL3 amino acid sequence of human anti-HER3 antibody U1-43

SEQ ID NO: 490 - Amino acid sequence of heavy chain CDRH1 of human anti-HER3 antibody U1-44

SEQ ID NO: 491 - Amino acid sequence of heavy chain CDRH2 of human anti-HER3 antibody U1-44

SEQ ID NO: 492 - Amino acid sequence of heavy chain CDRH3 of human anti-HER3 antibody U1-44

SEQ ID NO: 493 - Light chain CDRL1 amino acid sequence of human anti-HER3 antibody U1-44

SEQ ID NO: 494 - Light chain CDRL2 amino acid sequence of human anti-HER3 antibody U1-44

SEQ ID NO: 495 - Light chain CDRL3 amino acid sequence of human anti-HER3 antibody U1-44

SEQ ID NO: 496 - Amino acid sequence of heavy chain CDRH1 of human anti-HER3 antibody U1-45

SEQ ID NO: 497 - Amino acid sequence of heavy chain CDRH2 of human anti-HER3 antibody U1-45

SEQ ID NO: 498 - Amino acid sequence of heavy chain CDRH3 of human anti-HER3 antibody U1-45

SEQ ID NO: 499 - Light chain CDRL1 amino acid sequence of human anti-HER3 antibody U1-45

SEQ ID NO: 500 - Amino acid sequence of CDRL2 light chain of human anti-HER3 antibody U1-45

SEQ ID NO: 501 - Light chain CDRL3 amino acid sequence of human anti-HER3 antibody U1-45

SEQ ID NO: 502 - Amino acid sequence of heavy chain CDRH1 of human anti-HER3 antibody U1-46

SEQ ID NO: 503 - Amino acid sequence of heavy chain CDRH2 of human anti-HER3 antibody U1-46

SEQ ID NO: 504 - Amino acid sequence of heavy chain CDRH3 of human anti-HER3 antibody U1-46

SEQ ID NO: 505 - Amino acid sequence of heavy chain CDRH1 of human anti-HER3 antibody U1-47

SEQ ID NO: 506 - Amino acid sequence of heavy chain CDRH2 of human anti-HER3 antibody U1-47

SEQ ID NO: 507 - Amino acid sequence of heavy chain CDRH3 of human anti-HER3 antibody U1-47

SEQ ID NO: 508 - Light chain CDRL1 amino acid sequence of human anti-HER3 antibody U1-47

SEQ ID NO: 509 - Light chain CDRL2 amino acid sequence of human anti-HER3 antibody U1-47

SEQ ID NO: 510 - Light chain CDRL3 amino acid sequence of human anti-HER3 antibody U1-47

SEQ ID NO: 511 - Amino acid sequence of heavy chain CDRH1 of human anti-HER3 antibody U1-48

SEQ ID NO: 512 - Amino acid sequence of heavy chain CDRH2 of human anti-HER3 antibody U1-48

SEQ ID NO: 513 - Amino acid sequence of heavy chain CDRH3 of human anti-HER3 antibody U1-48

SEQ ID NO: 514 - Amino acid sequence of heavy chain CDRH1 of human anti-HER3 antibody U1-49

SEQ ID NO: 515 - Amino acid sequence of heavy chain CDRH2 of human anti-HER3 antibody U1-49

SEQ ID NO: 516 - Amino acid sequence of heavy chain CDRH3 of human anti-HER3 antibody U1-49

SEQ ID NO: 517 - Light chain CDRL1 amino acid sequence of human anti-HER3 antibody U1-49

SEQ ID NO: 518 - Light chain CDRL2 amino acid sequence of human anti-HER3 antibody U1-49

SEQ ID NO: 519 - Light chain CDRL3 amino acid sequence of human anti-HER3 antibody U1-49

SEQ ID NO: 520 - Amino acid sequence of heavy chain CDRH1 of human anti-HER3 antibody U1-50

SEQ ID NO: 521 - Amino acid sequence of heavy chain CDRH2 of human anti-HER3 antibody U1-50

SEQ ID NO: 522 - Amino acid sequence of heavy chain CDRH3 of human anti-HER3 antibody U1-50

SEQ ID NO: 523 - Light chain CDRL1 amino acid sequence of human anti-HER3 antibody U1-50

SEQ ID NO: 524 - Light chain CDRL2 amino acid sequence of human anti-HER3 antibody U1-50

SEQ ID NO: 525 - Light chain CDRL3 amino acid sequence of human anti-HER3 antibody U1-50

SEQ ID NO: 526 - Amino acid sequence of heavy chain CDRH1 of human anti-HER3 antibody U1-51

SEQ ID NO: 527 - Amino acid sequence of heavy chain CDRH2 of human anti-HER3 antibody U1-51

SEQ ID NO: 528 - Amino acid sequence of heavy chain CDRH3 of human anti-HER3 antibody U1-51

SEQ ID NO: 529 - Light chain CDRL1 amino acid sequence of human anti-HER3 antibody U1-51

SEQ ID NO: 530 - Light chain CDRL2 amino acid sequence of human anti-HER3 antibody U1-51

SEQ ID NO: 531 - Light chain CDRL3 amino acid sequence of human anti-HER3 antibody U1-51

SEQ ID NO: 532 - Amino acid sequence of heavy chain CDRH1 of human anti-HER3 antibody U1-52

SEQ ID NO: 533 - Amino acid sequence of heavy chain CDRH2 of human anti-HER3 antibody U1-52

SEQ ID NO: 534 - Amino acid sequence of heavy chain CDRH3 of human anti-HER3 antibody U1-52

SEQ ID NO: 535 - Light chain CDRL1 amino acid sequence of human anti-HER3 antibody U1-52

SEQ ID NO: 536 - Light chain CDRL2 amino acid sequence of human anti-HER3 antibody U1-52

SEQ ID NO: 537 - Light chain CDRL3 amino acid sequence of human anti-HER3 antibody U1-52

SEQ ID NO: 538 - Amino acid sequence of heavy chain CDRH1 of human anti-HER3 antibody U1-53

SEQ ID NO: 539 - Amino acid sequence of heavy chain CDRH2 of human anti-HER3 antibody U1-53

SEQ ID NO: 540 - Amino acid sequence of heavy chain CDRH3 of human anti-HER3 antibody U1-53

SEQ ID NO: 541 - Light chain CDRL1 amino acid sequence of human anti-HER3 antibody U1-53

SEQ ID NO: 542 - Light chain CDRL2 amino acid sequence of human anti-HER3 antibody U1-53

SEQ ID NO: 543 - Light chain CDRL3 amino acid sequence of human anti-HER3 antibody U1-53

SEQ ID NO: 544 - Amino acid sequence of heavy chain CDRH1 of human anti-HER3 antibody U1-55.1

SEQ ID NO: 545 - Amino acid sequence of heavy chain CDRH2 of human anti-HER3 antibody U1-55.1

SEQ ID NO: 546 - Amino acid sequence of heavy chain CDRH3 of human anti-HER3 antibody U1-55.1

SEQ ID NO: 547 - Light chain CDRL1 amino acid sequence of human anti-HER3 antibody U1-55

SEQ ID NO: 548 - Light chain CDRL2 amino acid sequence of human anti-HER3 antibody U1-55

SEQ ID NO: 549 - Light chain CDRL3 amino acid sequence of human anti-HER3 antibody U1-55

SEQ ID NO: 550 - Light chain CDRL1 amino acid sequence of human anti-HER3 antibody U1-57.1

SEQ ID NO: 551 - Light chain CDRL2 amino acid sequence of human anti-HER3 antibody U1-57.1

SEQ ID NO: 552 - Light chain CDRL3 amino acid sequence of human anti-HER3 antibody U1-57.1

SEQ ID NO: 553 - Amino acid sequence of heavy chain CDRH1 of human anti-HER3 antibody U1-57

SEQ ID NO: 554 - Amino acid sequence of heavy chain CDRH2 of human anti-HER3 antibody U1-57

SEQ ID NO: 555 - Amino acid sequence of heavy chain CDRH3 of human anti-HER3 antibody U1-57

SEQ ID NO: 556 - Amino acid sequence of heavy chain CDRH1 of human anti-HER3 antibody U1-58

SEQ ID NO: 557 - Amino acid sequence of heavy chain CDRH2 of human anti-HER3 antibody U1-58

SEQ ID NO: 558 - Amino acid sequence of heavy chain CDRH3 of human anti-HER3 antibody U1-58

SEQ ID NO: 559 - Light chain CDRL1 amino acid sequence of human anti-HER3 antibody U1-58

SEQ ID NO: 560 - Light chain CDRL2 amino acid sequence of human anti-HER3 antibody U1-58

SEQ ID NO: 561 - Light chain CDRL3 amino acid sequence of human anti-HER3 antibody U1-58

SEQ ID NO: 562 - Amino acid sequence of heavy chain CDRH1 of human anti-HER3 antibody U1-59

SEQ ID NO: 563 - Amino acid sequence of heavy chain CDRH2 of human anti-HER3 antibody U1-59

SEQ ID NO: 564 - Amino acid sequence of heavy chain CDRH3 of human anti-HER3 antibody U1-59

SEQ ID NO: 565 - Light chain CDRL1 amino acid sequence of human anti-HER3 antibody U1-59

SEQ ID NO: 566 - Light chain CDRL2 amino acid sequence of human anti-HER3 antibody U1-59

SEQ ID NO: 567 - Light chain CDRL3 amino acid sequence of human anti-HER3 antibody U1-59

SEQ ID NO: 568 - Amino acid sequence of heavy chain CDRH1 of human anti-HER3 antibody U1-61.1

SEQ ID NO: 569 - Amino acid sequence of heavy chain CDRH2 of human anti-HER3 antibody U1-61.1

SEQ ID NO: 570 - Amino acid sequence of heavy chain CDRH3 of human anti-HER3 antibody U1-61.1

SEQ ID NO: 571 - Light chain CDRL1 amino acid sequence of human anti-HER3 antibody U1-61.1

SEQ ID NO: 572 - Light chain CDRL2 amino acid sequence of human anti-HER3 antibody U1-61.1

SEQ ID NO: 573 - Light chain CDRL3 amino acid sequence of human anti-HER3 antibody U1-61.1

SEQ ID NO: 574 - Amino acid sequence of heavy chain CDRH1 of human anti-HER3 antibody U1-61

SEQ ID NO: 575 - Amino acid sequence of heavy chain CDRH2 of human anti-HER3 antibody U1-61

SEQ ID NO: 576 - Amino acid sequence of heavy chain CDRH3 of human anti-HER3 antibody U1-61

SEQ ID NO: 577 - Amino acid sequence of heavy chain CDRH1 of human anti-HER3 antibody U1-62

SEQ ID NO: 578 - Amino acid sequence of heavy chain CDRH2 of human anti-HER3 antibody U1-62

SEQ ID NO: 579 - Amino acid sequence of heavy chain CDRH3 of human anti-HER3 antibody U1-62

SEQ ID NO: 580 - Light chain CDRL1 amino acid sequence of human anti-HER3 antibody U1-62

SEQ ID NO: 581 - Light chain CDRL2 amino acid sequence of human anti-HER3 antibody U1-62

SEQ ID NO: 582 - Light chain CDRL3 amino acid sequence of human anti-HER3 antibody U1-62

SEQ ID NO: 583 - Full length amino acid sequence of the heavy chain of human anti-HER3 antibody U1-59

SEQ ID NO: 584 - Full length amino acid sequence of the light chain of human anti-HER3 antibody U1-59

[Sequence Listing]

[FINAL]PD20-9009WO_Seq.txt

--->

SEQUENCE LIST

<110> Daiichi Sankyo Company, Limited

<120> ANTIBODY-DRUG CONJUGATE ANTI HER3

<130>PD20-9009WO

<150>JP2014-081454

<151> 2014-04-10

<160> 584

<170>PatentIn version 3.5

<210> 1

<211> 342

<212> DNA

<213> Artificial

<220>

<223> Antibody

<400> 1

gaggtgcagc tggtggagtc tggaggaggc ttgatccagc ctggggggtc cctgagactc 60

tcctgtgcag cctctgggtt caccgtcagt agcaactaca tgagctgggt ccgccaggct 120

ccagggaagg ggctggattg ggtctcagtt atttatagcg gtggtagcac atactacgca 180

gactccgtga agggccgatt caccatctcc agagacaatt ccaagaacac gctgtatctt 240

caaatgaaca gcctgagagc cgaggacacg gccgtgtatt actgtgcgag agggcagtgg 300

ctggacgtct ggggccaagg gaccacggtc accgtctcct ca 342

<210> 2

<211> 114

<212> Protein

<213> Artificial

<220>

<223> Antibody

<400> 2

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Ile Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Val Ser Ser Asn

20 25 30

Tyr Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Asp Trp Val

35 40 45

Ser Val Ile Tyr Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Gly Gln Trp Leu Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val

100 105 110

Ser Ser

<210> 3

<211> 336

<212> DNA

<213> Artificial

<220>

<223> Antibody

<400> 3

gatattgtga tgactcagtc tccactctcc ctgcccgtca cccctggaga gccggcctcc 60

atctcctgca ggtcaagtca gagcctcctg catagtaatg gatacaacta tttggattgg 120

tacctgcaga ggccagggca gtctccacaa ctcctgttct atttgggttt tcatcgggcc 180

tccggggtcc ctgacaggtt cagtggcagt ggatcaggca cagattttac actgaaaatc 240

agcagagtgg aggctgagga tgttggggtt tattactgca ggcaagctct acaaactccg 300

ctcactttcg gcggagggac caaggtggag atcaaa 336

<210> 4

<211> 112

<212> Protein

<213> Artificial

<220>

<223> Antibody

<400> 4

Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly

1 5 10 15

Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu His Ser

20 25 30

Asn Gly Tyr Asn Tyr Leu Asp Trp Tyr Leu Gln Arg Pro Gly Gln Ser

35 40 45

Pro Gln Leu Leu Phe Tyr Leu Gly Phe His Arg Ala Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Arg Gln Ala

85 90 95

Leu Gln Thr Pro Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105 110

<210> 5

<211> 374

<212> DNA

<213> Artificial

<220>

<223> Antibody

<400> 5

caggtgcagc tgcaggagtc gggccgga ctggtgaagc cttcacagac cctgtccctc 60

acctgtactg tctctggtgg ctccatcagc agtggtggtt actactggag ctggatccgc 120

cagcacccag ggaagggcct ggagtggatt gggtacatct attccagtgg gagcacctac 180

tacaacccgt ccctcaagag tcgagttacc atatcagtag acacgtctaa gaaccagttc 240

tccctgaagc tgagctctgt gactgccgcg gacacggccg tgtattactg tgcgagagat 300

agggaactgg aactttacta ctactactac ggtatggacg tctggggcca agggaccacg 360

gtcaccgtct cctc 374

<210> 6

<211> 124

<212> Protein

<213> Artificial

<220>

<223> Antibody

<400> 6

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Gly

20 25 30

Gly Tyr Tyr Trp Ser Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu

35 40 45

Trp Ile Gly Tyr Ile Tyr Ser Ser Gly Ser Thr Tyr Tyr Asn Pro Ser

50 55 60

Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe

65 70 75 80

Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr

85 90 95

Cys Ala Arg Asp Arg Glu Leu Glu Leu Tyr Tyr Tyr Tyr Tyr Gly Met

100 105 110

Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser

115 120

<210> 7

<211> 336

<212> DNA

<213> Artificial

<220>

<223> Antibody

<400> 7

gatattgtga tgactcagtc tccactctcc ctgcccgtca cccctggaga gccggcctcc 60

atctcctgca ggtctagtca gagcctcctg tatagtaatg gatacaacta tttggattgg 120

tacctgcaga agccaggggca gtctccacag ctcctgatct atttgggttc taatcgggcc 180

tccggggtcc ctgacaggtt cagtggcagt ggatcaggca cagattttac actgaaaatc 240

agcagagtgg aggctgagga tgttgggatt tattactgca tgcaagctct acaaactccg 300

ctcactttcg gcggagggac caaggtggag atcaaa 336

<210> 8

<211> 112

<212> Protein

<213> Artificial

<220>

<223> Antibody

<400> 8

Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly

1 5 10 15

Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu Tyr Ser

20 25 30

Asn Gly Tyr Asn Tyr Leu Asp Trp Tyr Leu Gln Lys Pro Gly Gln Ser

35 40 45

Pro Gln Leu Leu Ile Tyr Leu Gly Ser Asn Arg Ala Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Ile Tyr Tyr Cys Met Gln Ala

85 90 95

Leu Gln Thr Pro Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105 110

<210> 9

<211> 357

<212> DNA

<213> Artificial

<220>

<223> Antibody

<400> 9

cagatcacct tgaaggagtc tggtcctacg ctggtgaaac ccacacagac cctcacgctg 60

acctgcacct tctctgggtt ctcactcagc actagtggag tgggtgtggg ctggatccgt

cagcccccag gaaaggccct ggactggctt gcactcattt attggaatga tgataagcgc 180

tacagcccat ctctgaagag caggctcacc atcaccaagg acacctccaa aaaccaggtg 240

gtccttacaa tgaccaacat ggatcttgtg gacacagcca catattactg tgtacacaga 300

gacgaagttc gagggtttga ctactggggc cagggaaccc tggtcaccgt ctcctca 357

<210> 10

<211> 119

<212> Protein

<213> Artificial

<220>

<223> Antibody

<400> 10

Gln Ile Thr Leu Lys Glu Ser Gly Pro Thr Leu Val Lys Pro Thr Gln

1 5 10 15

Thr Leu Thr Leu Thr Cys Thr Phe Ser Gly Phe Ser Leu Ser Thr Ser

20 25 30

Gly Val Gly Val Gly Trp Ile Arg Gln Pro Pro Gly Lys Ala Leu Asp

35 40 45

Trp Leu Ala Leu Ile Tyr Trp Asn Asp Asp Lys Arg Tyr Ser Pro Ser

50 55 60

Leu Lys Ser Arg Leu Thr Ile Thr Lys Asp Thr Ser Lys Asn Gln Val

65 70 75 80

Val Leu Thr Met Thr Asn Met Asp Leu Val Asp Thr Ala Thr Tyr Tyr

85 90 95

Cys Val His Arg Asp Glu Val Arg Gly Phe Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 11

<211> 336

<212> DNA

<213> Artificial

<220>

<223> Antibody

<400> 11

gatgttgtga tgactcagtc tccactctcc ctgcccgtca cccttggaca gccggcctcc 60

atctcctgca ggtctagtca aagcctcgta tacagtgatg gatacaccta cttgcattgg 120

tttcagcaga ggccaggcca atctccaagg cgccttattt ataaggtttc taactgggac 180

tctggggtcc cagacagatt cagcggcagt gggtcaggca ctgatttcac actgaaaatc 240

agcagggtgg aggctgagga tgttggggtt tattactgca tgcaaggtgc acactggccg 300

atcaccttcg gccaagggac acgactggag attaaa 336

<210> 12

<211> 112

<212> Protein

<213> Artificial

<220>

<223> Antibody

<400> 12

Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Leu Gly

1 5 10 15

Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val Tyr Ser

20 25 30

Asp Gly Tyr Thr Tyr Leu His Trp Phe Gln Gln Arg Pro Gly Gln Ser

35 40 45

Pro Arg Arg Leu Ile Tyr Lys Val Ser Asn Trp Asp Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln Gly

85 90 95

Ala His Trp Pro Ile Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys

100 105 110

<210> 13

<211> 374

<212> DNA

<213> Artificial

<220>

<223> Antibody

<400> 13

caggtgcagc tgcaggagtc gggcccagga ctggtgaagc cttcacagac cctgtccctc 60

acctgcactg tctctggtgg ctccatcagc agtggtgggt actactggag ctggatccgc 120

cagcacccag ggaagggcct ggagtggatt gggtacatct attacagtgg gagcacctac 180

tacaacccgt ccctcaagag tcgagttacc atatcagtag acacgtctaa gaaccagttc 240

tccctgaagc tgagctctgt gactgccgcg gacacggccg tgtatttctg tgcgagagat 300

cgggaacttg agggttactc caactactac ggtgtggacg tctggggcca agggaccacg 360

gtcaccgtct cctc 374

<210> 14

<211> 124

<212> Protein

<213> Artificial

<220>

<223> Antibody

<400> 14

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Gly

20 25 30

Gly Tyr Tyr Trp Ser Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu

35 40 45

Trp Ile Gly Tyr Ile Tyr Tyr Ser Gly Ser Thr Tyr Tyr Asn Pro Ser

50 55 60

Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe

65 70 75 80

Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Phe

85 90 95

Cys Ala Arg Asp Arg Glu Leu Glu Gly Tyr Ser Asn Tyr Tyr Gly Val

100 105 110

Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser

115 120

<210> 15

<211> 321

<212> DNA

<213> Artificial

<220>

<223> Antibody

<400> 15

gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60

atcacttgcc gggcaagtca ggccattagc aactatttaa attggtatca gcagaaacca 120

gggaaagccc ctaagctcct gatctatgct gcatccagtt tgcaaagtgg ggtcccatca 180

aggttcagtg gcagtggatc tgggacagat ttcactctca ccatcagcag cctgcagcct 240

gaagattttg caacttatta ctgtcaacag aataatagtc tcccgatcac cttcggccaa 300

gggacacgac tggagattaa a 321

<210> 16

<211> 107

<212> Protein

<213> Artificial

<220>

<223> Antibody

<400> 16

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ala Ile Ser Asn Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Asn Asn Ser Leu Pro Ile

85 90 95

Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys

100 105

<210> 17

<211> 354

<212> DNA

<213> Artificial

<220>

<223> Antibody

<400> 17

gaggtgcagc tggtgcagtc tggagcagag gtgaaaaagc ccggggagtc tctgaagatc 60

tcctgtaagg gttctggata cagctttacc agctactgga tcggctgggt gcgccagatg 120

cccgggaaag gcctggagtg gatggggatc atctatcctg gtgactctga taccagatac 180

agcccgtcct tccaaggcca ggtcaccatc tcagccgaca agtccatcag caccgcctac 240

ctgcagtgga gcagcctgaa ggcctcggac accgccatgt attactgtgc gagacatgaa 300

aactacggtg actacaacta ctggggccag ggaaccctgg tcaccgtctc ctca 354

<210> 18

<211> 118

<212> Protein

<213> Artificial

<220>

<223> Antibody

<400> 18

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu

1 5 10 15

Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr Ser Phe Thr Ser Tyr

20 25 30

Trp Ile Gly Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp Met

35 40 45

Gly Ile Ile Tyr Pro Gly Asp Ser Asp Thr Arg Tyr Ser Pro Ser Phe

50 55 60

Gln Gly Gln Val Thr Ile Ser Ala Asp Lys Ser Ile Ser Thr Ala Tyr

65 70 75 80

Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys

85 90 95

Ala Arg His Glu Asn Tyr Gly Asp Tyr Asn Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser

115

<210> 19

<211> 321

<212> DNA

<213> Artificial

<220>

<223> Antibody

<400> 19

gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtcggaga cagagtcacc 60

atcacttgcc gggcaagtca gagcattcgc agctatttaa attggtatca gcagaaacca 120

gggaaagccc ctaagctcct gatctatgct gcttccagtt tgcaaagtgg ggtcccatca 180

aggttcagtg gcagtggatc tgggacagat ttcactctca ccatcagcag tctgcaacct 240

gaagattttg cactttactg ctgtcaacag agtaacggtt ccccgctcac tttcggcgga 300

gggaccaagg tggagatcaa a 321

<210> 20

<211> 107

<212> Protein

<213> Artificial

<220>

<223> Antibody

<400> 20

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Arg Ser Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Leu Tyr Cys Cys Gln Gln Ser Asn Gly Ser Pro Leu

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 21

<211> 380

<212> DNA

<213> Artificial

<220>

<223> Antibody

<400> 21

caggtgcagc tgcaggagtc gggcccagga ctggtgaagc cttcacagac cctgtccctc 60

acctgcactg tctctggtgg ctccatcagc agtggtggtt actactggag ctggatccgc 120

cagcacccag ggaagggcct ggagtggatt gggtacatct attacagtgg gagcacctac 180

tacaacccgt ccctcaggag tcgagttacc atatcagtag acacgtctaa gaaccagttc 240

tccctgaagc tgagctctgt gactgccgcg gacacggccg tgtattactg tgcgagagat 300

aggagagag agtgggatga ttacggtgac ccccaaggta tggacgtctg gggccaaggg 360

accacggtca ccgtctcctc 380

<210> 22

<211> 126

<212> Protein

<213> Artificial

<220>

<223> Antibody

<400> 22

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Gly

20 25 30

Gly Tyr Tyr Trp Ser Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu

35 40 45

Trp Ile Gly Tyr Ile Tyr Tyr Ser Gly Ser Thr Tyr Tyr Asn Pro Ser

50 55 60

Leu Arg Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe

65 70 75 80

Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr

85 90 95

Cys Ala Arg Asp Arg Glu Arg Glu Trp Asp Asp Tyr Gly Asp Pro Gln

100 105 110

Gly Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser

115 120 125

<210> 23

<211> 321

<212> DNA

<213> Artificial

<220>

<223> Antibody

<400> 23

gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60

atcacttgcc gggcaagtca gagcattagc agctatttac attggtatca gcagaaacca 120

gggaaagccc ctaagctcct gatccatgct gcatccagtt tacaaagtgg ggtcccatca 180

aggttcagtg gcagtggatc tgggacagat ttcactctca ccatcagtag tctgcaacct 240

gaagattttg caacttacta ctgtcaacag agttacagta acccgctcac tttcggcgga 300

gggaccaagg tggagatcca a 321

<210> 24

<211> 107

<212> Protein

<213> Artificial

<220>

<223> Antibody

<400> 24

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr

20 25 30

Leu His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

His Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Asn Pro Leu

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Gln

100 105

<210> 25

<211> 354

<212> DNA

<213> Artificial

<220>

<223> Antibody

<400> 25

gaggtgcagc tggtgcagtc tggagcagag gtgaaaaagc ccggggagtc tctgaagatc 60

tcctgtaagg gttctggata cagctttacc agctactgga tcggctgggt gcgccagatg 120

cccgggaaag gcctggagtg gatggggatc atctggcctg gtgactctga taccatatac 180

agcccgtcct tccaaggcca ggtcaccatc tcagccgaca agtccatcag caccgcctac 240

ctgcagtgga gcagcctgaa ggcctcggac accgccatgt attactgtgc gagacatgaa 300

aactacggtg actacaacta ctggggccag ggaaccctgg tcaccgtctc ctca 354

<210> 26

<211> 118

<212> Protein

<213> Artificial

<220>

<223> Antibody

<400> 26

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu

1 5 10 15

Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr Ser Phe Thr Ser Tyr

20 25 30

Trp Ile Gly Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp Met

35 40 45

Gly Ile Ile Trp Pro Gly Asp Ser Asp Thr Ile Tyr Ser Pro Ser Phe

50 55 60

Gln Gly Gln Val Thr Ile Ser Ala Asp Lys Ser Ile Ser Thr Ala Tyr

65 70 75 80

Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys

85 90 95

Ala Arg His Glu Asn Tyr Gly Asp Tyr Asn Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser

115

<210> 27

<211> 321

<212> DNA

<213> Artificial

<220>

<223> Antibody

<400> 27

gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtgggaga cagagtcacc 60

atcacttgcc gggcaagtca gagcattcga agttatttaa attggtatca gcagaaaccg 120

gggaatgccc ctaaactcct gatctatgct gcatccagtt tgcaaagtgg ggtcccatca 180

aggttcagtg gcagtggatc tgggacagat ttcactctca ccatcagcag tctgcaacct 240

gaagattttg cactttacta ctgtcaacag agtatcagtt ccccgctcac tttcggcgga 300

gggaccaagg tggagatcaa a 321

<210> 28

<211> 107

<212> Protein

<213> Artificial

<220>

<223> Antibody

<400> 28

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Arg Ser Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Asn Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Leu Tyr Tyr Cys Gln Gln Ser Ile Ser Ser Pro Leu

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 29

<211> 374

<212> DNA

<213> Artificial

<220>

<223> Antibody

<400> 29

caggtgcagc tggtgcagtc tggggctgag gtgaagaagc ctggggcctc agtgaaggtc 60

tcctgcaagg cttctggata caccttcacc agttatgata tcaactgggt gcgacaggcc 120

actggacaag ggcttgagtg gatgggatgg atgaacccta acagtggtga cactggctat 180

gcacaggtgt tccaggggcag agtcaccatg acctggaaca cctccataag cacagcctac 240

atggaactga gcagcctgag atctgaggac acggccgtgt attactgtgc gagatttggg 300

gatctcccgt atgactacag ttactacgaa tggttcgacc cctggggcca gggaaccctg 360

gtcaccgtct cctc 374

<210> 30

<211> 124

<212> Protein

<213> Artificial

<220>

<223> Antibody

<400> 30

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Asp Ile Asn Trp Val Arg Gln Ala Thr Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Trp Met Asn Pro Asn Ser Gly Asp Thr Gly Tyr Ala Gln Val Phe

50 55 60

Gln Gly Arg Val Thr Met Thr Trp Asn Thr Ser Ile Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Phe Gly Asp Leu Pro Tyr Asp Tyr Ser Tyr Tyr Glu Trp Phe

100 105 110

Asp Pro Trp Gly Gln Gly Thr Leu Val Thr Val Ser

115 120

<210> 31

<211> 321

<212> DNA

<213> Artificial

<220>

<223> Antibody

<400> 31

gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60

atcacttgcc gggcaagcca gagcattagc agctatttaa attggtatca gcagagacca 120

gggaaagccc ctaagctcct gatctatgca gcatccagtt tgcaaagtgg ggtcccatca 180

aggttcagtg gcagtggatc tgggacagat ttcactctca ccatcagcag tctgcaacct 240

gaagattttg caacttacta ctgtcaacag agttacagta ccccgctcac tttcggcgga 300

gggaccaagg tggagatcaa a 321

<210> 32

<211> 107

<212> Protein

<213> Artificial

<220>

<223> Antibody

<400> 32

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Arg Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Pro Leu

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 33

<211> 386

<212> DNA

<213> Artificial

<220>

<223> Antibody

<400> 33

caggtacagc tgcagcagtc aggtcagga ctggtgaagc cctcgcagac cctctcactc 60

acctgtgcca tctccggggga cagtgtctct agcaacagtg ctgcttggaa ctggatcagg 120

cagtccccat cgagaggcct tgagtggctg ggaaggacat actacaggtc caagtggtat 180

aatgattatg cagtatctgt gaaaagtcga ataaccatca acccagacac atccaagaac 240

cagttctccc tgcagctgaa ctctgtgact cccgaggaca cggctgtgta ttactgtgca 300

agagatctct acgatttttg gagtggttat ccctactact acggtatgga cgtctggggc 360

caagggacca cggtcaccgt ctcctc 386

<210> 34

<211> 128

<212> Protein

<213> Artificial

<220>

<223> Antibody

<400> 34

Gln Val Gln Leu Gln Gln Ser Gly Pro Gly Leu Val Lys Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Ile Ser Gly Asp Ser Val Ser Ser Asn

