Anti-her3 antibody-drug conjugate

Abstract

To provide an antitumor drug having excellent therapeutic effect, which is excellent in terms of antitumor effect and safety. Provided is an antibody-drug conjugate in which an antitumor compound represented by the following formula is conjugated to an anti-HER3 antibody via a linker having a structure represented by the formula: L1-L2-L-NH-(CH2)n1-La-(CH2)n2 C(=0)- or - - (the anti-HER3 antibody is connected to the terminal of L1, the antitumor compound is connected to the carbonyl group of -(CH2)n2-C(=0)- moiety or the C terminal of LP, with the nitrogen atom of the amino group at position 1 as a connecting position). NH2 Me O N F N HO Me 20 24 20 45 02 28 J un 2 02 4 A B S T R A C T 2 0 2 4 2 0 4 5 0 2 2 8 2 0 2 4 J u n . . . . N H M e 0 N F N O H O M e

Description

Title of Invention: ANTI-HER3 ANTIBODY-DRUG CONJUGATE Technical Field

[0001] The present invention relates to an antibody-drug conjugate having an anti-HER3 antibody and an antitumor drug conjugated to each other via a linker structure moiety, the conjugate being useful as an antitumor drug. Background Art

[0002] An antibody-drug conjugate (ADC) having a drug with cytotoxicity conjugated to an antibody which binds to an antigen expressed on a surface of cancer cells and capable of cellular internalization (the antibody which binds to the angtigen is also capable of cellular internalization), can deliver the drug selectively to the cancer cells and is thus expected to cause accumulation of the drug in the cancer cells and to kill the cancer cells (see, Non Patent Literatures 1 to 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. Further, 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). The drugs contained in ADCs which have been approved until now target DNA or tubulin.

[0003] As an antitumor, low-molecular-weight compounds, camptothecin derivatives, which inhibit topoisomerase I to exhibit an antitumor effect, are known. Among them, an antitumor compound represented by the formula below (exatecan, chemical name: (lS,9S)-l-amino-9-ethyl-5-fluoro-2,3-dihydro-9-hydroxy-4-methyl-lH,12H-benzo[de] pyrano[3',4':6,7]indolizino[l,2-b]quinolin-10,13(9H,15H)-dione) is a water soluble derivative of camptothecin (Patent Literature 1 and 2).

[0004] [Chern. 1] Me 2024204502   28 Jun 2024

[0005] Unlike irinotecan currently used in clinical settings, this compound does not require activation by an enzyme for exerting its antitumor effect. Further, compared to SN-38 as a main pharmaceutically active ingredient of irinotecan and topotecan also used in clinical settings, it has higher inhibitory activity on topoisomerase I and has higher cytocidal activity in vitro against various cancer cells. In particular, it exhibits the effect against cancer cells which have resistance to SN-38 or the like due to expression of P-glycoprotein. Further, in a mouse model with a human tumor subcutaneously transplanted, it exhibited a potent antitumor effect, and thus has undergone the clinical studies, but has not been put on the market yet (see, Non Patent Literatures 5 to 10). It remains unclear whether or not exatecan functions effectively as an ADC.

[0006] DE-310 is a complex in which exatecan is conjugated to a biodegradable car boxymethyldextran polyalcohol polymer via a GGFG peptide spacer (Patent Literature 3). By making exatecan into a form of a polymer prodrug, a high blood retention property can be maintained and also a high penetration property to a tumor area is passively increased by utilizing the increased permeability of newly formed tumor vessels and retention property in tumor tissues. With DE-310, the peptide spacer is cleaved by an enzyme to continuously release exatecan as a main active ingredient and exatecan with glycine bonded to an amino group, and as a result, the pharmacokinetics are improved. According to various tumor evaluation models in non-clinical studies, it was found that higher effectiveness was obtained by DE-310 than exatecan administered alone even though the total amount of exatecan contained therein is lower than the case of administration of exatecan alone. A clinical study was conducted for DE-310, and effective cases were confirmed. There is also a report suggesting that the main active ingredient accumulates in a tumor than in normal tissues. However, there is also a report indicating that the accumulation of DE-310 and the main active ingredient in a tumor is not much different from the accumulation in normal tissues, and thus no passive targeting is observed in humans (see, Non Patent Literatures 11 to 14). As a result, DE-310 was not also commercialized, and it remains unclear whether or not exatecan effectively functions as a drug oriented for such targeting.

[0007] As a compound relating to DE-310, a complex in which a structure moiety represented by -NH-(CH2)4-C(=O)- is inserted between -GGFG -spacer and exatecan to form -GGFG-NH-(CH2)4-C(=O)- used as a spacer structure is also known (Patent Literature 4). However, the antitumor effect of the complex is not known at all.

[0008] The human epidermal growth factor receptor 3 (also known as HER3 and ErbB3) is a receptor protein tyrosine kinase and belongs to the epidermal growth factor receptor (EGFR) subfamily of receptor protein tyrosine kinases, which also includes HER1 (also known EGFR), HER2, and HER4 (see Non Patent Literatures 15 to 17). As with the prototypical epidermal growth factor receptor, the transmembrane receptor HER3 2024204502   28 Jun 2024 consists of an extracellular ligand-binding domain (ECD), a dimerization domain within the ECD, a transmembrane domain, and a carboxyl-terminal phosphorylation domain. HER1, HER2, and HER4 carry an intracellular protein tyrosine kinase domain (TKD) in addition to these domains, while HER3 lacks this domain and is thus unable to be autophosphorylated. The ligand Heregulin (HRG) binds to the extracellular domain of HER3 and activates the receptor-mediated signaling pathway by promoting dimerization with other human epidermal growth factor receptor (HER) family members and transphosphorylation of its intracellular domain. The dimer formation of HER3 with other HER family members expands the signaling potential of HER3 and serves as 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 HER family members. HER3 is overexpressed in several types of cancers such as breast, gastrointestinal, and pancreatic cancers. Interestingly, a correlation between the expression of HER2 / HER3 and the progression from a non-invasive stage to an invasive stage has been shown (see Non Patent Literatures 18 to 20). Accordingly, agents that interfere with HER3-mediated signaling are desirable. Anti-HER3 antibodies and immunoconjugates thereof have been reported in, for example, Patent Literatures 5 to 10, respectively. Citation List Patent Literature

[0009] [PTL 1] Japanese Patent Laid-Open No. 5-59061 [PTL 2] Japanese Patent Laid-Open No. 8-337584 [PTL 3] International Publication No. WO 1997 / 46260 [PTL 4] International Publication No. WO 2000 / 25825 [PTL 5] U.S. Patent No. 5968511 [PTL 6] U.S. Patent No. 5480968 [PTL 7] International Publication No. WO 2003 / 013602 [PTL 8] International Publication No. WO 2007 / 077028 [PTL 9] International Publication No. WO 2008 / 100624 [PTL 10] International Publication No. WO 2012 / 019024 Non Patent Literature

[0010] [NPL 1] Ducry, L., et al., Bioconjugate Chern. (2010) 21, 5-13. [NPL 2] Alley, S. C., et al., Current Opinion in Chemical Biology (2010) 14, 529-537. [NPL 3] Damle N.K. Expert Opin. Biol. Ther. (2004) 4, 1445-1452. [NPL 4] Senter P. D., et al., Nature Biotechnology (2012) 30, 631-637. 2024204502   28 Jun 2024 [NPL 5] Kumazawa, E., Tohgo, A., Exp. Opin. Invest. Drugs (1998) 7, 625-632. [NPL 6] Mitsui, I., et al., Jpn J. Cancer Res. (1995) 86, 776-786. [NPL 7] Takiguchi, S., et al., Jpn J. Cancer Res. (1997) 88, 760-769. [NPL 8] Joto, N. et al., Int J Cancer (1997) 72, 680-686. [NPL 9] Kumazawa, E. et al., Cancer Chemother. Pharmacol. (1998) 42, 210-220. [NPL 10] De Jager, R„ et al., Ann N Y Acad Sci (2000) 922, 260-273. [NPL 11] Inoue, K. et al. Polymer Drugs in the Clinical Stage, Edited by Maeda et al., (2003)145-153. [NPL 12] Kumazawa, E. et al., Cancer Sci (2004) 95, 168-175. [NPL 13] Soepenberg, O. et al., Clinical Cancer Research, (2005) 11, 703-711. [NPL 14] Wente M. N. et al., Investigational New Drugs (2005) 23, 339-347. [NPL 15] Plowman, et al., Proc. Natl. Acad. Sci. U.S.A. (1990) 87, 4905-4909. [NPL 16] Kraus et al., Proc. Natl. Acad. Sci. U.S.A. (1989) 86, 9193-9197. [NPL 17] Kraus et al., Proc. Natl. Acad. Sci. U.S.A. (1993) 90, 2900-2094. [NPL 18] Alimandi et al., Oncogene (1995) 10, 1813-1821. [NPL 19] DeFazio et al., Int. J. Cancer (2000) 87, 487-498. [NPL 20] Nadiu et al., Br. J. Cancer (1998) 78, 1385-1390. Summary of Invention Technical Problem

[0011] With regard to the treatment of tumor using an antibody, an insufficient antitumor effect may be observed even when the antibody recognizes an antigen and binds to tumor cells, and thus a more effective antitumor antibody is sometimes needed. Further, many antitumor low-molecular-weight compounds have a problem in safety like side effect and toxicity even the compounds have an excellent antitumor effect. As such, it remains as a subject to achieve a superior therapeutic effect by further enhancing the safety. Thus, an object of the present invention is to provide an antitumor drug having an excellent therapeutic effect, which is excellent in terms of antitumor effect and safety. Solution to Problem

[0012] The inventors thought that, since the anti-HER3 antibody is an antibody capable of targeting tumor cells, that is, it is an antibody having a property of recognizing tumor cells, a property of binding to tumor cells, a property of internalizing in tumor cells, a cytocidal activity against tumor cells, or the like, when exatecan as an antitumor compound is converted into an antibody-drug conjugate by conjugation to the antibody via a linker structure moiety, the antitumor compound can be more surely delivered to tumor cells to specifically exhibit the antitumor effect of the compound in tumor cells, and thus the antitumor effect can be surely exhibited and also an enhanced cytocidal 2024204502   28 Jun 2024 effect of the anti-HER3 antibody is expected, and a dose of the antitumor compound can be reduced compared to a case of administering the compound alone, and thus an influence of the antitumor compound on normal cells can be alleviated so that higher safety can be achieved. In this connection, the inventors created a linker with a specific structure and succeeded in obtaining an antibody-drug conjugate in which the anti-HER3 antibody and exatec an are conjugated to each other via the linker, and confirmed an excellent antitumor effect exhibited by the conjugate to thereby complete the present invention.

[0013] Specifically, the present invention relates to the followings. [1] An antibody-drug conjugate wherein an antitumor compound represented by the following formula [Chern. 2] /  0 Me is conjugated to an anti-HER3 antibody by a thioether bond which is formed at a disulfide bond moiety present in a hinge part of the anti-HER3 antibody via a linker having a structure represented by the following formula: -L1-L2-Lp-NH-(CH2)n1-L“-(CH2)n2-C(=O)- or -L1-L2-Lp-.

[0014] Here, the anti-HER3 antibody is connected to the terminal of L1, the antitumor compound is connected to the carbonyl group of -(CH2)n2-C(=O)- moiety or the C terminal of Lp, with the nitrogen atom of the amino group at position 1 as a connecting position. In the formula, n1 represents an integer of 0 to 6, n2 represents an integer of 0 to 5, L1 represents -(Succinimid-3-yl-N)-(CH2)n3-C(=O)-, wherein n3 represents an integer of 2 to 8, L2 represents -NH-(CH2CH2-O)n4-CH2CH2-C(=O)- or a single bond, wherein n4 represents an integer of 1 to 6, Lp represents a peptide residue consisting of 2 to 7 amino acids, La represents -O- or a single bond, -(Succinimid-3-yl-N)- has a structure represented by the following formula: 2024204502   28 Jun 2024 [Chern. 3] 0 0 which is connected to the anti-HER3 antibody at position 3 thereof and is connected on the nitrogen atom at position 1 to a methylene group in the linker structure containing this structure.

[0015] The present invention further relates to each of the followings. [2] The antibody-drug conjugate according to [1], wherein the peptide residue of Lp is a peptide residue comprising an amino acid selected from phenylalanine, glycine, valine, lysine, citrulline, serine, glutamic acid, and aspartic acid. [3] The antibody-drug conjugate according to [1] or [2], wherein Lp is a peptide residue selected from the following group -GGF-, -DGGF-, -(D-)D-GGF-, -EGGF-, -GGFG-, -SGGF-, -KGGF-, -DGGFG-, -GGFGG-, -DDGGFG-, -KDGGFG-, and -GGFGGGF-; (wherein, "(D-)D" represents D-aspartic acid). [4] The antibody-drug conjugate according to [1] or [2], wherein Lp is a peptide residue comprising 4 or 5 amino acids. [5] The antibody-drug conjugate according to any one of [1] to [4], wherein Lp is -GGFG- or -DGGFG-. [6] The antibody-drug conjugate according to any one of [1] to [4], wherein Lp is -GGFG-.

[0016] [7] The antibody-drug conjugate according to any one of [1] to [6], wherein n3 is an integer of 2 to 5 and L2 is a single bond. [8] The antibody-drug conjugate according to any one of [1] to [7], wherein the 2024204502   28 Jun 2024 linker is -L1-L2-Lp-NH-(CH2)n1-La-(CH2)n2-C(=O)-. [9] The antibody-drug conjugate according to [8], wherein n3 is an integer of 2 to 5, L2 is -NH-(CH2CH2-O)n4-CH2CH2-C(=O)-, and n4 is 2 or 4.

[10] The antibody-drug conjugate according to [8] or [9], wherein -NH-(CH2)n'-La -(CH2)n2-C(=O)- is a partial structure having chain length of 4 to 7 atoms.

[11] The antibody-drug conjugate according to [8] or [9], wherein -NH-(CH2)n'-La -(CH2)n2-C(=O)- is a partial structure having chain length of 5 or 6 atoms.

[12] The antibody-drug conjugate described in

[10] or

[11] , wherein -NH-(CH2)n'-La -(CH2)n2-C(=O)- is -NH-CH2CH2-C(=O)-, -NH-CH2CH2CH2-C(=O)-, -NH-CH2CH2CH2CH2-C(=O)-, -NH-CH2CH2CH2CH2CH2-C(=O)-, -NH-CH2-O-CH2-C(=O)-, or -NH-CH2CH2-O-CH2-C(=O)-.

[13] The antibody-drug conjugate according to

[12] , wherein -NH-(CH2)n'-La-(CH2)n2 -C(=O)- is any one of the followings: -NH-CH2CH2CH2-C(=O)-, -NH-CH2-O-CH2-C(=O)-, or -NH-CH2CH2-O-CH2-C(=O)-.

[14] The antibody-drug conjugate according to any one of [1] to [5], wherein the linker is -U-L2-! / -.

[15] The antibody-drug conjugate according to

[14] , wherein Lp is -DGGFG-.

[16] The antibody-drug conjugate according to

[15] , wherein n3 is an integer of 2 to 5 and L2 is a single bond.

[0017]

[17] The antibody-drug conjugate according to [1], wherein the drug-linker structure moiety in which a drug is bound to -L1-L2-Lp-NH-(CH2)n1-La-(CH2)n2-C(=O)- or -L'-L2 -Lp- is one drug-linker structure selected from the following group: -(Succinimid-3-yl-N)-CH2CH2-C(=O)-GGFG-NH-CH2CH2-C(=O)-(NH-DX), -(Succinimid-3-yl-N)-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-(NH-DX), -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2-C(=O)-(NH- DX), -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-( NH-DX), -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2CH2CH2- C(=0)-(NH-DX), -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2-O-CH2-C(=O)-( NH-DX), 2024204502   28 Jun 2024 -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2-O-CH2-C(=O) -(NH-DX), -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-DGGFG-NH-CH2CH2-C(=O)-(NH-DX), -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-DGGFG-NH-CH2CH2CH2-C(=O)-( NH-DX), -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-DGGFG-NH-CH2CH2CH2CH2CH2-C(=O)-(NH-DX), -(Succinimid-3-yl-N)-CH2CH2-C(=O)-NH-CH2CH2-O-CH2CH2-O-CH2CH2-C(=O)-GG FG-NH-CH2CH2-C(=O)-(NH-DX), -(Succinimid-3-yl-N)-CH2CH2-C(=O)-NH-CH2CH2-O-CH2CH2-O-CH2CH2-C(=O)-GG FG-NH-CH2CH2CH2-C(=O)-(NH-DX), -(Succinimid-3-yl-N)-CH2CH2-C(=O)-NH-CH2CH2-O-CH2CH2-O-CH2CH2-O-CH2CH2 -O-CH2CH2-C(=O)-GGFG-NH-CH2CH2-C(=O)-(NH-DX), -(Succinimid-3-yl-N)-CH2CH2-C(=O)-NH-CH2CH2-O-CH2CH2-O-CH2CH2-O-CH2CH2 -O-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-(NH-DX), -(Succinimid-3-yl-N)-CH2CH2-C(=O)-GGFG-(NH-DX), -(Succinimid-3-yl-N)-CH2CH2-C(=O)-DGGFG-(NH-DX), -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-(NH-DX), -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-DGGFG-(NH-DX).

[0018] In the above, -(Succinimid-3-yl-N)- has a structure represented by the following formula: [Chern. 4] O which is connected to the anti-HER3 antibody at position 3 and is connected to a methylene group in the linker structure containing it on the nitrogen atom at position 1, -(NH-DX) represents a group represented by the following formula, wherein the nitrogen atom of the amino group at position 1 is the connectig position, 2024204502   28 Jun 2024 [Chem.5] Me -GGFG- represents a tetrapeptide residue of -Gly-Gly-Phe-Gly- and -DGGFG-represents a pentapeptide residue of -Asp-Gly-Gly-Phe-Gly-.

[0019]

[18] The antibody-drug conjugate described in [1], wherein the drug-linker structure moiety having a drug bonded to -L1-L2-Lp-NH-(CH2)n1-La-(CH2)n2-C(=O)- is one druglinker structure selected from the following group: -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-( NH-DX), -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-DGGFG-NH-CH2CH2CH2-C(=O) -(NH-DX), -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2-O-CH2-C(=O)-( NH-DX), -(Succinimid-3-yl-N)-CH2CH2-C(=O)-NH-CH2CH2-O-CH2CH2-O-CH2CH2-C(=O)-G GFG-NH-CH2CH2CH2-C(=O)-(NH-DX), -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-DGGFG-(NH-DX).

[0020] Here, -(Succinimid-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: 2024204502   28 Jun 2024 [Chern. 6] Me conjugated to an anti-HER3 antibody by a thioether bond which is formed at a disulfide bond moiety present in a hinge part of the anti-HER3 antibody via a linker having a structure represented by the following formula: -L1-L2-Lp-NH-(CH2)n1-La-(CH2)n2-C(=O)-. Here, the anti-HER3 antibody is connected to the terminal of L1 and the antitumor compound is connected to the carbonyl group of -(CH2)n2-C(=O)- moiety. In the formula, n1 represents an integer of 0 to 6, n2 represents an integer of 0 to 5, L1 represents -(Succinimid-3-yl-N)-(CH2)n3-C(=O)-, wherein n3 represents an integer of 2 to 8, L2 represents -NH-(CH2CH2-O)n4-CH2CH2-C(=O)- or a single bond, wherein n4 represents an integer of 1 to 6, Lp represents a tetrapeptide residue of -GGFG-, La represents -O- or a single bond, -(Succinimid-3-yl-N)- has a structure represented by the following formula: [Chern. 7] which is connected to the anti-HER3 antibody at position 3 thereof and binds on the nitrogen atom at position 1 to a methylene group in a linker structure containing this structure.

[0022]

[20] The antibody-drug conjugate according to

[19] , wherein n1 is 3, n2 is 0, n3 is 2, L 2 is -NH-(CH2CH2-O)n4-CH2CH2-C(=O)-, n4 is 2, and La is a single bond, or n1 is 1, n2 is 1, n3 is 5, L2 is a single bond, and La is -O-, or 2024204502   28 Jun 2024 n1 is 2, n2 is 1, n3 is 5, L2 is a single bond, and La is -O-.

[21] The antibody-drug conjugate according to

[19] or

[20] , wherein n3 is 2 or 5 and L2 is a single bond.

[22] The antibody-drug conjugate according to

[19] or

[20] , wherein n3 is 2 or 5, L2 is -NH-(CH2CH2-O)n4-CH2CH2-C(=O)-, and n4 is 2 or 4.

[23] The antibody-drug conjugate described in any one of

[19] to

[22] , wherein -NH-(CH2)n1-La-(CH2)n2-C(=O)- is -NH-CH2CH2CH2-C(=O)-, -NH-CH2-O-CH2-C(=O)-, or -NH-CH2CH2-O-CH2-C(=O)-.