20 25 30

Ser Ala Ala Trp Asn Trp Ile Arg Gln Ser Pro Ser Arg Gly Leu Glu

35 40 45

Trp Leu Gly Arg Thr Tyr Tyr Arg Ser Lys Trp Tyr Asn Asp Tyr Ala

50 55 60

Val Ser Val Lys Ser Arg Ile Thr Ile Asn Pro Asp Thr Ser Lys Asn

65 70 75 80

Gln Phe Ser Leu Gln Leu Asn Ser Val Thr Pro Glu Asp Thr Ala Val

85 90 95

Tyr Tyr Cys Ala Arg Asp Leu Tyr Asp Phe Trp Ser Gly Tyr Pro Tyr

100 105 110

Tyr Tyr Gly Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser

115 120 125

<210> 35

<211> 383

<212> DNA

<213> Artificial

<220>

<223> Antibody

<400> 35

caggtacagc tgcagcagtc aggtcagga ctggtgaagc cctcgcagac cctctcactc 60

acctgtgcca tctccggggga cagtgtctct agcaacagtg ctgcttggaa ctggatcagg 120

cagtccccat cgagaggcct tgagtggctg ggaaggacat actacaggtc caagtggtat 180

aatgattatg cagtatctgt gaaaagtcga ataaccatca acccagacac atccaagaac 240

cagttctccc tgcagctgaa ctctgtgact cccgaggaca cggctgtgta ttactgtgca 300

agagattact atggttcggg gagtttctac tactactacg gtatggacgt ctggggccaa 360

ggaccacgg tcaccgtctc ctc 383

<210> 36

<211> 127

<212> Protein

<213> Artificial

<220>

<223> Antibody

<400> 36

Gln Val Gln Leu Gln Gln Ser Gly Pro Gly Leu Val Lys Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Ile Ser Gly Asp Ser Val Ser Ser Asn

20 25 30

Ser Ala Ala Trp Asn Trp Ile Arg Gln Ser Pro Ser Arg Gly Leu Glu

35 40 45

Trp Leu Gly Arg Thr Tyr Tyr Arg Ser Lys Trp Tyr Asn Asp Tyr Ala

50 55 60

Val Ser Val Lys Ser Arg Ile Thr Ile Asn Pro Asp Thr Ser Lys Asn

65 70 75 80

Gln Phe Ser Leu Gln Leu Asn Ser Val Thr Pro Glu Asp Thr Ala Val

85 90 95

Tyr Tyr Cys Ala Arg Asp Tyr Tyr Gly Ser Gly Ser Phe Tyr Tyr Tyr

100 105 110

Tyr Gly Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser

115 120 125

<210> 37

<211> 321

<212> DNA

<213> Artificial

<220>

<223> Antibody

<400> 37

gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60

atcacttgcc gggcaagtca gagcattagc agctatttaa attggtatca gcagaaacca 120

gggaaagccc ctaaggtcct gatctatgct gcatccaatt tgcaaagtgg ggtcccatca 180

aggttcagtg gcagtggatc tgggacagat ttcactctca ccatcagcag tctgcaacct 240

gaagattttg caacttacta ctgtcaacag agttacagta cccctcggac gttcggccaa 300

gggaccaagg tggaaatcaa a 321

<210> 38

<211> 107

<212> Protein

<213> Artificial

<220>

<223> Antibody

<400> 38

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Val Leu Ile

35 40 45

Tyr Ala Ala Ser Asn Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Pro Arg

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210> 39

<211> 371

<212> DNA

<213> Artificial

<220>

<223> Antibody

<400> 39

caggtgcagc tgcaggagtc gggcccagga ctggtgaagc cttcggagac cctgtccctc 60

acctgcactg tctctggtgg ctccatcagt agttactact ggagctggat ccggcagccc 120

gccgggaagg gactggagtg gattgggcat atctatacca gtgggagcac caactacaac 180

ccctccctca agagtcgagt caccatgtca gtagacacgt ccaagaacca gttctccctg 240

aagctgagct ctgtgaccgc cgcggacacg gccgtgtatt actgtgcgag agaagcgatt 300

tttggagtgg gcccctacta ctactacggt atggacgtct ggggccaagg gaccacggtc 360

accgtctcct c 371

<210> 40

<211> 123

<212> Protein

<213> Artificial

<220>

<223> Antibody

<400> 40

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Tyr

20 25 30

Tyr Trp Ser Trp Ile Arg Gln Pro Ala Gly Lys Gly Leu Glu Glu Trp Ile

35 40 45

Gly His Ile Tyr Thr Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys

50 55 60

Ser Arg Val Thr Met Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu

65 70 75 80

Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Glu Ala Ile Phe Gly Val Gly Pro Tyr Tyr Tyr Tyr Gly Met Asp

100 105 110

Val Trp Gly Glyn Gly Thr Thr Val Thr Val Ser

115 120

<210> 41

<211> 377

<212> DNA

<213> Artificial

<220>

<223> Antibody

<400> 41

caggtgcagc tggtgcagtc tggggctgag gtgaagaagc ctggggcctc agtgaaggtc 60

tcctgcaagg cttctggata caccttcacc ggctactata tgcactgggt gcgacaggcc 120

cctggacaag ggcttgagtg gatgggatgg atcaacccta atattggtgg cacaaactgt 180

gcacagaagt ttcaggggcag ggtcaccatg accagggaca cgtccatcag cacagcctac 240

atggagctga gcaggctgag atctgacgac acggccgtgt attactgtgc gagaggggga 300

cggtatagca gcagctggtc ctactactac tacggtatgg acgtctgggg ccaagggacc 360

acggtcaccg tctcctc 377

<210> 42

<211> 125

<212> Protein

<213> Artificial

<220>

<223> Antibody

<400> 42

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Gly Tyr

20 25 30

Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Glu Trp Met

35 40 45

Gly Trp Ile Asn Pro Asn Ile Gly Gly Thr Asn Cys Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Gly Arg Tyr Ser Ser Ser Trp Ser Tyr Tyr Tyr Tyr Gly

100 105 110

Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser

115 120 125

<210> 43

<211> 336

<212> DNA

<213> Artificial

<220>

<223> Antibody

<400> 43

gatattctga tgacccagac tccactctct ctgtccgtca cccctggaca gccggcctcc 60

atctcctgca agtctagtca gagcctcctg cttagtgatg gagggaccta tttgtattgg 120

tacctgcaga agccaggcca gcctccacag ctcctgatct atgaagtttc caaccggttc 180

tctggagtgc cagataggtt cagtggcagc gggtcaggga cagatttcac actgaaaatc 240

agccgggtgg aggctgagga tgttggggtt tattactgca tgcaaagtat gcagcttccg 300

atcaccttcg gccaagggac acgactggaa attaaa 336

<210> 44

<211> 112

<212> Protein

<213> Artificial

<220>

<223> Antibody

<400> 44

Asp Ile Leu Met Thr Gln Thr Pro Leu Ser Leu Ser Val Thr Pro Gly

1 5 10 15

Gln Pro Ala Ser Ile Ser Cys Lys Ser Ser Gln Ser Leu Leu Leu Ser

20 25 30

Asp Gly Gly Thr Tyr Leu Tyr Trp Tyr Leu Gln Lys Pro Gly Gln Pro

35 40 45

Pro Gln Leu Leu Ile Tyr Glu Val Ser Asn Arg Phe Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln Ser

85 90 95

Met Gln Leu Pro Ile Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys

100 105 110

<210> 45

<211> 380

<212> DNA

<213> Artificial

<220>

<223> Antibody

<400> 45

caggtgcagc tgcaggagtc gggcccagga ctggtgaagc cttcggagac cctgtccctc 60

acctgcactg tctctggtgg ctccgtcagc agtggtggtt actactggag ctggatccgg 120

cagcccccag ggaagggact ggagtggatt gggtatatct attacagtgg gagcaccaac 180

tacaacccct ccctcaagag tcgagtcacc atatcagtag acacgtccaa gaaccagttc 240

tccctgaagc tgagctctgt gaccgctgcg gacacggccg tgtattactg tgcgagaggg 300

ggggacagta actacgagga ttactactac tactacggta tggacgtctg gggccaaggg 360

accacggtca ccgtctcctc 380

<210> 46

<211> 126

<212> Protein

<213> Artificial

<220>

<223> Antibody

<400> 46

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Val Ser Ser Gly

20 25 30

Gly Tyr Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu

35 40 45

Trp Ile Gly Tyr Ile Tyr Tyr Ser Gly Ser Thr Asn Tyr Asn Pro Ser

50 55 60

Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe

65 70 75 80

Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr

85 90 95

Cys Ala Arg Gly Gly Asp Ser Asn Tyr Glu Asp Tyr Tyr Tyr Tyr Tyr

100 105 110

Gly Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser

115 120 125

<210> 47

<211> 321

<212> DNA

<213> Artificial

<220>

<223> Antibody

<400> 47

gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60

atcacttgcc gggcaagtca gagcattagc atctatttac attggtatca gcagaaacca 120

gggaaagccc ctaagctctt gatctctgct gcatccagtt tgcaaagtgg ggtcccgtca 180

aggttcagtg gcagtggatc tgggacagat ttcactctca ccatcagaag tctgcaacct 240

gaagattttg caacttacta ctgtcaacag agttacactt ccccgatcac cttcggccaa 300

gggacacgac tggagattaa a 321

<210> 48

<211> 107

<212> Protein

<213> Artificial

<220>

<223> Antibody

<400> 48

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ile Tyr

20 25 30

Leu His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Ser Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Arg Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Thr Ser Pro Ile

85 90 95

Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys

100 105

<210> 49

<211> 380

<212> DNA

<213> Artificial

<220>

<223> Antibody

<400> 49

caggtgcagc tgcaggagtc gggcccagga ctggtgaagc cttcggagac cctgtccctc 60

acctgcactg tctctggtgg ctccatcagt agttactact ggagctggat ccggcagccc 120

ccagggaagg gactggagtg gattgggtat atctattaca gtgggagcac caactacaac 180

ccctccctca agagtcgagt caccatatca gtagacacgt ccaagcacca gttctccctg 240

aagctgagct ctgtgaccgc tgcggacacg gccgtgtatt actgtgcgag agattcgagt 300

tactatgata gtagtggtta ttacttatac tactacgcta tggacgtctg gggccaaggg 360

accacggtca ccgtctcctc 380

<210> 50

<211> 126

<212> Protein

<213> Artificial

<220>

<223> Antibody

<400> 50

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Tyr

20 25 30

Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Tyr Ile Tyr Tyr Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys

50 55 60

Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys His Gln Phe Ser Leu

65 70 75 80

Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Asp Ser Ser Tyr Tyr Asp Ser Ser Gly Tyr Tyr Leu Tyr Tyr Tyr

100 105 110

Ala Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser

115 120 125

<210> 51

<211> 339

<212> DNA

<213> Artificial

<220>

<223> Antibody

<400> 51

gacatcgtga tgacccagtc tccagactcc ctggctgtgt ctctgggcga gagggccacc 60

atcaactgca agtccagcca gagtgtttta tacagctcca acaataagaa ctacttagct 120

tggtaccagc agaaaccagg acagcctcct aagctgctca ttttcctgggc atctacccgg 180

gaatccgggg tccctgaccg attcagtggc agcgggtctg ggacagattt cactctcacc 240

atcagcagcc tgcaggctga agatgtggca gtttattact gtcagcaata ttatactact 300

cctctcactt tcggccctgg gaccaaagtg gatatcaaa 339

<210> 52

<211> 113

<212> Protein

<213> Artificial

<220>

<223> Antibody

<400> 52

Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Val Leu Tyr Ser

20 25 30

Ser Asn Asn Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln

35 40 45

Pro Pro Lys Leu Leu Ile Ser Trp Ala Ser Thr Arg Glu Ser Gly Val

50 55 60

Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln

85 90 95

Tyr Tyr Thr Pro Leu Thr Phe Gly Pro Gly Thr Lys Val Asp Ile

100 105 110

Lys

<210> 53

<211> 357

<212> DNA

<213> Artificial

<220>

<223> Antibody

<400> 53

gaggtgcaac tggtggagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc 60

tcctgtgcag cctctggatt caccttcagt atctatagca tgaactgggt ccgccaggct 120

ccagggaagg ggctggagtg ggtttcatac attagtagta gtagtagtac catatactac 180

gcagactctg tgaagggccg attcaccatc tccagagaca atgccaagaa ctcactgtat 240

ctgcaaatga acagcctgag agacgaggac acggctgtgt attactgtgc gagagatagg 300

ggtgacttcg atgcttttga tatctggggc caagggacaa tggtcaccgt ctcttca 357

<210> 54

<211> 119

<212> Protein

<213> Artificial

<220>

<223> Antibody

<400> 54

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ile Tyr

20 25 30

Ser Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Tyr Ile Ser Ser Ser Ser Ser Thr Ile Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Asp Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asp Arg Gly Asp Phe Asp Ala Phe Asp Ile Trp Gly Gln Gly

100 105 110

Thr Met Val Thr Val Ser Ser

115

<210> 55

<211> 321

<212> DNA

<213> Artificial

<220>

<223> Antibody

<400> 55

gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60

atcacttgcc aggcgagtca ggacattacc aactatttga attggtatca gcagaaacca 120

gggaaagccc ctaagctcct gatctacgat gcatccaatt tggaaacagg ggtcccatca 180

aggttcagtg gaagtggatc tgggacagat tttactttca ccatcagcag cctgcagcct 240

gaagatattg caacatataa ctgtcaacag tgtgaaaatt tcccgatcac cttcggccaa 300

gggacacgac tggagattaa a 321

<210> 56

<211> 107

<212> Protein

<213> Artificial

<220>

<223> Antibody

<400> 56

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Gln Ala Ser Gln Asp Ile Thr Asn Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Asp Ala Ser Asn Leu Glu Thr Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Ile Ala Thr Tyr Asn Cys Gln Gln Cys Glu Asn Phe Pro Ile

85 90 95

Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys

100 105

<210> 57

<211> 336

<212> DNA

<213> Artificial

<220>

<223> Antibody

<400> 57

gatattgtga tgactcagtc tccactctcc ctgcccgtca cccctggaga gccggcctcc 60

atctcctgca ggtctagtca gagcctcctg tatagtaatg gatacaagta tttggattgg 120

tacctgcaga agccaggggca gtctccacag ctcctgatct atttgggttc taatcgggcc 180

tccggggtcc ctgacaggtt cagtggcagt ggatcaggca cagattttac actgaaaatc 240

agcagagtgg aggctgagga tgttggggtt tattattgca tgcaggctct acaaactccg 300

atcaccttcg gccaagggac acgactggag attaaa 336

<210> 58

<211> 112

<212> Protein

<213> Artificial

<220>

<223> Antibody

<400> 58

Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly

1 5 10 15

Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu Tyr Ser

20 25 30

Asn Gly Tyr Lys Tyr Leu Asp Trp Tyr Leu Gln Lys Pro Gly Gln Ser

35 40 45

Pro Gln Leu Leu Ile Tyr Leu Gly Ser Asn Arg Ala Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln Ala

85 90 95

Leu Gln Thr Pro Ile Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys

100 105 110

<210> 59

<211> 374

<212> DNA

<213> Artificial

<220>

<223> Antibody

<400> 59

caggtgcagc tgcaggagtc gggcccagga ctggtgaagc cttcggagac cctgtccctc 60

acctgcactg tctctggtgg ctccgtcagc agtggtggtt actactggaa ctggatccgg 120

cagcccccag ggaagggact ggagtggatt gggtatatca attacagtgg gagcaccaac 180

tacaacccct ccctcaagag tcgagtcacc atatcagtag acacgtccaa gaaccagttc 240

tccctgaagc tgagctctgt gaccgctgcg gacacggccg tgtattactg tgcgagagat 300

cgagaactgg aactttacta ctactactac ggtatggacg tctggggcca agggaccacg 360

gtcaccgtct cctc 374

<210> 60

<211> 124

<212> Protein

<213> Artificial

<220>

<223> Antibody

<400> 60

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Val Ser Ser Gly

20 25 30

Gly Tyr Tyr Trp Asn Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu

35 40 45

Trp Ile Gly Tyr Ile Asn Tyr Ser Gly Ser Thr Asn Tyr Asn Pro Ser

50 55 60

Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe

65 70 75 80

Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr

85 90 95

Cys Ala Arg Asp Arg Glu Leu Glu Leu Tyr Tyr Tyr Tyr Tyr Gly Met

100 105 110

Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser

115 120

<210> 61

<211> 374

<212> DNA

<213> Artificial

<220>

<223> Antibody

<400> 61

caggtgcagc tgcaggagtc gggcccagga ctggtgaagc cttctgagac cctgtccctc 60

acctgcactg tctctggtgg ctccgtcagc agtggtggtt actactggaa ctggatccgg 120

cagcccccag ggaagggact ggagtggatt gggtatatca attacagtgg gagcaccaac 180

tacaacccct ccctcaagag tcgagtcacc atatcagtag acacgtccaa gaaccagttc 240

tccctgaagc tgagctctgt gaccgctgcg gacacggccg tgtattactg tgcgagagat 300

cgagaactgg aactttacta ctactactac ggtatggacg tctggggcca agggaccacg 360

gtcaccgtct cctc 374

<210> 62

<211> 124

<212> Protein

<213> Artificial

<220>

<223> Antibody

<400> 62

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Val Ser Ser Gly

20 25 30

Gly Tyr Tyr Trp Asn Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu

35 40 45

Trp Ile Gly Tyr Ile Asn Tyr Ser Gly Ser Thr Asn Tyr Asn Pro Ser

50 55 60

Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe

65 70 75 80

Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr

85 90 95

Cys Ala Arg Asp Arg Glu Leu Glu Leu Tyr Tyr Tyr Tyr Tyr Gly Met

100 105 110

Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser

115 120

<210> 63

<211> 336

<212> DNA

<213> Artificial

<220>

<223> Antibody

<400> 63

gatattgtga tgactcagtc tccactctcc ctgcccgtca cccctggaga gccggcctcc 60

atctcctgca ggtctagtca gagcctcctg tatagtaatg gatacaagta tttggattgg 120

tacctgcaga agccaggggca gtctccacag ctcatgatct atttgggttc taatcgggcc 180

tccggggtcc ctgacaggtt cagtggcagt ggatcaggca cagattttac actgaaaatc 240

agcagagtgg aggctgagga tgttggggtt tattattgca tgcaggctct acaaactccg 300

atcaccttcg gccaagggac acgactggag attaaa 336

<210> 64

<211> 112

<212> Protein

<213> Artificial

<220>

<223> Antibody

<400> 64

Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly

1 5 10 15

Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu Tyr Ser

20 25 30

Asn Gly Tyr Lys Tyr Leu Asp Trp Tyr Leu Gln Lys Pro Gly Gln Ser

35 40 45

Pro Gln Leu Met Ile Tyr Leu Gly Ser Asn Arg Ala Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln Ala

85 90 95

Leu Gln Thr Pro Ile Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys

100 105 110

<210> 65

<211> 363

<212> DNA

<213> Artificial

<220>

<223> Antibody

<400> 65

caggtgcagc tggtggagtc tgggggaggc gtggtccagc ctgggaggtc cctgagactc 60

tcctgtgcag cgtctggatt caccttcagt agctatggca tgcactgggt ccgccaggct 120

ccaggcaagg ggctggagtg ggtggcagtt atatggtatg atggaagtaa taaatactat 180

gcagactccg tgaagggccg attcaccatc tccagagaca attccaagaa cacgctgtat 240

ctgcaaatga acagcctgag agccgaggac acggctgtgt attactgtgc gagagcagct 300

cgccttgact actactacgg tatggacgtc tggggccaag ggaccacggt caccgtctcc 360

tca 363

<210> 66

<211> 121

<212> Protein

<213> Artificial

<220>

<223> Antibody

<400> 66

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Val Ile Trp Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ala Ala Arg Leu Asp Tyr Tyr Tyr Gly Met Asp Val Trp Gly

100 105 110

Gln Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 67

<211> 321

<212> DNA

<213> Artificial

<220>

<223> Antibody

<400> 67

gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtctcc 60

atcacttgcc gggcaagtca gagcattaac agctatttaa attggtttca gcagaagcca 120

gggaaagccc ctcagctcct gatctttggt gcatccggtt tgcaaagtgg ggtcccatca 180

aggttcagtg gcagtggatc tgggacagat ttcactctca ccatcaacag tctgcaacct 240

gaagattttg caacttacta ctgtcaacag agttacagtt ccccgctcac cttcggccaa 300

gggacacgac tggagattaa a 321

<210> 68

<211> 107

<212> Protein

<213> Artificial

<220>

<223> Antibody

<400> 68

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Ser Ile Thr Cys Arg Ala Ser Gln Ser Ile Asn Ser Tyr

20 25 30

Leu Asn Trp Phe Gln Gln Lys Pro Gly Lys Ala Pro Gln Leu Leu Ile

35 40 45

Phe Gly Ala Ser Gly Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Asn Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Ser Pro Leu

85 90 95

Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys

100 105

<210> 69

<211> 351

<212> DNA

<213> Artificial

<220>

<223> Antibody

<400> 69

caggtgcagc tacagcagtg gggcgcagga ctgttgaagc cttcggagac cctgtccctc 60

acctgcgctg tctatggtgg gtccttcagt ggttactact ggagctggat ccgccagccc 120

ccagggaagg ggctggagtg gattggggaa atcaatcata gtggaagcac caactacaac 180

ccgtccctca agagtcgagt caccatatca gtagaaacgt ccaagaacca gttctccctg 240

aagctgagct ctgtgaccgc cgcggacacg gctgtgtatt actgtgcgag agataagtgg 300

acctggtact tcgatctctg gggccgtggc accctggtca ctgtctcctc a 351

<210> 70

<211> 117

<212> Protein

<213> Artificial

<220>

<223> Antibody

<400> 70

Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Ser Gly Tyr

20 25 30

Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Glu Ile Asn His Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys

50 55 60

Ser Arg Val Thr Ile Ser Val Glu Thr Ser Lys Asn Gln Phe Ser Leu

65 70 75 80

Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Asp Lys Trp Thr Trp Tyr Phe Asp Leu Trp Gly Arg Gly Thr Leu

100 105 110

Val Thr Val Ser Ser

115

<210> 71

<211> 339

<212> DNA

<213> Artificial

<220>

<223> Antibody

<400> 71

gacatcgaga tgacccagtc tccagactcc ctggctgtgt ctctgggcga gagggccacc 60

atcaactgca ggtccagcca gagtgtttta tacagctcca gcaataggaa ctacttagct 120

tggtaccagc agaacccagg acagcctcct aagctgctca tttactgggc ttctacccgg 180

gaatccgggg tccctgaccg attcagtggc agcgggtctg ggacagattt cactctcacc 240

atcagcagcc tgcaggctga agatgtggca gtttattact gtcagcaata ttatagtact 300

cctcggacgt tcggccaagg gaccaaggtg gaaatcaaa 339

<210> 72

<211> 113

<212> Protein

<213> Artificial

<220>

<223> Antibody

<400> 72

Asp Ile Glu Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Glu Arg Ala Thr Ile Asn Cys Arg Ser Ser Gln Ser Val Leu Tyr Ser

20 25 30

Ser Ser Asn Arg Asn Tyr Leu Ala Trp Tyr Gln Gln Asn Pro Gly Gln

35 40 45

Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val

50 55 60

Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln

85 90 95

Tyr Tyr Ser Thr Pro Arg Thr Phe Gly Gln Gly Thr Lys Val Glu Ile

100 105 110

Lys

<210> 73

<211> 374

<212> DNA

<213> Artificial

<220>

<223> Antibody

<400> 73

caggtgcagc tgcaggagtc gggcccagga ctggtgaagc cttcacagac cctgtccctc 60

acctgcactg tctctggtgg ctccatcagc agtggtggtt actactggag ctggatccgc 120

cagcacccag ggaagggcct ggagtggatg gggaacatct attacagtgg gagcacctac 180

tacaacccgt ccctcaagag tcgagttacc atatcagtag acacgtctga gaaccagttc 240

tccctgaagc tgaactctgt gactgccgcg gacacggccg tatattactg tgcgagaggg 300

ggaactggaa ccaattacta ctactactac ggtatggacg tctggggcca agggaccacg 360

gtcaccgtct cctc 374

<210> 74

<211> 124

<212> Protein

<213> Artificial

<220>

<223> Antibody

<400> 74

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Gly

20 25 30

Gly Tyr Tyr Trp Ser Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu

35 40 45

Trp Met Gly Asn Ile Tyr Tyr Ser Gly Ser Thr Tyr Tyr Asn Pro Ser

50 55 60

Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Glu Asn Gln Phe

65 70 75 80

Ser Leu Lys Leu Asn Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr

85 90 95

Cys Ala Arg Gly Gly Thr Gly Thr Asn Tyr Tyr Tyr Tyr Tyr Gly Met

100 105 110

Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser

115 120

<210> 75

<211> 324

<212> DNA

<213> Artificial

<220>

<223> Antibody

<400> 75

gaaattgtgt tgacgcagtc tccaggcacc ctgtctttgt ctccagggga aagagccacc 60

ctctcctgca gggccagtca gagtgttagc agcagctact tagcctggta ccagcagaaa 120

cctggccagg ctcccaggct cctcatctat ggtgcatcca gctgggccac tggcatccca 180

aacagggttca gtggcagtgg gtctgggaca gacttcactc tcaccatcag cagactggag 240

cctgaagatt ttgcagtgta ttactgtcag cagtatggta gctcaccgct cactttcggc 300

ggagggacca aggtggagat caaa 324

<210> 76

<211> 108

<212> Protein

<213> Artificial

<220>

<223> Antibody

<400> 76

Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Ser

20 25 30

Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu

35 40 45

Ile Tyr Gly Ala Ser Ser Trp Ala Thr Gly Ile Pro Asn Arg Phe Ser

50 55 60

Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu

65 70 75 80

Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Gly Ser Ser Pro

85 90 95

Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 77

<211> 377

<212> DNA

<213> Artificial

<220>

<223> Antibody

<400> 77

caggtgcagc tgcaggagtc gggcccagga ctggtgaagc cttcacagac cctgtccctc 60

acctgcactg tctctggtgt ctccatcagc agtggtggtt actactggag ctggatccgc 120

cagcacccag ggatgggcct ggagtggatt gggtacatct attacagtgg gagcacctac 180

tacaacccgt ccctcaagag tcgagtcacc atatcagaag acacgtctaa gaaccagttc 240

tccctgaagc tgagctctgt gactgccgcg gacacggccg tgtattactg tgcgagagat 300

tccgagtccg agtatagcag ctcgtcgaac tacggtatgg acgtctgggg ccaagggacc 360

acggtcaccg tctcctc 377

<210> 78

<211> 125

<212> Protein

<213> Artificial

<220>

<223> Antibody

<400> 78

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Val Ser Ile Ser Ser Gly

20 25 30

Gly Tyr Tyr Trp Ser Trp Ile Arg Gln His Pro Gly Met Gly Leu Glu

35 40 45

Trp Ile Gly Tyr Ile Tyr Tyr Ser Gly Ser Thr Tyr Tyr Asn Pro Ser

50 55 60

Leu Lys Ser Arg Val Thr Ile Ser Glu Asp Thr Ser Lys Asn Gln Phe

65 70 75 80

Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr

85 90 95

Cys Ala Arg Asp Ser Glu Ser Glu Tyr Ser Ser Ser Ser Asn Tyr Gly

100 105 110

Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser

115 120 125

<210> 79

<211> 377

<212> DNA

<213> Artificial

<220>

<223> Antibody

<400> 79

caggtgcagc tgcaggagtc gggcccagga ctggtgaagc cttcacagac cctgtccctc 60

acctgcactg tctctggtgt ctccatcagc agtggtggtt actactggag ctggatccgc 120

cagcacccag ggatgggcct ggagtggatt gggtacatct attacagtgg gagcacctac 180

tacaacccgt ccctcaagag tcgagtcacc atatcagaag acacgtctaa gaaccagttc 240

tccctgaagc tgagctctgt gactgccgcg gacacggccg tgtattactg tgcgagagat 300

tccgagtccg agtatagcag ctcgtcgaac tacggtatgg acgtctgggg ccaagggacc 360

acggtcaccg tctcctc 377

<210> 80

<211> 125

<212> Protein

<213> Artificial

<220>

<223> Antibody

<400> 80

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Val Ser Ile Ser Ser Gly

20 25 30

Gly Tyr Tyr Trp Ser Trp Ile Arg Gln His Pro Gly Met Gly Leu Glu

35 40 45

Trp Ile Gly Tyr Ile Tyr Tyr Ser Gly Ser Thr Tyr Tyr Asn Pro Ser

50 55 60

Leu Lys Ser Arg Val Thr Ile Ser Glu Asp Thr Ser Lys Asn Gln Phe

65 70 75 80

Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr

85 90 95

Cys Ala Arg Asp Ser Glu Ser Glu Tyr Ser Ser Ser Ser Asn Tyr Gly

100 105 110

Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser

115 120 125

<210> 81

<211> 321

<212> DNA

<213> Artificial

<220>

<223> Antibody

<400> 81

gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagaatcacc 60

atcacttgcc gggcaagtca gaccattagc agctatttaa attggtatca gcagaaacca 120

gggaaagccc ctaagctcct gatctatgct gcatccagtt tgcaaggtgg ggtcccatca 180

aggttcagtg gcagtgtatc tgggacagat ttcaccctca ccgtcagcag tctgcaacct 240

gaagattttg caacttacta ctgtcaacag agttacagta acccgctcac tttcggcgga 300

gggaccaagg tggagatcaa a 321

<210> 82

<211> 107

<212> Protein

<213> Artificial

<220>

<223> Antibody

<400> 82

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Ile Thr Ile Thr Cys Arg Ala Ser Gln Thr Ile Ser Ser Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ala Ala Ser Ser Leu Gln Gly Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Val Ser Gly Thr Asp Phe Thr Leu Thr Val Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Asn Pro Leu

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 83

<211> 371

<212> DNA

<213> Artificial

<220>

<223> Antibody

<400> 83

gaggtgcagc tggtgcagtc tggagcagag gtgaaaaagc ccggggagtc tctgaagatc 60

tcctgtaagg gttctggata cagttttacc agctactgga tcggctgggt gcgccagatg 120

cccgggaaag gcctggagtg gatggggatc atctatcctg gtgactctga taccagatac 180

agcccgtcct tccaaggcca ggtcaccatg tcagccgaca agtccatcag taccgcctac 240

ctgcagctga gcagccatga aggcctcgga caccgccatg tattactgtg cgagacagat 300

ggctggaaac tacgtacatc acgggtgatc gagacgtcct ggggccaagg gaccacggtc 360

accgtctcct c 371

<210> 84

<211> 123

<212> Protein

<213> Artificial

<220>

<223> Antibody

<400> 84

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu

1 5 10 15

Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr Ser Phe Thr Ser Tyr

20 25 30

Trp Ile Gly Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp Met

35 40 45

Gly Ile Ile Tyr Pro Gly Asp Ser Asp Thr Arg Tyr Ser Pro Ser Phe

50 55 60

Gln Gly Gln Val Thr Met Ser Ala Asp Lys Ser Ile Ser Thr Ala Tyr

65 70 75 80

Leu Gln Leu Ser Ser His Glu Gly Leu Gly His Arg His Val Leu Leu

85 90 95

Cys Glu Thr Asp Gly Trp Lys Leu Arg Thr Ser Arg Val Ile Glu Thr

100 105 110

Ser Trp Gly Gln Gly Thr Thr Val Thr Val Ser

115 120

<210> 85

<211> 324

<212> DNA

<213> Artificial

<220>

<223> Antibody

<400> 85

gaaattgtgt tgacgcagtc tccaggcacc ctgtctttgt ctccagggga aagagccacc 60

ctctcctgca gggccagtca gagtgttatc agcatctact tagcctggta ccagcagaaa 120

cctggccagg ctcccaggct cctcatctat ggtgcatcca gcagggccac tggcatccca 180

gacaggttca gtggcagtgg gtctgggaca gacttcactc tcaccatcag cagactggag 240

cctgaagatt ttgcagtgta ttactgtcag cagtatggta gctcaccgtg cagttttggc 300

caggggacca aactggagat caaa 324

<210> 86

<211> 108

<212> Protein

<213> Artificial

<220>

<223> Antibody

<400> 86

Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ile Ser Ile

20 25 30

Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu

35 40 45

Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser

50 55 60

Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu

65 70 75 80

Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Gly Ser Ser Pro

85 90 95

Cys Ser Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 87

<211> 366

<212> DNA

<213> Artificial

<220>

<223> Antibody

<400> 87

caggtgcagc tgcaggagtc gggcccagga ctggtgaagc cttcacagac cctgtccctc 60

acctgcactg tctctggtgg ctccatcagc agtggtgatt actactggag ctggatccgc 120

cagcacccag ggaagggcct ggagtggatt gggtacatct attacagtgg gagcacctac 180

tacaacccgt ccctcaggag tcgagttacc atatcagtag acacgtctaa gaaccagttc 240

tccctgaagc tgagctctgt gactgccgcg gacacggccg tgtattactg tgcgagagcg 300

gattacgatt tttggagtgg ttatttttgac tactggggcc agggaaccct ggtcaccgtc 360

tcctca 366

<210> 88

<211> 122

<212> Protein

<213> Artificial

<220>

<223> Antibody

<400> 88

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Gly

20 25 30

Asp Tyr Tyr Trp Ser Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu

35 40 45

Trp Ile Gly Tyr Ile Tyr Tyr Ser Gly Ser Thr Tyr Tyr Asn Pro Ser

50 55 60

Leu Arg Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe

65 70 75 80

Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr

85 90 95

Cys Ala Arg Ala Asp Tyr Asp Phe Trp Ser Gly Tyr Phe Asp Tyr Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 89

<211> 322

<212> DNA

<213> Artificial

<220>

<223> Antibody

<400> 89

gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60

atcacttgcc gggcaagtca gggcattaga aatgatttag gctggtatca gcagatacct 120

gggaaagccc ctaagcgcct gatctatgct gcatccagtt tgcaaagtgg ggtcccatca 180

aggttcagcg gcagtggatc tgggacagaa ttcactctca caatcaacag cctgcagcct 240

gaagattttg caacttatta ctgtctacag cataatggtt acccgtggac gttcggccaa 300

gggaccaagg tggaaatcaa ac 322

<210> 90

<211> 107

<212> Protein

<213> Artificial

<220>

<223> Antibody

<400> 90

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Arg Asn Asp

20 25 30

Leu Gly Trp Tyr Gln Gln Ile Pro Gly Lys Ala Pro Lys Arg Leu Ile

35 40 45

Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Asn Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln His Asn Gly Tyr Pro Trp

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210> 91

<211> 366

<212> DNA

<213> Artificial

<220>

<223> Antibody

<400> 91

caggtgcagc tgcaggagtc gggcccagga ctggtgaagc cttcacagac cctgtccctc 60

acctgcactg tctctggtgg ctccatcagc agtggtgatt actactggag ctggatccgc 120

cagcacccag ggaagggcct ggagtggatt ggatacatct attacagtgg gagcacctac 180

tacaacccgt ccctcaagag tcgagttacc atatcagtag acacgtctaa gaaccagttc 240

tccctgaagc tgagctctgt gactgccgcg gacacggccg tgtattactg tgcgagagcg 300

gattacgatt tttggagtgg ttatttttgac tactggggcc agggaaccct ggtcaccgtc 360

tcctca 366

<210> 92

<211> 122

<212> Protein

<213> Artificial

<220>

<223> Antibody

<400> 92

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Gly

20 25 30

Asp Tyr Tyr Trp Ser Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu

35 40 45

Trp Ile Gly Tyr Ile Tyr Tyr Ser Gly Ser Thr Tyr Tyr Asn Pro Ser

50 55 60

Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe

65 70 75 80

Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr

85 90 95

Cys Ala Arg Ala Asp Tyr Asp Phe Trp Ser Gly Tyr Phe Asp Tyr Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 93

<211> 322

<212> DNA

<213> Artificial

<220>

<223> Antibody

<400> 93

gacttccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60

atcacttgcc gggcaagtca ggacattcga aatgatttag gctggtatcg gcagaaacct 120

gggaaagccc ctaagcgcct gatctatgct gcatccagtt tgcaaagtgg ggtcccatca 180

aggttcagcg gcagtggatc tgggacagaa ttcactctca caatcagcag cctgcagcct 240

gaagattttg caacttatta ctgtctacag cataatagtt acccgtggac gttcggccaa 300

gggaccaagg tggaaatcaa ac 322

<210> 94

<211> 107

<212> Protein

<213> Artificial

<220>

<223> Antibody

<400> 94

Asp Phe Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Arg Asn Asp

20 25 30

Leu Gly Trp Tyr Arg Gln Lys Pro Gly Lys Ala Pro Lys Arg Leu Ile

35 40 45

Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln His Asn Ser Tyr Pro Trp

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210> 95

<211> 366

<212> DNA

<213> Artificial

<220>

<223> Antibody

<400> 95

caggtgcagc tgcaggagtc gggcccagga ctggtgaagc cttcacagac cctgtccctc 60

acctgcactg tctctggtgg ctccatcagc agtggtgatt actactggag ctggatccgc 120

cagcacccag ggaagggcct ggagtggatt ggatacatct attacagtgg gagcacctac 180

tacaacccgt ccctcaagag tcgagttacc atatcaatag acacgtctaa gaaccagttc 240

tccctgaagc tgagctctgt gactgccgcg gacacggccg tgtattactg tgcgagagcg 300

gattacgatt tttggaatgg ttatttttgac tactggggcc agggaaccct ggtcaccgtc 360

tcctca 366

<210> 96

<211> 122

<212> Protein

<213> Artificial

<220>

<223> Antibody

<400> 96

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Gly

20 25 30

Asp Tyr Tyr Trp Ser Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu

35 40 45

Trp Ile Gly Tyr Ile Tyr Tyr Ser Gly Ser Thr Tyr Tyr Asn Pro Ser

50 55 60

Leu Lys Ser Arg Val Thr Ile Ser Ile Asp Thr Ser Lys Asn Gln Phe

65 70 75 80

Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr

85 90 95

Cys Ala Arg Ala Asp Tyr Asp Phe Trp Asn Gly Tyr Phe Asp Tyr Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 97

<211> 321

<212> DNA

<213> Artificial

<220>

<223> Antibody

<400> 97

gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60

atcacttgcc gggcaagtca ggacattaga aatgatttag gctggtatcg gcagaaacct 120

gggaaagccc ctaagcgcct gatctatgct gcatccagtt tgcaaagtgg ggtcccatca 180

aggttcagcg gcagtggatc tgggacagaa ttcactctca caatcagcag cctgcagcct 240

gaagattttg caacttatta ctgtctacag cataatagtt acccgtggac gttcggccaa 300

gggaccaagg tggaaatcaa a 321

<210> 98

<211> 107

<212> Protein

<213> Artificial

<220>

<223> Antibody

<400> 98

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Arg Asn Asp

20 25 30

Leu Gly Trp Tyr Arg Gln Lys Pro Gly Lys Ala Pro Lys Arg Leu Ile

35 40 45

Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln His Asn Ser Tyr Pro Trp

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210> 99

<211> 366

<212> DNA

<213> Artificial

<220>

<223> Antibody

<400> 99

caggtgcagc tgcaggagtc gggcccagga ctggtgaagc ctacacagac cctgtccctc 60

acctgcactg tctctggtgg ctccatcagc agtggtgatt actactggag ctggatccgc 120

cagcacccag ggaagggcct ggagtggatt gggtacatct attacagtgg gagcacctac 180

tacaacccgt ccctcaagag tcgacttacc atatcagtag acacgtctaa gaaccagttc 240

tccctgaagc tgagctctgt gactgccgcg gacacggccg tgtattactg tgcgagagca 300

gattacgatt tttggagtgg ttactttgac tactggggcc agggaaccct ggtcaccgtc 360

tcctca 366

<210> 100

<211> 122

<212> Protein

<213> Artificial

<220>

<223> Antibody

<400> 100

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Thr Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Gly

20 25 30

Asp Tyr Tyr Trp Ser Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu

35 40 45

Trp Ile Gly Tyr Ile Tyr Tyr Ser Gly Ser Thr Tyr Tyr Asn Pro Ser

50 55 60

Leu Lys Ser Arg Leu Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe

65 70 75 80

Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr

85 90 95

Cys Ala Arg Ala Asp Tyr Asp Phe Trp Ser Gly Tyr Phe Asp Tyr Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 101

<211> 321

<212> DNA

<213> Artificial

<220>

<223> Antibody

<400> 101

gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60

atcacttgcc gggcaagtca gggcattaga aatgatttag gctggtatca gcagaaacca 120

gggaaagccc ctaagcgcct gatctatgct gcatccagtt tgcaaagtgg ggtcccatca 180

aggttcagcg gcagtggatc tgggacagaa ttcactctca caatcagcag cctgcagcct 240

gaagattttg caacttatta ctgtctacag cataataatt acccgtggac gttcggccaa 300

gggaccaagg tggaaatcaa a 321

<210> 102

<211> 107

<212> Protein

<213> Artificial

<220>

<223> Antibody

<400> 102

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Arg Asn Asp

20 25 30

Leu Gly Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Arg Leu Ile

35 40 45

Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln His Asn Asn Tyr Pro Trp

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210> 103

<211> 366

<212> DNA

<213> Artificial

<220>

<223> Antibody

<400> 103

caggtgcagc tgcaggagtc gggcccagga ctggtgaagc cttcacagac cctgtccctc 60

acctgcactg tctctggtgg ctccatcagt agtggtgatt actactggag ctggatccgc 120

cagcacccag ggaagggcct ggagtggatt gggtacatct attacagtgg gagcacctac 180

tacaacccgt ccctcaagag tcgagttacc atatcagtag acacgtctaa gaaccagttc 240

tccctgaagt tgagctctgt gactgccgcg gacacggccg tgtattactg tgcgagagcc 300

gattacgatt tttggagtgg ttatttttgac tactggggcc agggaaccct ggtcaccgtc 360

tcctca 366

<210> 104

<211> 122

<212> Protein

<213> Artificial

<220>

<223> Antibody

<400> 104

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Gly

20 25 30

Asp Tyr Tyr Trp Ser Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu

35 40 45

Trp Ile Gly Tyr Ile Tyr Tyr Ser Gly Ser Thr Tyr Tyr Asn Pro Ser

50 55 60

Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe

65 70 75 80

Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr

85 90 95

Cys Ala Arg Ala Asp Tyr Asp Phe Trp Ser Gly Tyr Phe Asp Tyr Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 105

<211> 321

<212> DNA

<213> Artificial

<220>

<223> Antibody

<400> 105

gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60

atcacttgcc gggcaagtca gggcattaga aatgatttag gctggtatca gcagaaacca 120

gggaaagccc ctaagcgcct gatctatgct gcatccagtt tgcaaagtgg ggtcccatca 180

aggttcagcg gcagtggatc tgggacagaa ttcactctca caatcagcag cctgcagcct 240

gaagattttg caacttatta ctgtctacag cataataatt acccgtggac gttcggccaa 300

gggaccaagg tggaaatcaa a 321

<210> 106

<211> 107

<212> Protein

<213> Artificial

<220>

<223> Antibody

<400> 106

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Arg Asn Asp

20 25 30

Leu Gly Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Arg Leu Ile

35 40 45

Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln His Asn Asn Tyr Pro Trp

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210> 107

<211> 351

<212> DNA

<213> Artificial

<220>

<223> Antibody

<400> 107

caggtgcagc tgcaggagtc gggcccagga ctggtgaagc cttcacagac cctgtccctc 60

acctgcactg tctctggtgg ctccatcagc agtggtggtt actactggag ctggatccgc 120

cagcacccag ggaagggcct ggagtggatt gggtacatct attacagtgg gagcacctac 180

tacaacccgt ccctcaagag tcgagttacc atatcagtag acacgtctaa gaaccagttc 240

tccctgaagc tgagctctgt gactgccgcg gacacggccg tgtattactg tgcgagagag 300

gacgacggta tggacgtctg gggccaaggg accacggtca ccgtctcctc a 351

<210> 108

<211> 117

<212> Protein

<213> Artificial

<220>

<223> Antibody

<400> 108

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Gly

20 25 30

Gly Tyr Tyr Trp Ser Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu

35 40 45

Trp Ile Gly Tyr Ile Tyr Tyr Ser Gly Ser Thr Tyr Tyr Asn Pro Ser

50 55 60

Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe

65 70 75 80

Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr

85 90 95

Cys Ala Arg Glu Asp Asp Gly Met Asp Val Trp Gly Gln Gly Thr Thr

100 105 110

Val Thr Val Ser Ser

115

<210> 109

<211> 336

<212> DNA

<213> Artificial

<220>

<223> Antibody

<400> 109

gatattgtga tgactcagtc tccactctcc ctgcccgtca cccctggaga gccggcctcc 60

120

tacctgcaga agccagggca gtccccacag ttcatgattt atttggggtc taatcgggcc 180

tccggggtcc ctgacaggtt cagtggcagt ggatcaggca cagattttac actgaaaatc 240

agcagagtgg aggctgagga tgttggggtt tattactgca tgcaagctct acaaactccg 300

atcaccttcg gccaagggac acgactggag attaaa 336

<210> 110

<211> 112

<212> Protein

<213> Artificial

<220>

<223> Antibody

<400> 110

Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly

1 5 10 15

Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu His Ser

20 25 30

Asn Gly Tyr Asn Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser

35 40 45

Pro Gln Phe Met Ile Tyr Leu Gly Ser Asn Arg Ala Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln Ala

85 90 95

Leu Gln Thr Pro Ile Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys

100 105 110

<210> 111

<211> 366

<212> DNA

<213> Artificial

<220>

<223> Antibody

<400> 111

caggtgcagc tgcaggagtc gggcccagga ctggtgaagc cttcacagac cctgtccctc 60

acctgcactg tctctggtgg ctccatcagc agtggtgatt actactggag ctggatccgc 120

cagtacccag ggaagggcct ggagtggatt gggtacatct attacagtgg gagcacctac 180

tacaacccgt ccctcaagag tcgagttacc atatcagtag acacgtctaa gaaccagttc 240

tccctgaagc tgaggtctgt gactgccgcg gacacggccg tgtattactg tgcgagagcg 300

gattacgatt tttggagtgg ttatttttgac tactggggcc agggaaccct ggtcaccgtc 360

tcctca 366

<210> 112

<211> 122

<212> Protein

<213> Artificial

<220>

<223> Antibody

<400> 112

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Gly

20 25 30

Asp Tyr Tyr Trp Ser Trp Ile Arg Gln Tyr Pro Gly Lys Gly Leu Glu

35 40 45

Trp Ile Gly Tyr Ile Tyr Tyr Ser Gly Ser Thr Tyr Tyr Asn Pro Ser

50 55 60

Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe

65 70 75 80

Ser Leu Lys Leu Arg Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr

85 90 95

Cys Ala Arg Ala Asp Tyr Asp Phe Trp Ser Gly Tyr Phe Asp Tyr Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 113

<211> 322

<212> DNA

<213> Artificial

<220>

<223> Antibody

<400> 113

gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60

atcacttgcc gggcaagtca gggcattaga aatgatttag gctggtatca gcagaaacca 120

gggaaagccc ctaagcgcct gatctatgct gcatccagtt tgcaaagtgg ggtcccatca 180

aggttcagcg gcagtggatc tgggacagaa ttcactctca caatcagcag cctgcagcct 240

gaagattttg caacttatta ctgtctacag cataatactt acccgtggac gttcggccaa 300

gggaccaagg tggaaatcaa ac 322

<210> 114

<211> 107

<212> Protein

<213> Artificial

<220>

<223> Antibody

<400> 114

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Arg Asn Asp

20 25 30

Leu Gly Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Arg Leu Ile

35 40 45

Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln His Asn Thr Tyr Pro Trp

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210> 115

<211> 372

<212> DNA

<213> Artificial

<220>

<223> Antibody

<400> 115

caggtgcagc tgcaggagtc gggcccagga ctggtgaagc cttcggagac cctgtccctc 60

acctgcactg tctctggtgg ctccgtcagc agtggtggtt actactggag ctggatccgg 120

cagcccccag ggaagggact ggagtggatt gggtatatct attacagtgg gagcaccaac 180

tacaacccct ccctcaagag tcgagtcacc atatcagtag acacgtccaa gaaccagttc 240

tccctgaagc tgagctctgt gaccgctgcg gacacggccg tgtattactg tgcgagagat 300

ggggacgtgg atacagctat ggtcgatgct tttgatatct ggggccaagg gacaatggtc 360

accgtctcct ca 372

<210> 116

<211> 124

<212> Protein

<213> Artificial

<220>

<223> Antibody

<400> 116

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Val Ser Ser Gly

20 25 30

Gly Tyr Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu

35 40 45

Trp Ile Gly Tyr Ile Tyr Tyr Ser Gly Ser Thr Asn Tyr Asn Pro Ser

50 55 60

Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe

65 70 75 80

Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr

85 90 95

Cys Ala Arg Asp Gly Asp Val Asp Thr Ala Met Val Asp Ala Phe Asp

100 105 110

Ile Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser

115 120

<210> 117

<211> 324

<212> DNA

<213> Artificial

<220>

<223> Antibody

<400> 117

gaaattgtat tgacgcagtc tccaggcacc ctgtctttgt ctccagggga aagagccacc 60

ctctcctgca gggccagtca gagtttaagc ggcaactact tagcctggta ccagcagaag 120

cctggccagg ctcccaggct catcatctgt ggtgcatcca gcagggccac tggcatccca 180

gacaggttca gtggcagtgg gtctgggaca gacttcactc tcaccatcac aagactggag 240

cctgaagatt ttgcagtgta ttactgtcag cagtatgata ggtcaccgct cactttcggc 300

ggagggacca aggtggagat caaa 324

<210> 118

<211> 108

<212> Protein

<213> Artificial

<220>

<223> Antibody

<400> 118

Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Leu Ser Gly Asn

20 25 30

Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Ile

35 40 45

Ile Cys Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser

50 55 60

Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Thr Arg Leu Glu

65 70 75 80

Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Asp Arg Ser Pro

85 90 95

Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 119

<211> 366

<212> DNA

<213> Artificial

<220>

<223> Antibody

<400> 119

caggtgcagc tgcaggagtc gggcccagga ctggtgaagc cttcacagac cctgtccctc 60

acctgcactg tctctggtgg ctccatcagc agtggtgatt actactggag ctggatccgc 120

cagcacccag ggaagggcct ggagtggatt gggtacatct attacagtgg gagcacctac 180

tacaacccgt ccctcaagag tcgagttacc atatcagtag acacgtctaa gaaccagttc 240

tccctgaagc tgagctctgt gactgccgcg gacacggccg tgtattactg tgcgagagga 300

gattacgatt tttggagtgg agagtttgac tactggggcc agggaaccct ggtcaccgtc 360

tcctca 366

<210> 120

<211> 122

<212> Protein

<213> Artificial

<220>

<223> Antibody

<400> 120

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Gly

20 25 30

Asp Tyr Tyr Trp Ser Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu

35 40 45

Trp Ile Gly Tyr Ile Tyr Tyr Ser Gly Ser Thr Tyr Tyr Asn Pro Ser

50 55 60

Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe

65 70 75 80

Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr

85 90 95

Cys Ala Arg Gly Asp Tyr Asp Phe Trp Ser Gly Glu Phe Asp Tyr Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 121

<211> 386

<212> DNA

<213> Artificial

<220>

<223> Antibody

<400> 121

caggtgcagc tgcaggagtc gggcccagga ctggtgaagc cttcacagac cctgtccctc 60

acctgcactg tctctggtgg ctccatcagc agtggtggtt actactggag ctggatccgc 120

cagcacccag ggaagggcct ggagtggatt gggtacatct atgacagtgg gagcacctac 180

tacaacccgt ccctcaagag tcgagttacc atatcagtag acacgtctaa gaaccagttc 240

tccctgaagc tgaggtctgt gactgccgcg gacacggccg tgtattactg tgcgagagat 300

caggggcagg acggatacag ctatggttac ggctactact acggtatgga cgtctggggc 360

caagggacca cggtcaccgt ctcctc 386

<210> 122

<211> 128

<212> Protein

<213> Artificial

<220>

<223> Antibody

<400> 122

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Gly

20 25 30

Gly Tyr Tyr Trp Ser Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu

35 40 45

Trp Ile Gly Tyr Ile Tyr Asp Ser Gly Ser Thr Tyr Tyr Asn Pro Ser

50 55 60

Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe

65 70 75 80

Ser Leu Lys Leu Arg Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr

85 90 95

Cys Ala Arg Asp Gln Gly Gln Asp Gly Tyr Ser Tyr Gly Tyr Gly Tyr

100 105 110

Tyr Tyr Gly Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser

115 120 125

<210> 123

<211> 321

<212> DNA

<213> Artificial

<220>

<223> Antibody

<400> 123

gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60

atcacttgcc aggcgagtca ggacattagc aattatttaa attggtatca gcagaaacca 120

gggaaagccc ctaaactcct gatctacgtt gcatccaatt tggaaacagg ggtcccatca 180

aggttcagtg gaagtggatc tgggacagat tttactttca ccatcagcag cctgcagcct 240

gaagatattg caacatatta ctgtcaacag tgtgataatc tccctctcac tttcggcgga 300

gggaccaagg tggagatcaa a 321

<210> 124

<211> 107

<212> Protein

<213> Artificial

<220>

<223> Antibody

<400> 124

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Gln Ala Ser Gln Asp Ile Ser Asn Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Val Ala Ser Asn Leu Glu Thr Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Cys Asp Asn Leu Pro Leu

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 125

<211> 365

<212> DNA

<213> Artificial

<220>

<223> Antibody

<400> 125

caggtgcagc tgcaggagtc gggcccagga ctggtgaagc cttcacagac cctgtccctc 60

acctgcactg tctctggtgg ctccatcagc agtggtgatt actactggag ctggatccgc 120

cagcacccag ggaagggcct ggagtggatt ggatacatct attacagtgg gagcacctac 180

tacaacccgt ccctcaagag tcgagttacc atatcagtag acacgtctaa gaaccagttc 240

tccctgaagc tgagctctgt gactgccgcg gacacggccg tgtattactg tgcgagagcg 300

gattacgatt tttggagtgg ttatttttgac tactggggcc agggaaccct ggtcaccgtc 360

tcctc 365

<210> 126

<211> 121

<212> Protein

<213> Artificial

<220>

<223> Antibody

<400> 126

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Gly

20 25 30

Asp Tyr Tyr Trp Ser Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu

35 40 45

Trp Ile Gly Tyr Ile Tyr Tyr Ser Gly Ser Thr Tyr Tyr Asn Pro Ser

50 55 60

Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe

65 70 75 80

Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr

85 90 95

Cys Ala Arg Ala Asp Tyr Asp Phe Trp Ser Gly Tyr Phe Asp Tyr Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser

115 120

<210> 127

<211> 322

<212> DNA

<213> Artificial

<220>

<223> Antibody

<400> 127

gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60

atcacttgcc gggcaagtca ggacattaga aatgatttag gctggtatcg gcagaaacct 120

gggaaagccc ctaagcgcct gatctatgct gcatcccgtt tgcaaagtgg ggtcccatca 180

aggttcagcg gcagtggatc tgggacagaa ttcactctca caatcagcag cctgcagcct 240

gaagattttg caacttatta ctgtctacag cataatagtt acccgtggac gttcggccaa 300

gggaccaagg tggaaatcaa ac 322

<210> 128

<211> 107

<212> Protein

<213> Artificial

<220>

<223> Antibody

<400> 128

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Arg Asn Asp

20 25 30

Leu Gly Trp Tyr Arg Gln Lys Pro Gly Lys Ala Pro Lys Arg Leu Ile

35 40 45

Tyr Ala Ala Ser Arg Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln His Asn Ser Tyr Pro Trp

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210> 129

<211> 366

<212> DNA

<213> Artificial

<220>

<223> Antibody

<400> 129

caggtgcagc tgcaggagtc gggcccagga ctggtgaagc cttcacagac cctgtccctc 60

acctgcactg tctctggtgg ctccatcagc agtggtgatt actactggag ctggatccgc 120

cagcacccag ggaagggcct ggagtggatt gggtacatct attacagtgg gagcacctac 180

tacaacccgt ccctcaagag tcgagttacc atatcagtag acacgtctaa gaaccagttc 240

tccctgaagc tgagctctgt gactgccgcg gacacggccg tgtattactg tgcgagagcc 300

gattacgatt tttggagtgg ttatttttgac tactggggcc agggaaccct ggtcaccgtc 360

tcctca 366

<210> 130

<211> 122

<212> Protein

<213> Artificial

<220>

<223> Antibody

<400> 130

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Gly

20 25 30

Asp Tyr Tyr Trp Ser Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu

35 40 45

Trp Ile Gly Tyr Ile Tyr Tyr Ser Gly Ser Thr Tyr Tyr Asn Pro Ser

50 55 60

Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe

65 70 75 80

Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr

85 90 95

Cys Ala Arg Ala Asp Tyr Asp Phe Trp Ser Gly Tyr Phe Asp Tyr Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 131

<211> 322

<212> DNA

<213> Artificial

<220>

<223> Antibody

<400> 131

gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60

atcacttgcc gggcaagtca gggcattaga aatgatttag gctggtatca gcagaaacca 120

gggaaagccc ctaagcgcct gatctatgct gcatccagtt tgcaaaatgg ggtcccatca 180

aggttcagcg gcagtggatc tgggacagaa ttcactctca caatcagcag cctgcagcct 240

gaagattttg caacttatta ctgtctacag cataatagtt acccgtggac gttcggccaa 300

gggaccaagg tggaaatcaa ac 322

<210> 132

<211> 107

<212> Protein

<213> Artificial

<220>

<223> Antibody

<400> 132

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Arg Asn Asp

20 25 30

Leu Gly Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Arg Leu Ile

35 40 45

Tyr Ala Ala Ser Ser Leu Gln Asn Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln His Asn Ser Tyr Pro Trp

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210> 133

<211> 365

<212> DNA

<213> Artificial

<220>

<223> Antibody

<400> 133

caggtgcagc tgcaggagtc gggcccagga ctggtgaagc cttcacagac cctgtccctc 60

acctgcactg tctctggtgg ctccatcagc agtggtgatt actactggag ctggatccgc 120

cagcacccag ggaagggcct ggagtggatt gggtacatct attacagtgg gagcacctac 180

tacaacccgt ccctcaagag tcgagttacc atatcagtag acacgtctaa gaaccagttc 240

tccctgaagc tgagctctgt gactgccgcg gacacggccg tgtattactg tgcgagagcg 300

gattacgatt tttggagtgg ttatttttgac tactggggcc agggaatcct ggtcaccgtc 360

tcctc 365

<210> 134

<211> 121

<212> Protein

<213> Artificial

<220>

<223> Antibody

<400> 134

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Gly

20 25 30

Asp Tyr Tyr Trp Ser Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu

35 40 45

Trp Ile Gly Tyr Ile Tyr Tyr Ser Gly Ser Thr Tyr Tyr Asn Pro Ser

50 55 60

Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe

65 70 75 80

Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr

85 90 95

Cys Ala Arg Ala Asp Tyr Asp Phe Trp Ser Gly Tyr Phe Asp Tyr Trp

100 105 110

Gly Gln Gly Ile Leu Val Thr Val Ser

115 120

<210> 135

<211> 322

<212> DNA

<213> Artificial

<220>

<223> Antibody

<400> 135

gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60

atcacttgcc gggcaagtca gggcattaga aatgatttag gctggtatca gcagaaacca 120

gggaaagccc ctaagcgcct gatttatgct gcatccagtt tgcaaagtgg ggtcccatca 180

aggttcagcg gcagtggatc tgggacagaa ttcactctca caatcagcag cctgcagcct 240

gaagattttg caacttatta ctgtctacag cataatagtt acccgtggac gttcggccaa 300

gggaccaagg tggaaatcaa ac 322

<210> 136

<211> 107

<212> Protein

<213> Artificial

<220>

<223> Antibody

<400> 136

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Arg Asn Asp

20 25 30

Leu Gly Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Arg Leu Ile

35 40 45

Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln His Asn Ser Tyr Pro Trp

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210> 137

<211> 366

<212> DNA

<213> Artificial

<220>

<223> Antibody

<400> 137

caggtgcagc tgcaggagtc gggcccagga ctggtgaagc cttcacagac cctgtccctc 60

acctgcactg tctctggtgg ctccatcagt agtggtgatt actactggag ctggatccgc 120

cagcacccag ggaagggcct ggagtggatt gggtacatct attacagtgg gagcacctac 180

tacaacccgt ccctcaagag tcgagttacc atatcagtag acacgtctaa gaaccagttc 240

tccctgaagt tgagctctgt gactgccgcg gacacggccg tgtattactg tgcgagagcc 300

gattacgatt tttggaatgg ttatttttgac tactggggcc agggaaccct ggtcaccgtc 360

tcctca 366

<210> 138

<211> 122

<212> Protein

<213> Artificial

<220>

<223> Antibody

<400> 138

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Gly

20 25 30

Asp Tyr Tyr Trp Ser Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu

35 40 45

Trp Ile Gly Tyr Ile Tyr Tyr Ser Gly Ser Thr Tyr Tyr Asn Pro Ser

50 55 60

Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe

65 70 75 80

Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr

85 90 95

Cys Ala Arg Ala Asp Tyr Asp Phe Trp Asn Gly Tyr Phe Asp Tyr Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 139