[0023]

[24] The antibody-drug conjugate described in any one of

[19] to

[23] , wherein the drug-linker structure moiety having a drug bonded to -L1-L2-Lp-NH-(CH2)n1-La-(CH2)n 2-C(=O)- is one drug-linker structure selected from the following group: -(Succinimid-3-yl-N)-CH2CH2-C(=O)-GGFG-NH-CH2CH2-C(=O)-(NH-DX), -(Succinimid-3-yl-N)-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-(NH-DX), -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2-C(=O)-(NH- DX), -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-( NH-DX), -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2CH2CH2- C(=0)-(NH-DX), -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2-O-CH2-C(=O)-( NH-DX), -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2-O-CH2-C(= O)-(NH-DX), -(Succinimid-3-yl-N)-CH2CH2-C(=O)-NH-CH2CH2-O-CH2CH2-O-CH2CH2-C(=O)-G GFG-NH-CH2CH2-C(=O)-(NH-DX), -(Succinimid-3-yl-N)-CH2CH2-C(=O)-NH-CH2CH2-O-CH2CH2-O-CH2CH2-C(=O)-G GFG-NH-CH2CH2CH2-C(=O)-(NH-DX), -(Succinimid-3-yl-N)-CH2CH2-C(=O)-NH-CH2CH2-O-CH2CH2-O-CH2CH2-O-CH2C H2-O-CH2CH2-C(=O)-GGFG-NH-CH2CH2-C(=O)-(NH-DX); -(Succinimid-3-yl-N)-CH2CH2-C(=O)-NH-CH2CH2-O-CH2CH2-O-CH2CH2-O-CH2C H2-O-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-(NH-DX),

[0024] In the above, -(Succinimid-3-yl-N)- has a structure represented by the following formula: 2024204502   28 Jun 2024 [Chem.8] 0 0 which is connected to the anti-HER3 antibody at position 3 thereof and is connected on the nitrogen atom at position 1 to a methylene group in a linker structure containing this structure. -(NH-DX) represents a group represented by the following formula, wherein the nitrogen atom of the amino group at position 1 is the connectig position: [Chem.9] / O Me -GGFG- represents a tetrapeptide residue of -Gly-Gly-Phe-Gly-.

[0025]

[25] The antibody-drug conjugate described in any one of

[19] to

[23] , wherein the drug-linker structure moiety having a drug connected to -L1-L2-Lp-NH-(CH2)n1-La-(CH 2)n2-C(=O)- is one drug-linker structure selected from the following group: -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2-O-CH2-C(=O)-( NH-DX), -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2-O-CH2-C(= O)-(NH-DX), -(Succinimid-3-yl-N)-CH2CH2-C(=O)-NH-CH2CH2-O-CH2CH2-O-CH2CH2-C(=O)-G GFG-NH-CH2CH2CH2-C(=O)-(NH-DX). In the above, -(Succinimid-3-yl-N)-, -(NH-DX), and -GGFG- are as defined above.

[0026]

[26] The antibody-drug conjugate according to any one of [1] to

[25] , wherein the average number of units of the selected one drug-linker structure conjugated per antibody is in a range of from 1 to 10.

[27] The antibody-drug conjugate according to any one of [1] to

[25] , wherein the 2024204502   28 Jun 2024 average number of units of the selected one drug-linker structure conjugated per antibody is in a range of from 2 to 8.

[28] The antibody-drug conjugate according to any one of [1] to

[25] , wherein the average number of units of the selected one drug-linker structure conjugated per antibody is in a range of from 3 to 8.

[0027]

[29] A medicine comprising the antibody-drug conjugate according to any one of [1] to

[28] , a salt thereof or a hydrate thereof.

[30] An antitumor medicine and / or anticancer medicine comprising the antibodydrug conjugate according to any one of [1] to

[28] , a salt thereof or a hydrate thereof.

[31] The antitumor medicine and / or anticancer medicine according to

[30] , which is applied to lung cancer, kidney cancer, urothelial cancer, colorectal cancer, prostate cancer, glioblastoma multiforme, ovarian cancer, pancreatic cancer, breast cancer, melanoma, liver cancer, bladder cancer, stomach 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, uterus cancer, salivary cancer, renal cancer, vulval cancer, thyroid cancer, hepatic carcinoma, anus carcinoma, or penis cancer.

[32] A pharmaceutical composition comprising the antibody-drug conjugate according to any one of [1] to

[28] , a salt thereof or a hydrate thereof as an active component, and a pharmaceutically acceptable formulation component.

[33] The pharmaceutical composition according to

[32] , which is applied to lung cancer, kidney cancer, urothelial cancer, colorectal cancer, prostate cancer, glioblastoma multiforme, ovarian cancer, pancreatic cancer, breast cancer, melanoma, liver cancer, bladder cancer, stomach 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, uterus cancer, salivary cancer, renal cancer, vulval cancer, thyroid cancer, hepatic carcinoma, anus carcinoma, or penis cancer.

[34] A method for treating a tumor and / or cancer comprising administering the antibody-drug conjugate according to any one of [1] to

[28] , a salt thereof or a hydrate thereof.

[35] The medicine according to

[29] , the antitumor medicine and / or anticancer medicine according to

[30] or

[31] , the pharmaceutical composition according to

[32] or

[33] , or the treatment method according to

[34] , which is used in administration in combination with an additional medicine.

[36] The pharmaceutical composition according to

[32] or

[33] , further comprising 2024204502   28 Jun 2024 even an additional medicine as an active ingredient.

[0028]

[35] A method for producing an antibody-drug conjugate comprising reacting a compound represented by the following formula: (maleimid-N-yl)-(CH2)n3-C(=O)-L2-Lp-NH-(CH2)n1-La-(CH2)n2-C(=O)-(NH-DX) or (maleimid-N-yl)-(CH2)n3-C(=O)-L2-Lp-(NH-DX) with an anti-HER3 antibody or a reactive derivative thereof and conjugating a druglinker moiety to the antibody by a method for forming a thioether bond on a disulfide bond moiety present at a hinge part of the antibody.

[0029] In the formula, n3 represents an integer of 2 to 8, L2 represents -NH-(CH2CH2-O)n4-CH2CH2-C(=O)- or a single bond wherein n4 represents an integer of 1 to 6, Lp represents a peptide residue consisting of 2 to 7 amino acids selected from phenylalanine, glycine, valine, lysine, citrulline, serine, glutamic acid, and aspartic acid, n1 represents an integer of 0 to 6, n2 represents an integer of 0 to 5, La represents -O- or a single bond, (maleimid-N-yl)- is a group represented by the following formula and has a nitrogen atom as a connecting position. [Chern. 10] -(NH-DX) represents a group represented by the following formula, wherein the nitrogen atom of the amino group at position 1 is the connectig position: [Chern. 11] Me 2024204502   28 Jun 2024

[0030]

[36] The production method described in

[35] , wherein the method for conjugating a drug-linker moiety to an anti-HER3 antibody is a method of reducing the antibody for conversion into a reactive derivative.

[0031]

[37] The production method described in

[35] or

[36] , wherein the average number of units of the selected one drug-linker structure conjugated per antibody is in the range of from 1 to 10.

[38] The production method described in

[35] or

[36] , wherein the average number of units of the selected one drug-linker structure conjugated per antibody is in the range of from 2 to 8.

[39] The production method described in

[35] or

[36] , wherein the average number of units of the selected one drug-linker structure conjugated per antibody is in the range of from 3 to 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 obtained by forming a thioether bond at a disulfide bond site present in a hinge part of an anti-HER3 antibody, wherein the anti-HER3 antibody is treated in a reducing condition and thereafter reacted with a compound selected from the compound group shown below: (maleimid-N-yl)-CH2CH2-C(=O)-GGFG-NH-CH2CH2-C(=O)-(NH-DX), (maleimid-N-yl)-CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2-C(=O)-(NH-DX), (maleimid-N-yl)-CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2-C(=O)-(NH-DX), (maleimid-N-yl)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2-C(=O)-(NH-DX), (maleimid-N-yl)-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-(NH-DX), (maleimid-N-yl)-CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-(NH-DX), (maleimid-N-yl)-CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-(NH-DX), (maleimid-N-yl)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-(NH- DX), (maleimid-N-yl)-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2CH2CH2-C(=O)-(NH-DX), (maleimid-N-yl)-CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2CH2CH2-C(=O)-(NH- DX), (maleimid-N-yl)-CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2CH2CH2-C(=O)-( NH-DX), (maleimid-N-yl)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2CH2CH2-C(= O)-(NH-DX), (maleimid-N-yl)-CH2CH2CH2CH2CH2-C(=O)-DGGFG-NH-CH2CH2-C(=O)-(NH-D X), (maleimid-N-yl)-CH2CH2CH2CH2CH2-C(=O)-DGGFG-NH-CH2CH2CH2 -C(=0)-(NH-DX), (maleimid-N-yl)-CH2CH2CH2CH2CH2-C(=O)-DGGFG-NH-CH2CH2CH2CH2CH2-C( 2024204502   28 Jun 2024 2024204502   28 Jun 2024 (maleimid-N-yl)-CH2CH2CH2CH2-C(=O)-GGFG-(NH-DX), (maleimid-N-yl)-CH2CH2CH2CH2CH2-C(=O)-GGFG-(NH-DX), (maleimid-N-yl)-CH2CH2-C(=O)-DGGFG-(NH-DX), (maleimid-N-yl)-CH2CH2CH2-C(=O)-DGGFG-(NH-DX), (maleimid-N-yl)-CH2CH2CH2CH2-C(=O)-DGGFG-(NH-DX), or (maleimid-N-yl)-CH2CH2CH2CH2CH2-C(=O)-DGGFG-(NH-DX).

[0033] In the above, (maleimid-N-yl)- is a group represented by the following formula: [Chern. 12] which has a nitrogen atom as a connecting position. -(NH-DX) represents a group represented by the following formula, the nitrogen atom of the amino group at position 1 being a connecting position. [Chern. 13] / O Me -GGFG- represents a tetrapeptide residue of -Gly-Gly-Phe-Gly- and -DGGFG-represents pentapeptide residue of -Asp-Gly-Gly-Phe-Gly-.

[0034]

[42] An antibody-drug conjugate obtained by forming a thioether bond at a disulfide bond site present in a hinge part of an anti-HER3 antibody, and characterized by treating the anti-HER3 antibody with a reducing condition and thereafter reacting with a compound selected from the compound group shown below: (maleimid-N-yl)-CH2CH2-C(=O)-NH-CH2CH2-O-CH2CH2-O-CH2CH2-C(=O)-GGFG -NH-CH2CH2CH2-C(=O)-(NH-DX), (maleimid-N-yl)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2-O-CH2-C(=O)-(NH-DX), or 2024204502   28 Jun 2024 (maleimid-N-yl)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2-O-CH2-C(=O)-(NH -DX). In the above, (maleimid-N-yl)-, -(NH-DX), and -GGFG- are as defined above.

[0035]

[43] The antibody-drug conjugate according to

[41] or

[42] , wherein an average conjugated number of the selected one drug-linker structure per antibody is in a range of from 1 to 10.

[44] The antibody-drug conjugate according to

[41] or

[42] , wherein an average conjugated number of the selected one drug-linker structure per antibody is in a range of from 2 to 8.

[45] The antibody-drug conjugate according to

[41] or

[42] , wherein an average conjugated number of the selected one drug-linker structure per antibody is in a range of from 3 to 8.

[46] A medicine comprising the antibody-drug conjugate according to any one of

[40] to

[45] , a salt thereof or a hydrate thereof.

[47] An antitumor medicine and / or anticancer medicine comprising the antibodydrug conjugate according to any one of

[40] to

[45] , a salt thereof or a hydrate thereof.

[48] The antitumor medicine and / or anticancer medicine according to

[47] , which is applied to lung cancer, kidney cancer, urothelial cancer, colorectal cancer, prostate cancer, glioblastoma multiforme, ovarian cancer, pancreatic cancer, breast cancer, melanoma, liver cancer, bladder cancer, stomach 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, uterus cancer, salivary cancer, renal cancer, vulval cancer, thyroid cancer, hepatic carcinoma, anus carcinoma, or penis cancer.

[49] A pharmaceutical composition comprising the antibody-drug conjugate according to any one of

[40] to

[45] , a salt thereof or a hydrate thereof as an active component, and a pharmaceutically acceptable formulation component.

[50] The pharmaceutical composition according to

[49] , which is applied to lung cancer, kidney cancer, urothelial cancer, colorectal cancer, prostate cancer, glioblastoma multiforme, ovarian cancer, pancreatic cancer, breast cancer, melanoma, liver cancer, bladder cancer, stomach 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, uterus cancer, salivary cancer, renal cancer, vulval cancer, thyroid cancer, hepatic carcinoma, anus carcinoma, or penis cancer.

[51] A method for treating a tumor and / or cancer comprising administering the 2024204502   28 Jun 2024 antibody-drug conjugate according to any one of

[40] to

[45] , a salt thereof or a hydrate thereof.

[52] The medicine according to

[46] , the antitumor medicine and / or anticancer medicine according to

[47] or

[48] , the pharmaceutical composition according to

[49] or

[50] , or the treatment method according to

[51] , which is used in administration in combination with an additional medicine.

[53] The pharmaceutical composition according to

[49] or

[50] , further comprising even an additional medicine as an active ingredient. Advantageous Effects of Invention

[0036] With an anti-HER3 antibody-drug conjugate having an antitumor compound exatecan conjugated via a linker with a specific structure, an excellent antitumor effect and safety can be achieved. Brief Description of Drawings

[0037] [fig.l]Figure 1 shows the full-length amino acid sequence of a heavy chain of anti-HER3 human antibody Ul-59 (SEQ ID NO: 583). [fig.2]Figure 2 shows the full-length amino acid sequence of a light chain of anti-HER3 human antibody Ul-59 (SEQ ID NO: 584). [fig.3]Figure 3 shows the mean fluorescence intensity of HCC1569 treated with serial dilutions of Ul-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 Ul-59 or varied antibody-drug conjugates. HER3 or phosphorylated HER3 was evaluated by Western blotting, panActin was detected as an electrophoresis control. [fig.5]Figure 5 shows an average value of reduction in HER3 expression on the surface of HCC1569 cells treated with Ul-59 or each antibody-drug conjugate (37C ("C" represents "degrees Celsius"), 1 hr). [fig.6]Figure 6 shows results of a test on the inhibition of mitogenic or survival signals by each HER3 antibody-drug conjugate in a human breast cancer line (HCC1569). Figure 6A shows cell growth or survival derived from the antibody-drug conjugate in the presence of 10% FBS. The data is indicated by mean + / - standard deviation of triplicates. The ordinate depicts a luminescence value indicating the ATP activity of each sample. The abscissa depicts the concentration of each antibody-drug conjugate. Figure 6B shows the rate of reduction in luminescence caused by antibody-drug conjugate treatment when the luminescence of an untreated group was defined as 100%. [fig.7]Figure 7 shows results of a test on the inhibition of mitogenic or survival signals by each HER3 antibody-drug conjugate in a human breast cancer line (MDA-MB 453). 2024204502   28 Jun 2024 Figure 7A shows cell growth or survival derived from the antibody-drug conjugate in the presence of 10% FBS. The ordinate depicts a luminescence value indicating the ATP activity of each sample. The abscissa depicts the concentration of each antibodydrug conjugate. The data is indicated by mean + / - standard deviation of triplicates. Figure 7B shows the rate of reduction in luminescence caused by antibody-drug conjugate treatment when the luminescence of an untreated group was defined as 100%. [fig.8]Figure 8 shows results of a test on the inhibition of mitogenic or survival signals by each HER3 antibody-drug conjugate in a human melanoma line (A375). Figure 8A shows cell growth or survival derived from the antibody-drug conjugate in the presence of 10% FBS. The ordinate depicts a luminescence value indicating the ATP activity of each sample. The abscissa depicts the concentration of each antibody-drug conjugate. The data is indicated by mean + / - standard deviation of triplicates. Figure 8B shows the rate of reduction in luminescence caused by antibody-drug conjugate treatment when the luminescence of an untreated group was defined as 100%. [fig.9]Figure 9 shows results of a test on the inhibition of mitogenic or survival signals by each HER3 antibody-drug conjugate in a human colorectal cancer line (HT29). Figure 9A shows cell growth or survival derived from the antibody-drug conjugate in the presence of 10% FBS. The ordinate depicts a luminescence value indicating the ATP activity of each sample. The abscissa depicts the concentration of each antibodydrug conjugate. The data is indicated by mean + / - standard deviation of triplicates. Figure 9B shows the rate of reduction in luminescence caused by antibody-drug conjugate treatment when the luminescence of an untreated group was defined as 100%. [fig. 10]Figure 10 shows results of a test on the inhibition of mitogenic or survival signals by each HER3 antibody-drug conjugate in a human lung cancer line (A549). Figure 10A shows cell growth or survival derived from the antibody-drug conjugate in the presence of 10% FBS. The ordinate depicts a luminescence value indicating the ATP activity of each sample. The abscissa depicts the concentration of each antibodydrug conjugate. The data is indicated by mean + / - standard deviation of triplicates. Figure 10B shows the rate of reduction in luminescence caused by antibody-drug conjugate treatment when the luminescence of an untreated group was defined as 100%. [fig. 1 l]Figure 11 shows results of comparing the rate of inhibition of cell growth or survival between the antibody-drug conjugate (3) and the antibody-drug conjugate (4). The left diagram shows the rate of inhibition of cell growth or survival derived from the antibody-drug conjugate in the presence of 10% FBS. The ordinate depicts luminescence indicating the ATP activity of each sample. The abscissa depicts the con- 2024204502   28 Jun 2024 centration of each antibody-drug conjugate. The data is indicated by mean + / - standard deviation of triplicates. The right diagram shows the comparison of the rate of reduction in luminescence caused by antibody-drug conjugate treatment between high drug loading (HDL) and middle drug loading (MDL) when the luminescence of an untreated group was defined as 100%. [fig. 12]Figure 12 shows results of comparing the rate of inhibition of cell growth or survival between the antibody-drug conjugate (10) and the antibody-drug conjugate (11). The left diagram shows the rate of inhibition of cell growth or survival derived from the antibody-drug conjugate in the presence of 10% FBS. The ordinate depicts luminescence indicating the ATP activity of each sample. The abscissa depicts the concentration of each antibody-drug conjugate. The data is indicated by mean + / - standard deviation of triplicates. The right diagram shows the comparison of the rate of reduction in luminescence caused by antibody-drug conjugate treatment between high drug loading (HDL) and middle drug loading (MDL) when the luminescence of an untreated group was defined as 100%. [fig.l3]Figure 13 shows results of comparing the rate of inhibition of cell growth or survival between the antibody-drug conjugate (13) and the antibody-drug conjugate (14). The left diagram shows the rate of inhibition of cell growth or survival derived from the antibody-drug conjugate in the presence of 10% FBS. The ordinate depicts luminescence indicating the ATP activity of each sample. The abscissa depicts the concentration of each antibody-drug conjugate. The data is indicated by mean + / - standard deviation of triplicates. The right diagram shows the comparison of the rate of reduction in luminescence caused by antibody-drug conjugate treatment between high drug loading (HDL) and middle drug loading (MDL) when the luminescence of an untreated group was defined as 100%. [fig. 14]Figure 14 shows results of a human breast cancer (HCC1569) antitumor test using the antibody-drug conjugate (3), (10), or (13). The ordinate depicts an average tumor volume. The abscissa depicts the number of days from cell transplantation. All values are indicated by mean + / - standard deviation. The initial tumor volume and the initial mouse weight were analyzed on the basis of descriptive data (mean and standard deviation) using Microsoft Excel 2009. [fig.l5]Figure 15 shows results of a human melanoma (HT-144) antitumor test using the antibody-drug conjugate (3), (10), or (13). The ordinate depicts an average tumor volume. The abscissa depicts the number of days from cell transplantation. All values are indicated by mean + / - standard deviation. The initial tumor volume and the initial mouse weight were analyzed on the basis of descriptive data (mean and standard deviation) using Microsoft Excel 2009. [fig. 16]Figure 16 shows results of a human breast cancer (MDA-MB-453) antitumor 2024204502   28 Jun 2024 test using the antibody-drug conjugate (3), (10), or (13). The ordinate depicts an average tumor volume. The abscissa depicts the number of days from administration. All values are indicated by mean + / - standard deviation. The initial tumor volume and the initial mouse weight were analyzed on the basis of descriptive data (mean and standard deviation) using Microsoft Excel 2009. [fig.l7]Figure 17 shows results of a human colorectal cancer line (HT-29) antitumor test using the antibody-drug conjugate (3), (10), or (13). The ordinate depicts an average tumor volume. The abscissa depicts the number of days from administration. All values are indicated by mean + / - standard deviation. The initial tumor volume and the initial mouse weight were analyzed on the basis of descriptive data (mean and standard deviation) using Microsoft Excel 2009. [fig.l8]Figure 18 shows results of a human lung cancer line (A549) antitumor test using the antibody-drug conjugate (3), (10), or (13). The ordinate depicts an average tumor volume. The abscissa depicts the number of days from cell transplantation. All values are indicated by mean + / - standard deviation. The initial tumor volume and the initial mouse weight were analyzed on the basis of descriptive data (mean and standard deviation) using Microsoft Excel 2009. [fig. 19]Figure 19 shows results of a human triple-negative breast cancer line (MDA-MB-468) antitumor test using the antibody-drug conjugate (13). The ordinate depicts an average tumor volume. The abscissa depicts the number of days from cell transplantation. All values are indicated by mean + / - standard deviation. The initial tumor volume and the initial mouse weight were analyzed on the basis of descriptive data (mean and standard deviation) using Microsoft Excel 2009. [fig.20]Figure 20 shows results of a human luminal breast cancer line (MCF-7) antitumor test using the antibody-drug conjugate (16a). The ordinate depicts an average tumor volume. The abscissa depicts the number of days from cell transplantation. All values are indicated by mean + / - standard deviation. The initial tumor volume and the initial mouse weight were analyzed on the basis of descriptive data (mean and standard deviation) using Microsoft Excel 2009. [fig.21]Figure 21 shows results of a human melanoma line (WM-266-4) antitumor test using the antibody-drug conjugate (16a). The ordinate depicts an average tumor volume. The abscissa depicts the number of days from cell transplantation. All values are indicated by mean + / - standard deviation. The initial tumor volume and the initial mouse weight were analyzed on the basis of descriptive data (mean and standard deviation) using Microsoft Excel 2009. [fig.22]Figure 22 shows results of a human ovarian cancer line (OVCAR-8) antitumor test using the antibody-drug conjugate (16a). The ordinate depicts an average tumor volume. The abscissa depicts the number of days from cell transplantation. All values 2024204502   28 Jun 2024 are indicated by mean + / - standard deviation. The initial tumor volume and the initial mouse weight were analyzed on the basis of descriptive data (mean and standard deviation) using Microsoft Excel 2009. [fig.23]Figure 23 shows results of a human bladder cancer line (SW-780) antitumor test using the antibody-drug conjugate (16a). The ordinate depicts an average tumor volume. The abscissa depicts the number of days from cell transplantation. All values are indicated by mean + / - standard deviation. The initial tumor volume and the initial mouse weight were analyzed on the basis of descriptive data (mean and standard deviation) using Microsoft Excel 2009. [fig.24]Figure 24 shows results of a human breast cancer line (MDA-MB-453) antitumor test using the antibody-drug conjugate (16a). The ordinate depicts an average tumor volume. The abscissa depicts the number of days from cell transplantation. All values are indicated by mean + / - standard deviation. The initial tumor volume and the initial mouse weight were analyzed on the basis of descriptive data (mean and standard deviation) using Microsoft Excel 2009. [fig.25]Figure 25 shows results of a human breast cancer line (MDA-MB-453) antitumor test using the antibody-drug conjugate (16a). The ordinate depicts an average tumor volume. The abscissa depicts the number of days from cell transplantation. All values are indicated by mean + / - standard deviation. The initial tumor volume and the initial mouse weight were analyzed on the basis of descriptive data (mean and standard deviation) using Microsoft Excel 2009. [fig.26]Figure 26 shows results of a human breast cancer line (JIMT-1) antitumor test using the antibody-drug conjugate (15). The ordinate depicts an average tumor volume. The abscissa depicts the number of days from cell transplantation. All values are indicated by mean + / - standard deviation. The initial tumor volume and the initial mouse weight were analyzed on the basis of descriptive data (mean and standard deviation) using Microsoft Excel 2009. [fig.27]Figure 27 shows results of a human lung cancer line (PC9) antitumor test using the antibody-drug conjugate (16a). The ordinate depicts an average tumor volume. The abscissa depicts the number of days from cell transplantation. All values are indicated by mean + / - standard deviation. The initial tumor volume and the initial mouse weight were analyzed on the basis of descriptive data (mean and standard deviation) using Microsoft Excel 2009. [fig.28]Figure 28 shows results of a human triple-negative breast cancer line (MDA-MB-468) antitumor test using the antibody-drug conjugate (16a). The ordinate depicts an average tumor volume. The abscissa depicts the number of days from cell transplantation. All values are indicated by mean + / - standard deviation. The initial tumor volume and the initial mouse weight were analyzed on the basis of descriptive 2024204502   28 Jun 2024 data (mean and standard deviation) using Microsoft Excel 2009. [fig.29]Figure 29 shows results of a human head and neck cancer line (Fadu) antitumor test using the antibody-drug conjugate (16a). The ordinate depicts an average tumor volume. The abscissa depicts the number of days from cell transplantation. All values are indicated by mean + / - standard deviation. The initial tumor volume and the initial mouse weight were analyzed on the basis of descriptive data (mean and standard deviation) using Microsoft Excel 2009. [fig.30]Figure 30 shows results of an antitumor test using a human stomach cancer patient-derived tumor section (NIBIO-G016) and the antibody-drug conjugate (16a). The ordinate depicts an average tumor volume. The abscissa depicts the number of days from cell transplantation. All values are indicated by mean + / - standard deviation. The initial tumor volume and the initial mouse weight were analyzed on the basis of descriptive data (mean and standard deviation) using Microsoft Excel 2009. Description of Embodiments