<211> 321

<212> DNA

<213> Artificial

<220>

<223> Antibody

<400> 139

gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60

atcacttgcc gggcaagtca gggcattaga aatgatttag gctggtatca gcagaaacca 120

gggaaagccc ctaagcgcct gatctatgct gcatccagtt tgcaaagtgg ggtcccatca 180

aggttcagcg gcagtggatc tgggacagaa ttcactctca caatcagcag cctgcagcct 240

gaagattttg caacttatta ctgtctacag cataataatt acccgtggac gttcggccaa 300

gggaccaagg tggaaatcaa a 321

<210> 140

<211> 107

<212> Protein

<213> Artificial

<220>

<223> Antibody

<400> 140

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Arg Asn Asp

20 25 30

Leu Gly Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Arg Leu Ile

35 40 45

Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln His Asn Asn Tyr Pro Trp

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210> 141

<211> 366

<212> DNA

<213> Artificial

<220>

<223> Antibody

<400> 141

caggtgcagc tgcaggagtc gggcccagga ctggtgaagc cttcacagac cctgtccctc 60

acctgcactg tctctggtgg ctccatcagc agtggtgatt actactggag ctggatccgc 120

cagcacccag ggaagggcct ggagtggatt gggtacatct attacagtgg gagcacctac 180

tacaacccgt ccctcaagag tcgagttacc atatcagtag acacgtctaa gaaccagttc 240

tccctgaagc tgagctctgt gactgccgcg gacacggccg tgtattactg tgcgagagcc 300

gattacgatt tttggagtgg ttatttttgac tactggggcc agggaaccct ggtcaccgtc 360

tcctca 366

<210> 142

<211> 122

<212> Protein

<213> Artificial

<220>

<223> Antibody

<400> 142

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Gly

20 25 30

Asp Tyr Tyr Trp Ser Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu

35 40 45

Trp Ile Gly Tyr Ile Tyr Tyr Ser Gly Ser Thr Tyr Tyr Asn Pro Ser

50 55 60

Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe

65 70 75 80

Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr

85 90 95

Cys Ala Arg Ala Asp Tyr Asp Phe Trp Ser Gly Tyr Phe Asp Tyr Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 143

<211> 322

<212> DNA

<213> Artificial

<220>

<223> Antibody

<400> 143

gacatccagc tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60

atcacttgcc gggcaagtca gggcattaga aatgatttag gctggtatca gcagaaacca 120

gggaaagccc ctaagcgcct gatctatgct gcatccagtt tgcaaaatgg ggtcccatca 180

aggttcagcg gcagtggatc tgggacagaa ttcactctca caatcagcag cctgcagcct 240

gaagattttg caacttatta ctgtctacag cataatagtt acccgtggac gttcggccaa 300

gggaccaagg tggaaatcaa ac 322

<210> 144

<211> 107

<212> Protein

<213> Artificial

<220>

<223> Antibody

<400> 144

Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Arg Asn Asp

20 25 30

Leu Gly Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Arg Leu Ile

35 40 45

Tyr Ala Ala Ser Ser Leu Gln Asn Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln His Asn Ser Tyr Pro Trp

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210> 145

<211> 365

<212> DNA

<213> Artificial

<220>

<223> Antibody

<400> 145

caggtgcagc tgcaggagtc gggcccagga ctggtgaagc cttcacagac cctgtccctc 60

acctgcactg tctctggtgg ctccatcagc agtggtgatt actactggag ctggatccgc 120

cagtacccag ggaagggcct ggagtggatt gggtacatct attacagtgg gagcacctac 180

tacaacccgt ccctcaagag tcgagttacc atatcagtag acacgtctaa gaaccagttc 240

tccctgaagc tgggctctgt gactgccgcg gacacggccg tgtatttctg tgcgagagcc 300

gattacgatt tttggagtgg ttatttttgac ttctggggcc agggaaccct ggtcaccgtc 360

tcctc 365

<210> 146

<211> 121

<212> Protein

<213> Artificial

<220>

<223> Antibody

<400> 146

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Gly

20 25 30

Asp Tyr Tyr Trp Ser Trp Ile Arg Gln Tyr Pro Gly Lys Gly Leu Glu

35 40 45

Trp Ile Gly Tyr Ile Tyr Tyr Ser Gly Ser Thr Tyr Tyr Asn Pro Ser

50 55 60

Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe

65 70 75 80

Ser Leu Lys Leu Gly Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Phe

85 90 95

Cys Ala Arg Ala Asp Tyr Asp Phe Trp Ser Gly Tyr Phe Asp Phe Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser

115 120

<210> 147

<211> 322

<212> DNA

<213> Artificial

<220>

<223> Antibody

<400> 147

gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60

atcacttgcc gggcaagtca gggcattaga aatgatttag gctggtatca gcagaaacca 120

gggaaagccc ctaagcgcct gatctatgct gcatccagtt tgcaaagtgg ggtcccatca 180

aggttcagcg gcagtggatc tgggacagaa ttcactctca caatcagcag cctgcagcct 240

gaagattttg caacttatta ctgtctacag cataatggtt acccgtggac gttcggccaa 300

gggaccaagg tggaaatcaa ac 322

<210> 148

<211> 107

<212> Protein

<213> Artificial

<220>

<223> Antibody

<400> 148

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Arg Asn Asp

20 25 30

Leu Gly Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Arg Leu Ile

35 40 45

Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln His Asn Gly Tyr Pro Trp

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210> 149

<211> 365

<212> DNA

<213> Artificial

<220>

<223> Antibody

<400> 149

caggtgcagc tgcaggagtc gggcccagga ctggtgaagc cttcacagac cctgtccctc 60

acctgcactg tctctggtgg ctccatcagc agtggtgatt actactggag ctggatccgc 120

cagtacccag ggaagggcct ggagtggatt gggtacatct attacagtgg gagcacctac 180

tacaacccgt ccctcaagag tcgagttacc atatcagtag acacgtctaa gaaccagttc 240

tccctgaagc tgggctctgt gactgccgcg gacacggccg tgtatttctg tgcgagagcc 300

gattacgatt tttggagtgg ttatttttgac ttctggggcc agggaaccct ggtcaccgtc 360

tcctc 365

<210> 150

<211> 121

<212> Protein

<213> Artificial

<220>

<223> Antibody

<400> 150

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Gly

20 25 30

Asp Tyr Tyr Trp Ser Trp Ile Arg Gln Tyr Pro Gly Lys Gly Leu Glu

35 40 45

Trp Ile Gly Tyr Ile Tyr Tyr Ser Gly Ser Thr Tyr Tyr Asn Pro Ser

50 55 60

Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe

65 70 75 80

Ser Leu Lys Leu Gly Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Phe

85 90 95

Cys Ala Arg Ala Asp Tyr Asp Phe Trp Ser Gly Tyr Phe Asp Phe Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser

115 120

<210> 151

<211> 322

<212> DNA

<213> Artificial

<220>

<223> Antibody

<400> 151

gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60

atcacttgcc gggcaagtca gggcattaga aatgatttag gctggtatca gcagaaacca 120

gggaaagccc ctaagcgcct gatctatgct gcatccagtt tgcaaagtgg ggtcccatca 180

aggttcagcg gcagtggatc tgggacagaa ttcactctca caatcagcag cctgcagcct 240

gaagattttg caacttatta ctgtctacag cataatggtt acccgtggac gttcggccaa 300

gggaccaagg tggaaatcaa ac 322

<210> 152

<211> 107

<212> Protein

<213> Artificial

<220>

<223> Antibody

<400> 152

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Arg Asn Asp

20 25 30

Leu Gly Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Arg Leu Ile

35 40 45

Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln His Asn Gly Tyr Pro Trp

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210> 153

<211> 366

<212> DNA

<213> Artificial

<220>

<223> Antibody

<400> 153

caggtgcagc tgcaggagtc gggcccagga ctggtgaagc cttcacagac cctgtccctc 60

acctgcactg tctctggtgg ctccatcagt agtggtgatt actactggag ctggatccgc 120

cagcacccag ggaagggcct ggagtggatt gggtacatct attacagtgg gagcacctac 180

tacaacccgt ccctcaagag tcgagttacc atatcagtag acacgtctaa gaaccagttc 240

tccctgaagc tgagctctgt gactgccgcg gacacggccg tgtattactg tgcgagagcg 300

gattacgatt tttggagtgg ttatttttgac tcctggggcc agggaaccct ggtcaccgtc 360

tcctca 366

<210> 154

<211> 122

<212> Protein

<213> Artificial

<220>

<223> Antibody

<400> 154

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Gly

20 25 30

Asp Tyr Tyr Trp Ser Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu

35 40 45

Trp Ile Gly Tyr Ile Tyr Tyr Ser Gly Ser Thr Tyr Tyr Asn Pro Ser

50 55 60

Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe

65 70 75 80

Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr

85 90 95

Cys Ala Arg Ala Asp Tyr Asp Phe Trp Ser Gly Tyr Phe Asp Ser Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 155

<211> 321

<212> DNA

<213> Artificial

<220>

<223> Antibody

<400> 155

gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60

atcacttgcc gggcaagtca gggcattaga aatgatttag gctggtatca gcagatacct 120

gggaaagccc ctaagcgcct gatctatgct gcatccagtt tgcaaagtgg ggtcccatca 180

aggttcagcg gcagtggatc tgggacagaa ttcactctca caatcagcag cctgcagcct 240

gaagattttg caacttatta ctgtctacag cataatggtt acccgtggac gttcggccaa 300

gggaccaagg tggaaatcaa a 321

<210> 156

<211> 107

<212> Protein

<213> Artificial

<220>

<223> Antibody

<400> 156

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Arg Asn Asp

20 25 30

Leu Gly Trp Tyr Gln Gln Ile Pro Gly Lys Ala Pro Lys Arg Leu Ile

35 40 45

Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln His Asn Gly Tyr Pro Trp

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210> 157

<211> 366

<212> DNA

<213> Artificial

<220>

<223> Antibody

<400> 157

caggttcagc tggtgcagtc tggagctgag gtgaagaagc ctggggcctc agtgaaggtc 60

tcctgcaagg cttctggtta cacctttacc aactatggta tcagctgggt gcggcaggcc 120

cctggacaag ggcttgagtg gatgggatgg atcagcgctt acgatggtta cagaaactat 180

gcacagaagc tccagggcag agtcaccatg accacagaca catccacgac cactgcctac 240

atggagctga ggagcctgag atctgacgac acggccgtgt attactgtgc gagagatgtt 300

caagactacg gtgactacga ctactttgac tactggggcc agggaaccct ggtcaccgtc 360

tcctca 366

<210> 158

<211> 122

<212> Protein

<213> Artificial

<220>

<223> Antibody

<400> 158

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr

20 25 30

Gly Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Trp Ile Ser Ala Tyr Asp Gly Tyr Arg Asn Tyr Ala Gln Lys Leu

50 55 60

Gln Gly Arg Val Thr Met Thr Thr Asp Thr Ser Thr Thr Thr Ala Tyr

65 70 75 80

Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asp Val Gln Asp Tyr Gly Asp Tyr Asp Tyr Phe Asp Tyr Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 159

<211> 321

<212> DNA

<213> Artificial

<220>

<223> Antibody

<400> 159

gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60

atcacttgcc gggcaagtca gagcattagc agttatttaa attggtatca gcagaaacca 120

gggaaagccc ctaacctcct gatctatgct gcatccagtt tgcaaagtgg ggtcccatca 180

agattcaggg gcagtggatc tgggacagat ttcactctca ccatcagcag tctgcaacct 240

gaagattttg caacttacta ctgtcaacag agttacagta cccccatcac cttcggccaa 300

gggacacgac tggagattaa a 321

<210> 160

<211> 107

<212> Protein

<213> Artificial

<220>

<223> Antibody

<400> 160

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Asn Leu Leu Ile

35 40 45

Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Arg Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Pro Ile

85 90 95

Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys

100 105

<210> 161

<211> 366

<212> DNA

<213> Artificial

<220>

<223> Antibody

<400> 161

caggtgcagc tgcaggagtc gggcccagga ctggtgaagc ctttacagac cctgtccctc 60

acctgcactg tctctggtgg ctccatcagc agtggtgatt actactggag ctggatccgc 120

cagcacccag ggaagggcct ggagtggatt gggtacatct attacagtgg gaccacctac 180

tacaacccgt ccctcaagag tcgagttacc atatcagtag acacgtctaa gaaccagttc 240

gccctgaagc tgaactctgt gactgccgcg gacacggccg tgtattactg tgcgagagcc 300

gattacgatt tttggagtgg ttatttttgac tactggggcc agggaaccct ggtcaccgtc 360

tcctca 366

<210> 162

<211> 122

<212> Protein

<213> Artificial

<220>

<223> Antibody

<400> 162

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Leu Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Gly

20 25 30

Asp Tyr Tyr Trp Ser Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu

35 40 45

Trp Ile Gly Tyr Ile Tyr Tyr Ser Gly Thr Thr Tyr Tyr Asn Pro Ser

50 55 60

Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe

65 70 75 80

Ala Leu Lys Leu Asn Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr

85 90 95

Cys Ala Arg Ala Asp Tyr Asp Phe Trp Ser Gly Tyr Phe Asp Tyr Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 163

<211> 322

<212> DNA

<213> Artificial

<220>

<223> Antibody

<400> 163

gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60

atcacttgcc gggcaggtca gggcattaga aatgatttag gctggtatca gcagaaacca 120

gggaaagccc ctcagcgcct gatctatgct gcatccagtt tgcaaagtgg ggtcccatca 180

aggttcagcg gcagtggatc tgggacagaa ttctctctca caatctccag cctgcagcct 240

gaagattttg caacttatta ctgtctacag cataatagtt acccgtggac gttcggccaa 300

gggaccaagg tggaaatcaa ac 322

<210> 164

<211> 107

<212> Protein

<213> Artificial

<220>

<223> Antibody

<400> 164

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Gly Gln Gly Ile Arg Asn Asp

20 25 30

Leu Gly Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Gln Arg Leu Ile

35 40 45

Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Glu Phe Ser Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln His Asn Ser Tyr Pro Trp

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210> 165

<211> 369

<212> DNA

<213> Artificial

<220>

<223> Antibody

<400> 165

caggtgcagc tggtggagtc tgggggaggc ttggtcaagc ctggagggtc cctgagactc 60

tcctgtgcag cctctggatt caccttcagt gactactaca tgagctggat ccgccaggct 120

ccagggaagg ggctggagtg ggtttcatat attagtagta gtggtaataa catataccac 180

gcagactctg tgaagggccg attcaccatc tccagggaca acgccaagaa ctcactgtat 240

ctgcaaatga acagcctgag agccgaggac acggccgtgt attactgtgc gagagagaga 300

tatagtggct acgacgaccc tgatggtttt gatatctgggg gccaagggac aatggtcacc 360

gtctcttca 369

<210> 166

<211> 123

<212> Protein

<213> Artificial

<220>

<223> Antibody

<400> 166

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr

20 25 30

Tyr Met Ser Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Tyr Ile Ser Ser Ser Gly Asn Asn Ile Tyr His Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Glu Arg Tyr Ser Gly Tyr Asp Asp Pro Asp Gly Phe Asp Ile

100 105 110

Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser

115 120

<210> 167

<211> 321

<212> DNA

<213> Artificial

<220>

<223> Antibody

<400> 167

gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60

atcacttgcc aggcgagtca ggacattagc aactatttaa gttggtttca gcagaaacca 120

gggaaagccc ctaagctcct gatccacgat gcatccaatt tggaaacagg ggtcccttca 180

aggttcagtg gaagtggatc tgggacagat tttactttca ccatcagcag cctgcagcct 240

gaagatattg caacatatta ctgtcaacag tatgataatc ccccgtgcag ttttggccag 300

gggaccaagc tggagatcaa a 321

<210> 168

<211> 107

<212> Protein

<213> Artificial

<220>

<223> Antibody

<400> 168

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Gln Ala Ser Gln Asp Ile Ser Asn Tyr

20 25 30

Leu Ser Trp Phe Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

His Asp Ala Ser Asn Leu Glu Thr Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Tyr Asp Asn Pro Pro Cys

85 90 95

Ser Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 169

<211> 366

<212> DNA

<213> Artificial

<220>

<223> Antibody

<400> 169

caggtgcagc tgcaggagtc gggcccagga ctggtgaagc cttcacagac cctgtccctc 60

acctgcactg tctctggtgg ctccatcagc agtggttatt actactggag ctggatccgc 120

cagcacccag ggaagggcct ggagtggatt gggtacatct attacagtgg gaccacctac 180

tacaatccgt ccttcaagag tcgagttacc atatcagtag acacgtctaa gaaccagttc 240

tccctgaaac tgagctctgt gactgccgcg gacacggccg tgtattactg tgcgagagcc 300

gattacgatt tttggagtgg tcactttgac tactggggcc agggaaccct ggtcaccgtc 360

tcctca 366

<210> 170

<211> 122

<212> Protein

<213> Artificial

<220>

<223> Antibody

<400> 170

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Gly

20 25 30

Tyr Tyr Tyr Trp Ser Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu

35 40 45

Trp Ile Gly Tyr Ile Tyr Tyr Ser Gly Thr Thr Tyr Tyr Asn Pro Ser

50 55 60

Phe Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe

65 70 75 80

Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr

85 90 95

Cys Ala Arg Ala Asp Tyr Asp Phe Trp Ser Gly His Phe Asp Tyr Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 171

<211> 321

<212> DNA

<213> Artificial

<220>

<223> Antibody

<400> 171

gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60

atcacttgcc gggcaagtca gggcattaga aatgatttag gctggtatca gcagaaacca 120

gggaaagccc ctaagcgcct gatctatgct gcatccagtt tgcaaagtgg ggtcccatca 180

aggttcagcg gcagtggatc tgggacagaa ttcactctca caatcagcag cctgcagcct 240

gaagattttg caacttatta ctgtctacag cataatagtt acccgtggac gttcggccaa 300

gggaccaagg tggaaatcaa a 321

<210> 172

<211> 107

<212> Protein

<213> Artificial

<220>

<223> Antibody

<400> 172

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Arg Asn Asp

20 25 30

Leu Gly Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Arg Leu Ile

35 40 45

Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln His Asn Ser Tyr Pro Trp

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210> 173

<211> 365

<212> DNA

<213> Artificial

<220>

<223> Antibody

<400> 173

caggttcagc tggtgcagtc tggagctgag gtgaagaagc ctggggcctc agtgaaggtc 60

tcctgcaagg cttctggtta cacctttacc agctatggta tcagctgggt gcgacaggcc 120

cctggacaag gacttgagtg gatgggatgg atcagcgctt acgatggtca cacaaactat 180

gcacagaagc tccaggggcag agtcaccatg accacagaca catccacgaa cacagcctac 240

atggagctga ggagcctgag atctgacgac acggccgttt attactgtgc gagagacccc 300

catgactaca gtaactacga ggcttttgac ttctggggcc agggaaccct ggtcaccgtc 360

tcctc 365

<210> 174

<211> 121

<212> Protein

<213> Artificial

<220>

<223> Antibody

<400> 174

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Gly Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Trp Ile Ser Ala Tyr Asp Gly His Thr Asn Tyr Ala Gln Lys Leu

50 55 60

Gln Gly Arg Val Thr Met Thr Thr Asp Thr Ser Thr Asn Thr Ala Tyr

65 70 75 80

Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asp Pro His Asp Tyr Ser Asn Tyr Glu Ala Phe Asp Phe Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser

115 120

<210> 175

<211> 519

<212> DNA

<213> Artificial

<220>

<223> Antibody

<400> 175

atgaggtccc ctgctcagct cctggggctc ctgctactct ggctccgagg tgccagatgt 60

gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 120

atcacttgcc gggcaagtca gagcattagc agttatttaa attggtatca gcagaaacca 180

gggaaagccc ctaacctcct gatctatgct gcatccagtt tgcaaagtgg ggtcccatca 240

agattcagtg gcagtggatc tgggacagat ttcactctca ccatcagcag tctgcaacct 300

gaagattttg caacttacta ctgtcaacag agttacagta cccccatcac cttcggccaa 360

gggacacgac tggagattaa acgaactgtg gctgcaccat ctgtcttcat cttcccgcca 420

tctgatgagc agttgaaatc tggaactgcc tctgttgtgt gcctgctgaa taacttctat 480

cccagagagg ccaaagtaca gtggaaggtg gataacgcc 519

<210> 176

<211> 107

<212> Protein

<213> Artificial

<220>

<223> Antibody

<400> 176

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Asn Leu Leu Ile

35 40 45

Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Pro Ile

85 90 95

Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys

100 105

<210> 177

<211> 534

<212> DNA

<213> Artificial

<220>

<223> Antibody

<400> 177

accatggact ggacctggag ggtccttttc ttggtggcag cagcaacagg tgcccactcc 60

caggttcagc tggtgcagtc tggagctgag gtgaagaagc ctggggcctc agtgaaggtc 120

tcctgcaagg cttctggtta cacctttacc aactatggta tcagctgggt gcggcaggcc 180

cctggacaag ggcttgagtg gatgggatgg atcagcgctt acgatggtta cagaaactat 240

gcacagaagc tccagggcag agtcaccatg accacagaca catccacgac cactgcctac 300

atggagctga ggagcctgag atctgacgac acggccgtgt attactgtgc gagagatgtt 360

caagactacg gtgactacga ctactttgac tactggggcc agggaaccct ggtcaccgtc 420

tcctcagctt ccaccaaggg cccatccgtc ttccccctgg tgccctgctc caggagcacc 480

tccgagagca cagccgccct gggctgcctg gtcaaggact acttccccga accg 534

<210> 178

<211> 122

<212> Protein

<213> Artificial

<220>

<223> Antibody

<400> 178

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr

20 25 30

Gly Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Trp Ile Ser Ala Tyr Asp Gly Tyr Arg Asn Tyr Ala Gln Lys Leu

50 55 60

Gln Gly Arg Val Thr Met Thr Thr Asp Thr Ser Thr Thr Thr Ala Tyr

65 70 75 80

Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asp Val Gln Asp Tyr Gly Asp Tyr Asp Tyr Phe Asp Tyr Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 179

<211> 504

<212> DNA

<213> Artificial

<220>

<223> Antibody

<400> 179

cagctcctgg ggctcctgct actctggctc cgaggtgcca gatgtgacat ccagatgacc 60

cagtctccat cctccctgtc tgcatctgta ggagacagag tcaccatcac ttgccgggca 120

agtcagagca ttagcagtta tttaaattgg tatcagcaga aaccagggaa agcccctaac 180

ctcctgatct atgctgcatc cagtttgcaa agtggggtcc catcaagatt cagtggcagt 240

ggatctggga cagatttcac tctcaccatc agcagtctgc aacctgaaga ttttgcaact 300

tactactgtc aacagagtta cagtaccccc atcaccttcg gccaagggac acgactggag 360

attaaacgaa ctgtggctgc accatctgtc ttcatcttcc cgccatctga tgagcagttg 420

aaatctggaa ctgcctctgt tgtgtgcctg ctgaataact tctatcccag agaggccaaa 480

gtacagtgga aggtggataa cgcc 504

<210> 180

<211> 107

<212> Protein

<213> Artificial

<220>

<223> Antibody

<400> 180

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Asn Leu Leu Ile

35 40 45

Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Pro Ile

85 90 95

Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys

100 105

<210> 181

<211> 493

<212> DNA

<213> Artificial

<220>

<223> Antibody

<400> 181

catctgtggt tcttcctcct gctggtggca gctcccagat gggtcctgtc ccaggtgcag 60

ctgcaggagt cgggcccagg actggtgaag ccttcacaga ccctgtccct cacctgcact 120

gtctctggtg gctccatcaa cagtggtgat tactactgga gctggatccg ccagcaccca 180

gggaagggcc tggagtggat tgggtacatc tattacagtg ggagcaccta ctacaacccg 240

tccctcaaga gtcgagttac catatcagta gacacgtcta agaaccagtt ctccctgaag 300

ctgagctctg tgactgccgc ggacacggcc gtgtattact gtgcgagagc agattacgat 360

ttttggagtg gttactttga ctactggggc cagggaaccc tggtcaccgt ctcctcagcc 420

tccaccaagg gcccatcggt cttccccctg gcaccctcct ccaagagcac ctctgggggc 480

acaacggccc tgg 493

<210> 182

<211> 122

<212> Protein

<213> Artificial

<220>

<223> Antibody

<400> 182

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Asn Ser Gly

20 25 30

Asp Tyr Tyr Trp Ser Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu

35 40 45

Trp Ile Gly Tyr Ile Tyr Tyr Ser Gly Ser Thr Tyr Tyr Asn Pro Ser

50 55 60

Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe

65 70 75 80

Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr

85 90 95

Cys Ala Arg Ala Asp Tyr Asp Phe Trp Ser Gly Tyr Phe Asp Tyr Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 183

<211> 518

<212> DNA

<213> Artificial

<220>

<223> Antibody

<400> 183

atgagggtcc ctgctcagct cctggggctc ctgctgctct ggttcccagg tgccaggtgt 60

gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 120

atcacttgcc gggcaagtca gggcattaga aatgatttag gctggtatca gcagaaacca 180

gggaaagccc ctaagcgcct gatctatgct gcatccagtt tgcaaagtgg ggtcccatca 240

aggttcagcg gcagtggatc tgggacagaa ttcactctca caatcagcag cctgcagcct 300

gaagattttg caacttatta ctgtctacag cataatagtt acccgtggac gttcggccaa 360

gggaccaagg tggaaatcaa acgaactgtg gctgcaccat ctgtcttcat cttcccgcca 420

tctgatgagc agttgaaatc tggaactgcc tctgttgtgt gcctgctgaa taacttctat 480

cccagagagg ccaaagtaca gtggaaggtg gataacgc 518

<210> 184

<211> 107

<212> Protein

<213> Artificial

<220>

<223> Antibody

<400> 184

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Arg Asn Asp

20 25 30

Leu Gly Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Arg Leu Ile

35 40 45

Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln His Asn Ser Tyr Pro Trp

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210> 185

<211> 436

<212> DNA

<213> Artificial

<220>

<223> Antibody

<400> 185

tggttcttcc ttctgctggt ggcagctccc agatgggtcc tgtcccaggt gcagctgcag 60

gagtcgggcc caggactggt gaagccttca cagaccctgt ccctcacctg cactgtctct 120

ggtggctcca tcagcagtgg tggttactac tggagctgga tccgccagca cccagggaag 180

ggcctggagt ggattgggta catctattac agtgggagca cctactacaa cccgtccctc 240

aagagtcgag ttaccatatc agtagacacg tctaagaacc agttctccct gaagctgagc 300

tctgtgactg ccgcggacac ggccgtgtat tactgtgcga gagatggcta tgatagtagt 360

ggttattacc acggctactt tgactactgg ggccagggaa ccctggtcac cgtctcctca 420

gcctccacca agggcc 436

<210> 186

<211> 125

<212> Protein

<213> Artificial

<220>

<223> Antibody

<400> 186

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Gly

20 25 30

Gly Tyr Tyr Trp Ser Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu

35 40 45

Trp Ile Gly Tyr Ile Tyr Tyr Ser Gly Ser Thr Tyr Tyr Asn Pro Ser

50 55 60

Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe

65 70 75 80

Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr

85 90 95

Cys Ala Arg Asp Gly Tyr Asp Ser Ser Gly Tyr Tyr His Gly Tyr Phe

100 105 110

Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120 125

<210> 187

<211> 521

<212> DNA

<213> Artificial

<220>

<223> Antibody

<400> 187

caggtcttca tttctctgtt gctctggatc tctggtgcct acggggacat cgtgatgacc 60

cagtctccag actccctggc tgtgtctctg ggcgagaggg ccaccatcaa ctgcaagtcc 120

agccagagtg ttttatacag ctccaacaat aagaactact tagcttggta ccagcagaaa 180

ccaggacagc ctcctaagct gctcatttac tgggcatcta cccgggaatc cggggtccct 240

gaccgattca gtggcagcgg gtctgggaca gatttcactc tcaccatcag cagcctgcag 300

gctgaagatg tggcagttta ttactgtcag caatattata gtactccgct cactttcggc 360

ggagggacca aggtggagat caaacgaact gtggctgcac catctgtctt catcttcccg 420

ccatctgatg agcagttgaa atctggaact gcctctgttg tgtgcctgct gaataacttc 480

tatccgag aggccaaagt acagtggaag gtggataacg c 521

<210> 188

<211> 113

<212> Protein

<213> Artificial

<220>

<223> Antibody

<400> 188

Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Val Leu Tyr Ser

20 25 30

Ser Asn Asn Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln

35 40 45

Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val

50 55 60

Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln

85 90 95

Tyr Tyr Ser Thr Pro Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile

100 105 110

Lys

<210> 189

<211> 455

<212> DNA

<213> Artificial

<220>

<223> Antibody

<400> 189

ctgtggttct tcctcctgct ggtggcagct cccagatggg tcctgtccca ggtgcagctg 60

caggagtcgg gcccaggact ggtgaagcct tcacagaccc tgtccctcac ctgcactgtc 120

tctggtggct ccatcagtag tggtgattac tactggagct ggatccgcca gcacccaggg 180

aagggcctgg agtggattgg gtacatctat tacagtggga gcacctacta caacccgtcc 240

ctcaagagtc gagttaccat atcagtagac acgtctaaga accagttctc cctgaagttg 300

agctctgtga ctgccgcgga cacggccgtg tattactgtg cgagagccga ttacgatttt 360

tggagtggtt attttgacta ctggggccag ggaaccctgg tcaccgtctc ctcagcctcc 420

accaagggcc catcggtctt ccccctggca ccctc 455

<210> 190

<211> 122

<212> Protein

<213> Artificial

<220>

<223> Antibody

<400> 190

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Gly

20 25 30

Asp Tyr Tyr Trp Ser Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu

35 40 45

Trp Ile Gly Tyr Ile Tyr Tyr Ser Gly Ser Thr Tyr Tyr Asn Pro Ser

50 55 60

Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe

65 70 75 80

Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr

85 90 95

Cys Ala Arg Ala Asp Tyr Asp Phe Trp Ser Gly Tyr Phe Asp Tyr Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 191

<211> 442

<212> DNA

<213> Artificial

<220>

<223> Antibody

<400> 191

gtgcccgctc agcgcctggg gctcctgctg ctctggttcc caggtgccag gtgtgacatc 60

cagatgaccc agtctccatc ctccctgtct gcatctgtag gagacagagt caccatcact 120

tgccgggcaa gtcagggcat tagaaatgat ttaggctggt atcagcagaa accagggaaa 180

gcccctaagc gcctgatcta tgctgcatcc agtttgcaaa gtggggtccc atcaaggttc 240

agcggcagtg gatctgggac agaattcact ctcacaatca gcagcctgca gcctgaagat 300

tttgcaactt attactgtct acagcataat aattacccgt ggacgttcgg ccaagggacc 360

aaggtggaaa tcaaacgaac tgtggctgca ccatctgtct tcatcttccc gccatctgat 420

gagcagttga aatctggaac tg 442

<210> 192

<211> 107

<212> Protein

<213> Artificial

<220>

<223> Antibody

<400> 192

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Arg Asn Asp

20 25 30

Leu Gly Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Arg Leu Ile

35 40 45

Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln His Asn Asn Tyr Pro Trp

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210> 193

<211> 427

<212> DNA

<213> Artificial

<220>

<223> Antibody

<400> 193

tggttcttcc ttctgctggt ggcagctccc agatgggtcc tgtcccaggt gcagctgcag 60

gagtcgggcc caggactggt gaagccttca cagaccctgt ccctcacctg cactgtctct 120

ggtggctcca tcagcagtgg tgattactac tggagctgga tccgccagca cccagggaag 180

ggcctggagt ggattgggta catctattac agtgggagca cctactacaa cccgtccctc 240

aagagtcgag ttaccatatc agtagacacg tctaagaacc agttctccct gaagctgagc 300

tctgtgactg ccgcggacac ggccgtgtat ttctgtgcga gagccgatta cgattttgg 360

agtggttatt ttgactactg gggccaggga accctggtca ccgtctcctc agcctccacc 420

aagggcc 427

<210> 194

<211> 122

<212> Protein

<213> Artificial

<220>

<223> Antibody

<400> 194

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Gly

20 25 30

Asp Tyr Tyr Trp Ser Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu

35 40 45

Trp Ile Gly Tyr Ile Tyr Tyr Ser Gly Ser Thr Tyr Tyr Asn Pro Ser

50 55 60

Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe

65 70 75 80

Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Phe

85 90 95

Cys Ala Arg Ala Asp Tyr Asp Phe Trp Ser Gly Tyr Phe Asp Tyr Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 195