[0038] Hereinbelow, the preferred embodiments for carrying out the present invention are explained in view of the drawings. Meanwhile, the embodiments explained below are the examples of the representative embodiments of the present invention and the scope of the present invention shall not be narrowly interpreted based on them.

[0039] The present invention provides HER3 binding protein-drug conjugate. Preferably, the HER3 binding protein of the invention is a scaffold protein having an antibody like binding activity or an antibody, i.e. an anti-HER3 antibody.

[0040] The anti-HER3 antibody-drug conjugate of the present invention is an antitumor medicine in which an anti-HER3 antibody is conjugated to an antitumor compound via a linker structure moiety and explained in detail hereinbelow. Within the context of the present invention, the term "scaffold protein", as used herein, means a polypeptide or protein with exposed surface areas in which amino acid insertions, substitutions or deletions are highly tolerable. Examples of scaffold proteins that can be used in accordance with the present invention are protein A from Staphylococcus aureus, the bilin binding protein from Pieris brassicae or other lipocalins, ankyrin repeat proteins, and human fibronectin (reviewed in Binz and Pluckthun, Curr Opin Biotechnol, 16, 459-69). Engineering of a scaffold protein can be regarded as grafting or integrating an affinity function onto or into the structural framework of a stably folded protein. Affinity function means a protein binding affinity according to the present invention. A scaffold can be structurally separable from the amino acid sequences conferring binding specificity. In general, proteins appearing suitable for the development of such artificial affinity reagents may be obtained by rational, or most commonly, combinatorial protein engineering techniques such as panning against 2024204502   28 Jun 2024 HER3, either purified protein or protein displayed on the cell surface, for binding agents in an artificial scaffold library displayed in vitro, skills which are known in the art (Skerra, J. Mol. Recog., 2000; Binz and Pluckthun, 2005). In addition, a scaffold protein having an antibody like binding activity can be derived from an acceptor polypeptide containing the scaffold domain, which can be grafted with binding domains of a donor polypeptide to confer the binding specificity of the donor polypeptide onto the scaffold domain containing the acceptor polypeptide. Said inserted binding domains may be, for example, the complementarity determining region (CDR) of an antibody, in particular an anti-HER3 antibody. Insertion can be accomplished by various methods known to those skilled in the art including, for example, polypeptide synthesis, nucleic acid synthesis of an encoding amino acid as well by various forms of recombinant methods well known to those skilled in the art.

[0041] {Antibody} Moreover, the term "antibody" or "anti-HER3 antibody", as used herein, means a monoclonal antibody, a polyclonal antibody, a recombinant antibody, a 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), a chimeric antibody (Morrison et al., Proc. Natl. Acad. Sci. U.S.A. 81 (1984), 6851-6855), a 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, p.3447-3448; Yoshida, H. et al., Animal Cell Technology: Basic and Applied Aspects vol. 10, p.69-73 (Kitagawa, Y., Matsuda, T. and Iijima, S. eds.), Kluwer Academic Publishers, 1999.; Tomizuka, K. et al., Proc. Natl. Acad. Sci. USA (2000) 97, p.722-727, International Publication No. WO 2007 / 077028, and so on), a multispecific antibody (e.g. a bispecific antibody) formed from at least two antibodies, or an antibody fragment thereof. The term "antibody fragment" comprises any portion of the afore-mentioned 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. U.S.A. 90 (1993), 6444-6448), single chain antibody molecules (Pluckthun in: The Pharmacology of Monoclonal Antibodies 113, Rosenburg and Moore, EDS, Springer Verlag, N.Y. (1994), 269-315) and other fragments as long as they exhibit the desired capability of binding to HER3.

[0042] In addition, the term "antibody" or "anti-HER3 antibody", as used herein, may include antibody-like molecules that contain engineered sub-domains of antibodies or naturally occurring antibody variants. These antibody-like molecules may be singledomain antibodies such as VH-only or VL-only domains derived either from natural sources such as camelids (Muyldermans et al., Reviews in Molecular Biotechnology 2024204502   28 Jun 2024 74, 277-302) or through in vitro display of libraries from humans, camelids or other species (Holt et al., Trends Biotechnol., 21, 484-90).

[0043] In accordance with the present invention, the "Fv fragment" is the minimum antibody fragment that contains a complete antigen-recognition and -binding site. This region consists of a dimer of one heavy- and one light-chain variable domain in tight, non-covalent association. It is in this configuration that the three CDRs of each variable domain interact to define an antigen-binding site on the surface of the VH-VL dimer. Collectively, the six CDR's confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three CDR's specific for an antigen) has the ability to recognize and bind the antigen, although usually at a lower affinity than the entire binding site. The "Fab fragment" also contains the constant domain of the light chain and the first constant domain (CHI) of the heavy chain. The "Fab fragment" differs from the "Fab1 fragment" by the addition of a few residues at the carboxy terminus of the heavy chain CHI domain including one or more cysteines from the antibody hinge region. The "F(ab')2 fragment" originally is produced as a pair of "Fab1 fragments" which have hinge cysteines between them. Methods of 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 invention is an anti-HER3 antibody directed against the extracellular domain (ECD) ofHER3.

[0045] The anti-HER3 antibody used in an anti-HER3 antibody-drug conjugate of the present invention may be derived from any species. Preferred examples of the species can include humans, rats, mice, and rabbits. The anti-HER3 antibody derived from other than human species is preferably chimerized or humanized using a well known technique. The antibody of the present invention may be a polyclonal antibody or a monoclonal antibody and is preferably a monoclonal antibody. The anti-HER3 antibody is may be those which are capable of targeting tumor cells and thus possesses the property of being capable of recognizing tumor cells, the property of being capable of binding to tumor cells, the property of being internalized into tumor cells, and cytocidal activity against tumor cells, etc. The anti-HER3 antibody can be conjugated with a compound having antitumor activity via a linker to form an antibody-drug conjugate. The binding activity of the antibody against tumor cells can be confirmed using flow cytometry. The internalization of the antibody into tumor cells can be confirmed using (1) an assay of visualizing an antibody incorporated in cells under a fluorescence microscope using a secondary antibody (fluorescently labeled) binding to the therapeutic antibody (Cell Death and Differentiation (2008) 15, 751-761), (2) an assay of 2024204502   28 Jun 2024 measuring the amount of fluorescence incorporated in cells using a secondary antibody (fluorescently labeled) binding to the therapeutic antibody (Molecular Biology of the Cell, Vol. 15, 5268-5282, December 2004), or (3) a Mab-ZAP assay using an immunotoxin binding to the 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 of a diphtheria toxin catalytic domain and protein G may be used as the immunotoxin. The antitumor activity of the antibody can be confirmed in vitro by determining inhibitory activity against cell growth. For example, a cancer cell line overexpressing a target protein for the antibody is cultured, and the antibody is added at varying concentrations into the culture system to determine inhibitory activity against focus formation, colony formation, and spheroid growth. The antitumor activity can be confirmed in vivo, for example, by administering the antibody to a nude mouse with a transplanted tumor cell line highly expressing the target protein, and determining change in the cancer (tumor) cells. Since the compound conjugated in the antibody-drug conjugate exerts an antitumor effect, it is preferred but not essential that the antibody itself should have an antitumor effect. For the purpose of exerting the cytotoxicity of the antitumor compound specifically and selectively for tumor cells, it is important and also preferred that the antibody should have the property of being internalized to migrate into tumor cells.

[0046] The anti-HER3 antibody can be obtained using a method usually carried out in the art, which involves immunizing animals with an antigenic polypeptide and collecting and purifying antibodies produced in vivo. The origin of the antigen is not limited to humans, and the animals may be immunized with an antigen derived from a nonhuman animal such as a mouse or a rat and the like. In this case, the cross-reactivity of antibodies binding to the obtained heterologous antigen with human antigens can be tested to screen for an antibody applicable to a human disease. Alternatively, antibody-producing cells which 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, p. 365-367, Plenum Press, N.Y. (1980)) to establish hybridomas, from which monoclonal antibodies can in turn be obtained. The antigen can be obtained by genetically engineering host cells to produce a gene encoding the antigenic protein. Specifically, vectors that permit expression of the antigen gene are prepared and transferred to host cells so that the gene is expressed. The antigen thus expressed can be purified. The antibody can also be obtained by use of a method which involves immunizing animals with the genetically engineered antigen-expressing cells or a cell line with an expressed antigen. 2024204502   28 Jun 2024 The anti-HER3 antibody can be obtained by means known in the art.

[0047] The anti-HER3 antibody that can be used in the present invention is not particularly limited and is desirably, for example, any of antibodies having properties as described below. (1) An anti-HER3 antibody having the following properties: (a) specifically binding to HER3, and / or (b) having the activity of being internalized into HER3-expressing cells through binding to HER3. (2) The antibody according to (1), wherein the antibody binds to the extracellular domain of HER3. (3) The antibody according to (1) or (2), wherein the antibody is a monoclonal antibody. (4) The antibody according to any of (1) to (3), wherein the antibody has antibodydependent cell-mediated cytotoxicity (ADCC) activity and / or complement-dependent cytotoxicity (CDC) activity. (5) The antibody according to any 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) The antibody according to any of (1) to (5), wherein the antibody is a humanized monoclonal antibody comprising a heavy chain comprising the amino acid sequence represented by SEQ ID NO: 1 and a light chain comprising the amino acid sequence represented by SEQ ID NO: 2. (7) The antibody according to any of (1) to (6), wherein the antibody lacks a lysine residue at the carboxy terminus of the heavy chain. (8) The antibody according to (7), wherein the antibody comprises a heavy chain variable region represented by the amino acid sequence represented by SEQ ID NO: 70 and a light chain variable region represented by the amino acid sequence represented by SEQ ID NO: 72. (9) An antibody obtained by a method for producing the antibody according to any of (1) to (8), the method comprising the steps of: culturing a host cell transformed with an expression vector comprising a polynucleotide encoding the antibody; and collecting the antibody of interest from the cultures obtained in the preceding step.

[0048] Hereinafter, the anti-HER3 antibody used in the present invention will be described. In the present specification, the terms "cancer" and "tumor" are used interchangeably. In the present specification, the term "gene" includes not only DNA but its mRNA, cDNA, and cRNA thereof. In the present specification, the term "polynucleotide" is used interchangeably with a nucleic acid and also includes DNA, RNA, probes, oligonucleotides, and primers. 2024204502   28 Jun 2024 In the present specification, the terms "polypeptide" and "protein" are used interchangeably. In the present specification, the term "cell" also includes cells in animal individuals and cultured cells. In the present specification, the term "HER3" is used interchangeably with HER3 protein. In the present specification, the term "CDR" means a complementarity determining region (CDR). An antibody molecule is known to have three CDRs in each of heavy and light chains. CDRs, also called hypervariable domains, are located in the variable regions of the antibody heavy and light chains. These sites have a particularly highly variable primary structure and are separated at three positions on the respective primary structures of heavy and light chain polypeptide strands. In the present specification, the antibody CDRs are referred to as CDRH1, CDRH2, and CDRH3 from the amino terminus of the heavy chain amino acid sequence as to heavy chain CDRs and as CDRL1, CDRL2, and CDRL3 from the amino terminus of the light chain amino acid sequence as to light chain CDRs. These sites are proximal to each other on the three-dimensional structure and determine specificity for the antigen to be bound. In the present invention, the phrase "hybridizing under stringent conditions" refers to hybridization at 68C in a commercially available hybridization solution ExpressHyb Hybridization Solution (manufactured by Clontech Laboratories, Inc.), or identifiable hybridization under conditions involving hybridization at 68C in the presence of 0.7 to 1.0 M NaCl using a DNA-immobilized filter, followed by washing at 68C using 0.1 to 2 ' SSC solution (1 ' SSC is composed of 150 mM NaCl and 15 mM sodium citrate), or hybridization under conditions equivalent thereto.

[0049] 1. HER3 The human epidermal growth factor receptor 3 (HER3, also known as ErbB3) is a receptor protein tyrosine kinase and belongs to the epidermal growth factor receptor (EGFR) subfamily of receptor protein tyrosine kinases, 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 within 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 several types of cancer such as breast, gastrointestinal and pancreatic cancers. A correlation between the expression of HER2 / HER3 and the progression from a non-invasive to an invasive stage has been shown. The HER3 protein used in the present invention can be used after direct purification from HER3-expressing human or non-human mammalian (rat, mouse, etc.) cells or can be used by preparing cell membrane fractions of the cells. Alternatively, HER3 may be 2024204502   28 Jun 2024 synthesized in vitro or may be produced from host cells by genetic engineering. In the genetic engineering, specifically, HER3 cDNA is integrated into vectors that permit expression thereof, and HER3 can then be expressed by synthesis in a solution containing enzymes necessary for transcription and translation, substrates, and energy substances or by transformation of other host prokaryotic cells or host eukaryotic cells to yield the protein. Alternatively, the genetically engineered HER3-expressing cells described above or a cell line with expressed HER3 may be used as the HER3 protein. An RNA sequence, a cDNA sequence, and an amino acid sequence of HER3 are available in public database, and can be referred to by an accession number such as AAA35979 (precursor including a signal sequence consisting of amino terminus 19 amino acid residue), M34309 (NCBI), for example. The above amino acid sequence of HER3 consists of an amino acid sequence which is subjected to replacements, deletions, additions and / or insertions of at least one amino acid, and proteins having a biological activity equivalent to that of the protein are also included in HER3.

[0050] 2. Production of anti HER3 antibody The antibody against HER3 of the present invention can be obtained by immunizing an animal with HER3 or an arbitrary polypeptide selected from the amino acid sequence of HER3, and collecting and purifying the antibody produced in vivo according to a method usually carried out in the art. The biological species of HER3 to be used as an antigen is not limited to being human, and an animal can be immunized with HER3 derived from an animal other than humans such as a mouse or a rat. In this case, by examining the cross-reactivity between an antibody binding to the obtained heterologous HER3 and human HER3, an antibody applicable to a human disease can be selected. Further, a monoclonal antibody can be obtained from a hybridoma established by fusing antibody-producing cells which produce an antibody against HER3 with myeloma cells according to a known method (for example, Kohler and Milstein, Nature, (1975) 256, pp. 495-497; Kennet, R. ed., Monoclonal Antibodies, pp. 365-367, Plenum Press, N.Y. (1980)). HER3 to be used as an antigen can be obtained by expressing HER3 gene in a host cell using genetic engineering. Specifically, a vector capable of expressing HER3 gene is produced, and the resulting vector is transfected into a host cell to express the gene, and then, the expressed HER3 is purified. It is also possible to use HER3 expressing cells obtained by the genetic engineering or a cell line expressing HER3 as HER3 protein. Hereinbelow, a method for obtaining an antibody against HER3 is explained specifically. 2024204502   28 Jun 2024

[0051] (1) Preparation of antigen Examples of the antigen to be used for producing the anti HER3 antibody include HER3, a polypeptide consisting of a partial amino acid sequence comprising at least 6 consecutive amino acids of HER3, and a derivative obtained by adding a given amino acid sequence or carrier thereto. HER3 can be purified directly from human tumor tissues or tumor cells and used. Further, HER3 can be obtained by synthesizing it in vitro or by producing it in a host cell by genetic engineering. With respect to the genetic engineering, specifically, after HER3 cDNA is integrated into a vector capable of expressing HER3 cDNA, HER3 can be obtained by synthesizing it in a solution containing an enzyme, a substrate and an energy substance required for transcription and translation, or by expressing HER3 in another prokaryotic or eucaryotic transformed host cell. Further, 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, to the constant region of an antibody in an appropriate host-vector system. HER3 cDNA can be obtained by, for example, a 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 performed using a cDNA library expressing HER3 cDNA as a template and primers which specifically amplify HER3 cDNA. As the in vitro synthesis of the polypeptide, for example, Rapid Translation System (RTS) manufactured by Roche Diagnostics, Inc. can be exemplified, but it is not limited thereto. Examples of the prokaryotic host cells include Escherichia coli and Bacillus subtilis. In order to transform the host cells with a target gene, the host cells are transformed by a plasmid vector comprising a replicon, i.e., a replication origin derived from a species compatible with the host, and a regulatory sequence. Further, the vector preferably has a sequence capable of imposing phenotypic selectivity on the transformed cell. Examples of the eucaryotic host cells include vertebrate cells, insect cells, and yeast cells. As the vertebrate cells, for example, simian COS cells (Gluzman, Y., Cell, (1981) 23, pp. 175-182, ATCC CRL-1650; ATCC: American Type Culture Collection), murine fibroblasts NIH3T3 (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 ovarian cells (CHO cells; ATCC: CCL-61); and the like are often used, however, the cells are not limited thereto. The thus obtained transformant can be cultured according to a method usually carried out in the art, and by the culturing of the transformant, a target polypeptide is produced intracellularly or extracellularly. 2024204502   28 Jun 2024 A suitable medium to be used for the culturing can be selected from various commonly used culture media depending on the employed host cells. If Escherichia coli is employed, for example, an LB medium supplemented with an antibiotic such as ampicillin or IPMG as needed can be used. A recombinant protein produced intracellularly or extracellularly by the transformant through such culturing can be separated and purified by any of various known separation methods utilizing the physical or chemical property of the protein. Specific examples of the methods include treatment with a common protein precipitant, 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 thereof. Further, by attaching a tag of six histidine residues to a recombinant protein to be expressed, the protein can be efficiently purified with a nickel affinity column. Alternatively, by attaching the IgG Fc region to a recombinant protein to be expressed, the protein can be efficiently purified with a protein A column. By combining the above-described methods, a large amount of a target polypeptide can be easily produced in high yield and high purity. It is also possible to use the aforementioned transformant itself as an antigen. It is also possible to use a cell line expressing HER3 as an antigen. Examples of the cell line include. However, as long as HER3 is expressed, it is not limited to those cell lines.

[0052] (2) Production of anti HER3 monoclonal antibody Examples of the antibody specific binding to HER3 include a monoclonal antibody specific binding to HER3, and a method of obtaining the antibody is as described below. The production of a monoclonal antibody generally requires the following operational steps of: (a) Purification of a biopolymer used as an antigen or preparation of cells expressing antigen; (b) preparing antibody-producing cells by immunizing an animal by injection of the antigen, collecting the blood, assaying its antibody titer to determine when the spleen is excised; (c) preparing myeloma cells (hereinafter referred to as "myeloma"); (d) fusing the antibody-producing cells with the myeloma; (e) screening a group of hybridomas producing a desired antibody; (f) dividing the hybridomas into single cell clones (cloning); (g) optionally, culturing the hybridoma or rearing an animal implanted with the hybridoma for producing a large amount of a monoclonal antibody; (h) examining the thus produced monoclonal antibody for biological activity and 2024204502   28 Jun 2024 binding specificity, or assaying the same for properties as a labeled reagent; and the like. Hereinafter, the method of producing a monoclonal antibody will be described in detail following the above steps, however, the method is not limited thereto, and, for example, antibody-producing cells other than spleen cells and myeloma can be used.