<211> 518

<212> DNA

<213> Artificial

<220>

<223> Antibody

<400> 195

atgagggtcc ccgctcagct cctggggctc ctgctgctct ggttcccagg tgccaggtgt 60

gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 120

atcacttgcc gggcaagtca gggcattaga aatgatttag gctggtatca gcagaaacca 180

gggaaagccc ctaagcgcct gatctatgct gcatccagtt tgcaaagtgg ggtcccatca 240

aggttcagcg gcagtggatc tgggacagaa ttcactctca caatcagcag cctgcagcct 300

gaagattttg caacttatta ctgtctacag cataatactt acccgtggac gttcggccaa 360

gggaccaagg tggaaatcaa acgaactgtg gctgcaccat ctgtcttcat cttcccgcca 420

tctgatgagc agttgaaatc tggaactgcc tctgttgtgt gcctgctgaa taacttctat 480

cccagagagg ccaaagtaca gtggaaggtg gataacgc 518

<210> 196

<211> 107

<212> Protein

<213> Artificial

<220>

<223> Antibody

<400> 196

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Arg Asn Asp

20 25 30

Leu Gly Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Arg Leu Ile

35 40 45

Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln His Asn Thr Tyr Pro Trp

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210> 197

<211> 428

<212> DNA

<213> Artificial

<220>

<223> Antibody

<400> 197

tggttcttcc ttctgctggt ggcagctccc agatgggtcc tgtcccaggt gcagctgcag 60

gagtcgggcc caggactggt gaagccttca cagaccctgt ccctcacctg cactgtctct 120

ggtggctcca tcagcagtgg tgattactac tggagctgga tccgccagca cccagggaag 180

ggcctggagt ggattgggta catctattac agtgggagca cctactacaa cccgtccctc 240

aagagtcgag ttaccatatc agtagacacg tctaagaacc agttctccct gaagctgagc 300

tctgtgactg ccgcggacac ggccgtgtat ttctgtgcga gagccgatta cgattttgg 360

aatggttatt ttgactactg gggccaggga accctggtca ccgtctcctc agcctccacc 420

aagggccc 428

<210> 198

<211> 122

<212> Protein

<213> Artificial

<220>

<223> Antibody

<400> 198

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Gly

20 25 30

Asp Tyr Tyr Trp Ser Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu

35 40 45

Trp Ile Gly Tyr Ile Tyr Tyr Ser Gly Ser Thr Tyr Tyr Asn Pro Ser

50 55 60

Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe

65 70 75 80

Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Phe

85 90 95

Cys Ala Arg Ala Asp Tyr Asp Phe Trp Asn Gly Tyr Phe Asp Tyr Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 199

<211> 519

<212> DNA

<213> Artificial

<220>

<223> Antibody

<400> 199

atgagggtcc ccgctcagct cctggggctc ctgctgctct ggttcccagg tgccaggtgt 60

gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 120

atcacttgcc gggcaagtca gggcattaga aatgatttag gctggtatca gcagaaacca 180

gggaaagccc ctaagcgcct gatctatgct gcttccagtt tgcaaagtgg ggtcccatca 240

aggttcagcg gcagtggatc tgggacagaa ttcactctca caatcagcag cctgcagcct 300

gaagattttg caacttatta ctgtctacag cataatactt acccgtggac gttcggccaa 360

gggaccaagg tggaaatcaa acgaactgtg gctgcaccat ctgtcttcat cttcccgcca 420

tctgatgagc agttgaaatc tggaactgcc tctgttgtgt gcctgctgaa taacttctat 480

cccagagagg ccaaagtaca gtggaaggtg gataacgcc 519

<210> 200

<211> 107

<212> Protein

<213> Artificial

<220>

<223> Antibody

<400> 200

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Arg Asn Asp

20 25 30

Leu Gly Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Arg Leu Ile

35 40 45

Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln His Asn Thr Tyr Pro Trp

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210> 201

<211> 398

<212> DNA

<213> Artificial

<220>

<223> Antibody

<400> 201

ttggtggcag cagctacagg cacccacgcc caggtccagc tggtacagtc tggggctgag 60

gtgaagaagc ctggggcctc agtgaaggtc tcctgcaagg tttccggata caccctcact 120

gaattatcca tgtactggggt gcgacaggct cctggaaaag ggcttgagtg gatgggaggt 180

tttgatcctg aagatggtga aacaatctac gcacagaagt tccaggggcag agtcaccatg 240

accgaggaca catctacaga cacagcctac atggagctga gcagcctgag atctgaggac 300

acggccgtgt attactgtgc aactgggtgg aactacgtct ttgactactg gggccaggga 360

accctggtca ccgtctcctc agcctccacc aagggccc 398

<210> 202

<211> 117

<212> Protein

<213> Artificial

<220>

<223> Antibody

<400> 202

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Val Ser Gly Tyr Thr Leu Thr Glu Leu

20 25 30

Ser Met Tyr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Glu Trp Met

35 40 45

Gly Gly Phe Asp Pro Glu Asp Gly Glu Thr Ile Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Met Thr Glu Asp Thr Ser Thr Asp Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Thr Gly Trp Asn Tyr Val Phe Asp Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser

115

<210> 203

<211> 388

<212> DNA

<213> Artificial

<220>

<223> Antibody

<400> 203

ggatccagtg gggatattgt gatgactcag tctccactct ccctgcccgt cacccctgga 60

gagccggcct ccatctcctg caggtccagt cagagcctcc tgcatagtaa tggatacaac 120

tatttggatt ggtacctgca gaagccaggg cagtctccac agctcctgat ctatttggat 180

tctcatcggg cctccggggt ccctgacagg ttcagtggca gtggatcagg cacagatttt 240

acactgaaaa tcagcagagt ggaggctgag gatgttgggg tttattactg catgcaagct 300

ctacaaactc cgctcacttt cggcggaggg accaaggtgg agatcaaacg aactgtggct 360

gcaccatctg tcttcatctt cccgccat 388

<210> 204

<211> 112

<212> Protein

<213> Artificial

<220>

<223> Antibody

<400> 204

Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly

1 5 10 15

Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu His Ser

20 25 30

Asn Gly Tyr Asn Tyr Leu Asp Trp Tyr Leu Gln Lys Pro Gly Gln Ser

35 40 45

Pro Gln Leu Leu Ile Tyr Leu Asp Ser His Arg Ala Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln Ala

85 90 95

Leu Gln Thr Pro Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105 110

<210> 205

<211> 446

<212> DNA

<213> Artificial

<220>

<223> Antibody

<400> 205

tggttcttcc ttctgctggt ggcagctccc agatgggtcc tgtcccaggt gcagctgcag 60

gagtcgggcc caggactggt gaagccttca cagaccctgt ccctcacctg cactgtctct 120

ggtggctcca tcagcagtgg tgattactac tggagctgga tccgccagca cccagggaag 180

ggcctggagt ggattgggta catctattac agtgggagca cctactacaa cccgtccctc 240

aagagtcgag ttaccatatc agtagacacg tctaagaacc agttctccct gaagctgagc 300

tctgtgactg ccgcggacac ggccgtgtat ttctgtgcga gagccgatta cgattttgg 360

agtggttatt ttgactactg gggccaggga accctggtca ccgtctcctc agcctccacc 420

aagggcccat cgagtcttcc ccctgg 446

<210> 206

<211> 122

<212> Protein

<213> Artificial

<220>

<223> Antibody

<400> 206

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Gly

20 25 30

Asp Tyr Tyr Trp Ser Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu

35 40 45

Trp Ile Gly Tyr Ile Tyr Tyr Ser Gly Ser Thr Tyr Tyr Asn Pro Ser

50 55 60

Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe

65 70 75 80

Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Phe

85 90 95

Cys Ala Arg Ala Asp Tyr Asp Phe Trp Ser Gly Tyr Phe Asp Tyr Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 207

<211> 519

<212> DNA

<213> Artificial

<220>

<223> Antibody

<400> 207

atgagggtcc ccgctcagct cctggggctc ctgctgctct ggttcccagg tgccaggtgt 60

gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 120

atcacttgcc gggcaagtca gggcattaga aatgatttag gctggtatca gcagaaacca 180

gggaaagccc ctaagcgcct gatctatgct gcatccagtt tgcaaagtgg ggtcccatca 240

aggttcagcg gcagtggatc tgggacaaaa ttcactctca ctatcagcag cctgcagcct 300

gaagattttg caacttatta ctgtctacag cataatactt acccgtggac gttcggccaa 360

gggaccaagg tggaaatcag acgaactgtg gctgcaccat ctgtcttcat cttcccgcca 420

tctgatgagc agttgaaatc tggaactgcc tctgttgtgt gcctgctgaa taacttctat 480

cccagagagg ccaaagtaca gtggaaggtg gataacgcc 519

<210> 208

<211> 107

<212> Protein

<213> Artificial

<220>

<223> Antibody

<400> 208

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Arg Asn Asp

20 25 30

Leu Gly Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Arg Leu Ile

35 40 45

Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Lys Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln His Asn Thr Tyr Pro Trp

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Arg

100 105

<210> 209

<211> 564

<212> DNA

<213> Artificial

<220>

<223> Antibody

<400> 209

accatgaaac atctgtggtt cttcctcctg ctggtggcag ctcccagatg ggtcctgtcc 60

120

acctgcactg tctctggtgg ctccatcagc agtggtgatt actactggag ctggatccgc 180

cagcacccag ggaagggcct ggagtggatt gggtacatct attacagtgg gagcacctac 240

tacaacccgt ccctcaagag tcgagttacc atatcagtag acacgtctaa gaaccagttc 300

tccctgaagc tgagctctgt gactgccgcg gacacggccg tgtattactg tgcgagagcg 360

gattacgatt tttggagtgg ttatttttgac tactggggcc agggaatcct ggtcaccgtc 420

tcctcagcct ccaccaaggg cccatcggtc ttccccctgg caccctcctc caagaacacc 480

tctggggggca cagcggccct gggctgcctg gtcaaggact acttccccga accggtgacg 540

gtgtcctgga actcaggcgc cctg 564

<210> 210

<211> 122

<212> Protein

<213> Artificial

<220>

<223> Antibody

<400> 210

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Gly

20 25 30

Asp Tyr Tyr Trp Ser Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu

35 40 45

Trp Ile Gly Tyr Ile Tyr Tyr Ser Gly Ser Thr Tyr Tyr Asn Pro Ser

50 55 60

Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe

65 70 75 80

Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr

85 90 95

Cys Ala Arg Ala Asp Tyr Asp Phe Trp Ser Gly Tyr Phe Asp Tyr Trp

100 105 110

Gly Gln Gly Ile Leu Val Thr Val Ser Ser

115 120

<210> 211

<211> 519

<212> DNA

<213> Artificial

<220>

<223> Antibody

<400> 211

atgagggtcc ccgctcagct cctggggctc ctgctgctct ggttcccagg tgccaggtgt 60

gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 120

atcacttgcc gggcaagtca gggcattaga aatgatttag gctggtatca gcagaaacca 180

gggaaagccc ctaagcgcct gatctatgct gcatccagtt tgcaaagtgg ggtcccatca 240

aggttcagcg gcagtggatc tgggacagaa ttcactctca caatcagcag cctgcagcct 300

gaagattttg caacttatta ctgtctacag cataatagtt acccgtggac gttcggccaa 360

gggaccaagg tggaaatcaa acgaactgtg gctgcaccat ctgtcttcat cttcccgcca 420

tctgatgagc agttgaaatc tggaactgcc tctgttgtgt gcctgctgaa taacttctat 480

cccagagagg ccaaagtaca gtggaaggtg gataacgcc 519

<210> 212

<211> 107

<212> Protein

<213> Artificial

<220>

<223> Antibody

<400> 212

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Arg Asn Asp

20 25 30

Leu Gly Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Arg Leu Ile

35 40 45

Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln His Asn Ser Tyr Pro Trp

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210> 213

<211> 432

<212> DNA

<213> Artificial

<220>

<223> Antibody

<400> 213

tggttcttcc ttctgctggt ggcagctccc agatgggtcc tgtcccaggt gcagctgcag 60

gagtcgggcc caggactggt gaagccttca cagaccctgt ccctcacctg cactgtctct 120

ggtggctcca tcagcagtgg tgattactac tggagctgga tccgccagca cccagggaag 180

ggcctggagt ggattggata catctattac agtgggagca cctactacaa ttcgtccctc 240

aagagtcgag ttaccatatc agtagacacg tctaagaacc agttctccct gaagctgagc 300

tctgtgactg ccgcggacac ggccgtgtat tactgtgcga gagcggatta cgatttttgg 360

agtggttatt ttgactactg gggccaggga accctggtca ccgtctcctc agcctccacc 420

aagggcccat cg 432

<210> 214

<211> 122

<212> Protein

<213> Artificial

<220>

<223> Antibody

<400> 214

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Gly

20 25 30

Asp Tyr Tyr Trp Ser Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu

35 40 45

Trp Ile Gly Tyr Ile Tyr Tyr Ser Gly Ser Thr Tyr Tyr Asn Ser Ser

50 55 60

Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe

65 70 75 80

Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr

85 90 95

Cys Ala Arg Ala Asp Tyr Asp Phe Trp Ser Gly Tyr Phe Asp Tyr Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 215

<211> 372

<212> DNA

<213> Artificial

<220>

<223> Antibody

<400> 215

ggtgccaggt gtgacatcca gatgacccag tctccatcct ccctgtctgc atctgtagga 60

gacagagtca ccatcacttg ccgggcaagt cagggcatta gaaatgattt aggctggtat 120

cagcagaaac ctgggaaagc ccctaagcgc ctgatctatg ctgcatccag tttgcaaagt 180

ggggtcccat caaggttcag cggcagtgga tctgggacag aattcactct cacaatcagc 240

agcctgcagc ctgaagattt tgcaacttat tactgtctac agcacaatag ttacccgtgg 300

acgttcggcc aagggaccaa ggtggaaatc aaacgaactg tggctgcacc atctgtcttc 360

atcttcccgc ca 372

<210> 216

<211> 107

<212> Protein

<213> Artificial

<220>

<223> Antibody

<400> 216

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Arg Asn Asp

20 25 30

Leu Gly Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Arg Leu Ile

35 40 45

Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln His Asn Ser Tyr Pro Trp

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210> 217

<211> 548

<212> DNA

<213> Artificial

<220>

<223> Antibody

<400> 217

aggttcttcc ttctgctggt ggcagctccc agatgggtcc tgtcccaggt gcagctgcag 60

gagtcgggcc caggactggt gaagccttca cagaccctgt ccctcacctg cactgtctct 120

ggtggctcca tcagcagtgg tgattactac tggagctgga tccgccagca cccagggaag 180

ggcctggagt ggattggata catctattac agtgggagca cctactacaa cccgtccctc 240

aagagtcgag ttaccatatc agtagacacg tctaagaacc agttctccct gaagctgagc 300

tctgtgactg ccgcggacac ggccgtgtat tactgtgcga gagccgatta cgatttttgg 360

agtggttatt ttgactactg gggccaggga accctggtca ccgtctcctc agcctccacc 420

aagggcccat cggtcttccc cctggcaccc tcctccaaga gcacctctgg gggcacagcg 480

gccctgggct gcctggtcaa ggactacttc cccgaaccgg tgacggtgtc gtggaactca 540

ggcgccct 548

<210> 218

<211> 122

<212> Protein

<213> Artificial

<220>

<223> Antibody

<400> 218

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Gly

20 25 30

Asp Tyr Tyr Trp Ser Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu

35 40 45

Trp Ile Gly Tyr Ile Tyr Tyr Ser Gly Ser Thr Tyr Tyr Asn Pro Ser

50 55 60

Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe

65 70 75 80

Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr

85 90 95

Cys Ala Arg Ala Asp Tyr Asp Phe Trp Ser Gly Tyr Phe Asp Tyr Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 219

<211> 517

<212> DNA

<213> Artificial

<220>

<223> Antibody

<400> 219

atgagggtcc ccgctcagct cctggggctc ctgctgctct ggttcccagg tgccaggtgt 60

gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 120

atcacttgcc gggcaagtca gggcattaga aatgatttag gctggtatca gcagaaacca 180

gggaaagccc ctaagcgcct gatctatgct gcatccagtt tgcaaagtgg ggtcccatca 240

aggttcagcg gcagtggatc tgggacagaa ttcactctca caatcagcag cctgcagcct 300

gaagattttg caacttatta ctgtctacag cataatagtt acccgtggac gttcggccaa 360

gggaccaagg tggaaatcaa acgaactgtg gctgcaccat ctgtcttcat cttcccgcca 420

tctgatgagc agttgaaatc tggaactgcc tctgttgtgt gcctgctgaa taacttctat 480

cccagagagg ccaaagtaca gtggaaggtg gataacg 517

<210> 220

<211> 107

<212> Protein

<213> Artificial

<220>

<223> Antibody

<400> 220

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Arg Asn Asp

20 25 30

Leu Gly Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Arg Leu Ile

35 40 45

Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln His Asn Ser Tyr Pro Trp

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210> 221

<211> 446

<212> DNA

<213> Artificial

<220>

<223> Antibody

<400> 221

ctgtggttct tccttctgct ggtggcagct cccagatggg tcctgtccca ggtgcagctg 60

caggagtcgg gcccaggact ggtgaagcct tcacagaccc tgtccctcac ctgcactgtc 120

tctggtggct ccatcagcag tggtgattac tactggagct ggatccgcca gcacccaggg 180

aagggcctgg agtggattgg gtacatctat tacagtggga gcacctacta caacccgtcc 240

ctcaagagtc gagttaccat gtcagtagac acgtctaaga accagttctc cctgaagctg 300

agctctgtga ctgccgcgga cacggccgtg tattactgtg cgagagccga ttacgatttt 360

tggagtggtc actttgactg ctggggccag ggaaccctgg tcaccgtctc ctcagcttcc 420

accaagggcc ccatccgtct tccccc 446

<210> 222

<211> 122

<212> Protein

<213> Artificial

<220>

<223> Antibody

<400> 222

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Gly

20 25 30

Asp Tyr Tyr Trp Ser Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu

35 40 45

Trp Ile Gly Tyr Ile Tyr Tyr Ser Gly Ser Thr Tyr Tyr Asn Pro Ser

50 55 60

Leu Lys Ser Arg Val Thr Met Ser Val Asp Thr Ser Lys Asn Gln Phe

65 70 75 80

Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr

85 90 95

Cys Ala Arg Ala Asp Tyr Asp Phe Trp Ser Gly His Phe Asp Cys Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 223

<211> 419

<212> DNA

<213> Artificial

<220>

<223> Antibody

<400> 223

atgagggtcc ccgctcagct cctggggctc ctgctgctct ggttcccagg tgccaggtgt 60

gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 120

atcacttgcc gggcaagtca gggcattaga gatgatttag gctggtatca gcagaaacca 180

gggaaagccc ctaagcgcct gatctatgct gaatccagtt tgcaaagtgg ggtcccatca 240

aggttcagcg gcagtggatc tgggacagaa ttcactctca caatcagcag cctgcagcct 300

gaagattttg caacttatta ctgtctacag catcatagtt acccgtggac gttcggccaa 360

gggaccaagg tggaaatcaa acgaactgtg gctgcaccat ctgtcttcat cttcccgcc 419

<210> 224

<211> 107

<212> Protein

<213> Artificial

<220>

<223> Antibody

<400> 224

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Arg Asp Asp

20 25 30

Leu Gly Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Arg Leu Ile

35 40 45

Tyr Ala Glu Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln His His Ser Tyr Pro Trp

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210> 225

<211> 504

<212> DNA

<213> Artificial

<220>

<223> Antibody

<400> 225

tggctgagct gggttttcct cgttgctctt ttaagaggtg tccagtgtca ggtgcagctg 60

gtggagtctg ggggaggcgt ggtccagcct gggaggtccc tgagactctc ctgtgcagcg 120

tctggattca ccttcaatag ctatgacatg cactgggtcc gccaggctcc aggcaagggg 180

ctggagtggg tggcagttat atggtatgat ggaagtaata aatactatgc agactccgtg 240

aagggccgat tcaccatctc tagagacaat tccaagaaca cgctgtatct gcaaatgaac 300

agcctgagag ccgaggacac ggctgtgtat tactgtgcga gagaccgctt gtgtactaat 360

ggtgtatgct atgaagacta cggtatggac gtctggggcc aagggaccac ggtcaccgtc 420

tcctcagctt ccaccaaggg cccatccgtc ttccccctgg cgccctgctc caggagcacc 480

tccgagagca cagccgccct gggc 504

<210> 226

<211> 126

<212> Protein

<213> Artificial

<220>

<223> Antibody

<400> 226

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Ser Tyr

20 25 30

Asp Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Val Ile Trp Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asp Arg Leu Cys Thr Asn Gly Val Cys Tyr Glu Asp Tyr Gly

100 105 110

Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser

115 120 125

<210> 227

<211> 504

<212> DNA

<213> Artificial

<220>

<223> Antibody

<400> 227

atgagggtcc ctgctcagct cctggggctc ctgctgctct ggctctcagg tgccagatgt 60

gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 120

atcacttgcc aggcgagtca ggacattagc aactatttaa attggtatca gcagaaacca 180

gggaaagccc ctaaggtcct gatctacgat gcatccaatt tggaaacagg ggtcccatca 240

aggttcagtg gaagtggatc tgggacagat tttactttca ccatcagcag cctgcagcct 300

360

gggaccaagg tggagatcaa acgaactgtg gctgcaccat ctgtcttcat cttcccgcca 420

tctgatgagc agttgaaatc tggaactgcc tctgttgtgt gcctgctgaa taacttctat 480

cccagagagg ccaaagtaca gtgg 504

<210> 228

<211> 107

<212> Protein

<213> Artificial

<220>

<223> Antibody

<400> 228

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Gln Ala Ser Gln Asp Ile Ser Asn Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Val Leu Ile

35 40 45

Tyr Asp Ala Ser Asn Leu Glu Thr Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Val Ala Thr Tyr Tyr Cys Gln His Tyr Asp Thr Leu Pro Leu

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 229

<211> 472

<212> DNA

<213> Artificial

<220>

<223> Antibody

<400> 229

ggactgtgca agaacatgaa acacctgtgg ttcttcctcc tgctggtggc agctcccaga 60

tgggtcctgt cccaggtgca gctgcaggag tcgggcccag gactggtgaa gcctttacag 120

accctgtccc tcacctgcac tgtctctggt ggctccatca gcagtggtga ttactactgg 180

agctggatcc gccagcaccc agggaagggc ctggagtgga ttgggtacat ctattacagt 240

gggaccacct actacaaccc gtccctcaag agtcgagtta ccatatcagt agacacgtct 300

aagaaccagt tcgccctgaa gctgaactct gtgactgccg cggacacggc cgtgtattac 360

tgtgcgagag ccgattacga ttttttggagt ggttatttttg actactgggg ccagggaacc 420

ctggtcaccg tctcctcagc ttccaccaag ggcccatccg tcttccccct gg 472

<210> 230

<211> 122

<212> Protein

<213> Artificial

<220>

<223> Antibody

<400> 230

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Leu Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Gly

20 25 30

Asp Tyr Tyr Trp Ser Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu

35 40 45

Trp Ile Gly Tyr Ile Tyr Tyr Ser Gly Thr Thr Tyr Tyr Asn Pro Ser

50 55 60

Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe

65 70 75 80

Ala Leu Lys Leu Asn Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr

85 90 95

Cys Ala Arg Ala Asp Tyr Asp Phe Trp Ser Gly Tyr Phe Asp Tyr Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 231

<211> 531

<212> DNA

<213> Artificial

<220>

<223> Antibody

<400> 231

atgagggtcc ctgctcagct cctggggctc ctgctgctct ggttcccagg tgccaggtgt 60

gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 120

atcacttgcc gggcaggtca gggcattaga aatgatttag gctggtatca gcagaaacca 180

gggaaagccc ctcagcgcct gatctatgct gcatccagtt tgcaaagtgg ggtcccatca 240

aggttcagcg gcagtggatc tgggacagaa ttctctctca caatctccag cctgcagcct 300

gaagattttg caacttatta ctgtctacag cataatagtt acccgtggac gttcggccaa 360

gggaccaagg tggaaatcaa acgaactgtg gctgcaccat ctgtcttcat cttcccgcca 420

tctgatgagc agttgaaatc tggaactgcc tctgttgtgt gcctgctgaa taacttctat 480

cccagagagg ccaaagtaca gtggaaggtg gataacgccc ttccaatcgg g 531

<210> 232

<211> 107

<212> Protein

<213> Artificial

<220>

<223> Antibody

<400> 232

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Gly Gln Gly Ile Arg Asn Asp

20 25 30

Leu Gly Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Gln Arg Leu Ile

35 40 45

Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Glu Phe Ser Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln His Asn Ser Tyr Pro Trp

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210> 233

<211> 26

<212> DNA

<213> Artificial

<220>

<223> Primer

<220>

<221> prim_transcript

<222> (1)..(26)

<400> 233

cgggatccat gtcctagcct aggggc 26

<210> 234

<211> 39

<212> DNA

<213> Artificial

<220>

<223> Primer

<220>

<221> prim_transcript

<222> (1)..(39)