[0053] (a) Purification of antigen As the antigen, HER3 prepared by the method as described above or a partial peptide thereof can be used. Further, a membrane fraction prepared from recombinant cells expressing HER3 or the recombinant cells expressing HER3 themselves, and also a partial peptide of the protein of the invention chemically synthesized by a method known to those skilled in the art can also be used as the antigen. Further, a cell line expressing HER3 can be also used as an antigen.

[0054] (b) Preparation of antibody-producing cells The antigen obtained in the step (a) is mixed with an adjuvant such as Freund's complete or incomplete adjuvant or aluminum potassium sulfate and the resulting mixture is used as an immunogen to immunize an experimental animal. In an alternative method, a test animal is immunized with cells expressing antigen as an immunogen. As the experimental animal, any animal used in a known hybridoma production method can be used without any trouble. Specifically, for example, a mouse, a rat, a goat, sheep, cattle, a horse, or the like can be used. However, from the viewpoint of ease of availability of myeloma cells to be fused with the extracted antibody-producing cells, a mouse or a rat is preferably used as the animal to be immunized. Further, the strain of a mouse or a rat to 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 can be used, and in the case of a rat, for example, Wistar, Low, Lewis, Sprague, Dawley, ACI, BN, Fischer and the like can be used. These mice and rats are commercially available from breeders / distributors of experimental animals, for example, CLEA Japan, Inc. and Charles River Laboratories Japan,Inc. As the animal to be immunized, in consideration of compatibility of fusing with myeloma cells described below, in the case of a mouse, BALB / c strain, and in the case of a rat, Wistar and Low strains are particularly preferred. Further, in consideration of antigenic homology between humans and mice, it is also preferred to use a mouse having decreased biological function to remove autoantibodies, that is, a mouse with an autoimmune disease. 2024204502   28 Jun 2024 The age of such mouse or rat at the time of immunization is preferably 5 to 12 weeks of age, more preferably 6 to 8 weeks of age. In order to immunize an animal with HER3 or a recombinant thereof, for example, a known method 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 can be used. Among these immunization methods, a preferred specific method in the invention is, for example, as follows. That is, first, a membrane protein fraction serving as the antigen or cells caused to express the antigen is / are intradermally or intraperitoneally administrated to an animal. However, the combination of both routes of administration is preferred for increasing the immunization efficiency, and when intradermal administration is performed in the first half and intraperitoneal administration is performed in the latter half or only at the last dosing, the immunization efficiency can be particularly increased. The administration schedule of the antigen varies depending on the type of animal to be immunized, individual difference or the like. However, in general, an administration schedule in which the frequency of administration of the antigen is 3 to 6 times and the dosing interval is 2 to 6 weeks is preferred, and an administration schedule in which the frequency of administration of the antigen is 3 to 4 times and the dosing interval is 2 to 4 weeks is more preferred. Further, the dose of the antigen varies depending on the type of animal, individual differences or the like, however, the dose is generally set to 0.05 to 5 mg, preferably about 0.1 to 0.5 mg. A booster immunization is performed 1 to 6 weeks, preferably 1 to 4 weeks, more preferably 1 to 3 weeks after the administration of the antigen as described above. When the immunogen is a cell, 1" 106 to 1 HO7 cells are used. The dose of the antigen at the time of performing the booster immunization varies depending on the type or size of animal or the like, however, in the case of, for example, a mouse, the dose is generally set to 0.05 to 5 mg, preferably 0.1 to 0.5 mg, more preferably about 0.1 to 0.2 mg. When the immunogen is a cell, ITO6 to ITO7 cells are used. Spleen cells or lymphocytes including antibody-producing cells are aseptically removed from the immunized animal 1 to 10 days, preferably 2 to 5 days, more preferably 2 to 3 days after the booster immunization. At this time, the antibody titer is measured, and if an animal having a sufficiently increased antibody titer is used as a supply source of the antibody-producing cells, the subsequent procedure can be carried out more efficiently. 2024204502   28 Jun 2024 Examples of the method of measuring the antibody titer to be used here include an RIA method and an ELISA method, but the method is not limited thereto. For example, if an ELISA method is employed, the measurement of the antibody titer in the invention can be carried out according to the procedures as described below. First, a purified or partially purified antigen is adsorbed to the surface of a solid phase such as a 96-well plate for ELISA, and the surface of the solid phase having no antigen adsorbed thereto is covered with a protein unrelated to the antigen such as bovine serum albumin (hereinafter referred to as "BSA"). After washing the surface, the surface is brought into contact with a serially-diluted sample (for example, mouse serum) as a primary antibody to allow the antibody in the sample to bind to the antigen. Further, as a secondary antibody, an antibody labeled with an enzyme against a mouse antibody is added and is allowed to bind to the mouse antibody. After washing, a substrate for the enzyme is added and a change in absorbance which occurs due to color development induced by degradation of the substrate or the like is measured and the antibody titer is calculated based on the measurement. The separation of the antibody-producing cells from the spleen cells or lymphocytes of the immunized animal can be carried out according to 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 in which the antibodyproducing cells are separated by homogenizing the spleen to yield the cells through filtration with a stainless steel mesh and suspending the cells in Eagle's Minimum Essential Medium (MEM) can be employed.

[0055] (c) Preparation of myeloma cells (hereinafter referred to as "myeloma") The myeloma cells to be used for cell fusion are not particularly limited and suitable cells can be selected from known cell lines. However, in consideration of convenience when a hybridoma is selected from fused cells, it is preferred to use an HGPRT (hypoxanthine-guanine phosphoribosyl transferase) deficient strain whose selection procedure has been established. More specifically, examples of the HGPRT-deficient strain include X63-Ag8(X63), NS1-ANS / 1(NS1), P3X63-Ag8.Ul(P3Ul), X63-Ag8.653(X63.653), SP2 / 0-Agl4(SP2 / 0), MPC11-45.6TG1.7(45.6TG), FO, S149 / 5XXO, and BU.l derived from mice; 21O.RSY3.Ag.l.2.3(Y3) derived from rats; and U266AR(SKO-007), GM1500xGTG-A12(GM1500), UC729-6, LICR-LOW-HMy2(HMy2) and 8226AR / NIP4-1(NP41) derived from humans. These HGPRT-deficient strains are available from, for example, ATCC or the like. These cell strains are subcultured in an appropriate medium such as an 8-azaguanine 2024204502   28 Jun 2024 medium [a medium obtained by adding 8-azaguanine to an RPMI 1640 medium supplemented with glutamine, 2-mercaptoethanol, gentamicin, and fetal calf serum (hereinafter referred to as "FBS")], Iscove's Modified Dulbecco's Medium; IMDM), or Dulbecco's Modified Eagle Medium (hereinafter referred to as "DMEM"). In this case, 3 to 4 days before performing cell fusion, the cells are subcultured in a normal medium [for example, an ASF104 medium (manufactured by Ajinomoto Co., Ltd.) containing 10% FCS] to ensure not less than 2 x 107 cells on the day of cell fusion.

[0056] (d) Cell fusion Fusion between the antibody-producing cells and the myeloma cells can be appropriately performed 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 conditions such that the survival rate of cells is not excessively reduced. As such a method, for example, a chemical method in which the antibody-producing cells and the myeloma cells are mixed in a solution containing a polymer such as polyethylene glycol at a high concentration, a physical method using electric stimulation, or the like can be used. Among these methods, a specific example of the chemical method is as described below. That is, in the case where polyethylene glycol is used in the solution containing a polymer at a high concentration, the antibody-producing cells and the myeloma cells are mixed in a solution of polyethylene glycol having a molecular weight of 1500 to 6000, more preferably 2000 to 4000 at a temperature of from 30 to 40C, preferably from 35 to 38C for 1 to 10 minutes, preferably 5 to 8 minutes.

[0057] (e) Selection of a group of hybridomas The method of selecting hybridomas obtained by the above-described cell fusion is not particularly limited. Usually, an HAT (hypoxanthine, aminopterin, thymidine) selection method (Kohler et al., Nature (1975), 256, p. 495; Milstein et al., Nature (1977), 266, p. 550) is used. This method is effective when hybridomas are obtained using the myeloma cells of an HGPRT-deficient strain which cannot survive in the presence of aminopterin. That is, by culturing unfused cells and hybridomas in an HAT medium, only hybridomas resistant to aminopterin are selectively allowed to survive and proliferate.

[0058] (f) Division into single cell clone (cloning) As a cloning method for hybridomas, a known method such as a methylcellulose method, a soft agarose method, or a limiting 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)). Among these 2024204502   28 Jun 2024 methods, particularly, a three-dimensional culture method such as a methylcellulose method is preferred. For example, the group of hybridomas produced by cell fusion are suspended in a methylcellulose medium such as ClonaCell-HY Selection Medium D (manufactured by StemCell Technologies, inc., #03804) and cultured. Then, the formed hybridoma colonies are collected, whereby monoclonal hybridomas can be obtained. The collected respective hybridoma colonies are cultured, and a hybridoma which has been confirmed to have a stable antibody titer in an obtained hybridoma culture supernatant is selected as an anti-HER3 monoclonal antibody-producing hybridoma strain.

[0059] (g) Preparation of monoclonal antibody by culturing hybridoma By culturing the thus selected hybridoma, a monoclonal antibody can be efficiently obtained. However, prior to culturing, it is preferred to perform screening of a hybridoma which produces a target monoclonal antibody. In such screening, a known method can be employed. The measurement of the antibody titer in the invention can be carried out by, for example, an ELISA method explained in item (b) described above. The hybridoma obtained by the method described above can be stored in a frozen state in liquid nitrogen or in a freezer at -80C or below. After completion of cloning, the medium is changed from an HT medium to a normal medium, and the hybridoma is cultured. Large-scale culture is performed by rotation culture using a large culture bottle or by spinner culture. From the supernatant obtained by the large-scale culture, a monoclonal antibody which specifically binds to the protein of the invention can be obtained by purification using a method known to those skilled in the art such as gel filtration. Further, the hybridoma is injected into the abdominal cavity of a mouse of the same strain as the hybridoma (for example, the above-described BALB / c) or a Nu / Nu mouse to proliferate the hybridoma, whereby the ascites containing a large amount of the monoclonal antibody of the invention can be obtained. In the case where the hybridoma is administrated in the abdominal cavity, if a mineral oil such as 2,6,10,14-tetramethyl pentadecane (pristane) is administrated 3 to 7 days prior thereto, a larger amount of the ascites can be obtained. For example, an immunosuppressant is previously injected into the abdominal cavity of a mouse of the same strain as the hybridoma to inactivate T cells. 20 days thereafter, 106 to 107 hybridoma clone cells are suspended in a serum-free medium (0.5 mL), and the suspension is administrated in the abdominal cavity of the mouse. In general, when the abdomen is expanded and filled with the ascites, the ascites is collected from the mouse. By this method, the monoclonal antibody can be obtained at a concentration which is about 100 times or much higher than that in the culture solution. 2024204502   28 Jun 2024 The monoclonal antibody obtained by the above-described method can be purified by a method described in, for example, Weir, D. M.: Handbook of Experimental Immunology Vol. I, II, III, Blackwell Scientific Publications, Oxford (1978). The thus obtained monoclonal antibody has high antigen specificity for HER3.

[0060] (h) Assay of monoclonal antibody The isotype and subclass of the thus obtained monoclonal antibody can be determined as follows. First, examples of the identification method include an Ouchterlony method, an ELISA method, and an RIA method. An Ouchterlony method is simple, but when the concentration of the monoclonal antibody is low, a condensation operation is required. On the other hand, when an ELISA method or an RIA method is used, by directly reacting the culture supernatant with an antigen-adsorbed solid phase and using antibodies corresponding to various types of immunoglobulin isotypes and subclasses as secondary antibodies, the isotype and subclass of the monoclonal antibody can be identified. In addition, as a simpler method, a commercially available identification kit (for example, Mouse Typer Kit manufactured by Bio-Rad Laboratories, Inc.) or the like can also be used. Further, the quantitative determination of a protein can be performed by the Folin Lowry method and a method of calculation based on the absorbance at 280 nm [1.4 (OD 280) = Immunoglobulin 1 mg / mL]. Further, even when the monoclonal antibody is separately and independently obtained by performing again the steps of (a) to (h) in (2), it is possible to yield an antibody having a cytotoxic activity equivalent to that of the anti-HER3 antibody. As one example of such an antibody, an antibody which binds to the same epitope as the anti-HER3 antibody can be exemplified. If a newly produced monoclonal antibody binds to a partial peptide or a 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. Further, by confirming the competition by the monoclonal antibody for binding of the anti-HER3 antibody to HER3 (binding between the anti-HER3 antibody and HER3 is interfered by the monoclonal antibody), it can be determined that the monoclonal antibody binds to the same epitope as the anti-HER3 antibody even though a specific sequence or structure of the epitope has not been identified. Once the epitope is confirmed to be the same, it is strongly expected that the monoclonal antibody has the same antigen binding capacity or biological activity as the anti-HER3 antibody.

[0061] (3) Other antibodie s 2024204502   28 Jun 2024 The antibody of the invention includes not only the above-described monoclonal antibody against HER3 but also a recombinant antibody obtained by artificial modification for the purpose of decreasing heterologous antigenicity to humans such as a chimeric antibody, a humanized antibody and a human antibody. These antibodies can be produced using a known method. As the chimeric antibody, an antibody in which antibody variable and constant regions are derived from different species, for example, a chimeric antibody in which a mouse-or rat-derived antibody variable region is connected to a human-derived constant region can be exemplified (see Proc. Natl. Acad. Sci. USA, 81, 6851-6855, (1984)). As the humanized antibody, an antibody obtained by integrating only a complementarity determining region (CDR) into a human-derived antibody (see Nature (1986) 321, pp. 522-525), and an antibody obtained by grafting a part of the amino acid residues of the framework as well as the CDR sequence to a human antibody by a CDR-grafting method (WO 90 / 07861) can be exemplified. The term "several" as used herein refers to 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 or 2.

[0062] In accordance with the present invention, it is to be understood, that the amino acid sequence of the binding protein of the invention is not limited to the twenty conventional amino acids (See Immunology - A Synthesis (2nd Edition, E. S. Golub and D. R. Gren, Eds., Sinauer Associates, Sunderland, Mass. (1991)), which is incorporated herein by reference). For example, the amino acids may include stereoisomers (e.g. D-amino acids) of the twenty conventional amino acids, unnatural amino acids such as alpha-,alpha-disubstituted amino acids, N-alkyl amino acids, lactic acid, and other unconventional amino acids. Examples of unconventional amino acids, which may also be suitable components for the binding protein of the invention, include: 4-hydroxyproline, gamma-carboxyglutamate, epsilon-N,N,N-trimethyllysine, epsilon-N-acetyllysine, O-phosphoserine, N-acetylserine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysine, sigma-N-methylarginine, and other similar amino acids and imino acids, e.g. 4-hydroxyproline.

[0063] As the amino acid substitution in this specification, a conservative amino acid substitution is preferred. The conservative amino acid substitution refers to a substitution occurring within a group of amino acids related to amino acid side chains. Preferred amino acid groups are as follows: an acidic group (aspartic acid and glutamic acid); a basic group (lysine, arginine, and histidine); a non-polar group (alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan); and an uncharged polar family (glycine, asparagine, glutamine, cysteine, serine, threonine, and tyrosine). More preferred amino acid groups are as follows: an aliphatic hydroxy group (serine and threonine); an amide-containing group (asparagine and glutamine); 2024204502   28 Jun 2024 an aliphatic group (alanine, valine, leucine, and isoleucine); and an aromatic group (phenylalanine, tryptophan, and tyrosine). Such an amino acid substitution is preferably performed within a range which does not impair the properties of a substance having the original amino acid sequence. When the heavy and light chains of the antibody of the present invention have glutamate as the N-terminal amino acid, it may be cyclized (in the form of pyroglutamate). In the present invention, such pyroglutamate is not differentiated from normal glutamine on amino acid sequences. In the heavy and light chains of the antibody of the present invention, cysteine may be in the form of cysteinyl. In the present invention, such a cysteinyl form is not differentiated from normal cysteine on amino acid sequences.

[0064] Further, the antibody of the invention includes a human antibody which binds to the HER3. An anti HER3 human antibody refers to a human antibody having only a gene sequence of an antibody derived from a human chromosome. The anti HER3 human antibody can be obtained by a method using a human antibody-producing mouse having a human chromosome fragment comprising heavy and light chain genes of a human antibody (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 created specifically as follows. A genetically modified animal in which endogenous immunoglobulin heavy and light chain gene loci have been disrupted, and instead, human immunoglobulin heavy and light chain gene loci have been introduced via a yeast artificial chromosome (YAC) vector or the like is created by producing a knockout animal and a transgenic animal and mating these animals. Further, according to a recombinant DNA technique, by using cDNAs encoding each of such a heavy chain and a light chain of a human antibody, and preferably a vector comprising such cDNAs, eukaryotic cells are transformed, and a transformant cell which produces a recombinant human monoclonal antibody is cultured, whereby the antibody can also be obtained from the culture supernatant. Here, as the host, for example, eukaryotic cells, preferably mammalian cells such as CHO cells, lymphocytes, or myeloma cells can be used. With regard to preparation of a human antibody, detailed descriptions are given in International Publication No. WO 2007 / 077028. The contents of International Publication No. WO 2007 / 077028 are incorporated herein by reference.

[0066] Further, a method of obtaining a phage display-derived human antibody selected from a human antibody library (see Wormstone, I. M. et al., Investigative Oph- 2024204502   28 Jun 2024 thalmology & 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.) is also known. For example, a phage display method in which a variable region of a human antibody is expressed on the surface of a phage as a single-chain antibody (scFv), and a phage which binds to an antigen is selected (Nature Biotechnology (2005), 23, (9), pp. 1105-1116)can be used. By analyzing the gene of the phage selected based on the binding to an antigen, a DNA sequence encoding the variable region of a human antibody which binds to an antigen can be determined. If the DNA sequence of scFv which binds to an antigen is determined, a human antibody can be obtained by preparing an expression vector comprising the sequence and introducing the vector into an appropriate host to express it (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, an isolated HER3-binding protein of the invention comprises a heavy chain variable region amino acid sequence comprising: (a) CDRH1 comprised in the amino acid sequence represented by 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, (b) CDRH2 comprised in the amino acid sequence represented by 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 (c) CDRH3 comprised in the amino acid sequence represented by 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 a light chain variable region amino acid sequence comprising: (d) CDRL1 comprisedin the amino acid sequence represented by 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, 192, 196, 200, 204, 208, 212, 216, 220, 224, 228 or 232, (e) CDRL2 comprised in the amino acid sequence represented by 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, 2024204502   28 Jun 2024 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 com prisedin the amino acid sequence represented by 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, 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) CDRH1 comprising the amino acid sequence represented by one selected from the group consisting of SEQ ID NOs: 236, 251, 252, and 256; (b) CDRH2 comprising the amino acid sequence represented by one selected from the group consisting of SEQ ID NOs: 258, 278, 280, and 282; and (c) CDRH3 comprising the amino acid sequence represented by one selected from the group consisting of SEQ ID NOs: 283, 285, 309, 313, and 315, and a light chain amino acid sequence comprising (d) CDRL1 cmprisingthe amino acid sequence represented by one selected from the group consisting of SEQ ID NOs: 320, 334, 337, and 340; (e) CDRL2 comprising the amino acid sequence represented by one selected from the group consisting of SEQ ID NOs: 343, 356, 351, and 344; and (f) CDRL3 comprising the amino acid sequence represented by one selected from the group consisting of SEQ ID NOs: 360, 381, 385, and 387.

[0069] In another embodiment of the present invention, an isolated binding protein of the 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, an isolated binding protein of the invention comprises a heavy chain variable region amino acid sequence and a light chain variable region amino acid sequence represented by SEQ ID NOs: 2 and 4, 6 and 8, 10 and 12, 14 and 16, 18 and 20, 22 and 24, 26 and 28, 30 and 32, 36 and 38, 42 and 44, 46 and 48, 50 and 52, 54 and 56, 60 and 58, 62 and 64, 66 and 68, 70 and 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 and 124, 126 and 128, 130 and 132, 134 and 136, 138 and 140, 142 and 144, 146 and 148, 150 and 152, 154 and 156, 158 and 160, 162 and 164, 166 and 168, 170 and 172, 174 and 176, 178 and 2024204502   28 Jun 2024 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 220, 222 and 224, 226 and 228 or 230 and 232, or, a heavy chain variable region amino acid sequence represented by SEQ ID NO: 34, 40, 60, 62 or 120 and a light chain variable region amino acid sequence represented by SEQ ID NO: 58 or 64, respectively. The isolated HER3-binding protein of the present invention more preferably comprises a heavy chain variable region amino acid sequence represented by SEQ ID NO: 42, 54, 70, 92, or 96 and a light chain variable region amino acid sequence represented by 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 represented by SEQ ID NOs: 2 and 4 is referred to as "Ul-39", an antibody comprising a heavy chain variable region amino acid sequence and a light chain variable region amino acid sequence represented by SEQ ID NOs: 6 and 8 is referred to as "Ul-40", an antibody comprising a heavy chain variable region amino acid sequence and a light chain variable region amino acid sequence represented by SEQ ID NOs: 10 and 12 is referred to as "Ul-38", an antibody comprising a heavy chain variable region amino acid sequence and a light chain variable region amino acid sequence represented by SEQ ID NOs: 14 and 16 is referred to as "Ul-41", an antibody comprising a heavy chain variable region amino acid sequence and a light chain variable region amino acid sequence represented by SEQ ID NOs: 18 and 20 is referred to as "UI-42", an antibody comprising a heavy chain variable region amino acid sequence and a light chain variable region amino acid sequence represented by SEQ ID NOs: 22 and 24 is referred to as "Ul-43", an antibody comprising a heavy chain variable region amino acid sequence and a light chain variable region amino acid sequence represented by SEQ ID NOs: 26 and 28 is referred to as "UI-44", an antibody comprising a heavy chain variable region amino acid sequence and a light chain variable region amino acid sequence represented by SEQ ID NOs: 30 and 32 is referred to as "Ul-45", an antibody comprising a heavy chain variable region amino acid sequence and a light chain variable region amino acid sequence represented by SEQ ID NOs: 36 and 38 is referred to as "Ul-47", an antibody comprising a heavy chain variable region amino acid sequence and a light chain variable region amino acid sequence represented by SEQ ID NOs: 42 and 44 is referred to as "Ul-49", an antibody comprising a heavy chain variable region amino acid sequence and a light chain variable region amino acid sequence represented by SEQ ID NOs: 46 and 48 is referred to as "Ul-50", an antibody comprising a heavy chain variable region amino acid sequence and a light chain variable region amino acid sequence represented by SEQ ID NOs: 50 and 52 is referred to as "Ul-51", an antibody comprising a heavy chain variable region amino acid sequence and a light 2024204502   28 Jun 2024 chain variable region amino acid sequence represented by SEQ ID NOs: 54 and 56 is referred to as "Ul-53", an antibody comprising a heavy chain variable region amino acid sequence and a light chain variable region amino acid sequence represented by SEQ ID NOs: 60 and 58 is referred to as "Ul-55", an antibody comprising a heavy chain variable region amino acid sequence and a light chain variable region amino acid sequence represented by SEQ ID NOs: 62 and 64 is referred to as "Ul-57", an antibody comprising a heavy chain variable region amino acid sequence and a light chain variable region amino acid sequence represented by SEQ ID NOs: 66 and 68 is referred to as "UI-58", an antibody comprising a heavy chain variable region amino acid sequence and a light chain variable region amino acid sequence represented by SEQ ID NOs: 70 and 72 is referred to as "Ul-59", an antibody comprising a heavy chain variable region amino acid sequence and a light chain variable region amino acid sequence represented by SEQ ID NOs: 74 and 76 is referred to as "Ul-52", an antibody comprising a heavy chain variable region amino acid sequence and a light chain variable region amino acid sequence represented by SEQ ID NOs: 78 and 82 is referred to as "Ul-61", an antibody comprising a heavy chain variable region amino acid sequence and a light chain variable region amino acid sequence represented by SEQ ID NOs: 80 and 82 is referred to as "Ul-61.1", an antibody comprising a heavy chain variable region amino acid sequence and a light chain variable region amino acid sequence represented by SEQ ID NOs: 84 and 86 is referred to as "Ul-62", an antibody comprising a heavy chain variable region amino acid sequence and a light chain variable region amino acid sequence represented by SEQ ID NOs: 88 and 90 is referred to as "UI-2", an antibody comprising a heavy chain variable region amino acid sequence and a light chain variable region amino acid sequence represented by SEQ ID NOs: 92 and 94 is referred to as "Ul-7", an antibody comprising a heavy chain variable region amino acid sequence and a light chain variable region amino acid sequence represented by SEQ ID NOs: 96 and 98 is referred to as "Ul-9", an antibody comprising a heavy chain variable region amino acid sequence and a light chain variable region amino acid sequence represented by SEQ ID NOs: 100 and 102 is referred to as "Ul-10", an antibody comprising a heavy chain variable region amino acid sequence and a light chain variable region amino acid sequence represented by SEQ ID NOs: 104 and 106 is referred to as "Ul-12", an antibody comprising a heavy chain variable region amino acid sequence and a light chain variable region amino acid sequence represented by SEQ ID NOs: 108 and 110 is referred to as "UI-13", an antibody comprising a heavy chain variable region amino acid sequence and a light chain variable region amino acid sequence represented by SEQ ID NOs: 112 and 114 is referred to as "UI-14", an antibody comprising a heavy chain variable region amino acid sequence and a light chain variable region amino acid sequence represented by 2024204502   28 Jun 2024 SEQ ID NOs: 116 and 118 is referred to as "UI-15", an antibody comprising a heavy chain variable region amino acid sequence and a light chain variable region amino acid sequence represented by SEQ ID NOs: 122 and 124 is referred to as "Ul-20", an antibody comprising a heavy chain variable region amino acid sequence and a light chain variable region amino acid sequence represented by SEQ ID NOs: 126 and 128 is referred to as "Ul-21", an antibody comprising a heavy chain variable region amino acid sequence and a light chain variable region amino acid sequence represented by SEQ ID NOs: 130 and 132 is referred to as "Ul-22", an antibody comprising a heavy chain variable region amino acid sequence and a light chain variable region amino acid sequence represented by SEQ ID NOs: 134 and 136 is referred to as "Ul-23", an antibody comprising a heavy chain variable region amino acid sequence and a light chain variable region amino acid sequence represented by SEQ ID NOs: 138 and 140 is referred to as "Ul-24", an antibody comprising a heavy chain variable region amino acid sequence and a light chain variable region amino acid sequence represented by SEQ ID NOs: 142 and 144 is referred to as "Ul-25", an antibody comprising a heavy chain variable region amino acid sequence and a light chain variable region amino acid sequence represented by SEQ ID NOs: 146 and 148 is referred to as "Ul-26", an antibody comprising a heavy chain variable region amino acid sequence and a light chain variable region amino acid sequence represented by SEQ ID NOs: 150 and 152 is referred to as "Ul-27", an antibody comprising a heavy chain variable region amino acid sequence and a light chain variable region amino acid sequence represented by SEQ ID NOs: 154 and 156 is referred to as "Ul-28", an antibody comprising a heavy chain variable region amino acid sequence and a light chain variable region amino acid sequence represented by SEQ ID NOs: 158 and 160 is referred to as "Ul-31", an antibody comprising a heavy chain variable region amino acid sequence and a light chain variable region amino acid sequence represented by SEQ ID NOs: 162 and 164 is referred to as "Ul-32", an antibody comprising a heavy chain variable region amino acid sequence and a light chain variable region amino acid sequence represented by SEQ ID NOs: 166 and 168 is referred to as "UI-35", an antibody comprising a heavy chain variable region amino acid sequence and a light chain variable region amino acid sequence represented by SEQ ID NOs: 170 and 172 is referred to as "Ul-36", an antibody comprising a heavy chain variable region amino acid sequence and a light chain variable region amino acid sequence represented by SEQ ID NOs: 174 and 176 is referred to as "Ul-37", an antibody comprising a heavy chain variable region amino acid sequence and a light chain variable region amino acid sequence represented by SEQ ID NOs: 178 and 180 is referred to as "Ul-34", an antibody comprising a heavy chain variable region amino acid sequence and a light chain variable region amino acid sequence represented by SEQ ID NOs: 182 and 184 is referred to as "Ul-1", an 2024204502   28 Jun 2024 antibody comprising a heavy chain variable region amino acid sequence and a light chain variable region amino acid sequence represented by SEQ ID NOs: 186 and 188 is referred to as "Ul-3", an antibody comprising a heavy chain variable region amino acid sequence and a light chain variable region amino acid sequence represented by SEQ ID NOs: 190 and 192 is referred to as "Ul-4", an antibody comprising a heavy chain variable region amino acid sequence and a light chain variable region amino acid sequence represented by SEQ ID NOs: 194 and 196 is referred to as "Ul-5", an antibody comprising a heavy chain variable region amino acid sequence and a light chain variable region amino acid sequence represented by SEQ ID NOs: 198 and 200 is referred to as "Ul-6", an antibody comprising a heavy chain variable region amino acid sequence and a light chain variable region amino acid sequence represented by SEQ ID NOs: 202 and 204 is referred to as "UI-8", an antibody comprising a heavy chain variable region amino acid sequence and a light chain variable region amino acid sequence represented by SEQ ID NOs: 206 and 208 is referred to as "UI-11", an antibody comprising a heavy chain variable region amino acid sequence and a light chain variable region amino acid sequence represented by SEQ ID NOs: 210 and 212 is referred to as "UI-16", an antibody comprising a heavy chain variable region amino acid sequence and a light chain variable region amino acid sequence represented by SEQ ID NOs: 214 and 216 is referred to as "UI-17", an antibody comprising a heavy chain variable region amino acid sequence and a light chain variable region amino acid sequence represented by SEQ ID NOs: 218 and 220 is referred to as "UI-18", an antibody comprising a heavy chain variable region amino acid sequence and a light chain variable region amino acid sequence represented by SEQ ID NOs: 222 and 224 is referred to as "Ul-33", an antibody comprising a heavy chain variable region amino acid sequence and a light chain variable region amino acid sequence represented by SEQ ID NOs: 226 and 228 is referred to as "Ul-29", an antibody comprising a heavy chain variable region amino acid sequence and a light chain variable region amino acid sequence represented by SEQ ID NOs: 230 and 232 is referred to as "Ul-30", an antibody comprising a heavy chain variable region amino acid sequence represented by SEQ ID NO: 34 is referred to as "Ul-46", an antibody comprising a heavy chain variable region amino acid sequence represented by SEQ ID NO: 40 is referred to as "Ul-48", an antibody comprising a heavy chain variable region amino acid sequence and a light chain variable region amino acid sequence represented by SEQ ID NOs: 60 and 58 is referred to as "Ul-55.1", an antibody comprising a heavy chain variable region amino acid sequence represented by SEQ ID NO: 120 is referred to as "UI-19", and an antibody comprising a heavy chain variable region amino acid sequence and a light chain variable region amino acid sequence represented by SEQ ID NOs: 62 and 64 is 2024204502   28 Jun 2024 referred to as "Ul-57.1". These antibodies are described in detail in Examples. The isolated HER3-binding protein of the present invention even more preferably comprises a heavy chain variable region amino acid sequence and a light chain variable region amino acid sequence represented by SEQ ID NOs: 42 and 44, respectively, a heavy chain variable region amino acid sequence and a light chain variable region amino acid sequence represented by SEQ ID NOs: 54 and 56, respectively, a heavy chain variable region amino acid sequence and a light chain variable region amino acid sequence represented by SEQ ID NOs: 70 and 72, respectively, a heavy chain variable region amino acid sequence and a light chain variable region amino acid sequence represented by SEQ ID NOs: 92 and 94, respectively, or a heavy chain variable region amino acid sequence and a light chain variable region amino acid sequence represented by SEQ ID NOs: 96 and 98, respectively, and still even more preferably, the HER3-binding proteins is Ul-49, Ul-53, Ul-59, Ul-7, or Ul-9, which are an anti-HER3 antibody.