<400> 234

gctctagatt aatgatgatg atgatgatgt tgtcctaaa 39

<210> 235

<211> 12

<212> Protein

<213> Artificial

<220>

<223> U1-1 CDR-1-h

<400> 235

Gly Gly Ser Ile Asn Ser Gly Asp Tyr Tyr Trp Ser

1 5 10

<210> 236

<211> 16

<212> Protein

<213> Artificial

<220>

<223> U1-1 CDR-2-h

<400> 236

Tyr Ile Tyr Tyr Ser Gly Ser Thr Tyr Tyr Asn Pro Ser Leu Lys Ser

1 5 10 15

<210> 237

<211> 12

<212> Protein

<213> Artificial

<220>

<223> U1-1 CDR-3-h

<400> 237

Ala Asp Tyr Asp Phe Trp Ser Gly Tyr Phe Asp Tyr

1 5 10

<210> 238

<211> 11

<212> Protein

<213> Artificial

<220>

<223> U1-1 CDR-1-l

<400> 238

Arg Ala Ser Gln Gly Ile Arg Asn Asp Leu Gly

1 5 10

<210> 239

<211> 7

<212> Protein

<213> Artificial

<220>

<223> U1-1 CDR-2-l

<400> 239

Ala Ala Ser Ser Leu Gln Ser

15

<210> 240

<211> 9

<212> Protein

<213> Artificial

<220>

<223> U1-1 CDR-3-l

<400> 240

Leu Gln His Asn Ser Tyr Pro Trp Thr

15

<210> 241

<211> 12

<212> Protein

<213> Artificial

<220>

<223> U 1 - 2-CDR-1h

<400> 241

Gly Gly Ser Ile Ser Ser Gly Asp Tyr Tyr Trp Ser

1 5 10

<210> 242

<211> 16

<212> Protein

<213> Artificial

<220>

<223> U 1 - 2-CDR-2h

<400> 242

Tyr Ile Tyr Tyr Ser Gly Ser Thr Tyr Tyr Asn Pro Ser Leu Arg Ser

1 5 10 15

<210> 243

<211> 12

<212> Protein

<213> Artificial

<220>

<223> U 1 - 2-CDR-3h

<400> 243

Ala Asp Tyr Asp Phe Trp Ser Gly Tyr Phe Asp Tyr

1 5 10

<210> 244

<211> 11

<212> Protein

<213> Artificial

<220>

<223> U 1 - 2-CDR-1l

<400> 244

Arg Ala Ser Gln Gly Ile Arg Asn Asp Leu Gly

1 5 10

<210> 245

<211> 7

<212> Protein

<213> Artificial

<220>

<223> U 1 - 2-CDR-2l

<400> 245

Ala Ala Ser Ser Leu Gln Ser

15

<210> 246

<211> 9

<212> Protein

<213> Artificial

<220>

<223> U 1 - 2-CDR-3l

<400> 246

Leu Gln His Asn Gly Tyr Pro Trp Thr

15

<210> 247

<211> 12

<212> Protein

<213> Artificial

<220>

<223> U 1 - 3-CDR-1-h

<400> 247

Gly Gly Ser Ile Ser Ser Gly Gly Tyr Tyr Trp Ser

1 5 10

<210> 248

<211> 16

<212> Protein

<213> Artificial

<220>

<223> U 1 - 3-CDR-2h

<400> 248

Tyr Ile Tyr Tyr Ser Gly Ser Thr Tyr Tyr Asn Pro Ser Leu Lys Ser

1 5 10 15

<210> 249

<211> 15

<212> Protein

<213> Artificial

<220>

<223> U 1 - 3-CDR-3h

<400> 249

Asp Gly Tyr Asp Ser Ser Gly Tyr Tyr His Gly Tyr Phe Asp Tyr

1 5 10 15

<210> 250

<211> 17

<212> Protein

<213> Artificial

<220>

<223> U 1 - 3-CDR-1l

<400> 250

Lys Ser Ser Gln Ser Val Leu Tyr Ser Ser Asn Asn Lys Asn Tyr Leu

1 5 10 15

Ala

<210> 251

<211> 7

<212> Protein

<213> Artificial

<220>

<223> U 1 - 3-CDR-2l

<400> 251

Trp Ala Ser Thr Arg Glu Ser

15

<210> 252

<211> 9

<212> Protein

<213> Artificial

<220>

<223> U 1 - 3-CDR-3l

<400> 252

Gln Gln Tyr Tyr Ser Thr Pro Leu Thr

15

<210> 253

<211> 12

<212> Protein

<213> Artificial

<220>

<223> U 1 - 4-CDR-1h

<400> 253

Gly Gly Ser Ile Ser Ser Gly Asp Tyr Tyr Trp Ser

1 5 10

<210> 254

<211> 16

<212> Protein

<213> Artificial

<220>

<223> U 1 - 4-CDR-2h

<400> 254

Tyr Ile Tyr Tyr Ser Gly Ser Thr Tyr Tyr Asn Pro Ser Leu Lys Ser

1 5 10 15

<210> 255

<211> 12

<212> Protein

<213> Artificial

<220>

<223> U 1 - 4-CDR-3h

<400> 255

Ala Asp Tyr Asp Phe Trp Ser Gly Tyr Phe Asp Tyr

1 5 10

<210> 256

<211> 11

<212> Protein

<213> Artificial

<220>

<223> U 1 - 4-CDR-1l

<400> 256

Arg Ala Ser Gln Gly Ile Arg Asn Asp Leu Gly

1 5 10

<210> 257

<211> 7

<212> Protein

<213> Artificial

<220>

<223> U 1 - 4-CDR-2l

<400> 257

Ala Ala Ser Ser Leu Gln Ser

15

<210> 258

<211> 9

<212> Protein

<213> Artificial

<220>

<223> U 1 - 4-CDR-3l

<400> 258

Leu Gln His Asn Asn Tyr Pro Trp Thr

15

<210> 259

<211> 12

<212> Protein

<213> Artificial

<220>

<223> U 1 - 5-CDR-1h

<400> 259

Gly Gly Ser Ile Ser Ser Gly Asp Tyr Tyr Trp Ser

1 5 10

<210> 260

<211> 16

<212> Protein

<213> Artificial

<220>

<223> U 1 - 5-CDR-2h

<400> 260

Tyr Ile Tyr Tyr Ser Gly Ser Thr Tyr Tyr Asn Pro Ser Leu Lys Ser

1 5 10 15

<210> 261

<211> 12

<212> Protein

<213> Artificial

<220>

<223> U 1 - 5-CDR-3h

<400> 261

Ala Asp Tyr Asp Phe Trp Ser Gly Tyr Phe Asp Tyr

1 5 10

<210> 262

<211> 11

<212> Protein

<213> Artificial

<220>

<223> U 1 - 5-CDR-1l

<400> 262

Arg Ala Ser Gln Gly Ile Arg Asn Asp Leu Gly

1 5 10

<210> 263

<211> 7

<212> Protein

<213> Artificial

<220>

<223> U 1 - 5-CDR-2l

<400> 263

Ala Ala Ser Ser Leu Gln Ser

15

<210> 264

<211> 9

<212> Protein

<213> Artificial

<220>

<223> U 1 - 5-CDR-3l

<400> 264

Leu Gln His Asn Thr Tyr Pro Trp Thr

15

<210> 265

<211> 12

<212> Protein

<213> Artificial

<220>

<223> U1-6-CDR-1h

<400> 265

Gly Gly Ser Ile Ser Ser Gly Asp Tyr Tyr Trp Ser

1 5 10

<210> 266

<211> 16

<212> Protein

<213> Artificial

<220>

<223> U1-6-CDR-2h

<400> 266

Tyr Ile Tyr Tyr Ser Gly Ser Thr Tyr Tyr Asn Pro Ser Leu Lys Ser

1 5 10 15

<210> 267

<211> 12

<212> Protein

<213> Artificial

<220>

<223> U1-6-CDR-3h

<400> 267

Ala Asp Tyr Asp Phe Trp Asn Gly Tyr Phe Asp Tyr

1 5 10

<210> 268

<211> 11

<212> Protein

<213> Artificial

<220>

<223> U1-6-CDR-1l

<400> 268

Arg Ala Ser Gln Gly Ile Arg Asn Asp Leu Gly

1 5 10

<210> 269

<211> 7

<212> Protein

<213> Artificial

<220>

<223> U1-6-CDR-2l

<400> 269

Ala Ala Ser Ser Leu Gln Ser

15

<210> 270

<211> 9

<212> Protein

<213> Artificial

<220>

<223> U1-6-CDR-3l

<400> 270

Leu Gln His Asn Thr Tyr Pro Trp Thr

15

<210> 271

<211> 12

<212> Protein

<213> Artificial

<220>

<223> U 1 - 7-CDR-1h

<400> 271

Gly Gly Ser Ile Ser Ser Gly Asp Tyr Tyr Trp Ser

1 5 10

<210> 272

<211> 16

<212> Protein

<213> Artificial

<220>

<223> U 1 - 7-CDR-2h

<400> 272

Tyr Ile Tyr Tyr Ser Gly Ser Thr Tyr Tyr Asn Pro Ser Leu Lys Ser

1 5 10 15

<210> 273

<211> 12

<212> Protein

<213> Artificial

<220>

<223> U 1 - 7-CDR-3h

<400> 273

Ala Asp Tyr Asp Phe Trp Ser Gly Tyr Phe Asp Tyr

1 5 10

<210> 274

<211> 11

<212> Protein

<213> Artificial

<220>

<223> U 1 - 7-CDR-1l

<400> 274

Arg Ala Ser Gln Asp Ile Arg Asn Asp Leu Gly

1 5 10

<210> 275

<211> 7

<212> Protein

<213> Artificial

<220>

<223> U 1 - 7-CDR-2l

<400> 275

Ala Ala Ser Ser Leu Gln Ser

15

<210> 276

<211> 9

<212> Protein

<213> Artificial

<220>

<223> U 1 - 7-CDR-3l

<400> 276

Leu Gln His Asn Ser Tyr Pro Trp Thr

15

<210> 277

<211> 10

<212> Protein

<213> Artificial

<220>

<223> U 1 - 8-CDR-1h

<400> 277

Gly Tyr Thr Leu Thr Glu Leu Ser Met Tyr

1 5 10

<210> 278

<211> 17

<212> Protein

<213> Artificial

<220>

<223> U 1 - 8-CDR-2h

<400> 278

Gly Phe Asp Pro Glu Asp Gly Glu Thr Ile Tyr Ala Gln Lys Phe Gln

1 5 10 15

gly

<210> 279

<211> 8

<212> Protein

<213> Artificial

<220>

<223> U 1 - 8-CDR-3h

<400> 279

Gly Trp Asn Tyr Val Phe Asp Tyr

15

<210> 280

<211> 16

<212> Protein

<213> Artificial

<220>

<223> U 1 - 8-CDR-1l

<400> 280

Arg Ser Ser Gln Ser Leu Leu His Ser Asn Gly Tyr Asn Tyr Leu Asp

1 5 10 15

<210> 281

<211> 7

<212> Protein

<213> Artificial

<220>

<223> U 1 - 8-CDR-2l

<400> 281

Leu Asp Ser His Arg Ala Ser

15

<210> 282

<211> 9

<212> Protein

<213> Artificial

<220>

<223> U 1 - 8-CDR-3l

<400> 282

Met Gln Ala Leu Gln Thr Pro Leu Thr

15

<210> 283

<211> 12

<212> Protein

<213> Artificial

<220>

<223> U 1 - 9-CDR-1h

<400> 283

Gly Gly Ser Ile Ser Ser Gly Asp Tyr Tyr Trp Ser

1 5 10

<210> 284

<211> 16

<212> Protein

<213> Artificial

<220>

<223> U 1 - 9-CDR-2h

<400> 284

Tyr Ile Tyr Tyr Ser Gly Ser Thr Tyr Tyr Asn Pro Ser Leu Lys Ser

1 5 10 15

<210> 285

<211> 12

<212> Protein

<213> Artificial

<220>

<223> U 1 - 9-CDR-3h

<400> 285

Ala Asp Tyr Asp Phe Trp Asn Gly Tyr Phe Asp Tyr

1 5 10

<210> 286

<211> 11

<212> Protein

<213> Artificial

<220>

<223> U 1 - 9-CDR-1l

<400> 286

Arg Ala Ser Gln Asp Ile Arg Asn Asp Leu Gly

1 5 10

<210> 287

<211> 7

<212> Protein

<213> Artificial

<220>

<223> U 1 - 9-CDR-2l

<400> 287

Ala Ala Ser Ser Leu Gln Ser

15

<210> 288

<211> 9

<212> Protein

<213> Artificial

<220>

<223> U 1 - 9-CDR-3l

<400> 288

Leu Gln His Asn Ser Tyr Pro Trp Thr

15

<210> 289

<211> 12

<212> Protein

<213> Artificial

<220>

<223> U1-10-CDR-1h

<400> 289

Gly Gly Ser Ile Ser Ser Gly Asp Tyr Tyr Trp Ser

1 5 10

<210> 290

<211> 16

<212> Protein

<213> Artificial

<220>

<223> U1-10-CDR-2h

<400> 290

Tyr Ile Tyr Tyr Ser Gly Ser Thr Tyr Tyr Asn Pro Ser Leu Lys Ser

1 5 10 15

<210> 291

<211> 12

<212> Protein

<213> Artificial

<220>

<223> U1-10-CDR-3h

<400> 291

Ala Asp Tyr Asp Phe Trp Ser Gly Tyr Phe Asp Tyr

1 5 10

<210> 292

<211> 11

<212> Protein

<213> Artificial

<220>

<223> U1-10-CDR-1l

<400> 292

Arg Ala Ser Gln Gly Ile Arg Asn Asp Leu Gly

1 5 10

<210> 293

<211> 7

<212> Protein

<213> Artificial

<220>

<223> U1-10-CDR-2l

<400> 293

Ala Ala Ser Ser Leu Gln Ser

15

<210> 294

<211> 9

<212> Protein

<213> Artificial

<220>

<223> U1-10-CDR-3l

<400> 294

Leu Gln His Asn Asn Tyr Pro Trp Thr

15

<210> 295

<211> 12

<212> Protein

<213> Artificial

<220>

<223> U 1 -11-CDR-1h

<400> 295

Gly Gly Ser Ile Ser Ser Gly Asp Tyr Tyr Trp Ser

1 5 10

<210> 296

<211> 16

<212> Protein

<213> Artificial

<220>

<223> U 1 -11-CDR-2h

<400> 296

Tyr Ile Tyr Tyr Ser Gly Ser Thr Tyr Tyr Asn Pro Ser Leu Lys Ser

1 5 10 15

<210> 297

<211> 12

<212> Protein

<213> Artificial

<220>

<223> U 1 -11-CDR-3h

<400> 297

Ala Asp Tyr Asp Phe Trp Ser Gly Tyr Phe Asp Tyr

1 5 10

<210> 298

<211> 11

<212> Protein

<213> Artificial

<220>

<223> U 1 -11-CDR-1l

<400> 298

Arg Ala Ser Gln Gly Ile Arg Asn Asp Leu Gly

1 5 10

<210> 299

<211> 7

<212> Protein

<213> Artificial

<220>

<223> U 1 -11-CDR-2l

<400> 299

Ala Ala Ser Ser Leu Gln Ser

15

<210> 300

<211> 9

<212> Protein

<213> Artificial

<220>

<223> U 1 -11-CDR-3l

<400> 300

Leu Gln His Asn Thr Tyr Pro Trp Thr

15

<210> 301

<211> 12

<212> Protein

<213> Artificial

<220>

<223> U 1 - 12-CDR-1h

<400> 301

Gly Gly Ser Ile Ser Ser Gly Asp Tyr Tyr Trp Ser

1 5 10

<210> 302

<211> 16

<212> Protein

<213> Artificial

<220>

<223> U 1 - 12-CDR-2h

<400> 302

Tyr Ile Tyr Tyr Ser Gly Ser Thr Tyr Tyr Asn Pro Ser Leu Lys Ser

1 5 10 15

<210> 303

<211> 12

<212> Protein

<213> Artificial

<220>

<223> U1-12-CDR-3h

<400> 303

Ala Asp Tyr Asp Phe Trp Ser Gly Tyr Phe Asp Tyr

1 5 10

<210> 304

<211> 11

<212> Protein

<213> Artificial

<220>

<223> U1-12-CDR-1l

<400> 304

Arg Ala Ser Gln Gly Ile Arg Asn Asp Leu Gly

1 5 10

<210> 305

<211> 7

<212> Protein

<213> Artificial

<220>

<223> U1-12-CDR-2l

<400> 305

Ala Ala Ser Ser Leu Gln Ser

15

<210> 306

<211> 9

<212> Protein

<213> Artificial

<220>

<223> U1-12-CDR-3l

<400> 306

Leu Gln His Asn Asn Tyr Pro Trp Thr

15

<210> 307

<211> 12

<212> Protein

<213> Artificial

<220>

<223> U 1 -13-CDR-1h

<400> 307

Gly Gly Ser Ile Ser Ser Gly Gly Tyr Tyr Trp Ser

1 5 10

<210> 308

<211> 16

<212> Protein

<213> Artificial

<220>

<223> U 1 -13-CDR-2h

<400> 308

Tyr Ile Tyr Tyr Ser Gly Ser Thr Tyr Tyr Asn Pro Ser Leu Lys Ser

1 5 10 15

<210> 309

<211> 7

<212> Protein

<213> Artificial

<220>

<223> U 1 -13-CDR-3h

<400> 309

Glu Asp Asp Gly Met Asp Val

15

<210> 310

<211> 16

<212> Protein

<213> Artificial

<220>

<223> U 1 -13-CDR-1l

<400> 310

Arg Ser Ser Gln Ser Leu Leu His Ser Asn Gly Tyr Asn Tyr Leu Glu

1 5 10 15

<210> 311

<211> 7

<212> Protein

<213> Artificial

<220>

<223> U 1 -13-CDR-2l

<400> 311

Leu Gly Ser Asn Arg Ala Ser

15

<210> 312

<211> 9

<212> Protein

<213> Artificial

<220>

<223> U 1 -13-CDR-3l

<400> 312

Met Gln Ala Leu Gln Thr Pro Ile Thr

15

<210> 313

<211> 12

<212> Protein

<213> Artificial

<220>

<223> U1-14-CDR-1h

<400> 313

Gly Gly Ser Ile Ser Ser Gly Asp Tyr Tyr Trp Ser

1 5 10

<210> 314

<211> 16

<212> Protein

<213> Artificial

<220>

<223> U1-14-CDR-2h

<400> 314

Tyr Ile Tyr Tyr Ser Gly Ser Thr Tyr Tyr Asn Pro Ser Leu Lys Ser

1 5 10 15

<210> 315

<211> 12

<212> Protein

<213> Artificial

<220>

<223> U1-14-CDR-3h

<400> 315

Ala Asp Tyr Asp Phe Trp Ser Gly Tyr Phe Asp Tyr

1 5 10

<210> 316

<211> 11

<212> Protein

<213> Artificial

<220>

<223> U1-14-CDR-1l

<400> 316

Arg Ala Ser Gln Gly Ile Arg Asn Asp Leu Gly

1 5 10

<210> 317

<211> 7

<212> Protein

<213> Artificial

<220>

<223> U1-14-CDR-2l

<400> 317

Ala Ala Ser Ser Leu Gln Ser

15

<210> 318

<211> 9

<212> Protein

<213> Artificial

<220>

<223> U1-14-CDR-3l

<400> 318

Leu Gln His Asn Thr Tyr Pro Trp Thr

15

<210> 319

<211> 12

<212> Protein

<213> Artificial

<220>

<223> U1-15-CDR-1h

<400> 319

Gly Gly Ser Val Ser Ser Gly Gly Tyr Tyr Trp Ser

1 5 10

<210> 320

<211> 16

<212> Protein

<213> Artificial

<220>

<223> U1-15-CDR-2h

<400> 320

Tyr Ile Tyr Tyr Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys Ser

1 5 10 15

<210> 321

<211> 14

<212> Protein

<213> Artificial

<220>

<223> U1-15-CDR-3h

<400> 321

Asp Gly Asp Val Asp Thr Ala Met Val Asp Ala Phe Asp Ile

1 5 10

<210> 322

<211> 12

<212> Protein

<213> Artificial

<220>

<223> U1-15-CDR-1l

<400> 322

Arg Ala Ser Gln Ser Leu Ser Gly Asn Tyr Leu Ala

1 5 10

<210> 323

<211> 7

<212> Protein

<213> Artificial

<220>

<223> U1-15-CDR-2l

<400> 323

Gly Ala Ser Ser Arg Ala Thr

15

<210> 324

<211> 9

<212> Protein

<213> Artificial

<220>

<223> U1-15-CDR-3l

<400> 324

Gln Gln Tyr Asp Arg Ser Pro Leu Thr

15

<210> 325

<211> 12

<212> Protein

<213> Artificial

<220>

<223> U1-16-CDR-1h

<400> 325

Gly Gly Ser Ile Ser Ser Gly Asp Tyr Tyr Trp Ser

1 5 10

<210> 326

<211> 16

<212> Protein

<213> Artificial

<220>

<223> U1-16-CDR-2h

<400> 326

Tyr Ile Tyr Tyr Ser Gly Ser Thr Tyr Tyr Asn Pro Ser Leu Lys Ser

1 5 10 15

<210> 327

<211> 12

<212> Protein

<213> Artificial

<220>

<223> U1-16-CDR-3h

<400> 327

Ala Asp Tyr Asp Phe Trp Ser Gly Tyr Phe Asp Tyr

1 5 10

<210> 328

<211> 11

<212> Protein

<213> Artificial

<220>

<223> U1-16-CDR-1l

<400> 328

Arg Ala Ser Gln Gly Ile Arg Asn Asp Leu Gly

1 5 10

<210> 329

<211> 7

<212> Protein

<213> Artificial

<220>

<223> U1-16-CDR-2l

<400> 329

Ala Ala Ser Ser Leu Gln Ser

15

<210> 330

<211> 9

<212> Protein

<213> Artificial

<220>

<223> U1-16-CDR-3l

<400> 330

Leu Gln His Asn Ser Tyr Pro Trp Thr

15

<210> 331

<211> 12

<212> Protein

<213> Artificial

<220>

<223> U1-17-CDR-1h

<400> 331

Gly Gly Ser Ile Ser Ser Gly Asp Tyr Tyr Trp Ser

1 5 10

<210> 332

<211> 16

<212> Protein

<213> Artificial

<220>

<223> U1-17-CDR-2h

<400> 332

Tyr Ile Tyr Tyr Ser Gly Ser Thr Tyr Tyr Asn Ser Ser Leu Lys Ser

1 5 10 15

<210> 333

<211> 12

<212> Protein

<213> Artificial

<220>

<223> U1-17-CDR-3h

<400> 333

Ala Asp Tyr Asp Phe Trp Ser Gly Tyr Phe Asp Tyr

1 5 10

<210> 334

<211> 11

<212> Protein

<213> Artificial

<220>

<223> U1-17-CDR-1l

<400> 334

Arg Ala Ser Gln Gly Ile Arg Asn Asp Leu Gly

1 5 10

<210> 335

<211> 7

<212> Protein

<213> Artificial

<220>

<223> U1-17-CDR-2l

<400> 335

Ala Ala Ser Ser Leu Gln Ser

15

<210> 336

<211> 9

<212> Protein

<213> Artificial

<220>

<223> U1-17-CDR-3l

<400> 336

Leu Gln His Asn Ser Tyr Pro Trp Thr

15

<210> 337

<211> 12

<212> Protein

<213> Artificial

<220>

<223> U1-18-CDR-1h

<400> 337

Gly Gly Ser Ile Ser Ser Gly Asp Tyr Tyr Trp Ser

1 5 10

<210> 338

<211> 16

<212> Protein

<213> Artificial

<220>

<223> U1-18-CDR-2h

<400> 338

Tyr Ile Tyr Tyr Ser Gly Ser Thr Tyr Tyr Asn Pro Ser Leu Lys Ser

1 5 10 15

<210> 339

<211> 12

<212> Protein

<213> Artificial

<220>

<223> U1-18-CDR-3h

<400> 339

Ala Asp Tyr Asp Phe Trp Ser Gly Tyr Phe Asp Tyr

1 5 10

<210> 340

<211> 11

<212> Protein

<213> Artificial

<220>

<223> U1-18-CDR-1l

<400> 340

Arg Ala Ser Gln Gly Ile Arg Asn Asp Leu Gly

1 5 10

<210> 341

<211> 7

<212> Protein

<213> Artificial

<220>

<223> U1-18-CDR-2l

<400> 341

Ala Ala Ser Ser Leu Gln Ser

15

<210> 342

<211> 9

<212> Protein

<213> Artificial

<220>

<223> U1-18-CDR-3l

<400> 342

Leu Gln His Asn Ser Tyr Pro Trp Thr

15

<210> 343

<211> 12

<212> Protein

<213> Artificial

<220>

<223> U1-19-CDR-1h

<400> 343

Gly Gly Ser Ile Ser Ser Gly Asp Tyr Tyr Trp Ser

1 5 10

<210> 344

<211> 16

<212> Protein

<213> Artificial

<220>

<223> U1-19-CDR-2h

<400> 344

Tyr Ile Tyr Tyr Ser Gly Ser Thr Tyr Tyr Asn Pro Ser Leu Lys Ser

1 5 10 15

<210> 345

<211> 12

<212> Protein

<213> Artificial

<220>

<223> U1-19-CDR-3h

<400> 345

Gly Asp Tyr Asp Phe Trp Ser Gly Glu Phe Asp Tyr

1 5 10

<210> 346

<211> 12

<212> Protein

<213> Artificial

<220>

<223> U1-20-CDR-1h

<400> 346

Gly Gly Ser Ile Ser Ser Gly Gly Tyr Tyr Trp Ser

1 5 10

<210> 347

<211> 16

<212> Protein

<213> Artificial

<220>

<223> U1-20-CDR-2h

<400> 347

Tyr Ile Tyr Asp Ser Gly Ser Thr Tyr Tyr Asn Pro Ser Leu Lys Ser

1 5 10 15

<210> 348

<211> 19

<212> Protein

<213> Artificial

<220>

<223> U1-20-CDR-3h

<400> 348

Asp Gln Gly Gln Asp Gly Tyr Ser Tyr Gly Tyr Gly Tyr Tyr Tyr Gly

1 5 10 15

Met Asp Val

<210> 349

<211> 11

<212> Protein

<213> Artificial

<220>

<223> U1-20-CDR-1l

<400> 349

Gln Ala Ser Gln Asp Ile Ser Asn Tyr Leu Asn

1 5 10

<210> 350

<211> 7

<212> Protein

<213> Artificial

<220>

<223> U1-20-CDR-2l

<400> 350

Val Ala Ser Asn Leu Glu Thr

15

<210> 351

<211> 9

<212> Protein

<213> Artificial

<220>

<223> U1-20-CDR-3l

<400> 351

Gln Gln Cys Asp Asn Leu Pro Leu Thr

15

<210> 352

<211> 12

<212> Protein

<213> Artificial

<220>

<223> U1-21-CDR-1h

<400> 352

Gly Gly Ser Ile Ser Ser Gly Asp Tyr Tyr Trp Ser

1 5 10

<210> 353

<211> 16

<212> Protein

<213> Artificial

<220>

<223> U1-21-CDR-2h

<400> 353

Tyr Ile Tyr Tyr Ser Gly Ser Thr Tyr Tyr Asn Pro Ser Leu Lys Ser

1 5 10 15

<210> 354

<211> 12

<212> Protein

<213> Artificial

<220>

<223> U1-21-CDR-3h

<400> 354

Ala Asp Tyr Asp Phe Trp Ser Gly Tyr Phe Asp Tyr

1 5 10

<210> 355

<211> 11

<212> Protein

<213> Artificial

<220>

<223> U1-21-CDR-1l

<400> 355

Arg Ala Ser Gln Asp Ile Arg Asn Asp Leu Gly

1 5 10

<210> 356

<211> 7

<212> Protein

<213> Artificial

<220>

<223> U1-21-CDR-2l

<400> 356

Ala Ala Ser Arg Leu Gln Ser

15

<210> 357

<211> 9

<212> Protein

<213> Artificial

<220>

<223> U1-21-CDR-3l

<400> 357

Leu Gln His Asn Ser Tyr Pro Trp Thr

15

<210> 358

<211> 12

<212> Protein

<213> Artificial

<220>

<223> U1-22-CDR-1h

<400> 358

Gly Gly Ser Ile Ser Ser Gly Asp Tyr Tyr Trp Ser

1 5 10

<210> 359

<211> 16

<212> Protein

<213> Artificial

<220>

<223> U1-22-CDR-2h

<400> 359

Tyr Ile Tyr Tyr Ser Gly Ser Thr Tyr Tyr Asn Pro Ser Leu Lys Ser

1 5 10 15

<210> 360

<211> 12

<212> Protein

<213> Artificial

<220>

<223> U1-22-CDR-3h

<400> 360

Ala Asp Tyr Asp Phe Trp Ser Gly Tyr Phe Asp Tyr

1 5 10

<210> 361

<211> 11

<212> Protein

<213> Artificial

<220>

<223> U1-22-CDR-1l

<400> 361

Arg Ala Ser Gln Gly Ile Arg Asn Asp Leu Gly

1 5 10

<210> 362

<211> 7

<212> Protein

<213> Artificial

<220>

<223> U1-22-CDR-2l

<400> 362

Ala Ala Ser Ser Leu Gln Asn

15

<210> 363

<211> 9

<212> Protein

<213> Artificial

<220>

<223> U1-22-CDR-3l

<400> 363

Leu Gln His Asn Ser Tyr Pro Trp Thr

15

<210> 364

<211> 12

<212> Protein

<213> Artificial

<220>

<223> U1-23-CDR-1h

<400> 364

Gly Gly Ser Ile Ser Ser Gly Asp Tyr Tyr Trp Ser

1 5 10

<210> 365

<211> 16

<212> Protein

<213> Artificial

<220>

<223> U1-23-CDR-2h

<400> 365

Tyr Ile Tyr Tyr Ser Gly Ser Thr Tyr Tyr Asn Pro Ser Leu Lys Ser

1 5 10 15

<210> 366

<211> 12

<212> Protein

<213> Artificial

<220>

<223> U1-23-CDR-3h

<400> 366

Ala Asp Tyr Asp Phe Trp Ser Gly Tyr Phe Asp Tyr

1 5 10

<210> 367

<211> 11

<212> Protein

<213> Artificial

<220>

<223> U1-23-CDR-1l

<400> 367

Arg Ala Ser Gln Gly Ile Arg Asn Asp Leu Gly

1 5 10

<210> 368

<211> 7

<212> Protein

<213> Artificial

<220>

<223> U1-23-CDR-2l

<400> 368

Ala Ala Ser Ser Leu Gln Ser

15

<210> 369

<211> 9

<212> Protein

<213> Artificial

<220>

<223> U1-23-CDR-3l

<400> 369

Leu Gln His Asn Ser Tyr Pro Trp Thr

15

<210> 370

<211> 12

<212> Protein

<213> Artificial

<220>

<223> U1-24-CDR-1h

<400> 370

Gly Gly Ser Ile Ser Ser Gly Asp Tyr Tyr Trp Ser

1 5 10

<210> 371

<211> 16

<212> Protein

<213> Artificial

<220>

<223> U1-24-CDR-2h

<400> 371

Tyr Ile Tyr Tyr Ser Gly Ser Thr Tyr Tyr Asn Pro Ser Leu Lys Ser

1 5 10 15

<210> 372

<211> 12

<212> Protein

<213> Artificial

<220>

<223> U1-24-CDR-3h

<400> 372

Ala Asp Tyr Asp Phe Trp Asn Gly Tyr Phe Asp Tyr

1 5 10

<210> 373

<211> 11

<212> Protein

<213> Artificial

<220>

<223> U1-24-CDR-1l

<400> 373

Arg Ala Ser Gln Gly Ile Arg Asn Asp Leu Gly

1 5 10

<210> 374

<211> 7

<212> Protein

<213> Artificial

<220>

<223> U1-24-CDR-2l

<400> 374

Ala Ala Ser Ser Leu Gln Ser

15

<210> 375

<211> 9

<212> Protein

<213> Artificial

<220>

<223> U1-24-CDR-3l

<400> 375

Leu Gln His Asn Asn Tyr Pro Trp Thr

15

<210> 376

<211> 12

<212> Protein

<213> Artificial

<220>

<223> U1-25-CDR-1h

<400> 376

Gly Gly Ser Ile Ser Ser Gly Asp Tyr Tyr Trp Ser

1 5 10

<210> 377

<211> 16

<212> Protein

<213> Artificial

<220>

<223> U1-25-CDR-2h

<400> 377

Tyr Ile Tyr Tyr Ser Gly Ser Thr Tyr Tyr Asn Pro Ser Leu Lys Ser

1 5 10 15

<210> 378

<211> 12

<212> Protein

<213> Artificial

<220>

<223> U1-25-CDR-3h

<400> 378

Ala Asp Tyr Asp Phe Trp Ser Gly Tyr Phe Asp Tyr

1 5 10

<210> 379

<211> 11

<212> Protein

<213> Artificial

<220>

<223> U1-25-CDR-1l

<400> 379

Arg Ala Ser Gln Gly Ile Arg Asn Asp Leu Gly

1 5 10

<210> 380

<211> 7

<212> Protein

<213> Artificial

<220>

<223> U1-25-CDR-2l

<400> 380

Ala Ala Ser Ser Leu Gln Asn

15

<210> 381

<211> 9

<212> Protein

<213> Artificial

<220>

<223> U1-25-CDR-3l

<400> 381

Leu Gln His Asn Ser Tyr Pro Trp Thr

15

<210> 382

<211> 12

<212> Protein

<213> Artificial

<220>

<223> U1-26-CDR-1h

<400> 382

Gly Gly Ser Ile Ser Ser Gly Asp Tyr Tyr Trp Ser

1 5 10

<210> 383

<211> 16

<212> Protein

<213> Artificial

<220>

<223> U1-26-CDR-2h

<400> 383

Tyr Ile Tyr Tyr Ser Gly Ser Thr Tyr Tyr Asn Pro Ser Leu Lys Ser

1 5 10 15

<210> 384

<211> 12

<212> Protein

<213> Artificial

<220>

<223> U1-26-CDR-3h

<400> 384

Ala Asp Tyr Asp Phe Trp Ser Gly Tyr Phe Asp Phe

1 5 10

<210> 385

<211> 11

<212> Protein

<213> Artificial

<220>

<223> U1-26-CDR-1l

<400> 385

Arg Ala Ser Gln Gly Ile Arg Asn Asp Leu Gly

1 5 10

<210> 386

<211> 7

<212> Protein

<213> Artificial

<220>