[0072] 2024204502   28 Jun 2024 [Chem.14] Sequence Listing Antibody Ul-39 1 Heavy Chain DMA: GAGGTGCAGCTGGTGGAGTCTGGAGGAGGCTTGATCCAGCCTGGGGGGTCCCTGAGACTC TCCTGTGCAGCCTCTGGGTTCACCGTCAGTAGCAACTACATGAGCTGGGTCCGCCAGGCT CCAGGGAAGGGGCTG GATTGGGT CTCAGTTATTTATAGCGGTGGTAGC ACATACTACGCA GACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTT CAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTGTATTACTGTGCGAGAGGGCAGTGG ctggacgtctggggccaagggaccacggtcaccgtctcctca 2 Heavy Chain Protein: evqlvesgggliqpggslrlscaasgftvssnymswvrqapgkgldwsviysggstyya D SVKG R FTIS RUNSKNTLYLQMNS LRAEDTAVY YCARGQWLD WGQGTTVTV S S 3 Light Chain DMA: GATATTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTGGAGAGCCGGCCTCC atctcctgcaggtcaagtcagagcctcctgcatagtaatggatacaactatttggattgg tacctgcagaggccagggcagtctccacaactcctgttctatttgggttttcatcgggcc tccggggtccctgacaggttcagtggcagtggatcaggcacagattttacactgaaaatc AGCAGAGTGGAGGCTGa GGATGTTGGGGTTTATTACTGCAGGCAAG CT CTACAAACTCC G CTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAA 4 Light Chain. Protein: DIVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQRPGQSPQLLFYLGFHRA sgvpdrfsgsgsgtdftlkisrveaedvgvyycrqalqtpltfgggtkveik Antibody UI-40 5 Heavy Chain DMA: CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCACAGACCCTGTCCCTC ACCTGTACTGTCTCTGGTGGCTCCATCAGCAGTGGTGGTTACTACTGGAGCTGGATCCGC CAGCACCCAGGGAAGGGCCTGGAGTGGATTGGGTACATCTATTCCAGTGGGAGCACCTAC TACAACCCGTCCCTCAAGAGTCGAGTTACCATATCAGTAGACACGTCTAAGAACCAGTTC TCCCTGAAGCTGAGCTCTGTGACTGCCGCGGACACGGCCGTGTATTACTGTGCGAGAGAT AGGGAACTGGAACTTTACTACTACTACTACGGTATGGACGTCTGGGGCCAAGGGACCACG GTCACCGTCTCCTC 6 Heavy Chain Protein: QVQLQESGPGLVKPSQTLSLTCTVSGG9ISSGGYYWSWIRQHPGKGLEWIGYIYSSGSTY YNPSLKS RVTISVDTSKNQFSLKLSSVTAADTAVYYCARDRELELYYYYYGMDVWGQGTT VTVS                                                            • 7 Light Chain DNA: GATATTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTGGAGAGCCGGCCTCC ATCTCCTGCAGGTCTAGTCAGAGC CTCCTGTATAGTAATGGATACAACTATTTGGATTGG TACCTGCAGAAGCCAGGGCAGTCTCCACAGCTCCTGATCTATTTGGGTTCTAATCGGGCC TCCGGGGTCCCTGACAGGTTCAGTGGCAGTGGATCAGGCACAGATTTTACACTGAAAATC AGCAGAGTGGAGGCTGAGGATGTTGGGATTTATTACTGCATGCAAGCTCTACAAACTCCG CTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAA 8 Light Chain Protein:                                           . DIVMTQSPLSLPVTPGEPASISCRSSQSLLYSMGYNYLDWYLQKPGQSPQLLIYLGSNRA SGVPDRFSGSGSGTDFTLKISRVEAEDVGIYYCMQALQTPLTFGGGTKVEIK Antibody UI-38 9 Heavy Chain DNA: CAGATCACCTTGAAGGAGTCTGGTCCTACGCTGGTGAAACCCACACAGACCCTCACGCTG ACCTGCACCTTCTCTGGGTTCTCACTCAGCACTAGTGGAGTGGGTGTGGGCTGGATCCGT CAGCCCCCAGGAAAGGCCCTGGACTGGCTTGCACTCATTTATTGGAATGATGATAAGCGC 2024204502   28 Jun 2024 10 11 12 TacAGCCCATCTCTGAAGAGCAGGCTCACCATCACCAAGGACACCTCCAAAAACCAGuiG GTCCTTACAATGACCAACATGGATCTTGTGGACACAGCCACATATTACTGTGTACACAGA gacgaagttcgagggtttgactactggggccagggaaccctggtcaccgtctcctca Heavy Chain Protein:                                ■ oitlkesgptlvkptqtltltctfsgfslstsgvgvgwirqppgkaldwlaliywnddkr ygpSLKSRLTITKDTSKNQVVLTMTNMDLVDTATYYCVHRDEVRGFDYWGQGTLVTVSS Light Chain DNA: GATGTTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCTTGGACAGCCGGCCTCC ATCTCCTGCAGGTCTAGTCAAAGCCTCGTATACAGTGATGGATACACCTACTTGCATTGG TTTCAGCAGAGGCCAGGCCAATCTCCAAGGCGCCTTATTTATAAGGTTTCTAACTGGGAC TCTGGGGTCCCAGACAGATTCAGCGGCAGTGGGTCAGGCACTGATTTCACACTGAAAATC AGCAGGGTGGAGGCTGAGGATGTTGGGGTTTATTACTGCATGCAAGGTGCACACTGGCCG ATCACCTTCGGCCAAGGGACACGACTGGAGATTAAA    - Light Chain Protein: DWMTQSPLSLPVTLGQPASISCRSSQSLVYSDGYTYLHWFQQRPGQSPRRLIYKVSNWD SGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQGAEWPITFGQGTRLEIK Antibody UI ■ 41 CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCACAGACCCTGTCCCTC a,CCTGCACTGTCTCTGGTGGCTCCATCAGCAGTGGTGGGTACTACTGGAGCTGGATCCGC CAGCACCCAGGGAAGGGCCTGGAGTGGATTGGGTACATCTATTACAGTGGGAGCACCTAC TACAA CCCGTCCCTCAAGAGTCGAGTTACCATATCAGTAGACACGTCTAAGAACCAGTTC TCCCTGAAGCTGAGCTCTGTGACTGCCGCGGACACGGCCGTGTATTTCTGTGCGAGAGAT CGGGAACTTGAGGGTTACTCCAACTACTACGGTGTGGACGTCTGGGGCCAAGGGACCACG GTCACCGTCTCCTC 14 Heavy Chain Protein: OVOLQESGPGLVKPSQTLSLTCTVSGGSISSGGYYWSWIRQHPGKGLEWIGYIYYSGSTY ynpslksrvtisvdtsknqfslklssvtaadtavyfcardrelegysnyygvdvwgqgtt VTVS 15 Light Chain DMAs GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACC atcacttgccgggcaagtcaggccattagcaactatttaaattggtatcagcagaaacca gggaaagcccctaagctcctgatctatgctgcatccagtttgcaaagtggggtcccatca aggttcagtggcagtggatctgggacagatttcactctcaccatcagcagcctgcagcct gaagattttgcaacttattactgtcaacagaataatagtctcccgatcaccttcggccaa gggacacgactggagattaaa 16 Light Chain Protein: DIQMTQSPSSLSASVGDRVTITCRASQAISNYLNWYQQKPGKAPKLLIYAASSLQSGVPS RFSGSGSGTDFTLTISSLQPEDFATYYCQQNNSLPITFGQGTRLEIK Antibody UI-42 17 Heavy Chain DNA: ' gaggtgcagctggtgcagtctggagcagaggtgaaaaagcccggggagtctctgaagatc TCCTGTAAGGGTTCTGGATACAGCTTTACCAG CTACTGGATCGGCTGGGTGCGCCAG ATG CCCGGGAAAGGCCTGGAGTGGATGGGGATCATCTATCCTGGTGACTCTGATACCAGATAC aGCCCGTCCTTCCAAGGCCAGGTCACCATCTCAGCCGACAAGTCCATCAGCACCGCCTAC CTGCAGTGGAGCAGCCTGAAGGCCTCGGACACCGCCATGTATTACTGTGCGAGACATGAA AACTA CGGTGACTACAACTACTGGGG CCAGGG AA CCCTGGTCACCGTCTCCTCA 18 Heavy Chain Protein: EVOLVQSGAEVKKPGESLKISCKGSGYSFTSYIHGWVRQMPGKGLEWMGIIYPGDSDTR^ SPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARHENYGDYNYWGQGTLVTVSS 19 Light Chain DNA: 2024204502   28 Jun 2024 20 21 22 23 24 25 26 27 OAGATCCAGATGACCCAGTCTCCATCCTCCCTGTCI'GCAtCtGtCGGAGACAGAGTCACC ATCACTTGCCGGGCAAGTCAGAGCATTCGCAGCTAT'TTAAATTGGTATCAGCAGAAACCA gggaaagcccctaagctcctgatctatgctgcttccagtttgcaaagtggggtcccatca bggttcagtggcagtggatctgggacagatttcactctcaccatcagcagtctgcaacc gggaccaaggtggagatcaaa rfsgsgsgtdftltisslqpedfalyccqqsngspltfgggtkveik Antibody UI-43 caggtgcagctgcaggagtcgggcccaggactggtgaagccttcacagaccctgtccctc acctgcactgtctctggtggctccatcagcagtggtggttactactggagctggatccgc fagScccagggaagggcctgga^ tacaacccgtccctcaggagtcgagttaccatatcagtagacacgtctaagaaccagttc X^gSgagc^ SaSagS^^ accacggtcaccgtctcctc nvOLOESGPGbVKPSQTLSLTCTVSGGSISSGGYYWSWIRQHPGKGLEWIGYIYYSGSTY TTVTVS GACATCWGATGACCCAGTCTCCATCCTCCCTGTCTCCATCTGTAGGAGACAGAGTCACC rrrAAAGCCCCTAAGCTCCTGATCCATGCTGCATCCAGTTTACAAAGlGGGGTCCCATCA aggSSgSgXggatctggga^^^^^ gaagattttgcaacttactactgtcaacagagttacagtaacccgctcactttcggcgga gggaccaaggtggagatccaa rfsgsgsgtdftltisslqpedfatyycqqsysnpltfgggtkveiq Antibody UI-44 gaggtgSgctggtgcagtctggagcagaggtgaaaaagcccggggagtctctgaagatc tcctgtaagggttctggatacagctttaccagctactggatcggctgggtgcgccagatg cccgggaaaggcctggagtggatggggatcatctggcctggtgactctgataccatatac agcccgtccttccaaggccaggtcaccatctcagccgacaagtccatcagcaccgcctac ctgcagtogagcagcctgaaggcctcggacaccgccatgtattactgtgcgagacatgaa aIctSgXcaactac^^ FVOLVQSGAEVKKPGESLKisCKGSGYSFTSYWIGWVRQMPGKGLEFJMGIIWPGDSDTIY XXTIsZsiSTAYLQWSSLmDT^^ GlSTcX.TGACckGTC^ atcacttgccgggcaagtcagagcattcgaagttatttaaattggtatcagcagaaaccg gggaatgcccctaaactcctgatctatgctgcatccagtttgcaaagtggggtcccatca acgttcagtggcagtggatctgggacagatttcactctcaccatcagcagtctgcaacct gSStgcXtactactgtc^^ gg GACCAAGGTGGAGATCAAA 2024204502   28 Jun 2024 28 Light Chain Protein: DIQMTQSPSSLSASVGDRVTITCRASQSIRSYLNWYQQKPGNAPKLLIYAASSLQSGVPS rFSGSGSGTDFTLTISSLQPEDFALYYCQQSISSPLTFGGGTKVEIK Antibody (UI-45) 29 Heavy Chain DNA:          ■' CAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAGGiC TCCTGCAAGGCTTCTGGATACACCTTCACCAGTTATGATATCAACTGGGTGCGACAGGCC actggacaagggcttgagtggatgggatggatgaaccctaacagtggtgacactggctat GCACAGGTGTTCCAGGG CAGAGTCACCATG ACCTGGAACA CCTCCATAAGCAC AG CCTAC ATGGAACTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGTGCGAGATTTGGG GATCTCCCGTATGACTACAGTTACTACGAATGGTTCGACCCCTGGGGCCAGGGAACCCTG GTCACCGTCTCCTC 30 Heavy Chain Protein: OVOLVQSGAEVKKPGASVKVSCKASGYTFTSYDINWVRQATGQGLEWMGWMNPWSGDTGY AQVFQGRVTMTWNTSISTAYMELSSLRSEDTAVYYCARFGDLPYDYSYYEWFDPWGQGTL VTVS 31- Light Chain DNA: GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACC ATCACTTGCCGGGCAAGCCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAGACCA gggaaagcccctaagctcctgatctatgcagcatccagtttgcaaagtggggtcccatca aggttcagtggcagtggatctgggacagatttcactctcaccatcagcagtctgcaacct gaagattttgcaacttactactgtcaacagagttacagtaccccgctcactttcggcgga gggaccaaggtggagatcaaa 3? Lj oht Chiaiii Pirotom* diqmtqspsslsasvgdrvtitcrasqsissylnwyqqrpgkapklliyaasslqsgvps rfsgsgsgtdftltisslqpedfatyycqqsystpltfgggtkveik Antibody (UI-46 33 Heavy Chain DMA:                                           ___ CAGGTACAGCTGCAGCAGTCAGGTCCAGGACTGGTGAAGCCCTCGCAGACCCTCTCACTC ACCTGTGCCATCTCCGGGGACAGTGTCTCTAGCAACAGTGCTGCTTGGAACTGGATCAGG C AGTCCC CATCGAGAGGCCTTGAGTGG CTGGGAAG GACATACT A CAGGT CC AAGTGGT AT AATGATTATGCAGTATCTCTGAAAAGTCGAATAACCATCAACCCAGACACATCCAAGAAC CAGTTCTCCCTGCAGCTGAACTCTGTGACTCCCGAGGACACGGCTGTGTATTACTGTGCA AGAGATCTCTACGATTTTTGGAGTGGTTATCCCTACTACTACGGTATGGACGTCTGGGGC CAAGGGACCACGGTCACCGTCTCCTC 34 Heavy Chain Protein: OVOLOOSGPGLVKPSQTLSLTCAISGDSVSSNSAAWNWIRQSPSRGLEWLGRTYYRSKWY ndyavsvksritinpdtsknqfslqlnsvtpedtavyycardlydfwsgypyyygmdvwg QGTTVTVS Antibody Ul-47 3 5 CAGGTACAGCTGCAGCAGTCAGGTCCAGGACTGGTGAAGCCCTCGCAGACCCTCTCACTC ACCTGTGCCATCTCCGGGGACAGTGTCTCTAGCAACAGTGCTGCTTGGAACTGGATCAGG CAGTCCCCATCGAGAGGCCTTGAGTGGCTGGGAAGGACATACTACAGGTCCAAG'TGGTAT AATGATTATGCAGTATCTGTGAAAAGTCGAATAACCATCAACCCAGACACATCCAAGAAC CAGTTCTCCCTGCAGCTGAACTCTGTGACTCCCGAGGACACGGCTGTGTATTACTGTGCA AGAGATTACTATGGTTCGGGGAGTTTCTACTACTACTACGGTATGGACGTCTGGGGCCAA gggaccacggtcaccgtctcctc 36 Heavy Chain Protein: QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSAAWNWIRQSPSRGLEWLGRTYYRSKh Y 2024204502   28 Jun 2024 37 33 39 40 MDYAVSVKSRITINPDTSKNQFSLQLNSVTPED'TAVYYCARDYYGSGS^YYYiGi-lDWGQ GTTVTVS gacatcSgatgacccagtctccatcctccctgtctgcatctgtaggagacagagtcacc atcacttgccgggcaagtcagagcattagcagctatttaaattggtatcagcagaaacca gggaaagcccctaaggtcctgatctatgctgcatccaatttgcaaagtggggtcccatca aggttcagtggcagtggatctgggacagatttcactctcaccatcagcagtctgcaacct gaagattttgcaacttactactgtcaacagagttacagtacccctcggacgttcggccaa gggaccaaggtggaaatcaaa Light Chain Protein: DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKVLIYAASNLQSGVPS rFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPRTFGQGTKVEIK Antibody UI-48 CAGGTG^GCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGACCCTGTCCCTC ACCTGCACTGTCTCTGGTGGCTCCATCAGTAGTTACTACTGGAGCTGGATCCGGCAGCCC gccgggaagggactggagtggattgggcatatctataccagtgggagcaccaactacaac ccctccctcaagagtcgagtcaccatgtcagtagacacgtccaagaaccagttctccctg aagctgagctctgtgaccgccgcggacacggccgtgtattactgtgcgagagaagcgatt tttggagtgggcccctactactactacggtatggacgtctggggccaagggaccacggtc accgtctcctc ovolqesgpglvkpsetlsltctvsggsissyywswirqpagkglewighiytsgstnyn TVS 41 42 43 44 Antibody Ul-49 caggtgcagctggtgcagtctggggctgaggtgaagaagcctggggcctcagtgaaggtc tcctgcaaggcttctggatacaccttcaccggctactatatgcactgggtgcgacaggcc cctggacaagggcttgagtggatgggatggatcaaccctaatattggtggcacaaactgt gcacagaagtttcagggcagggtcaccatgaccagggacacgtccatcagcacagcctac atggagctgagcaggctgagatctgacgacacggccgtgtattactgtgcgagaggggga cggtatagcagcagctggtcctactactactacggtatggacgtctggggccaagggacc acggtcaccgtctcctc Heavy Chain Protein: „ qvqlvqsgaevkkpgasvkvsckasgytftgyymhwrqapgqglewgwinpniggtnc AQKFQGRVTMTRDTSISTAYMELSRLR SDDTAVYYCARGGRY S S SWSYYYYGMDVWGQGT TVTVS GATATTCTGATGACCCAGACTCCACTCTCTCTGTCCGTCACCCCTGGACAGCCGGCCTCC ATCTCCTGCAAGTCTAGTCAGAGCCTCCTGCTTAGTGATGGAGGGACCTATTTGTATTGG Scctgcagaagccaggccagcctccacagctcctgatctatgaagtttccaaccggttc tctggagtgccagataggttcagtggcagcgggtcagggacagatttcacactgaaaatc agccgggtggaggctgaggatgttggggtttattactgcatgcaaagtatgcagcttccg atcaccttcggccaagggacacgactggaaattaaa gGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQSMQLPITFGQGTRLEIK Antibody UI-50 45 Heavy Chain DNA: 2024204502   28 Jun 2024 46 47 43 49 CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGACCCTGTCCCLC ACCTGCACTGTCTCTGGTGGCTCCGTCAGCAGTGGTGGTTACTACTGGAGCTGGATCCGG CAGCCCCCAGGGAAGGGACTGGAGTGGATTGGGTATATCTATTACAGTGGGAGCACCAAC TACAACCCCTCCCTCAAGAGTCGAGTCACCATATCAGTAGACACGTCCAAGAACCAGTTC TCCCTGAAGCTGAGCTCTGTGACCGCTGCGGACACGGCCGTGTATTACTGTGCGAGAGGG GGGGACAGTAACTACGAGGATTACTACTACTACTACGGTATGGACGTCTGGGGCCAAGGG ACCACGGTCACCGTCTCCTC Heavy Chain Protein: OVOLQESGPGLVKPSETLSLTCTVSGGSVSSGGYYWSWIRQPPGKGLEWIGYIYYSGSTN YNPSLKSRVTISVDTS KNQF SLKLS SVTAADTAVYYCARGGDSNYEDYYYYYGMDVWGQG TTVTVS Light Chain DNA: GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACC ATCACTTGCCGGGCAAGTCAGAGCATTAGCATCTATTTACATTGGTATCAGCAGAAACCA GGGAAAGCCCCTAAGCTCTTGATCTCTGCTGCATCCAGTTTGCAAAGTGGGGTCCCGTCA AGGTTCAGTGG CAGTGGATCTGGGACAGATTTCACTCTCACCATCAGAAGTCTG CAACCT GAAGATTTTGCAACTTACTACTGTCAACAGAGTTACACTTCCCCGATCACCTTCGGCCAA gggacacgactggagattaaa Light Chain Protein: DIQMTQSPSSLSASVGDRVTITCRASQSISIYLHWYQQKPGKAPKLLISAASSLQSGVPS RFSGSGSGTDFTLTIRSLQPEDPATYYCQQSYTSPITFGQGTRLEIK Antibody Ul-51 Heavy Chain DNA: „„„„ CAGGT GCAGCTGCAGGAG TCGGGCCCAGGACTGGTGAAGCCTTCGGAGAC CCTGTCCCT C ACCTG CACTGTCTCTGGTGGCTCCATCAGTAGTTACTACTGGAGCTGGAT CCGGCAGCCC CCAGGGAAGGGACTGGAGTGGATTGGGTATATCTATTACAGTGGGAGCACCAACTACAAC CCCTCCCTCAAGAGTCGAGTCACCATATCAGTAGACACGTCCAAGCACCAGTTCTCCCTG aagctgagctctgtgaccgctgcggacacggccgtgtattactgtgcgagagattcgagt tactatgatagtagtggttattacttatactactacgctatggacgtctggggccaaggg ACCACGGTCACCGTCTCCTC 50 Heavy Chain Protein: ovqlqesgpglvkpsetlsltctvsggsissyywswirqppgkglewigyiyysgstnyn PSLKSRVTISVDTSKHQFSDKLSSVTAADTAVYYCARDSSYYDSSGYYLYYYAMDVWGQG TTVTVS 51 Light Chain DNA:                                           __ gacatcgtgatgacccagtctccagactccctggctgtgtctctgggcgagagggccacc atcaactgcaagtccagccagagtgttttatacagctccaacaataagaactacttagct tggtaccagcagaaaccaggacagcctcctaagctgctcatttcctgggcatctacccgg gaatccggggtccctgaccgattcagtggcagcgggtctgggacagatttcactctcacc atcagcagcctgcaggctgaagatgtggcagtttattactgtcagcaatattatactact cctctcactttcggccctgggaccaaagtggatatcaaa 52 Light Chain Protein:                                        ___ DIVMTQSPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQPPKLLISWASTR E SGVPDRF S GS GS GTDFTLTIS SLQAEDVAVYY CQQYYTTPLTFGPGTKVDIK Antibody Ul-53 53 Heavy Chain DNA: GAGGTGCAACTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTC -PCCTGTGCAGCCTCTGGATTCACCTTCAGTATCTATAGCATGAACTGGGTCCGCCAGGCT CCAGGGAAGGGGCTGGAGTGGCTTTCATACATTAGTAGTAGTAGTAGTACCATATACTAC GCAGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAATGCCAAGAACTCACTGTAT CTGCAAATGAACAGCCTGAGAGACGAGGACACGGCTGTGTATTACTGTGCGAGAGATAGG 2024204502   28 Jun 2024 GGTGACTTCGATGCTTTTGATATCTGGGGCCAAGGGACAATGGTCACGGTC1C11GA 54 Heavy Chain Protein:                                         . EVQLVESGGGLVQPGGSLRLSCAASGFTFSIYSMNWVRQAPGKGLEWVSYISSSSSTIZY ADSVKGRFTISRDNAKNSLYLQMNSLRDEDTAVYYCARDRGDFDAFDIWGQGTMVTVSS 55 Light Chain DNA: GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACC ATCACTTGCCAGGCGAGTCAGGACATTACCAACTATTTGAATTGGTATCAGCAGAAACCA gggaaagcccctaagctcctgatctacgatgcatccaatttggaaacaggggtcccatca aggttcagtggaagtggatctgggacagattttactttcaccatcagcagcctgcagcct GAAGATATTGCAACATATAACTGTCAACAGTGTGAAAATTTCCCGATCACCTTCGGCCAA gggacacgactggagattaaa 56 Light Chain Protein: diqmtqspsslsasvgdrvtitcqasqditnylnwyqqkpgkapklliydasnletgvps rfsgsgsgtdftftisslqpediatyncqqcenfpitfgqgtrleik Antibody Ul-55 57 GATATTOTGATCACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTGGAGAGCCGGCCTCC atctcctgcaggtctagtcagagcctcctgtatagtaatggatacaagtatttggattgg tacctgcagaagccagggcagtctccacaggtcctgatctatttgggttctaatcgggcc TCCGGGGTCCCTGACAGGTTCAGTGGCAGTGGATCAGGCACAGATTTTACACTGAAAATC agcagagtggaggctgaggatgttggggtttattattgcatgcaggctctacaaactccg atcaccttcggccaagggacacgactggagattaaa 58 Light Chain Protein: DIVMTQSPLSLPVTPGEPASISCRSSQSLLYSNGYKYLDWYLQKPGQSPQLLIYLGSNRA sgvpdrfsgsgsgtdftlkisrveaedvgvyycmqalqtpitfgqgtrleik Antibody (UI-55.1) 59 Heavy Chain DNA:                                       „„„„„„ CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGACCCTGTCCCTC ACCTGCACTGTCTCTGGTGGCTCCGTCAGCAGTGGTGGTTACTACTGGAACTGGATCCGG CAGCCCCCAGGGAAGGGACTGGAGTGGATTGGGTATATCAATTACAGTGGGAGCACCAAC TACAACCCCTCCCTCAAGAGTCGAGTCACCATATCAGTAGACACGTCCAAGAACCAGTTC TCCCTGAAGCTGAGCTCTGTGACCGCTGCGGACACGGCCGTGTATTACTGTGCGAGAGAT CGAGAACTGGAACTTTACTACTACTACTACGGTATGGACGTCTGGGGCCAAGGGACCACG GTCACCGTCTCCTC 60 Heavy Chain Protein: OVQLQESGPGLVKPSETLSLTCTVSGGSVSSGGYYWNWIRQPPGKGLEWIGYINYSGSTN ynpslksrvtisvdtsknqfslklssvtaadtavyycardrelelyyyyygmdvwgqgtt VTVS Antibody (UI-57) 61 Heavy Chain DNA: CAGGTGCAG CTG CAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCTGAG AC CCTGTCCCTC ACCTGCACTGTCTCTGGTGGCTCCGTCAGCAGTGGTGGTTACTACTGGAACTGGATCCGG CAG CC CCCA GG GAAGGGACTGGAGT GGATTGGGTATATCAATTACAGTGGGAGCACCAAC TACAA CCCCTCCCTC AAGAGTCG AGTCAC CATAT CACTAGACACGTCCAAGAACCAGTTC TCCCTGAAG CTGAGCTCTGTGACCG CTGCGGACA CGG CCGTGTATTACTG TGCGAGAGAT CGAGAACTGGAACTTTACTACTACTACTACGGTATGGACGTCTGGGGCCAAGGGACCACG GTCACCGTCTCCTC 62 Heavy Chain Protein: OVQLQESGPGLVKPSETLSLTCTVSGGSVSSGGYY'WNWIRQPPGKGLEWIGYINYSGSTN ynpslksrvtisvdtsknqfslklssvtaadtavyycardrelelyyyyygmdvwgqgtt 2024204502   28 Jun 2024 64 65 66 67 68 69 70 71 Antibody UI-57.1 Light Chain DNA: GATATTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTGGAGAGCCGGCCTCC ATCTCCTGCAGGTCTAGTCAGAGCCTCCTGTATAGTAATGGATACAAGTATTTGGATTGG TACCTGCAGAAGCCAGGGCAGTCTCCACAGCTCATtSATCTATTTGGGTTCTAATCGGGCC TCCGGGGTCCCTGACAGGTTCAGTGGCAGTGGATCAGGCACAGATTTTACACTGAAAATC AGCAGAGTGGAGGCTGAGGATGTTGGGGTTTATTATTGCATGCAGGCTCTACAAACTCCG ATCACCTTCGGCCAAGGGACACGACTGGAGATTAAA Light Chain Protein: DIVMTQSPLSLPVTPGEPASISCRSSQSLLYSNGYKYLDWYLQKPGQSPQLMIYLGSNRA SGVPDRFS GSGSGTDFTL KIS RVEAEDVGVYYCMQALQT PITFGQGTRLEIK Antibody.Ul-58 Heavy Chain DNA: ' CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTC TCCTGTGCAGCG TCTGGATTCA CCTTCAGTAGCTATGGCATGCACTGGGTCCGCCAGGCT CCAGGCAAGGGGCTGGAGTGGGTGGCAGTTATATGGTATGATGGAAGTAATAAATACTAT GCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTAT CTGCAAATGAACAGCCTGAGAG CCGAGGACACGGCTGTGTATTACTGT GCGAGAG C AGCT CGCCTTGACTACTACTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCC TCA Heavy Chain Protein: QVOLVE SGGG WQPGR SLRL S CAASG FT F S S YGMHWVR QA PGKGLEWVAVIWYD G S NKYY AD SVKGRFTISRDNS KNT LYLQMNSLRAEDTAVYYCARAARLDYYYGMDVWGQGTTVTVS S                                 . Light Chain DNA:        . GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCTCC ATCACTTGCCGGGCAAGTCAGAGCATTAACAGCTATTTAAATTGGTTTCAGCAGAAGCCA GGGAAAGCCCCTCAGCTCCTGATCTTTGGTGCATCCGGTTTGCAAAGTGGGGTCCCATCA AGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAACAGTCTGCAACCT GAAG ATTTTGCAACTTACTA CTGTCAACAG AGTTACAGTTCCCCGCTCACCTTCGGCCAA GGGACACGACTGGAGATTAAA Light Chain Protein: DIQMTQSPSSLSASVGDRVSITCRASQSINSYLNWFQQKPGKAPQLLIFGASGLQSGVPS RFSGSGSGTDFTLTINSLQPEDPATYYCQQSYSSPLTFGQGTRLEIK Antibody UI-59 Heavy Chain DNA:                                      ____ CAGGTGCAGCTACAGCAGTGGGGCGCAGGACTGTTGAAGCCTTCGGAGACCCTGTCCCTC acctgcgctgtctatggtgggtccttcagtggttactactggagctggatccgccagccc CCAGGGAAGGGGCTGGAGTGGATTGGGGAAATCAATCATAGTGGAAGCACCAACTACAAC CCGTCCCTCAAGAGTCGAGTCACCATATCAGTAGAAACGTCCAAGAACCAGTTCTCCCTG AAGCTGAGCTCTGTGACCGCCGCGGACACGGCTGTGTATTACTGTGCGAGAGATAAGTGG ACCTGGTACTTCGATCTCTGGGGCCGTGGCACCCTGGTCACTGTCTCCTCA Heavy Chain Protein: qvqlqqwgagllkpsetlsltcavyggsfsgyywswirqppgkglewigeinhsgstnyn PSLKSRVTISVETSKNQFSLKLSSVTAADTAVYYCARDKWTWYFDLWGRGTLVTVSS Light Chain DNA: GACATCGAGATGACCCAGTCTCCAGACTCCCTGGCTGTGTCTCTGGGCGAGAGGGCCACC ATCAACTGCAGGTCCAGCCAGAGTGTTTTATACAGCTCCAGCAATAGGAACTACTTAGCT TGGTACCAGCAGAACCCAGGACAGCCTCCTAAGCTGCTCATTTACTGGGCTTCTACCCGG 2024204502   28 Jun 2024 72 73 74 75 76 GAATCCGGGGTCCCTGACCGATTCAGTGGCAGCGGGTCTGGGACAGATTTCACTCTCACC ATCAGCAGCCTGCAGGCTGAAGATGTGGCAGTTTATTACTGTCAGCAATATTATAGTACT CCTCGGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAA Light Chain Protein: DIEMTQSPDSLAVSLGERATINCRSSQSVLYSSSNRMYLAWYQQNPGQPPKLLIYWASTR ES'GVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSTPRTFGQGTKVEIK Antibody Ul-52 Heavy Chain DMA: CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCACAGACCCTGTCCCTC ACCTGCACTGTCTCTGGTGGCTCCATCAGCAGTGGTGGTTACTACTGGAGCTGGATCCGC CAGCACCCAGGG AAGGGCCTGGAGTGGATGGGGAACAT CTATTACAGTGGGAGCACCTAC TACAACCCGTCCCTCAAGAGTCGAGTTACCATATCAGTAGACACGTCTGAGAACCAGTTC TCCCTGAAGCTGAACTCTGTGACTGCCGCGGACACGGCCGTATATTACTGTGCGAGAGGG GGAACTGGAACCAATTACTACTACTACTACGGTATGGACGTCTGGGGCCAAGGGACCACG GTCACCGTCTCCTC Heavy Chain Protein: ovqlqesgpglvkpsqtlsltctvsggsissggyywswirqhpgkglewmgniyysgsty YNPSLKSRVTISVDTSENQFSLKLNSVTAADTAVYYCARGGTGTWYYYYYGMDVWGQGTT VTVS Light Chain DNA: gaaattgtgttgacgcagtctccaggcaccctgtctttgtctccaggggaaagagccacc CTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCAGCTACTTAGCCTGGTACCAGCAGAAA CCTGGCCAGGCTCCCAGGCTCCTCATCTATGGTGCATCCAGCTGGGCCACTGGCATCCCA AACAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGACTGGAG CCTGAAGATTTTGCAGTGTATTACTGTCAGCAGTATGGTAGCTCACCGCTCACTTTCGGC GGAGGGACCAAGGTGGAGATCAAA Light Chain Protein: EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSWATGIP nr F SG S GSGTDFTLTIS RLE PEDFAVYYCQQYGS S PLT FGGGTKVEIK 77 78 Antibody UI-61 Heavy Chain DNA:                                          __ CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCACAGACCCTGTCCCTC AC CTGCACTGTCTCTGGTGTCTCCATCAG CAGTGGTGGTTACTACTGGAG CTGGAT CCGC CAGCACCCAGGGATGGGCCTGGAGTGGATTGGGTACATCTATTACAGTGGGAGCACCTAC TA CA ACCCGTCCCTCAAG AGTCGAGTCA CCATATCAGAAG ACACGTCTAA G AACCAGTTC TCCCTGAAGCTGAGCTCTGTGACTGCCGCGGACACGGCCGTGTATTACTGTGCGAGAGAT TC CGAGTCCGAGTATAGCAGCTCGTCGAA CTACGGTATGGACGTCTGGGG CCAAGGGACC ACGGTCACCGTCTCCTC Heavy Chain Protein: QVQLQESGPGLVKPSQTLSLTCTVSGVSISSGGYYWSWIRQHPGMGLEWIGYIYYSGSTY YNPSLKSRVTISEDTSKNQFSLKLSSVTAADTAVYYCARDSESEYSSSSNYGMDVWGQGT TVTVS 79 Antibody UI-61.1 Heavy Chain DMA:                                        ___ CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCACAGACCCTGTCCCTC ACCTGCACTGTCTCTGGTGTCTCCATCAGCAGTGGTGGTTACTACTGGAGCTGGATCCGC CAGCACCCAGGGATGGGCCTGGAGTGGATTGGGTACATCTATTACAGTGGGAGCACCTAC TACAACCCGTCCCTCAAGAGTCGAGTCACCATATCAGAAGACACGTCTAAGAACCAGTTC TCCCTGAAGCTGAGCTCTGTGACTGCCGCGGACACGGCCGTGTATTACTGTGCGAGAGAT TCCGAGTCCGAGTATAGCAG CTCGTCGAACTACGGTATGGACGTCTGGGGCCAAGGGACC 2024204502   28 Jun 2024 81 82 83 84 85 86 87 88 acggtcaccgtctcctc Heavy Chain Protein: ISSGGYYWSWIRQHPGMGLEWIGYIYYSGSTY TVTVS CACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAATCACC atcacttgccgggcaagtcagaccattagcagctatttaaattggtatcagcagaaacca gggaaagcccctaagctcctgatctatgctgcatccagtttgcaaggtggggtcccatca gaagattttgcaacttactactgtcaacagagttacagtaacccgctcactttcggcgg. gggaccaaggtggagatcaaa MQmQSPSSLSASVGDRITITCRASQTISSYLNWYQQKPGKAPKLMYAASSLQGGVPS rFSGSVSGTDFTLTVSSLQPEDFATYYCQQSYSNPLTFGGGTKVEIK Antibody Ul-62 (2.9.1) rarrw'rACCTGGTGCAGTCTGGAGCAGAGGTGAAAAAGCCCGGGGAGTCTCTGAAGATC TCCTGTAAGGGTTCTGGATACAGTTTTACCAGCTACTGGATCGGCTGGGTGCGCCAGATG CCCGGGAAAGGCCTGGAGTGGATGGGGATCATCTATCCTGGTGACTCTGATACCAGATAC AGCCCGTCCTTCCAAGGCCAGGTCACCATGTCAGCCGACAAGTCCATCAGTACCGCCTAC ctgcagctgagcagccatgaaggc^^ ggctSctac^^ accgtctcctc , fvqS / oSSvkkpgeslkisckgsgysftsywigwvrqmpgkglewmgiiypgdsdtry Sgqvtmsadksistaymlssheglghr^ TVS ctctcctgcagggccagtcagagtgttatcagcatctacttagcctggtaccagcagaaa cctggccaggct cccaggctcctcat ctatggtg catccagcagggccactggcatccca gaSggSSSggXgggtc^ cctgaagattttgcagtgtattactgtcagcagtatggtagctcaccgtgcagttttggc CAGGGGACCAAACTGGAGATCAAA drfsgsgsgtdftltisrlepedfavyycqqygsspcsfgqgtkgeik Antibody UI-2 Sgctgcagc?gcaggagtcgggcccaggactggtgaagccttcacagaccctgtccctc acctgcactgtctctggtggctccatcagcagtggtgattactactggagctggatccgc cagcacccagggaagggcctggagtggattgggtacatctattacagtgggagcacctac tacaacccgtccctcaggagtcgagttaccatatcagtagacacgtctaagaaccagttc tccctgaagctgagctctgtgactgccgcggacacggccgtgtattactgtgcgagagcg gattacgatttttggagtggttattttgactactggggccagggaaccctggtcaccgt . tcctca            , qvqSeSpglvkpsqtlsltctvsggsissgdywswirqhpgkglewigyiyysgsty ?NPSLRSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARADYDFWSGYFDYWGQGTbVrV ss 2024204502   28 Jun 2024 89 90 91 92 93 94 95 96 97 Light Chain DNA:                                   —-c-™ gacatccagatgacccagtctccatcctccctgtctgcatctgtaggagacagagicacc atcacttgccgggcaagtcagggcattagaaatgatttaggctggtatcagcagatacct gggaaagcccctaagcgcctgatctatgctgcatccagtttgcaaagtggggtcccatca AGGTT CAGCGGCAGTGGATCTGGG ACAG AATTCACTCTCACAATCA ACAG C CTGCAGCCT GAAGATTTTGCAACTTATTACTGTCTACAGCATAATGGTTACCCGTGGACGTTCGGCCAA GGGACCAAGGTGGAAATCAAAC DIQMTQSPSSLSASVGDRVTITCRASQGIRMDLGWYQQIPGKAPKRLIYAASSLQSGVPS rfsgsgsgteftltinslqpedfatyyclqhngypwtfgqgtkveik Antibody UI-7 acctgcactgtS^ cagcacccagggaagggcctggagtggattggatacatctattacagtgggagcacctac Sc cXcCTCAAGAG^^^ ScSgmgctgagctctgtgactgccgcggacacggccgtgtattactgtgcgagagcg GATTA CGATTTTTGGAGTGGTTATTTTGACTACTGGGGCCAGGGA ACCCTGGTCACCGTC TCCTCA qvqlqesgpglvkpsqtlsltctvsggsissgdyywswirqhpgkglewigyiyysgsty ynpslksrvtisvdtsknqfslklssvtaadtavyycaradydfwsgyfdywgqgtlvtv ss               . gaotccmatca^ atcacttgccgggcaagtcaggacattcgaaatgatttaggctggtatcggcagaaacct GGGAAAGCCCCTAAGCGCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCA agg^agcggcagtggatctgggacagaattcactctcacaatcagcagcctgcagcct gaagattttgcaacttattactgtctacagcataatagttacccgtggacgttcggccaa gggaccaaggtggaaatcaaac SfqmtqspsslsaXdrvtitcrasq rfsgsgsgteftltisslqpedfatyyclqhnsypwtfgqgtkveik Antibody UI-9 caggtgcagctgcaggagtcgggcccaggactggtgaagccttcacagaccctgtccctc acctgcactgtctctggtggctccatcagcagtggtgattactactggagctggatccgc cagcacccagggaagggcctggagtggattggatacatctattacagtgggagcacctac tacaacccgtccctcaagagtcgagttaccatatcaatagacacgtctaagaaccagttc TCCCTGAAG CTGAGCTCTGTGACTGCCGCGGACACGGCCGTGTATTACTGTGCGAGAGCG gatScgatttttggaatggttattttgactactggggccagggaaccctggtcaccgtc TCCTCA OVQLQESGPGLVKPSQTLSLTCTVSGGSISSGDYYWSWIRQHPGKGLEWIGYIYYSGSTY SSrvtiswt^^ ss GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACC ATCACTTGCCGGGCAAGTCAGGACATTAGAAATGATTTAGGCTGGTATCGGCAGAAACCT GGGAAAGCCCCTAAGCGCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGG GTC C CATCA agg™gcggcagtggatctgggacagaattcactctcacaatcagcagcctgcagcct 2024204502   28 Jun 2024 gaagattttgcaacttattactgtctacagcataatagttacccgtggacgttcggccaa gggaccaaggtggaaatcaaa 98 Light Chain Protein: diqmtqspsslsasvgdrvtitcrasqdirndlgwyrqkpgkapkrliyaasslqsgvps rfsgsgsgteftltisslqpedfatyyclqhnsypwtfgqgtkveik Antibody UI-10 99 Heavy Chain DNA: caggtgcagctgcaggagtcgggcccaggactggtgaagcctacacagaccctgtccctc acctgcactgtctctggtggctccatcagcagtggtgattactactggagctggatccgc cagcacccagggaagggcctggagtggattgggtacatctattacagtgggagcacctac tacaacccgtccctcaagagtcgacttaccatatcagtagacacgtctaagaaccagttc tccctgaagctgagctctgtgactgccgcggacacggccgtgtattactgtgcgagagca gattacgatttttggagtggttactttgactactggggccagggaaccctggtcaccgtc TCCTCA 100 Heavy Chain Protein: qvqlqesgpglvkptqtlsltctvsggsissgdyywswirqhpgkglewigyiyysgsty ynpslksrltisvdtsknqfslklssvtaadtavyycaradydfwsgyfdywgqgtlvtv 101 102 103 104 105 106 SS Light Chain DNA: GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACC atcacttgccgggcaagtcagggcattagaaatgatttaggctggtatcagcagaaacca gggaaagcccctaagcgcctgatctatgctgcatccagtttgcaaagtggggtcccatca aggttcagcggcagtggatctgggacagaattcactctcacaatcagcagcctgcagcct gaagattttgcaacttattactgtctacagcataataattacccgtggacgttcggccaa gggaccaaggtggaaatcaaa Light Chain Protein: diqmtqspsslsasvgdrvtitcrasqgirndlgwyqqkpgkapkrliyaasslqsgvps rfsgsgsgteftltisslqpedfatyyclqennypwtfgqgtkveik Antibody Ui-12 Heavy Chain DNA                                           __„ CAGGTGCAG CTG CAGG AGTCGGGCCCAGGACTGGTG AAGC CTTC AC AG ACCCTGTCCCTC acctgcactgtctctggtggctccatcagtagtggtgattactactggagctggatccgc cagcacccagggaagggcctggagtggattgggtacatctattacagtgggagcacctac tacaacccgtccctcaagagtcgagttaccatatcagtagacacgtctaagaaccagttc tccctgaagttgagctctgtgactgccgcggacacggccgtgtattactgtgcgagagcc gattacgatttttggagtggttattttgactactggggccagggaaccctggtcaccgtc TCCTCA Heavy Chain protein: ovqlqesgpglvkpsqtlsltctvsggsissgdyywswirqhpgkglewigyiyysgsty ynpslksrvtisvdtsknqfslklssvtaadtavyycaradydfwsgyfdywgqgtlvtv SS Light Chain DNA: gacatccagatgacccagtctccatcctccctgtctgcatctgtaggagacagagtcacc atcacttgccgggcaagtcagggcattagaaatgatttaggctggtatcagcagaaacca gggaaagcccctaagcgcctgatctatgctgcatccagtttgcaaagtggggtcccatca aggttcagcggcagtggatctgggacagaattcactctcacaatcagcagcctgcagcct gaagattttgcaacttattactgtctacagcataataattacccgtggacgttcggccaa gggaccaaggtggaaatcaaa Light Chain Protein: diqmtqspsslsasvgdrvtitcrasqgirndlgwyqqkpgkapkrliyaasslqsgvps rfsgsgsgteftltisslqpedfatyyclqhnnypwtfgqgtkveik 2024204502   28 Jun 2024 Antibody UI- 13 107 Heavy Chain DNA:                                           __ caggtgcagctgcaggagtcgggcccaggactggtgaagccttcacagaccctgtccctc acctgcactgtctctggtggctccatcagcagtggtggttactactggagctggatccgc cagcacccagggaagggcctggagtggattgggtacatctattacagtgggagcacctac tacaacccgtccctcaagagtcgagttaccatatcagtagacacgtctaagaaccagttc tccctgaagctgagctctgtgactgccgcggacacggccgtgtattactgtgcgagagag gacgacggtatggacgtctggggccaagggaccacggtcaccgtctcctca 10S Heavy Chain. Protein:                                        ___ qvqlqesgpglvkpsqtlsltctvsggsissggyywswirqhpgkglewigyiyysgsty ynpslksrvtisvdtsknqfslklssvtaadtavyycareddgmdvwgqgttvtvss 109 Light Chain DNA:                                        _____ gatattgtgatgactcagtctccact ctccctgcccgtcacccctggagagccgg cctcc atttcctgcaggtctagtcagagcctcctgcatagtaatggatacaactatttggaatgg tacctgcagaagccagggcagtccccacagttcatgatttatttggggtctaatcgggcc TCCGGGGTCCCTGACAGGTTCAGTGGCAGTGGATCAGGCACAGATTTTACACTGAAAATC AGCAGAGTGGAGGCTGAGGATGTTGGGGTTTATTACTGCATGCAAGCTCTACAAACTCCG ATCACCTTCGGCCAAGGGACACGACTGGAGATTAAA 110 Light Chain Protein: dxvmtqsplslpvtpgepasiscrssqsllhsngynylewylqkpgqspqfmiylgsnra SGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQADQTPITFGQGTRLEIK 111 112 Antibody UI-14 Heavy Chain DNA: CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCACAGACCCTGTCCCTC acctg cactgtct CTGGTGGCTCCATCagcagtggtgattactactggagctggatccg c cagtacccagggaagggcctggagtggattgggtacatctattacagtgggagcacctac tacaacccgtccctcaagagtcgagttaccatatcagtagacacgtctaagaaccagttc tccctgaagctgaggtctgtgactgccgcggacacggccgtgtattactgtgcgagagcg GATTACGATTTTTGGAGTGGTTATTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTC TCCTCA Heavy Chain Protein: OVQLQESGPGLVKPSQTLSLTCTVSGGSISSGDYYWSWIRQYPGKGLEWIGYIYYSGSTY ynpslksrvtisvdtsknqfslklrsvtaadtavyycaradydfwsgyfdywgqgtlvtv ss 113 Light Chain DNA: GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACC ATCACTTG CCGGGCAAGTCAGGGCATTAGAAATGATTTAG GCTGGTATCAG CAGAAACCA GGGAAAGCCCCTAAGCGCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCA aggttcagcggcagtggatctgggacagaattcactctcacaatcagcagcctgcagcct gaagattttgcaacttattactgtctacagcataatacttacccgtggacgttcggccaa gggaccaaggtggaaatcaaac 114 Light Chain Protein: diqmtqspsslsasvgdrvtitcrasqgirndlgwyqqkpgkapkrliyaasslqsgvps RFSGSGSGTEFTLTISSLQPEDFATYYCLQHNTYPWTFGQGTKVEIK 115 Antibody Ui-15 Heavy Chain DNA: CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGACCCTGTCCCTC ACCTGCACTGTCTCTGGTGGCTCCGTCAGCAGTGGTGGl’TACTACTGGAGCTGGATCCGG CAGCCCCCAGGGAAGGGACTGGAGTGGATTGGGTATATCTATTACAGTGGGAGCACCAAC TACAACCCCTCCCTCAAGAGTCGAGTCACCATATCAGTAGACACG'TCCAAGAACCAGTTC 2024204502   28 Jun 2024 tccctgaagctgagctctgtgaccgctgcggacacggccgtgtattactgtgcgagagat ggggacgtggatacagctatggtcgatgcttttgatatctggggccaagggacaatggtc accgtctcctca 116 qvOLQESGPGLVKPSETLSLTCTVSGGSVSSGGYYWSWIRQPPGKGLEWIGYIYYSGSTN SlkStjsvdtskwslklssvtaadtavyycardgd^ TVSS 117 g^ATTGTATTGACGCAGTCTCCAGGCACCC^ CTCTCCTGCAGGGCCAGTCAGAGTTTAAGCGGCAACTACTTAGCCTGGTACCAGCAGAAG cctggccaggctcccaggctcatcatctgtggtgcatccagcagggccactggcatccca gacaggttcagtggcsgtgcgtctcggacagacttcactctcaccatcacaagactggag cctgaagattttgcagtgtattactgtcagcagtatgataggtcaccgctcactttcggc GGAGGGACCAAGGTGGAGATCAAA 113 EIVLTQSPGTLSLSPGERATLSCRASQSLSGNYLAWYQQKPGQAPRLIICGASSRATGIP DRFSGSGSGTDFTLTITRLEPEDFAVYYCQQYDRSPLTFGGGTKVEIK Antibody UI-19 119 CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCACAGACCCTGTCCCTC acctgcactgtc™ cagcacccagggaagggcctggagtggattgggtacatctattacagtgggagcacctac tacaacccgtccctcaagagtcgagttaccatatcagtagacacgtctaagaaccagttc tccctgaagctgagctctgtgactgccgcggacacggccgtgtattactgtgcgagagga TCCTCA 120 qvQLQESGPGLVKPSQTLSLTCTVSGGSTSSGDYYWSWIRQHPGKGLEWIGYIYYSGSTY SSXtisvdtsknqfslklssvt^^ S3 Antibody Ul-20 CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCACAGACCCTGTCCCTC acctgcactgtctctggtggctccatcagcagtggtggttactactggagctggatccgc cagcacccagggaagggcctggagtggattgggtacatctatgacagtgggagcacctac tacScccgtccctcaagagtcgagttaccatatcagtagacacgtctaagaaccagttc tccctSgctgaggtctgtgactgccgcggacacggccgtgtattactgtgcgagagat SggggSgg^^ CAAGGGACCACGGTCACCGTCTCCTC                    ■ 122 QVqSeSGPGLVKPSQTLSLTCTVSGGSISSGGYYWSWIRQHPG SXtisvdtswfslklrsvtaad^ QGTTVTVS 123 gacatccagatgacccagtctccatcctccctgtctgcatctgtaggagacagagtcacc atcacttgccaggcgagtcaggacattagcaattatttaaattggtatcagcagaaacca gggaaagcccctaaactcctgatctacgttgcatccaatttggaaacaggggtcccatca AGGTTCAGTGGAAGTGGATCTGGGACAGATTTTACTTTCACCATCAGCAGCCTGCAGCC'P gSgatattgcaacatattactgtcaacagtgtgataatctccctctcactttcggcgga gggaccaaggtggagatcaaa 124 Light Chain Protein: 2024204502   28 Jun 2024 124 RFSGSGSGTDFTFTISSLQPEDIATYYCQQCDNLPLTFGGGTKVEIK Antibody UI-21 120 CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCACAGACCCTGTCCCTC acSgcactgtctcXggc^^^^^ CAGCACCCAGGGAAGGGCCTGGAGTGGATTGGATACATCTATTACAGTGGGAGCACCTAC ScScCCG^C^ TCCCTGAAGCTGAGCTCTGTGACTGCCGCGGACACGGCCGTGTATTACTGTGCGAGAGCG GATTACGATTTTT GG AGTGGTTATTTTGACTACTGGGG CCAGGGAACC CTGGT CACCGT C TCCTC 126 QVQLQESGPGLVKPSQTLSLTCTVSGGSISSGDYYWSWIRQHPGKGLEWIGYIYYSGSTY SsSSvtiZtsknqfslk^ s 127 gwStccStgacccagtctccatcctccctgtctgcatctgtaggagacagagtcacc atcacttgccgggcaagtcaggacattagaaatgatttaggctggtatcggcagaaacct GGGAAAGCCCCTAAGCGCCTGATCTATGCTGCATCCCGTTTGCAAAGTGGGGrCCCATCA aggttcagcggcagtggatctgggacagaattcactctcacaatcagcagcctgcagcct gStttgcScttattac^^^ gggaccaaggtggaaatcaaac 128 dxqmTQSPSSLSASVGDRVTITCRASQDIRNBIjGWYRQKPGKAPKRLIYAASRLQSGVPS RFSGSGSGTEFTLTISSLQPEDFATYYCLQHNSYPWTFGQGTKVEIK Antibody Ul-22 129 Sg^Sg^Xactcggg^ ScScTGTCTCTGGTGGCTCCAKM^^ CAGCACCCAGGGAAGGGCCTGGAGTGGATTGGGTACATCTATTACAGTGGGAGCACCTAC tacaacccgtccctcaagagtcgagttaccatatcagtagacacgtctaagaaccagttc TCCCTGAAGCTGAGCTCTGTGACTGCCGCGGACACGGCCGTGTATTACTGTGCGAGAGCC GATTACGATTTTTGGA GTGG TTATTTTGACTACTGGGG CCAGGGAA CCCTGGTCACCGTC TCCTCA            . 