<223> U1-26-CDR-2l

<400> 386

Ala Ala Ser Ser Leu Gln Ser

15

<210> 387

<211> 9

<212> Protein

<213> Artificial

<220>

<223> U1-26-CDR-3l

<400> 387

Leu Gln His Asn Gly Tyr Pro Trp Thr

15

<210> 388

<211> 12

<212> Protein

<213> Artificial

<220>

<223> U1-27-CDR-1h

<400> 388

Gly Gly Ser Ile Ser Ser Gly Asp Tyr Tyr Trp Ser

1 5 10

<210> 389

<211> 16

<212> Protein

<213> Artificial

<220>

<223> U1-27-CDR-2h

<400> 389

Tyr Ile Tyr Tyr Ser Gly Ser Thr Tyr Tyr Asn Pro Ser Leu Lys Ser

1 5 10 15

<210> 390

<211> 12

<212> Protein

<213> Artificial

<220>

<223> U1-27-CDR-3h

<400> 390

Ala Asp Tyr Asp Phe Trp Ser Gly Tyr Phe Asp Phe

1 5 10

<210> 391

<211> 11

<212> Protein

<213> Artificial

<220>

<223> U1-27-CDR-1l

<400> 391

Arg Ala Ser Gln Gly Ile Arg Asn Asp Leu Gly

1 5 10

<210> 392

<211> 7

<212> Protein

<213> Artificial

<220>

<223> U1-27-CDR-2l

<400> 392

Ala Ala Ser Ser Leu Gln Ser

15

<210> 393

<211> 9

<212> Protein

<213> Artificial

<220>

<223> U1-27-CDR-3l

<400> 393

Leu Gln His Asn Gly Tyr Pro Trp Thr

15

<210> 394

<211> 12

<212> Protein

<213> Artificial

<220>

<223> U1-28-CDR-1h

<400> 394

Gly Gly Ser Ile Ser Ser Gly Asp Tyr Tyr Trp Ser

1 5 10

<210> 395

<211> 16

<212> Protein

<213> Artificial

<220>

<223> U1-28-CDR-2h

<400> 395

Tyr Ile Tyr Tyr Ser Gly Ser Thr Tyr Tyr Asn Pro Ser Leu Lys Ser

1 5 10 15

<210> 396

<211> 12

<212> Protein

<213> Artificial

<220>

<223> U1-28-CDR-1l

<400> 396

Ala Asp Tyr Asp Phe Trp Ser Gly Tyr Phe Asp Ser

1 5 10

<210> 397

<211> 11

<212> Protein

<213> Artificial

<220>

<223> U1-28-CDR-1l

<400> 397

Arg Ala Ser Gln Gly Ile Arg Asn Asp Leu Gly

1 5 10

<210> 398

<211> 7

<212> Protein

<213> Artificial

<220>

<223> U1-28-CDR-2l

<400> 398

Ala Ala Ser Ser Leu Gln Ser

15

<210> 399

<211> 9

<212> Protein

<213> Artificial

<220>

<223>U1-28-CDR-3l

<400> 399

Leu Gln His Asn Gly Tyr Pro Trp Thr

15

<210> 400

<211> 10

<212> Protein

<213> Artificial

<220>

<223> U1-29-CDR-1h

<400> 400

Gly Phe Thr Phe Asn Ser Tyr Asp Met His

1 5 10

<210> 401

<211> 17

<212> Protein

<213> Artificial

<220>

<223> U1-29-CDR-2h

<400> 401

Val Ile Trp Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val Lys

1 5 10 15

gly

<210> 402

<211> 17

<212> Protein

<213> Artificial

<220>

<223> U1-29-CDR-3h

<400> 402

Asp Arg Leu Cys Thr Asn Gly Val Cys Tyr Glu Asp Tyr Gly Met Asp

1 5 10 15

Val

<210> 403

<211> 11

<212> Protein

<213> Artificial

<220>

<223> U1-29-CDR-1l

<400> 403

Gln Ala Ser Gln Asp Ile Ser Asn Tyr Leu Asn

1 5 10

<210> 404

<211> 7

<212> Protein

<213> Artificial

<220>

<223> U1-29-CDR-2l

<400> 404

Asp Ala Ser Asn Leu Glu Thr

15

<210> 405

<211> 9

<212> Protein

<213> Artificial

<220>

<223> U1-29-CDR-3l

<400> 405

Gln His Tyr Asp Thr Leu Pro Leu Thr

15

<210> 406

<211> 12

<212> Protein

<213> Artificial

<220>

<223> U1-30-CDR-1h

<400> 406

Gly Gly Ser Ile Ser Ser Gly Asp Tyr Tyr Trp Ser

1 5 10

<210> 407

<211> 16

<212> Protein

<213> Artificial

<220>

<223> U1-30-CDR-2h

<400> 407

Tyr Ile Tyr Tyr Ser Gly Thr Thr Tyr Tyr Asn Pro Ser Leu Lys Ser

1 5 10 15

<210> 408

<211> 12

<212> Protein

<213> Artificial

<220>

<223> U1-30-CDR-3h

<400> 408

Ala Asp Tyr Asp Phe Trp Ser Gly Tyr Phe Asp Tyr

1 5 10

<210> 409

<211> 11

<212> Protein

<213> Artificial

<220>

<223> U1-30-CDR-1l

<400> 409

Arg Ala Gly Gln Gly Ile Arg Asn Asp Leu Gly

1 5 10

<210> 410

<211> 7

<212> Protein

<213> Artificial

<220>

<223> U1-30-CDR-2l

<400> 410

Ala Ala Ser Ser Leu Gln Ser

15

<210> 411

<211> 9

<212> Protein

<213> Artificial

<220>

<223> U1-30-CDR-3l

<400> 411

Leu Gln His Asn Ser Tyr Pro Trp Thr

15

<210> 412

<211> 10

<212> Protein

<213> Artificial

<220>

<223> U1-31-CDR-1h

<400> 412

Gly Tyr Thr Phe Thr Asn Tyr Gly Ile Ser

1 5 10

<210> 413

<211> 17

<212> Protein

<213> Artificial

<220>

<223> U1-31-CDR-2h

<400> 413

Trp Ile Ser Ala Tyr Asp Gly Tyr Arg Asn Tyr Ala Gln Lys Leu Gln

1 5 10 15

gly

<210> 414

<211> 13

<212> Protein

<213> Artificial

<220>

<223> U1-31-CDR-3h

<400> 414

Asp Val Gln Asp Tyr Gly Asp Tyr Asp Tyr Phe Asp Tyr

1 5 10

<210> 415

<211> 11

<212> Protein

<213> Artificial

<220>

<223> U1-31-CDR-1l

<400> 415

Arg Ala Ser Gln Ser Ile Ser Ser Tyr Leu Asn

1 5 10

<210> 416

<211> 7

<212> Protein

<213> Artificial

<220>

<223> U1-31-CDR-2l

<400> 416

Ala Ala Ser Ser Leu Gln Ser

15

<210> 417

<211> 9

<212> Protein

<213> Artificial

<220>

<223> U1-31-CDR-3l

<400> 417

Gln Gln Ser Tyr Ser Thr Pro Ile Thr

15

<210> 418

<211> 12

<212> Protein

<213> Artificial

<220>

<223> U1-32-CDR-1h

<400> 418

Gly Gly Ser Ile Ser Ser Gly Asp Tyr Tyr Trp Ser

1 5 10

<210> 419

<211> 16

<212> Protein

<213> Artificial

<220>

<223> U1-32-CDR-2h

<400> 419

Tyr Ile Tyr Tyr Ser Gly Thr Thr Tyr Tyr Asn Pro Ser Leu Lys Ser

1 5 10 15

<210> 420

<211> 12

<212> Protein

<213> Artificial

<220>

<223> U1-32-CDR-3h

<400> 420

Ala Asp Tyr Asp Phe Trp Ser Gly Tyr Phe Asp Tyr

1 5 10

<210> 421

<211> 11

<212> Protein

<213> Artificial

<220>

<223> U1-32-CDR-1l

<400> 421

Arg Ala Gly Gln Gly Ile Arg Asn Asp Leu Gly

1 5 10

<210> 422

<211> 7

<212> Protein

<213> Artificial

<220>

<223> U1-32-CDR-2l

<400> 422

Ala Ala Ser Ser Leu Gln Ser

15

<210> 423

<211> 9

<212> Protein

<213> Artificial

<220>

<223> U1-32-CDR-3l

<400> 423

Leu Gln His Asn Ser Tyr Pro Trp Thr

15

<210> 424

<211> 12

<212> Protein

<213> Artificial

<220>

<223> U1-33-CDR-1h

<400> 424

Gly Gly Ser Ile Ser Ser Gly Asp Tyr Tyr Trp Ser

1 5 10

<210> 425

<211> 16

<212> Protein

<213> Artificial

<220>

<223> U1-33-CDR-2h

<400> 425

Tyr Ile Tyr Tyr Ser Gly Ser Thr Tyr Tyr Asn Pro Ser Leu Lys Ser

1 5 10 15

<210> 426

<211> 12

<212> Protein

<213> Artificial

<220>

<223> U1-33-CDR-3h

<400> 426

Ala Asp Tyr Asp Phe Trp Ser Gly His Phe Asp Cys

1 5 10

<210> 427

<211> 11

<212> Protein

<213> Artificial

<220>

<223> U1-33-CDR-1l

<400> 427

Arg Ala Ser Gln Gly Ile Arg Asp Asp Leu Gly

1 5 10

<210> 428

<211> 7

<212> Protein

<213> Artificial

<220>

<223> U1-33-CDR-2l

<400> 428

Ala Glu Ser Ser Leu Gln Ser

15

<210> 429

<211> 9

<212> Protein

<213> Artificial

<220>

<223> U1-33-CDR-3l

<400> 429

Leu Gln His His Ser Tyr Pro Trp Thr

15

<210> 430

<211> 10

<212> Protein

<213> Artificial

<220>

<223> U1-34-CDR-1h

<400> 430

Gly Tyr Thr Phe Thr Asn Tyr Gly Ile Ser

1 5 10

<210> 431

<211> 17

<212> Protein

<213> Artificial

<220>

<223> U1-34-CDR-2h

<400> 431

Trp Ile Ser Ala Tyr Asp Gly Tyr Arg Asn Tyr Ala Gln Lys Leu Gln

1 5 10 15

gly

<210> 432

<211> 13

<212> Protein

<213> Artificial

<220>

<223> U1-34-CDR-3h

<400> 432

Asp Val Gln Asp Tyr Gly Asp Tyr Asp Tyr Phe Asp Tyr

1 5 10

<210> 433

<211> 11

<212> Protein

<213> Artificial

<220>

<223> U1-34-CDR-1l

<400> 433

Arg Ala Ser Gln Ser Ile Ser Ser Tyr Leu Asn

1 5 10

<210> 434

<211> 7

<212> Protein

<213> Artificial

<220>

<223> U1-34-CDR-2l

<400> 434

Ala Ala Ser Ser Leu Gln Ser

15

<210> 435

<211> 9

<212> Protein

<213> Artificial

<220>

<223> U1-34-CDR-3l

<400> 435

Gln Gln Ser Tyr Ser Thr Pro Ile Thr

15

<210> 436

<211> 10

<212> Protein

<213> Artificial

<220>

<223> U1-35-CDR-1h

<400> 436

Gly Phe Thr Phe Ser Asp Tyr Tyr Met Ser

1 5 10

<210> 437

<211> 17

<212> Protein

<213> Artificial

<220>

<223> U1-35-CDR-2h

<400> 437

Tyr Ile Ser Ser Ser Gly Asn Asn Ile Tyr His Ala Asp Ser Val Lys

1 5 10 15

gly

<210> 438

<211> 14

<212> Protein

<213> Artificial

<220>

<223> U1-35-CDR-3h

<400> 438

Glu Arg Tyr Ser Gly Tyr Asp Asp Pro Asp Gly Phe Asp Ile

1 5 10

<210> 439

<211> 11

<212> Protein

<213> Artificial

<220>

<223> U1-35-CDR-1l

<400> 439

Gln Ala Ser Gln Asp Ile Ser Asn Tyr Leu Ser

1 5 10

<210> 440

<211> 7

<212> Protein

<213> Artificial

<220>

<223> U1-35-CDR-2l

<400> 440

Asp Ala Ser Asn Leu Glu Thr

15

<210> 441

<211> 9

<212> Protein

<213> Artificial

<220>

<223> U1-35-CDR-3l

<400> 441

Gln Gln Tyr Asp Asn Pro Pro Cys Ser

15

<210> 442

<211> 12

<212> Protein

<213> Artificial

<220>

<223> U1-36-CDR-1h

<400> 442

Gly Gly Ser Ile Ser Ser Gly Tyr Tyr Tyr Trp Ser

1 5 10

<210> 443

<211> 16

<212> Protein

<213> Artificial

<220>

<223> U1-36-CDR-2h

<400> 443

Tyr Ile Tyr Tyr Ser Gly Thr Thr Tyr Tyr Asn Pro Ser Phe Lys Ser

1 5 10 15

<210> 444

<211> 12

<212> Protein

<213> Artificial

<220>

<223> U1-36-CDR-3h

<400> 444

Ala Asp Tyr Asp Phe Trp Ser Gly His Phe Asp Tyr

1 5 10

<210> 445

<211> 11

<212> Protein

<213> Artificial

<220>

<223> U1-36-CDR-1l

<400> 445

Arg Ala Ser Gln Gly Ile Arg Asn Asp Leu Gly

1 5 10

<210> 446

<211> 7

<212> Protein

<213> Artificial

<220>

<223> U1-36-CDR-2l

<400> 446

Ala Ala Ser Ser Leu Gln Ser

15

<210> 447

<211> 9

<212> Protein

<213> Artificial

<220>

<223> U1-36-CDR-3l

<400> 447

Leu Gln His Asn Ser Tyr Pro Trp Thr

15

<210> 448

<211> 10

<212> Protein

<213> Artificial

<220>

<223> U1-37-CDR-1h

<400> 448

Gly Tyr Thr Phe Thr Ser Tyr Gly Ile Ser

1 5 10

<210> 449

<211> 17

<212> Protein

<213> Artificial

<220>

<223> U1-37-CDR-2h

<400> 449

Trp Ile Ser Ala Tyr Asp Gly His Thr Asn Tyr Ala Gln Lys Leu Gln

1 5 10 15

gly

<210> 450

<211> 13

<212> Protein

<213> Artificial

<220>

<223> U1-37-CDR-3h

<400> 450

Asp Pro His Asp Tyr Ser Asn Tyr Glu Ala Phe Asp Phe

1 5 10

<210> 451

<211> 11

<212> Protein

<213> Artificial

<220>

<223> U1-37-CDR-1l

<400> 451

Arg Ala Ser Gln Ser Ile Ser Ser Tyr Leu Asn

1 5 10

<210> 452

<211> 7

<212> Protein

<213> Artificial

<220>

<223> U1-37-CDR-2l

<400> 452

Ala Ala Ser Ser Leu Gln Ser

15

<210> 453

<211> 9

<212> Protein

<213> Artificial

<220>

<223> U1-37-CDR-3l

<400> 453

Gln Gln Ser Tyr Ser Thr Pro Ile Thr

15

<210> 454

<211> 12

<212> Protein

<213> Artificial

<220>

<223> U1-38-CDR-1h

<400> 454

Gly Phe Ser Leu Ser Thr Ser Gly Val Gly Val Gly

1 5 10

<210> 455

<211> 16

<212> Protein

<213> Artificial

<220>

<223> U1-38-CDR-2h

<400> 455

Leu Ile Tyr Trp Asn Asp Asp Lys Arg Tyr Ser Pro Ser Leu Lys Ser

1 5 10 15

<210> 456

<211> 9

<212> Protein

<213> Artificial

<220>

<223> U1-38-CDR-3h

<400> 456

Arg Asp Glu Val Arg Gly Phe Asp Tyr

15

<210> 457

<211> 16

<212> Protein

<213> Artificial

<220>

<223> U1-38-CDR-1l

<400> 457

Arg Ser Ser Gln Ser Leu Val Tyr Ser Asp Gly Tyr Thr Tyr Leu His

1 5 10 15

<210> 458

<211> 7

<212> Protein

<213> Artificial

<220>

<223> U1-38-CDR-2l

<400> 458

Lys Val Ser Asn Trp Asp Ser

15

<210> 459

<211> 9

<212> Protein

<213> Artificial

<220>

<223> U1-38-CDR-3l

<400> 459

Met Gln Gly Ala His Trp Pro Ile Thr

15

<210> 460

<211> 10

<212> Protein

<213> Artificial

<220>

<223> U1-39-CDR-1h

<400> 460

Gly Phe Thr Val Ser Asn Tyr Met Ser

1 5 10

<210> 461

<211> 16

<212> Protein

<213> Artificial

<220>

<223> U1-39-CDR-2h

<400> 461

Val Ile Tyr Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys Gly

1 5 10 15

<210> 462

<211> 6

<212> Protein

<213> Artificial

<220>

<223> U1-39-CDR-3h

<400> 462

Gly Gln Trp Leu Asp Val

15

<210> 463

<211> 16

<212> Protein

<213> Artificial

<220>

<223> U1-39-CDR-1l

<400> 463

Arg Ser Ser Gln Ser Leu Leu His Ser Asn Gly Tyr Asn Tyr Leu Asp

1 5 10 15

<210> 464

<211> 7

<212> Protein

<213> Artificial

<220>

<223> U1-39-CDR-2l

<400> 464

Leu Gly Phe His Arg Ala Ser

15

<210> 465

<211> 9

<212> Protein

<213> Artificial

<220>

<223> U1-39-CDR-3l

<400> 465

Arg Gln Ala Leu Gln Thr Pro Leu Thr

15

<210> 466

<211> 12

<212> Protein

<213> Artificial

<220>

<223> U1-40-CDR-1h

<400> 466

Gly Gly Ser Ile Ser Ser Gly Gly Tyr Tyr Trp Ser

1 5 10

<210> 467

<211> 16

<212> Protein

<213> Artificial

<220>

<223> U1-40-CDR-2h

<400> 467

Tyr Ile Tyr Ser Ser Gly Ser Thr Tyr Tyr Asn Pro Ser Leu Lys Ser

1 5 10 15

<210> 468

<211> 15

<212> Protein

<213> Artificial

<220>

<223> U1-40-CDR-3h

<400> 468

Asp Arg Glu Leu Glu Leu Tyr Tyr Tyr Tyr Tyr Gly Met Asp Val

1 5 10 15

<210> 469

<211> 16

<212> Protein

<213> Artificial

<220>

<223> U1-40-CDR-1l

<400> 469

Arg Ser Ser Gln Ser Leu Leu Tyr Ser Asn Gly Tyr Asn Tyr Leu Asp

1 5 10 15

<210> 470

<211> 7

<212> Protein

<213> Artificial

<220>

<223> U1-40-CDR-2l

<400> 470

Leu Gly Ser Asn Arg Ala Ser

15

<210> 471

<211> 9

<212> Protein

<213> Artificial

<220>

<223> U1-40-CDR-3l

<400> 471

Met Gln Ala Leu Gln Thr Pro Leu Thr

15

<210> 472

<211> 12

<212> Protein

<213> Artificial

<220>

<223> U1-41-CDR-1h

<400> 472

Gly Gly Ser Ile Ser Ser Gly Gly Tyr Tyr Trp Ser

1 5 10

<210> 473

<211> 16

<212> Protein

<213> Artificial

<220>

<223> U1-41-CDR-2h

<400> 473

Tyr Ile Tyr Tyr Ser Gly Ser Thr Tyr Tyr Asn Pro Ser Leu Lys Ser

1 5 10 15

<210> 474

<211> 15

<212> Protein

<213> Artificial

<220>

<223> U1-41-CDR-3h

<400> 474

Asp Arg Glu Leu Glu Gly Tyr Ser Asn Tyr Tyr Gly Val Asp Val

1 5 10 15

<210> 475

<211> 11

<212> Protein

<213> Artificial

<220>

<223> U1-41-CDR-1l

<400> 475

Arg Ala Ser Gln Ala Ile Ser Asn Tyr Leu Asn

1 5 10

<210> 476

<211> 7

<212> Protein

<213> Artificial

<220>

<223> U1-41-CDR-2l

<400> 476

Ala Ala Ser Ser Leu Gln Ser

15

<210> 477

<211> 9

<212> Protein

<213> Artificial

<220>

<223> U1-41-CDR-3l

<400> 477

Gln Gln Asn Asn Ser Leu Pro Ile Thr

15

<210> 478

<211> 10

<212> Protein

<213> Artificial

<220>

<223> U1-42-CDR-1h

<400> 478

Gly Tyr Ser Phe Thr Ser Tyr Trp Ile Gly

1 5 10

<210> 479

<211> 17

<212> Protein

<213> Artificial

<220>

<223> U1-42-CDR-2h

<400> 479

Ile Ile Tyr Pro Gly Asp Ser Asp Thr Arg Tyr Ser Pro Ser Phe Gln

1 5 10 15

gly

<210> 480

<211> 9

<212> Protein

<213> Artificial

<220>

<223> U1-42-CDR-3h

<400> 480

His Glu Asn Tyr Gly Asp Tyr Asn Tyr

15

<210> 481

<211> 11

<212> Protein

<213> Artificial

<220>

<223> U1-42-CDR-1l

<400> 481

Arg Ala Ser Gln Ser Ile Arg Ser Tyr Leu Asn

1 5 10

<210> 482

<211> 7

<212> Protein

<213> Artificial

<220>

<223> U1-42-CDR-2l

<400> 482

Ala Ala Ser Ser Leu Gln Ser

15

<210> 483

<211> 9

<212> Protein

<213> Artificial

<220>

<223> U1-42-CDR-3l

<400> 483

Gln Gln Ser Asn Gly Ser Pro Leu Thr

15

<210> 484

<211> 12

<212> Protein

<213> Artificial

<220>

<223> U1-43-CDR-1h

<400> 484

Gly Gly Ser Ile Ser Ser Gly Gly Tyr Tyr Trp Ser

1 5 10

<210> 485

<211> 16

<212> Protein

<213> Artificial

<220>

<223> U1-43-CDR-2h

<400> 485

Tyr Ile Tyr Tyr Ser Gly Ser Thr Tyr Tyr Asn Pro Ser Leu Arg Ser

1 5 10 15

<210> 486

<211> 17

<212> Protein

<213> Artificial

<220>

<223> U1-43-CDR-3h

<400> 486

Asp Arg Glu Arg Glu Trp Asp Asp Tyr Gly Asp Pro Gln Gly Met Asp

1 5 10 15

Val

<210> 487

<211> 11

<212> Protein

<213> Artificial

<220>

<223> U1-43-CDR-1l

<400> 487

Arg Ala Ser Gln Ser Ile Ser Ser Tyr Leu His

1 5 10

<210> 488

<211> 7

<212> Protein

<213> Artificial

<220>

<223> U1-43-CDR-2l

<400> 488

Ala Ala Ser Ser Leu Gln Ser

15

<210> 489

<211> 9

<212> Protein

<213> Artificial

<220>

<223> U1-43-CDR-3l

<400> 489

Gln Gln Ser Tyr Ser Asn Pro Leu Thr

15

<210> 490

<211> 10

<212> Protein

<213> Artificial

<220>

<223> U1-44-CDR-1h

<400> 490

Gly Tyr Ser Phe Thr Ser Tyr Trp Ile Gly

1 5 10

<210> 491

<211> 17

<212> Protein

<213> Artificial

<220>

<223> U1-44-CDR-2h

<400> 491

Ile Ile Trp Pro Gly Asp Ser Asp Thr Ile Tyr Ser Pro Ser Phe Gln

1 5 10 15

gly

<210> 492

<211> 9

<212> Protein

<213> Artificial

<220>

<223> U1-44-CDR-3h

<400> 492

His Glu Asn Tyr Gly Asp Tyr Asn Tyr

15

<210> 493

<211> 11

<212> Protein

<213> Artificial

<220>

<223> U1-44-CDR-1l

<400> 493

Arg Ala Ser Gln Ser Ile Arg Ser Tyr Leu Asn

1 5 10

<210> 494

<211> 7

<212> Protein

<213> Artificial

<220>

<223> U1-44-CDR-2l

<400> 494

Ala Ala Ser Ser Leu Gln Ser

15

<210> 495

<211> 9

<212> Protein

<213> Artificial

<220>

<223> U1-44-CDR-3l

<400> 495

Gln Gln Ser Ile Ser Ser Pro Leu Thr

15

<210> 496

<211> 10

<212> Protein

<213> Artificial

<220>

<223> U1-45-CDR-1h

<400> 496

Gly Tyr Thr Phe Thr Ser Tyr Asp Ile Asn

1 5 10

<210> 497

<211> 17

<212> Protein

<213> Artificial

<220>

<223> U1-45-CDR-2h

<400> 497

Trp Met Asn Pro Asn Ser Gly Asp Thr Gly Tyr Ala Gln Val Phe Gln

1 5 10 15

gly

<210> 498

<211> 16

<212> Protein

<213> Artificial

<220>

<223> U1-45-CDR-3h

<400> 498

Phe Gly Asp Leu Pro Tyr Asp Tyr Ser Tyr Tyr Glu Trp Phe Asp Pro

1 5 10 15

<210> 499

<211> 11

<212> Protein

<213> Artificial

<220>

<223> U1-45-CDR-1l

<400> 499

Arg Ala Ser Gln Ser Ile Ser Ser Tyr Leu Asn

1 5 10

<210> 500

<211> 7

<212> Protein

<213> Artificial

<220>

<223> U1-45-CDR-2l

<400> 500

Ala Ala Ser Ser Leu Gln Ser

15

<210> 501

<211> 9

<212> Protein

<213> Artificial

<220>

<223> U1-45-CDR-3l

<400> 501

Gln Gln Ser Tyr Ser Thr Pro Leu Thr

15

<210> 502

<211> 12

<212> Protein

<213> Artificial

<220>

<223> U1-46-CDR-1h

<400> 502

Gly Asp Ser Val Ser Ser Asn Ser Ala Ala Trp Asn

1 5 10

<210> 503

<211> 18

<212> Protein

<213> Artificial

<220>

<223> U1-46-CDR-2h

<400> 503

Arg Thr Tyr Tyr Arg Ser Lys Trp Tyr Asn Asp Tyr Ala Val Ser Val

1 5 10 15

Lys Ser

<210> 504

<211> 17

<212> Protein

<213> Artificial

<220>

<223> U1-46-CDR-3h

<400> 504

Asp Leu Tyr Asp Phe Trp Ser Gly Tyr Pro Tyr Tyr Tyr Gly Met Asp

1 5 10 15

Val

<210> 505

<211> 12

<212> Protein

<213> Artificial

<220>

<223> U1-47-CDR-1h

<400> 505

Gly Asp Ser Val Ser Ser Asn Ser Ala Ala Trp Asn

1 5 10

<210> 506

<211> 18

<212> Protein

<213> Artificial

<220>

<223> U1-47-CDR-2h

<400> 506

Arg Thr Tyr Tyr Arg Ser Lys Trp Tyr Asn Asp Tyr Ala Val Ser Val

1 5 10 15

Lys Ser

<210> 507

<211> 16

<212> Protein

<213> Artificial

<220>

<223> U1-47-CDR-3h

<400> 507

Asp Tyr Tyr Gly Ser Gly Ser Phe Tyr Tyr Tyr Tyr Gly Met Asp Val

1 5 10 15

<210> 508

<211> 11

<212> Protein

<213> Artificial

<220>

<223> U1-47-CDR-1l

<400> 508

Arg Ala Ser Gln Ser Ile Ser Ser Tyr Leu Asn

1 5 10

<210> 509

<211> 7

<212> Protein

<213> Artificial

<220>

<223> U1-47-CDR-2l

<400> 509

Ala Ala Ser Asn Leu Gln Ser

15

<210> 510

<211> 9

<212> Protein

<213> Artificial

<220>

<223> U1-47-CDR-3l

<400> 510

Gln Gln Ser Tyr Ser Thr Pro Arg Thr

15

<210> 511

<211> 10

<212> Protein

<213> Artificial

<220>

<223> U1-48-CDR-1h

<400> 511

Gly Gly Ser Ile Ser Ser Tyr Tyr Trp Ser

1 5 10

<210> 512

<211> 16

<212> Protein

<213> Artificial

<220>

<223> U1-48-CDR-2h

<400> 512

His Ile Tyr Thr Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys Ser

1 5 10 15

<210> 513

<211> 16

<212> Protein

<213> Artificial

<220>

<223> U1-48-CDR-3h

<400> 513

Glu Ala Ile Phe Gly Val Gly Pro Tyr Tyr Tyr Tyr Gly Met Asp Val

1 5 10 15

<210> 514

<211> 10

<212> Protein

<213> Artificial

<220>

<223> U1-49-CDR-1h

<400> 514

Gly Tyr Thr Phe Thr Gly Tyr Tyr Met His

1 5 10

<210> 515

<211> 17

<212> Protein

<213> Artificial

<220>

<223> U1-49-CDR-2h

<400> 515

Trp Ile Asn Pro Asn Ile Gly Gly Thr Asn Cys Ala Gln Lys Phe Gln

1 5 10 15

gly

<210> 516

<211> 17

<212> Protein

<213> Artificial

<220>

<223> U1-49-CDR-3h

<400> 516

Gly Gly Arg Tyr Ser Ser Ser Trp Ser Tyr Tyr Tyr Tyr Gly Met Asp

1 5 10 15

Val

<210> 517

<211> 16

<212> Protein

<213> Artificial

<220>

<223> U1-49-CDR-1l

<400> 517

Lys Ser Ser Gln Ser Leu Leu Leu Ser Asp Gly Gly Thr Tyr Leu Tyr

1 5 10 15

<210> 518

<211> 7

<212> Protein

<213> Artificial

<220>

<223> U1-49-CDR-2l

<400> 518

Glu Val Ser Asn Arg Phe Ser

15

<210> 519

<211> 9

<212> Protein

<213> Artificial

<220>

<223> U1-49-CDR-3l

<400> 519

Met Gln Ser Met Gln Leu Pro Ile Thr

15

<210> 520

<211> 12

<212> Protein

<213> Artificial

<220>

<223> U1-50-CDR-1h

<400> 520

Gly Gly Ser Val Ser Ser Gly Gly Tyr Tyr Trp Ser

1 5 10

<210> 521

<211> 16

<212> Protein

<213> Artificial

<220>

<223> U1-50-CDR-2h

<400> 521

Tyr Ile Tyr Tyr Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys Ser

1 5 10 15

<210> 522

<211> 17

<212> Protein

<213> Artificial

<220>

<223> U1-50-CDR-3h

<400> 522

Gly Gly Asp Ser Asn Tyr Glu Asp Tyr Tyr Tyr Tyr Tyr Gly Met Asp

1 5 10 15

Val

<210> 523

<211> 11

<212> Protein

<213> Artificial

<220>

<223> U1-50-CDR-1l

<400> 523

Arg Ala Ser Gln Ser Ile Ser Ile Tyr Leu His

1 5 10

<210> 524

<211> 7

<212> Protein

<213> Artificial

<220>

<223> U1-50-CDR-2l

<400> 524

Ala Ala Ser Ser Leu Gln Ser

15

<210> 525

<211> 9

<212> Protein

<213> Artificial

<220>

<223> U1-50-CDR-3l

<400> 525

Gln Gln Ser Tyr Thr Ser Pro Ile Thr

15

<210> 526

<211> 10

<212> Protein

<213> Artificial

<220>

<223> U1-51-CDR-1h

<400> 526

Gly Gly Ser Ile Ser Ser Tyr Tyr Trp Ser

1 5 10

<210> 527

<211> 16

<212> Protein

<213> Artificial

<220>

<223> U1-51-CDR-2h

<400> 527

Tyr Ile Tyr Tyr Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys Ser

1 5 10 15

<210> 528

<211> 19

<212> Protein

<213> Artificial

<220>

<223> U1-51-CDR-3h

<400> 528

Asp Ser Ser Tyr Tyr Asp Ser Ser Gly Tyr Tyr Leu Tyr Tyr Tyr Ala

1 5 10 15

Met Asp Val

<210> 529

<211> 17

<212> Protein

<213> Artificial

<220>

<223> U1-51-CDR-1l

<400> 529

Lys Ser Ser Gln Ser Val Leu Tyr Ser Ser Asn Asn Lys Asn Tyr Leu

1 5 10 15

Ala

<210> 530

<211> 7

<212> Protein

<213> Artificial

<220>

<223> U1-51-CDR-2l

<400> 530

Trp Ala Ser Thr Arg Glu Ser

15

<210> 531

<211> 9

<212> Protein

<213> Artificial

<220>

<223> U1-51-CDR-3l

<400> 531

Gln Gln Tyr Tyr Thr Thr Pro Leu Thr

15

<210> 532

<211> 12

<212> Protein

<213> Artificial

<220>

<223> U1-52-CDR-1h

<400> 532

Gly Gly Ser Ile Ser Ser Gly Gly Tyr Tyr Trp Ser

1 5 10

<210> 533

<211> 16

<212> Protein

<213> Artificial

<220>

<223> U1-52-CDR-2h

<400> 533

Asn Ile Tyr Tyr Ser Gly Ser Thr Tyr Tyr Asn Pro Ser Leu Lys Ser

1 5 10 15

<210> 534

<211> 15

<212> Protein

<213> Artificial

<220>

<223> U1-52-CDR-3h

<400> 534

Gly Gly Thr Gly Thr Asn Tyr Tyr Tyr Tyr Tyr Gly Met Asp Val

1 5 10 15

<210> 535

<211> 12

<212> Protein

<213> Artificial

<220>

<223> U1-52-CDR-1l

<400> 535

Arg Ala Ser Gln Ser Val Ser Ser Ser Tyr Leu Ala

1 5 10

<210> 536

<211> 7

<212> Protein

<213> Artificial

<220>

<223> U1-52-CDR-2l

<400> 536

Gly Ala Ser Ser Trp Ala Thr

15

<210> 537

<211> 9

<212> Protein

<213> Artificial

<220>

<223> U1-52-CDR-3l

<400> 537

Gln Gln Tyr Gly Ser Ser Pro Leu Thr

15

<210> 538

<211> 10

<212> Protein

<213> Artificial

<220>

<223> U1-53-CDR-1h

<400> 538

Gly Phe Thr Phe Ser Ile Tyr Ser Met Asn

1 5 10

<210> 539

<211> 17

<212> Protein

<213> Artificial

<220>

<223> U1-53-CDR-2h

<400> 539

Tyr Ile Ser Ser Ser Ser Ser Thr Ile Tyr Tyr Ala Asp Ser Val Lys

1 5 10 15

gly

<210> 540

<211> 10

<212> Protein

<213> Artificial

<220>

<223> U1-53-CDR-3h

<400> 540

Asp Arg Gly Asp Phe Asp Ala Phe Asp Ile

1 5 10

<210> 541

<211> 11

<212> Protein

<213> Artificial

<220>

<223> U1-53-CDR-1l

<400> 541

Gln Ala Ser Gln Asp Ile Thr Asn Tyr Leu Asn

1 5 10

<210> 542

<211> 7

<212> Protein

<213> Artificial

<220>

<223> U1-53-CDR-2l

<400> 542

Asp Ala Ser Asn Leu Glu Thr

15

<210> 543

<211> 9

<212> Protein

<213> Artificial

<220>

<223> U1-53-CDR-3l

<400> 543

Gln Gln Cys Glu Asn Phe Pro Ile Thr

15

<210> 544

<211> 12

<212> Protein

<213> Artificial

<220>

<223> U1-55.1-CDR-1h

<400> 544

Gly Gly Ser Val Ser Ser Gly Gly Tyr Tyr Trp Asn

1 5 10

<210> 545

<211> 16

<212> Protein

<213> Artificial

<220>

<223> U1-55.1-CDR-2h

<400> 545

Tyr Ile Asn Tyr Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys Ser

1 5 10 15

<210> 546

<211> 15

<212> Protein

<213> Artificial

<220>

<223> U1-55.1-CDR-3h

<400> 546

Asp Arg Glu Leu Glu Leu Tyr Tyr Tyr Tyr Tyr Gly Met Asp Val

1 5 10 15

<210> 547

<211> 16

<212> Protein

<213> Artificial

<220>

<223> U1-55-CDR-1l

<400> 547

Arg Ser Ser Gln Ser Leu Leu Tyr Ser Asn Gly Tyr Lys Tyr Leu Asp

1 5 10 15

<210> 548

<211> 7

<212> Protein

<213> Artificial

<220>

<223> U1-55-CDR-2l

<400> 548

Leu Gly Ser Asn Arg Ala Ser

15

<210> 549

<211> 9

<212> Protein

<213> Artificial

<220>

<223> U1-55-CDR-3l

<400> 549

Met Gln Ala Leu Gln Thr Pro Ile Thr

15

<210> 550

<211> 16

<212> Protein

<213> Artificial

<220>

<223> U1-57.1-CDR-1l

<400> 550

Arg Ser Ser Gln Ser Leu Leu Tyr Ser Asn Gly Tyr Lys Tyr Leu Asp

1 5 10 15

<210> 551

<211> 7

<212> Protein

<213> Artificial

<220>

<223> U1-57.1-CDR-2l

<400> 551

Leu Gly Ser Asn Arg Ala Ser

15

<210> 552

<211> 9

<212> Protein

<213> Artificial

<220>

<223> U1-57.1-CDR-3l

<400> 552

Met Gln Ala Leu Gln Thr Pro Ile Thr

15

<210> 553

<211> 12

<212> Protein

<213> Artificial

<220>

<223> U1-57_CDR-1h

<400> 553

Gly Gly Ser Val Ser Ser Gly Gly Tyr Tyr Trp Asn

1 5 10

<210> 554

<211> 16

<212> Protein

<213> Artificial

<220>

<223> U1-57_CDR-2h

<400> 554

Tyr Ile Asn Tyr Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys Ser

1 5 10 15

<210> 555

<211> 15

<212> Protein

<213> Artificial

<220>

<223> U1-57_CDR-3h

<400> 555

Asp Arg Glu Leu Glu Leu Tyr Tyr Tyr Tyr Tyr Gly Met Asp Val

1 5 10 15

<210> 556

<211> 10

<212> Protein

<213> Artificial

<220>

<223> U1-58-CDR-1h

<400> 556

Gly Phe Thr Phe Ser Ser Tyr Gly Met His

1 5 10

<210> 557

<211> 17

<212> Protein

<213> Artificial

<220>

<223> U1-58-CDR-2h

<400> 557

Val Ile Trp Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val Lys

1 5 10 15

gly

<210> 558

<211> 12

<212> Protein

<213> Artificial

<220>

<223> U1-58-CDR-3h

<400> 558

Ala Ala Arg Leu Asp Tyr Tyr Tyr Gly Met Asp Val

1 5 10

<210> 559

<211> 11

<212> Protein

<213> Artificial

<220>

<223> U1-58-CDR-1l

<400> 559

Arg Ala Ser Gln Ser Ile Asn Ser Tyr Leu Asn

1 5 10

<210> 560

<211> 7

<212> Protein

<213> Artificial

<220>

<223> U1-58-CDR-2l

<400> 560

Gly Ala Ser Gly Leu Gln Ser

15

<210> 561

<211> 9

<212> Protein

<213> Artificial

<220>

<223> U1-58-CDR-3l

<400> 561

Gln Gln Ser Tyr Ser Ser Pro Leu Thr

15

<210> 562

<211> 10

<212> Protein

<213> Artificial

<220>

<223> U1-59-CDR-1h

<400> 562

Gly Gly Ser Phe Ser Gly Tyr Tyr Trp Ser

1 5 10

<210> 563

<211> 16

<212> Protein

<213> Artificial

<220>

<223> U1-59-CDR-2h

<400> 563

Glu Ile Asn His Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys Ser

1 5 10 15

<210> 564

<211> 9

<212> Protein

<213> Artificial

<220>

<223> U1-59-CDR-3h

<400> 564

Asp Lys Trp Thr Trp Tyr Phe Asp Leu

15

<210> 565

<211> 17

<212> Protein

<213> Artificial

<220>

<223> U1-59-CDR-1l

<400> 565

Arg Ser Ser Gln Ser Val Leu Tyr Ser Ser Ser Asn Arg Asn Tyr Leu

1 5 10 15

Ala

<210> 566

<211> 7

<212> Protein

<213> Artificial

<220>

<223> U1-59-CDR-2l

<400> 566

Trp Ala Ser Thr Arg Glu Ser

15

<210> 567

<211> 9

<212> Protein

<213> Artificial

<220>

<223> U1-59-CDR-3l

<400> 567

Gln Gln Tyr Tyr Ser Thr Pro Arg Thr

15

<210> 568

<211> 12

<212> Protein

<213> Artificial

<220>

<223> U1-61.1-CDR-1h

<400> 568

Gly Val Ser Ile Ser Ser Gly Gly Tyr Tyr Trp Ser

1 5 10

<210> 569

<211> 16

<212> Protein

<213> Artificial

<220>

<223> U1-61.1-CDR-2h

<400> 569

Tyr Ile Tyr Tyr Ser Gly Ser Thr Tyr Tyr Asn Pro Ser Leu Lys Ser

1 5 10 15

<210> 570

<211> 16

<212> Protein

<213> Artificial

<220>

<223> U1-61.1-CDR-3h

<400> 570

Asp Ser Glu Ser Glu Tyr Ser Ser Ser Ser Asn Tyr Gly Met Asp Val

1 5 10 15

<210> 571

<211> 11

<212> Protein

<213> Artificial

<220>

<223> U1-61.1-CDR-1l

<400> 571

Arg Ala Ser Gln Thr Ile Ser Ser Tyr Leu Asn

1 5 10

<210> 572

<211> 7

<212> Protein

<213> Artificial

<220>

<223> U1-61.1-CDR-2l

<400> 572

Ala Ala Ser Ser Leu Gln Gly

15

<210> 573

<211> 9

<212> Protein

<213> Artificial

<220>

<223> U1-61.1-CDR-3l

<400> 573

Gln Gln Ser Tyr Ser Asn Pro Leu Thr

15

<210> 574

<211> 12

<212> Protein

<213> Artificial

<220>

<223> U1-61-CDR-1h

<400> 574

Gly Val Ser Ile Ser Ser Gly Gly Tyr Tyr Trp Ser

1 5 10

<210> 575

<211> 16

<212> Protein

<213> Artificial

<220>

<223> U1-61-CDR-2h

<400> 575

Tyr Ile Tyr Tyr Ser Gly Ser Thr Tyr Tyr Asn Pro Ser Leu Lys Ser

1 5 10 15

<210> 576

<211> 16

<212> Protein

<213> Artificial

<220>

<223> U1-61-CDR-3h

<400> 576

Asp Ser Glu Ser Glu Tyr Ser Ser Ser Ser Asn Tyr Gly Met Asp Val

1 5 10 15

<210> 577

<211> 10

<212> Protein

<213> Artificial

<220>

<223> U1-62-CDR-1h

<400> 577

Gly Tyr Ser Phe Thr Ser Tyr Trp Ile Gly

1 5 10

<210> 578

<211> 17

<212> Protein

<213> Artificial

<220>

<223> U1-62-CDR-2h

<400> 578

Ile Ile Tyr Pro Gly Asp Ser Asp Thr Arg Tyr Ser Pro Ser Phe Gln

1 5 10 15

gly

<210> 579

<211> 12

<212> Protein

<213> Artificial

<220>

<223> U1-62-CDR-3h

<400> 579

Gln Met Ala Gly Asn Tyr Tyr Tyr Gly Met Asp Val

1 5 10

<210> 580

<211> 12

<212> Protein

<213> Artificial

<220>

<223> U1-62-CDR-1l

<400> 580

Arg Ala Ser Gln Ser Val Ile Ser Ile Tyr Leu Ala

1 5 10

<210> 581

<211> 7

<212> Protein

<213> Artificial

<220>

<223> U1-62-CDR-2l

<400> 581

Gly Ala Ser Ser Arg Ala Thr

15

<210> 582

<211> 9

<212> Protein

<213> Artificial

<220>

<223> U1-62-CDR-3l

<400> 582

Gln Gln Tyr Gly Ser Ser Pro Cys Ser

15

<210> 583

<211> 447

<212> Protein

<213> Faux Sequence

<220>

<223> Antibody

<400> 583

Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Ser Gly Tyr

20 25 30

Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Glu Ile Asn His Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys

50 55 60

Ser Arg Val Thr Ile Ser Val Glu Thr Ser Lys Asn Gln Phe Ser Leu

65 70 75 80

Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Asp Lys Trp Thr Trp Tyr Phe Asp Leu Trp Gly Arg Gly Thr Leu

100 105 110

Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu

115 120 125

Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys

130 135 140

Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser

145 150 155 160

Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser

165 170 175

Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser

180 185 190

Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn

195 200 205

Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His

210 215 220

Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val

225 230 235 240

Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr

245 250 255

Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu

260 265 270

Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

275 280 285

Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser

290 295 300

Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys

305 310 315 320

Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile

325 330 335

Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro

340 345 350

Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu

355 360 365

Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn

370 375 380

Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser

385 390 395 400

Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg

405 410 415

Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu

420 425 430

His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

435 440 445

<210> 584

<211> 220

<212> Protein

<213> Faux Sequence

<220>

<223> Antibody

<400> 584

Asp Ile Glu Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Glu Arg Ala Thr Ile Asn Cys Arg Ser Ser Gln Ser Val Leu Tyr Ser

20 25 30

Ser Ser Asn Arg Asn Tyr Leu Ala Trp Tyr Gln Gln Asn Pro Gly Gln

35 40 45

Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val

50 55 60

Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln

85 90 95

Tyr Tyr Ser Thr Pro Arg Thr Phe Gly Gln Gly Thr Lys Val Glu Ile

100 105 110

Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp

115 120 125

Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn

130 135 140

Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu

145 150 155 160

Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp

165 170 175

Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr

180 185 190

Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser

195 200 205

Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215 220

<---

Claims (108)

1. A method for producing an antibody-drug conjugate, comprising reacting a compound represented by the following formula: (maleimide-N-yl)-(CH ₂ )n ³ -C(=O)-L ² -L ^P -NH-(CH ₂ )n ¹ -L ^a -(CH ₂ )n ² -C(=O )-(NH-DX) or (maleimide-N-yl)-(CH ₂ )n ³ -C(=O)-L ² -L ^P -(NH-DX), with an anti-HER3 antibody or a reactive derivative thereof and conjugating the drug-linker fragment with the antibody in a manner to form a thioether bond at the disulfide bond site present in the hinge portion of the antibody, Where n ³ is an integer having a value from 2 to 8, L ² is -NH-(CH ₂ CH ₂ -O)n ⁴ -CH ₂ CH ₂ -C(=O)- or a single bond, where n ⁴ is an integer value from 1 to 6, L ^P is a peptide residue consisting of 2-7 amino acids selected from phenylalanine, glycine, valine, lysine, citrulline, serine, glutamic acid and aspartic acid, n ¹ is an integer having a value from 0 to 6, n ² is an integer having a value from 0 to 5, L ^a is -O- or a single bond, (maleimide-N-yl)- is a group represented by the following formula: , where the nitrogen atom represents the binding position, and -(NH-DX) is a group represented by the following formula: where the nitrogen atom of the amino group in position 1 represents the binding position. 2. The method of claim 1, wherein L ^P is a -GGFG- tetrapeptide residue. 3. The method according to claim 1, where the compound is selected from the following group: (maleimid-N-yl)-CH ₂ CH ₂ -C (= O) -GGFG-NH-CH ₂ CH ₂ -C (= O) - (NH-DX), (maleimid-N-yl)-CH ₂ CH ₂ CH ₂ -C (= O) -GGFG-NH-CH ₂ CH ₂ -C (= O) - (NH-DX), (maleimid-N-yl)-CH ₂ CH ₂ CH ₂ CH ₂ -C (= O) -GGFG-NH-CH ₂ CH ₂ -C (= O) - (NH-DX), (maleimid-N-yl)-CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ -C (= O) -GGFG-NH-CH ₂ CH ₂ -C (= O) - (NH-DX), (maleimid-N-yl)-CH ₂ CH ₂ -C (= O) -GGFG-NH-CH ₂ CH ₂ CH ₂ -C (= O) - (NH-DX), (maleimid-N-yl)-CH ₂ CH ₂ CH ₂ -C (= O) -GGFG-NH-CH ₂ CH ₂ CH ₂ -C (= O) - (NH-DX), (maleimid-N-yl)-CH ₂ CH ₂ CH ₂ CH ₂ -C (= O) -GGFG-NH-CH ₂ CH ₂ CH ₂ -C (= O) - (NH-DX), (maleimid-N-yl)-CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ -C (= O) -GGFG-NH-CH ₂ CH ₂ CH ₂ -C (= O) - (NH-DX), (maleimid-N-yl)-CH ₂ CH ₂ -C (= O) -GGFG-NH-CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ -C (= O) - (NH-DX), (maleimid-N-yl)-CH ₂ CH ₂ CH ₂ -C (= O) -GGFG-NH-CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ -C (= O) - (NH-DX), (maleimid-N-yl)-CH ₂ CH ₂ CH ₂ CH ₂ -C (= O) -GGFG-NH-CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ -C (= O) - (NH-DX), (maleimid-N-yl) -CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ -C (= O) -GGFG-NH-CH ₂ CH ₂ CH 2 CH ₂ CH _{2 -C} (= O) - (NH-DX ), (maleimid-N-yl)-CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ -C (= O) -DGGFG-NH-CH ₂ CH ₂ -C (= O) - (NH-DX), (maleimid-N-yl)-CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ -C (= O) -DGGFG-NH-CH ₂ CH ₂ CH ₂ -C (= O) - (NH-DX), (maleimid-N-yl)-CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ -C (= O) -DGGFG-NH-CH ₂ CH 2 CH ₂ CH ₂ CH _{2 -C} (= O) - (NH-DX ), (maleimid-N-yl)-CH ₂ CH ₂ -C(=O)-GGFG-NH-CH ₂ -O-CH ₂ -C(=O)-(NH-DX), (maleimid-N-yl)-CH ₂ CH ₂ CH ₂ -C(=O)-GGFG-NH-CH ₂ -O-CH ₂ -C(=O)-(NH-DX), (maleimid-N-yl)-CH ₂ CH ₂ CH ₂ CH ₂ -C (= O) -GGFG-NH-CH ₂ -O-CH ₂ -C (= O) - (NH-DX), (maleimid-N-yl)-CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ -C (= O) -GGFG-NH-CH ₂ -O-CH ₂ -C (= O) - (NH-DX), (maleimid-N-yl)-CH ₂ CH ₂ -C (= O) -GGFG-NH-CH ₂ CH ₂ -O-CH ₂ -C (= O) - (NH-DX), (maleimid-N-yl)-CH ₂ CH ₂ CH ₂ -C (= O) -GGFG-NH-CH ₂ CH ₂ -O-CH ₂ -C (= O) - (NH-DX), (maleimid-N-yl)-CH ₂ CH ₂ CH ₂ CH ₂ -C (= O) -GGFG-NH-CH ₂ CH ₂ -O-CH ₂ -C (= O) - (NH-DX), (maleimid-N-yl)-CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ -C (= O) -GGFG-NH-CH ₂ CH ₂ -O-CH ₂ -C (= O) - (NH-DX) , (maleimid-N-yl) -CH ₂ CH ₂ -C (= O) -NH-CH ₂ CH ₂ -O-CH ₂ CH ₂ -O-CH ₂ CH ₂ -C (= O) -GGFG-NH- CH ₂ CH ₂ -C (= O) - (NH-DX), (maleimid-N-yl)-CH ₂ CH ₂ -C (= O) -NH-CH ₂ CH ₂ -O-CH ₂ CH ₂ -O-CH ₂ CH ₂ O-CH ₂ CH ₂ -C (= O )-GGFG-NH-CH ₂ CH ₂ -C (= O) - (NH-DX), (maleimid-N-yl)-CH ₂ CH ₂ -C (= O) -NH-CH ₂ CH ₂ -O-CH ₂ CH ₂ -O-CH ₂ CH ₂ -O-CH ₂ CH ₂ -O-CH ₂ CH ₂ -C (= O) -GGFG-NH-CH ₂ CH ₂ -C (= O) - (NH-DX), (maleimid-N-yl) -CH ₂ CH ₂ -C (= O) -NH-CH ₂ CH ₂ -O-CH ₂ CH ₂ -O-CH ₂ CH ₂ -C (= O) -GGFG-NH- CH ₂ CH ₂ CH ₂ -C (= O) - (NH-DX), (maleimid-N-yl)-CH ₂ CH ₂ -C (= O) -NH-CH ₂ CH ₂ -O-CH ₂ CH ₂ -O-CH ₂ CH ₂ -O-CH ₂ CH ₂ -C (= O)-GGFG-NH-CH ₂ CH ₂ CH ₂ -C (= O) - (NH-DX), (maleimid-N-yl)-CH ₂ CH ₂ -C (= O) -NH-CH ₂ CH ₂ -O-CH ₂ CH ₂ -O-CH ₂ CH ₂ -O-CH ₂ CH ₂ -O-CH ₂ CH ₂ -C (= O) -GGFG-NH-CH ₂ CH ₂ CH ₂ -C (= O) - (NH-DX), (maleimid-N-yl) -CH ₂ CH ₂ -C (= O) -NH-CH ₂ CH ₂ -O-CH ₂ CH ₂ -O-CH ₂ CH ₂ -C (= O) -GGFG-NH- CH ₂ -O-CH ₂ -C (= O) - (NH-DX), (maleimid-N-yl)-CH ₂ CH ₂ -C (= O) -NH-CH ₂ CH ₂ -O-CH ₂ CH ₂ -O-CH ₂ CH ₂ -O-CH ₂ CH ₂ -C (= O)-GGFG-NH-CH ₂ -O-CH ₂ -C (= O) - (NH-DX), (maleimid-N-yl)-CH ₂ CH ₂ -C (= O) -NH-CH ₂ CH ₂ -O-CH ₂ CH ₂ -O-CH ₂ CH ₂ -O-CH ₂ CH ₂ -O-CH ₂ CH ₂ -C(=O)-GGFG-NH-CH ₂ -O-CH ₂ -C(=O)-(NH-DX), (maleimid-N-yl) -CH ₂ CH ₂ -C (= O) -NH-CH ₂ CH ₂ -O-CH ₂ CH ₂ -O-CH ₂ CH ₂ -C (= O) -GGFG-NH- CH ₂ CH ₂ -O-CH ₂ -C (= O) - (NH-DX), (maleimid-N-yl)-CH ₂ CH ₂ -C (= O) -NH-CH ₂ CH ₂ -O-CH ₂ CH ₂ -O-CH ₂ CH ₂ -O-CH ₂ CH ₂ -C (= O)-GGFG-NH-CH ₂ CH ₂ -O-CH ₂ -C (= O) - (NH-DX), (maleimid-N-yl)-CH ₂ CH ₂ -C (= O) -NH-CH ₂ CH ₂ O-CH ₂ CH ₂ O-CH ₂ CH ₂ O-CH ₂ CH ₂ O-CH ₂ CH ₂ - C(=O)-GGFG-NH-CH ₂ CH ₂ -O-CH ₂ -C(=O)-(NH-DX), (maleimide-N-yl)-CH ₂ CH ₂ -C (= O) -GGFG-(NH-DX), (maleimid-N-yl)-CH ₂ CH ₂ CH ₂ -C (= O) -GGFG- (NH-DX), (maleimid-N-yl)-CH ₂ CH ₂ CH ₂ CH ₂ -C (= O) -GGFG- (NH-DX), (maleimid-N-yl)-CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ -C (= O) -GGFG-(NH-DX), (maleimide-N-yl)-CH ₂ CH ₂ -C (= O) -DGGFG-(NH-DX), (maleimid-N-yl)-CH ₂ CH ₂ CH ₂ -C (= O) -DGGFG- (NH-DX), (maleimid-N-yl)-CH ₂ CH ₂ CH ₂ CH ₂ -C(=O)-DGGFG-(NH-DX) and (maleimid-N-yl)-CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ -C(=O)-DGGFG-(NH-DX). 4. The method according to claim 3, where the compound is selected from the following group: (maleimid-N-yl)-CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ -C (= O) -GGFG-NH-CH ₂ CH ₂ CH ₂ -C (= O) - (NH-DX), (maleimid-N-yl)-CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ -C (= O) -DGGFG-NH-CH ₂ CH ₂ CH ₂ -C (= O) - (NH-DX), (maleimid-N-yl)-CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ -C (= O) -GGFG-NH-CH ₂ -O-CH ₂ -C (= O) - (NH-DX), (maleimid-N-yl) -CH ₂ CH ₂ -C (= O) -NH-CH ₂ CH ₂ -O-CH ₂ CH ₂ -O-CH ₂ CH ₂ -C (= O) -GGFG-NH- CH ₂ CH ₂ CH ₂ -C (= O) - (NH-DX) and (maleimid-N-yl)-CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ -C(=O)-DGGFG-(NH-DX). 5. The method of claim 4, wherein the compound is (maleimide-N-yl)-CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ -C(=O)-GGFG-NH-CH ₂ CH ₂ CH ₂ -C( =O)-(NH-DX). 6. The method of claim 4 wherein the structure of the drug-linker group is (maleimide-N-yl)-CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ -C(=O)-DGGFG-NH-CH ₂ CH ₂ CH ₂ -C(=O)-(NH-DX). 7. The method of claim 4 wherein the structure of the drug-linker group is (maleimide-N-yl)-CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ -C(=O)-GGFG-NH-CH ₂ -O -CH ₂ -C(=O)-(NH-DX). 8. The method of claim 4, wherein the structure of the drug-linker group is (maleimide-N-yl)-CH ₂ CH ₂ -C(=O)-NH-CH ₂ CH ₂ -O-CH ₂ CH ₂ - O-CH ₂ CH ₂ -C(=O)-GGFG-NH-CH ₂ CH ₂ CH ₂ -C(=O)-(NH-DX). 9. The method of claim 4 wherein the structure of the drug-linker group is (maleimide-N-yl)-CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ -C(=O)-DGGFG-(NH-DX). 10. The method of claim 1, wherein the antibody-drug conjugate comprises a drug-linker structure represented by the following formula: -(succinimide-3-yl-N)-(CH ₂ )n ³ -C(=O)-L ² -L ^P -NH-(CH ₂ )n ¹ -L ^a -(CH ₂ )n ² -C (=O)-(NH-DX) or -(succinimide-3-yl-N)-(CH ₂ )n ³ -C(=O)-L ² -L ^P -(NH-DX), which is conjugated to an anti-HER3 antibody via a thioether bond that is formed at the site of a disulfide bond present in the hinge portion of the anti-HER3 antibody. 11. The method of claim 5, wherein the antibody-drug conjugate comprises a drug-linker structure represented by the following formula: -(succinimide-3-yl-N)-CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ -C (= O) -GGFG-NH-CH ₂ CH ₂ CH ₂ -C (= O) - (NH-DX) , which is conjugated to an anti-HER3 antibody via a thioether bond that is formed at the site of a disulfide bond present in the hinge portion of the anti-HER3 antibody. 12. The method of claim 6, wherein the antibody-drug conjugate comprises a drug-linker structure represented by the following formula: -(succinimide-3-yl-N)-CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ -C (= O) -DGGFG-NH-CH ₂ CH ₂ CH ₂ -C (= O) - (NH-DX) , which is conjugated to an anti-HER3 antibody via a thioether bond that is formed at the site of a disulfide bond present in the hinge portion of the anti-HER3 antibody. 13. The method of claim 7, wherein the antibody-drug conjugate comprises a drug-linker structure represented by the following formula: -(succinimide-3-yl-N)-CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ -C (= O) -GGFG-NH-CH ₂ -O-CH ₂ -C (= O) - (NH-DX ), which is conjugated to an anti-HER3 antibody via a thioether bond that is formed at the site of a disulfide bond present in the hinge portion of the anti-HER3 antibody. 14. The method of claim 8, wherein the antibody-drug conjugate comprises a drug-linker structure represented by the following formula: -(succinimide-3-yl-N)-CH ₂ CH ₂ -C(=O)-NH-CH ₂ CH ₂ -O-CH ₂ CH ₂ -O-CH ₂ CH ₂ -C(=O)-GGFG -NH-CH ₂ CH ₂ CH ₂ -C (= O) - (NH-DX), which is conjugated to an anti-HER3 antibody via a thioether bond that is formed at the site of a disulfide bond present in the hinge portion of the anti-HER3 antibody. 15. The method of claim 9, wherein the antibody-drug conjugate comprises a drug-linker structure represented by the following formula: -(succinimide-3-yl-N)-CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ -C (= O) -DGGFG- (NH-DX), which is conjugated to an anti-HER3 antibody via a thioether bond that is formed at the site of a disulfide bond present in the hinge portion of the anti-HER3 antibody. 16. The method according to any one of claims 1-15, wherein the anti-HER3 antibody comprises CDRH1 - CDRH3 and CDRL1 - CDRL3 U1-49, U1-53, U1-59, U1-7 or U1-9 in the heavy and light chains, respectively . 17. The method of any one of claims 1-15, wherein the anti-HER3 antibody comprises a heavy chain variable region and a light chain variable region U1-49, U1-53, U1-59, U1-7 or U1-9 in heavy and light chains, respectively. 18. The method according to any one of claims 1-15, wherein the anti-HER3 antibody comprises the amino acid sequences shown in SEQ ID Nos: 42 and 44, SEQ ID Nos: 54 and 56, SEQ ID Nos: 70 and 72, SEQ ID Nos. : 92 and 94 or SEQ ID Nos: 96 and 98 in the heavy and light chains, respectively. 19. The method of any one of claims 1-15, wherein the anti-HER3 antibody comprises the amino acid sequences shown in SEQ ID Nos: 583 and 584 in the heavy and light chains, respectively. 20. The method of claim 19, wherein the anti-HER3 antibody does not contain a lysine residue at the carboxyl terminus of the heavy chain. 21. The method according to any one of claims 1 to 20, wherein the average number of conjugated units of the selected single drug-linker structure per antibody is between 2 and 8. 22. The method according to any one of claims 1 to 20, wherein the average number of conjugated units of the selected single drug-linker structure per antibody is between 3 and 8. 23. A method for producing an antibody-drug conjugate represented by the following formula: where n is the average number of drug-linker conjugated units conjugated to the anti-HER3 antibody, and where the anti-HER3 antibody comprises the amino acid sequences represented by SEQ ID Nos: 583 and 584 of the heavy and light chains, respectively, comprising the step of treating the anti-HER3 antibody under reducing conditions and then reacting the anti-HER3 antibody with a compound of the following formula: . 24. The method of claim 23, wherein the anti-HER3 antibody does not contain a lysine residue on the heavy chain carboxyl ring. 25. The method according to any one of claims 23 or 24, where n takes values from the range from 2 to 8. 26. The method according to any one of claims 23 or 24, where n takes values from the range from 3 to 8.