130 QVQLQESGPGLVKPSQTLSLTCTVSGGSISSGDYYWSWIRQHPGKGLEWIGYIYYSGSTY SX^XnSWFSLKLSSVTAADTA^^^ ss 131 gS£cSga?g^ ScacSgS^ gggaaagcccctaagcgcctgatctatgctgcatccagtttgcaaaatggggtcccatca ScaSXtgga^^ gaagattttgcaacttattactgtctacagcataatagttacccgtggacgttcggccaa gggaccaaggtggaaatcaaac 13" niriMTQSPSSLSASVGDRVTITCRASQGIRNDLGWYQQKPGKAPKRLIYAASSLQNGVPS rFSGSGSGTEFTLTISSLQPEDFATYYCLQHNSYPWTFGQGTKVEIK Antibody UI-23 2024204502   28 Jun 2024 Antibody UI-23 133 caggtgcagctgcaggagtcgggcccaggactggtgaa.gccttcacagaccctgtccctc ACCTGCACTGTCTCTGGTGGCTCCATCAGCAGTGGTGATTACTACTGGAGCTGGATCCGC cagcacccagggaagggcctggagtggattgggtacatctattacagtgggagcacctac TACAACCCGTCCCTCAAGAGTCGAGTTACCATA'TCAGTAGACACGTCTAAGAACCAGTTC tccctgaagctgagctctgtgactgccgcggacacggccgtgtattactgtgcgagagcg GATTACGATTTTTGGAGTGGTTATTTTGACTACTGGGGCCAGGGAATCCTGGTCACCGTC TCCTC 134 X^e^XIStXctvsg^^ Sksrvtisvotsknqfslklssv^ s ■" ========= gaagattttgcaacttattactgtctacagcataatagttacccgtggacgttcggccaa GGGACCAAGGTGGAAATCAAAC 136 DIQMTQSPSSLSASVGDRVTITCRASQGIRNDLGWYQQKPGKAPKRLIYAASSLQSGVPS RFSGSGSGTEFTLTISSWPEDFATYYCLQHNSYPWTFGQGTKVEIK Antibody Ui-24 137 CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCACAGACCCTGTCCCTC acc?gcac?gtctXg^ CAGCACCCAGGGAAGGGCCTGGAGTGGATTGGGTACATCTATTACAGTGGGAGCACCTAC taCAACCCGTCCCTCAAGAGTCGAGTTACCATATCAGTAGACACG'I'CTAAGAACCAGTTC tccctgaagttgagctctgtgactgccgcggacacggccgtgtattactgtgcgagagcc GATTACGATTTTTGGAATGGTTATTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTC TCCTCA 13 8 QVQLQESGPGLVKPSQTLSLTCTVSGGSISSGDYYWSWIRQHPGKGLEWIGYIYYSGSTY ss 139 ScacSgccgggcaagtcagggcattagaaatgatttaggctggtatcagcagaaacca gggaaagcccctaagcgcctgatctatgctgcatccagtttgcaaagtggggtcccatca agXSgcggcagtggatc^^ gaagattttgcaacttattactgtctacagcataataattacccgtggacgttcggccaa gggaccaaggtggaaatcaaa 140 dwmtqspsslsasvgdrvtitc^^s rfsgsgsgteftltisslqpedfatyyclqhnnypwtfgqgtkveik Antibody UI-25 141 cagSgSgctgcaggagtcgggcccaggactggtgaagccttcacagaccctgtccctc ACCTG CACTGTCTCTGGTG GCTCCATCAGCAGTGGTGATTACTACTGG AG CTGG ATCCG C CAGCACCCAGGGAAGGGCCTGGAGTGGATTGGGTACATCTATTACAGTGGGAGCACCTAC 2024204502   28 Jun 2024 TACAACCCGTCCCTCAAGAGTCGAGTTACCATATCAGTAGACACGTCTAAGAACCAGTTC TCCCTGAAGCTGAGCTCTGTGACTGCCGCGGACACGGCCGTGTATTACTGTGCGAGAGCC GATTACGATTTPTGGAGTGGTTATTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTC TCCTCA                                                        • 142 Heavy Chain Protein: QVQLQESGPGLVKPSQTLSLTCTVSGGSISSGDYYWSWIRQHPGKGLEWIGYIYYSGSTY ■ yNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARADYDFWSGYFDYWGQGTLVTV SS 143 Light Chain DNA:                                           ___ GACATCCAGCTGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACC ATCACTTGCCGGGCAAGTCAGGGCATTAGAAATGATTTAGGCTGGTATCAGCAGAAACCA GGGAAAGCCCCTAAGCGCCTGATCTATGCTGCATCCAGTTTGCAAAATGGGGTCCCATCA AGGTTCAGCGGCAGTGGATCTGGGACAGAATTCACTCTCACAATCAGCAGCCTGCAGCCT gaagattttgcaacttattactgtctacagcataatagttacccgtggacgttcggccaa GGGACCAAGGTGGAAATCAAAC 144 Light Chain Protein: DIQLTQSPSSLSASVGDRVTITCRASQGIRNDLGWYQQKPGKAPKRLIYAASSLQNGVPS RFSGSGSGTEFTLTISSLQPEDFATYYCLQHNSYPWTFGQGTKVEIK Antibody UI-26 145 Heavy Chain DNA: caggtgcagctgcaggagtcgggcccaggactggtgaagccttcacagaccctgtccctc ACCTGCACTGTCTCTGGTGGCTCCA'TCAGCAGTGGTGATTACTACTGGAGCTGGATCCGC CAGTACCCAGGGAAGGGCCTGGAGTGGATTGGGTACATCTATTACAGTGGGAGCACCTAC tacaacccgtccctcaagagtcgagttaccatatcagtagacacgtctaagaaccagttc tccctgaagctgggctctgtgactgccgcggacacggccgtgtatttctgtgcgagagcc GATTACGATTTTTGGAGTGGTTATTTTGACTTCTGGGGCCAGGGAACCCTGGTCACCGTC TCC.TC 146 147 148 149 150 Heavy Chain Protein: qvqlqesgpglvkpsqtlsltctvsggsissgdyywswirqypgkglewigyiyysgsty yNPSLKSRVTISVDTSKNQFSLKLGSVTAADTAVYFCARADYDFWSGYFDFWGQGTLVTV S Light Chain DMA: GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACC atcacttgccgggcaagtcagggcattagaaatgatttaggctggtatcagcagaaacca gggaaagcccctaagcgcctgatctatgctgcatccagtttgcaaagtggggtcccatca aggttcagcggcagtggatctgggacagaattcactctcacaatcagcagcctgcagcct gaagattttgcaacttattactgtctacagcataatggttacccgtggacgttcggccaa GGGACCAAGGTGGAAATCAAAC Light Chain. Protein: DIQMTQSPSSLSASVGDRVTITCRASQGIMTOLGWYQQKPGKAPKRLIYAASSLQSGVPS RFSGSGSGTEFTLTISSLQPEDFATYYCLQHNGYPWTFGQGTKVEIK Antibody UI-27 Heavy Chain DMA: CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCACAGACCCTGTCCCTC ACCTGCACTGTCTCTGGTGGCTCCATCAGCAGTGGTGATTACTACTGGAGCTGGATCCGC CAGTACCCAGGGAAGGGCCTGGAGTGGATTGGGTACATCTATTACAGTGGGAGCACCTAC TACAACCCGTCCCTCAAGAGTCGAGTTACCATATCAGTAGACACGTCTAAGAACCAGTTC TCCCTGAAGCTGGGCTCTGTGACTGCCGCGGACACGGCCGTGTATTTCTGTGCGAGAGCC GATTACGATTTTTGGAGTGGTTATTTTGACTTCTGGGGCCAGGGAACCCTGGTCACCGTC TCCTC Heavy Chain Protein: 2024204502   28 Jun 2024 qvqlqesgpglvkpsqtlsltctvsggsissgdyywsvhrqypgkglewigyiyysgsty yiJslksrvtisvdtsknqfslklgsvtaadtavyfcara^^ s 151 GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCT^ atcacttgccgggcaagtcagggcattagaaatgatttaggctggtatcagcagaaacca AGGTTCAGCGGCAGTGGATCTGGGACAGAATTCACTCTCACAATCAGCAGCCTGCAGCCT gaagSSgcaacttattactgtctacagcataatggtta^ gggaccaaggtggaaatcaaac 152 diqmtqspsslsasvgdrvtitcrasqgiritolgwyqqkpg^ RFSGSGSGTEFTbTISSLQPEDFATYYCLQHNGYPWTFGQGTKVEIK 153 154 155 Antibody Ui-28 CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCACAGACCCTGTCCCTC ScStgtcStggtgg^ tccctgmgctgagc^^ gattacgatttttggagtggttattttgactcctggggccagggaaccctggtcaccgtc TCCTCA ovoloesgpglvkpsqtlsltctvsggsissgdyywswirqhpgkglewigyiyysgsty ?Xlksrvtisvdtsoqfslklssvtaadtavyycarmydfwsgyfdswgqgtlvtv ss GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACC ATCACTTGCCGGGCAAGTCAGGGCATTAGAAATGATTTAGGCTGGTATCAGCAGATACC1 gggaSgS^ AGGTTCAGCGGCAGTGGATCTGGGACAGAATTCACTCTCACAATCAGCAGCCTGCAGCCT gggaccaaggtggaaatcaaa 156 rf sgsgsgteftltis s lqp edfatyyclqhngypwt fgqgtkveik Antibody UI-31 157 caggttcagctggtgcagtctggagctgaggtgaagaagcctggggcctcagtgaaggtc tcctgcaaggcttctggttacacctttaccaactatggtatcagctgggtgcggcaggcc cctggacaagggcttgagtggatgggatggatcagcgcttacgatggttacagaaactat gcaoagaagctccagggcagagtcaccatgaccacagacacatccacgaccactgcctac atggagctgaggagcctgagatctgacgacacggccgtgtattactgtgcgagagatgtt TCCTCA            , 158 ovolvqsgaevkkpgasvkvsckasgytftnygiswvrqapgqglewmgwisaydgyrny iXXwTTDTSTTTAYMELRSLRSDDTAVYYCARDVQDYG^ S3 159 gacatccagatgacccagtctccatcctccctgtctgcatctgtaggagacagagtcacc 2024204502   28 Jun 2024 atcacttgc cgggcaagtcagagcatta gcagttatttaaattggtatcagcag aa acc a gggaaagcccctaacctcctgatctatgctgcatccagtttgcaaagtggggtcccatca agattcaggggcagtggatctgggacagatttcactctcaccatcagcagtctgcaacct gaagattttgcaacttactactgtcaacagagttacagtacccccatcaccttcggccaa gggacacgactggagattaaa 160 Light Chain Protein: dtqmtqspsslsasvgdrvtitcrasqsissylnwyqqkpgkapnlliyaasslqsgvps RFRGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPITFGQGTRLEIK Antibody UI-32 161 Heavy Chain DNA: caggtgcagctgcaggagtcgggcccaggactggtgaagcctttacagaccctgtccctc acctgcactgtctctggtggctccatcagcagtggtgattactactggagctggatccgc cagcacccagggaagggcctggagtggattgggtacatctattacagtgggaccacctac tacaacccgtccctcaagagtcgagttaccatatcagtagacacgtctaagaaccagttc gccctgaagctgaactctgtgactgccgcggacacggccgtgtattactgtgcgagagcc gattacgatttttggagtggttattttgactactggggccagggaaccctggtcaccgtc tcctca 162 Heavy Chain Protein: qvqlqesgpglvkplqtlsltctvsggsissgdyywswirqhpgkglewigyiyysgtty ynpslksrvtisvdtsknqfalklnsvtaadtavyycaradydfwsgyfdywgqgtlvtv ss 163 Light Chain DNA: gacatccagatgacccagtctccatcctccctgtctgcatctgtaggagacagagtcacc atcacttgccgggcaggtcagggcattagaaatgatttaggctggtatcagcagaaacca gggaaagcccctcagcgcctgatctatgctgcatccagtttgcaaagtggggtcccatca aggttcagcggcagtggatctgggacagaattctctctcacaatctccagcctgcagcct GAAG ATTTTG CAACTTATTACTGT CT AC AGCATAATAGTT ACCCGTGGACGTTCGG CCAA GGGACCAAGGTGGAAATCAAAC 154 Light Chain Protein: DIQMTQSPSSLSASVGDRVTITCRAGQGIRUDLGWYQQKPGKAPQRLIYAASSLQSGVPS RFSGSGSGTEFSLTTSSLQPEDFATYYCLQHNSYPWTFGQGTKVEIK Antibody UI-35 165 Heavy Chain DMA: CAGGTG CAGCTGGTGGAGTCTGGGGGAGGCTTGGTCAAGCCTGGAGGGTCCCTGAGACT C TCCTGTGCAGCCTCTGGATTCACCTTCAGTGACTACTACATGAGCTGGATCCGCCAGGCT CCAGGGAAGGGGCTGGAGTGGGTTTCATATATTAGTAGTAGTGGTAATAACATATACCAC GCAGACTCTGTGAAGGGCCGATTCACCATCTCCAGGGACAACGCCAAGAACTCACTGTAT CTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTGTATTACTGTGCGAGAGAGAGA TATAGTGGCTACGACGACCCTGATGGTTTTGATATCTGGGGCCAAGGGACAATGGTCACC GTCTCTTCA 166 Heavy Chain Protein: QVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMSWIRQAPGKGLEWVSYISSSGNNIYH ADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARERYSGYDDPDGFDIWGQGTMVT VSS 167 Light Chain DNA: GACATCCAGATGACC CAGTCT CCATCCT CCCTGTCTGCAT CTGTAGGA GA CAGAGTCACC ATCACTTGCCAGGCGAGTCAGGACATTAGCAACTATTTAAGTTGGTTTCAGCAGAAACCA GGGAAAGCCCCTAAGCTCCTGATCCACGATGCATCCAATTTGGAAACAGGGGTCCCTTCA AGGTTCAGTGGAAGTGGATCTGGGACAGATTTTACTTTCACCATCAGCAGCCTGCAGCCT GAAGATATTGCAACATATTACTGTCAACAGTATGATAATCCCCCGTGCAGTTTTGGCCAG GGGACCAAGCTGGAGATCAAA 2024204502   28 Jun 2024 168 Light Chain Protein: diq^spsslsasvgdrvtitcqasqdi™ rfsgsgsgtdftftisslqpediatyycqqydnppcsfgqgtkleik Antibody Ul-36 169 CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCACAGACCCTGTCCCTC ACCTGCA CT GT CTCTGGTGGCTCCATCAGC AGTGGTTATT ACTACTGG AG CTGGATC CGC CAGCACCCAGGGAAGGGCCTGGAGTGGATTGGGTACATCTATTACAGTGGGACCACCTAC tacStccgtccttcaagagtcgagttaccatatcagtagacacgtctaagaaccagttc tccctgaaactgagctctgtgactgccgcggacacggccgtgtattactgtgcgagagcc gattacgatttttggagtggtcactttgactactggggccagggaaccctggtcaccgtc TCCTCA 170 qvqlqesgpglvspsqtlsltctvsggsissgyyywswirqhpgkglewigyiyysgtty YNP SFKS RVTISVDT S KNQF S L KLS SVTAADTAVYYCARADYDFWSGH FDWGQGTLVTV SS 171 gacatccagatgacccagtctccatcctccctgtctgcatctgtaggagacagagtcacc atcacttgccgggcaagtcagggcattagaaatgatttaggctggtatcagcagaaacca gggaaagcccctaagcgcctgatctatgctgcatccagtttgcaaagtggggtcccatca agSgcggSgtggatctgggacagaat^^^ gaagattttgcaacttattactgtctacagcataatagttacccgtggacgttcggccaa gggaccaaggtggaaatcaaa 17 OIQMTQSPSSLSASVGDRVTITCRASQGIRNDLGWYQQKPGKAPKRLIYAASSLQSGVPS rfsgsgsgteftltisslqpedfatyyclqhnsypwtfgqgtkveik Antibody Ul-37 173 XScagctggtgcagtctggagctgaggtgaagaag tcctgcaaggcttctggttacacctttaccagctatggtatcagctgggtgcgacaggcc CCTGGACAAGGACTTGAGTGGATGGGATGGATCAGCGCTTACGATGGTCACACAAACTAT rCACAGAAGCTCCAGGGCAGAGTCACCATGACCACAGACACATCCACGAACACAGCCTAC atcgagctgaggagcctgagatctgacgaca^ catgactacagtaactacgaggcttttgacttctggggccagggaaccctggtcaccgtc TCCTC 1  oVOLVQSGAEVKKPGASVKVSCKASGYTFTSyGlSWVRQAPGQGLEWMGWISAYDGHTNY aqklqgrvtmttdtstntaymelrslrsddtavyycardphdysnyeafdfwgqgtlvtv s 175 Light Chain DNA atgaggtcccctgctcagctcctggggctcctgctactctggctccgaggtgccagatgtg acatccagatgacccagtctccatcctccctgtctgcatctgtaggagacagagtcaccat cacttgccgggcaagtcagagcattagcagttatttaaattggtatcagcagaaaccaggg aaagcccctaacctcctgatctatgctgcatccagtttgcaaagtggggtccoatcaagat tcagtggcagtggatctgggacagatttcactctcaccatcagcagtctgcaacctgaaga ttttgcaacttactactgtcaacagagttacagtacccccatcaccttcggccaagggaca cgactggagattaaacgaactgtggctgcaccatctgtcttcatcttcccgccatctgatg agcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagaga ggccaaagtacagtggaaggtggataacgcc 2024204502   28 Jun 2024 175 Diom^SSLsXDRWITCR^Q^ FSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPE'TFGQGTRLEIK Antibody UI'34 177 Heavy Chain DNA: accatggactggacctggagggtccttttcttggtggcagcagoaacaggtgcccactccca ggttcagctggtgcagtctggagctgaggtgaagaagcctggggcctcagtgaaggtctcct gcaaggcttctggttacacctttaccaactatggtatcagctgggtgcggcaggcccctgga caagggcttgagtggatgggatggatcagcgcttacgatggttaaagaaactatgcacagaa gctccagggcagagtoaccatgaccacagacacatccacgaccactgcctacatggagctga ggagcctgagatctgacgacacggccgtgtattactgtgcgagagatgttcaagactacggt gactacgactactttgactactggggccagggaaccctggtcaccgtctcctcagcttccac caagggcccatccgtcttccccctggtgccctgctccaggagcacctccgagagcacagccg ccctgggctgcctggtcaaggactacttccccgaaccg 173 JJqlvqsgaevkkpgasvkvsckasgytftnygiswvrqapgqglewgwisaydgyrnya 2k£qgrv™™^^ 179 Liaht Chain DNA: cagctcctggggctcctgctactctggctccgaggtgccagatgtgacatccagatgacco agtctccatcctccctgtctgcatctgtaggagacagagtcaccatcacttgccgggcaag tcagagcattagcagttatttaaattggtatcagcagaaaccagggaaagcccctaacctc ctgatctatgctgoatccagtttgcaaagtggggtcccatcaagattcagtggcagtggat ctgggacagatttcactctcaccatcagcagtctgcaacctgaagattttgcaacttacta ctgtcaacagagttacagtaccccoatcaccttcggccaagggacacgactggagattaaa cgaactgtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttgaaatctg gaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtg gaagg tgga taacg cc 180 DiQMTQSPSSLSASVGDRVTITCRASQSISSYLimQQKPGKAPNLLIYAASSLQSGVPSR fsgsgsgtdftltisslqpedfatyycqqsystpitfgqgtrleik Antibody Ul-1 1 al Hfaw Chain DNA:                                          , catctgtggttcttcctcctgctggtggcagctcccagatgggtcctgtcccaggtgcagc tgcaggagtcgggcccaggactggtgaagccttcacagaccctgtccctcacctgcactgt ctctggtggctcaatcaacagtggtgattactactggagctggatccgccagcacccaggg ctotgtgactgccgcggacacggccgtgtattactgtgcgagagcagattacgatttttgg agtggttactttgactactggggccagggaaccctggtcaccgtctcctcagcctccaoca agggcccatcggtcttccccctggcaccctcctccaagagcacctctgggggcacaacggc cctgg 2024204502   28 Jun 2024 192 Heavy Chain Protein                                          , QVQLQESGPGLVKPSQTLSLTCTVSGGSINSGDYYWSWIRQHPGKGLEWIGYIYYSGSTYY NPSLKSRVTISVD'TSKNQFSLKLSSVTAADTAVYYCARADYDFWSGYFDYWGQGTLVTVSS 103 Light Chain DNA: atgagggtccctgctcagctcctggggctcctgctgctctggttcccaggtgccaggtgtga catccagatgacccagtctccatcctccctgtctgcatctgtaggagacagagtcaccatca c t tgccgggcaag tcagggca t tagaaa tgatttaggctggtatcagcagaaaccagggaaa gcccctaagcgcctgatctatgctgcatccagtttgcaaagtggggtcccatcaaggttcag cggcagtggatctgggacagaattcaotctcacaatcagcagcctgcagcctgaagattttg caacttattactgtctacagcataatagttacccgtggacgttcggccaagggaccaaggtg gaaatcaaacgaactgtggctgcaccatctgtcttcatcttcccgccatctgatgagcagtt gaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaag tacagtggaaggtggataacgc 184 Light Chain Protein diqmtqspsslsasvgdrvtitcrasqgirndlgwyqqkpgkapkrliyaasslqsgvpsr fsgsgsgteftltisslqpedfatyyclqhnsypwtfgqgtkveik Antibody UI-3 185 Heavy Chain DNA: tggttcttccttctgctggtggcagctcccagatgggtcctgtcccaggtgcagctgcagga gtcgggcccaggactggtgaagccttcacagaccctgtccctcacctgcactgtctctggtg gctccatcagcagtggtggttactactggagctggatccgccagcacccagggaagggcctg gagtggattgggtacatctattacagtgggagcacctactacaacccgtccctcaagagtcg agttaccatatcagtagacacgtctaagaaccagttctccctgaagctgagctctgtgactg ccgcggacacggccgtgtattactgtgcgagagatggctatgatagtagtggttattaccac ggctactttgactactggggccagggaaccctggtcaccgtctcctcagcctccaccaaggg cc 186 Heavy Chain Protein                              . ovolqesgpglvkpsqtlsltctvsggsissggyywswirqhpgkglewigyiyysgstyy NPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARDGYDSSGYYHGYFDYWGQGTLVT vss                           ■ 187 Light Chain DNA: H3_13O_1N1K caggtctteatttctctgttgctctggatctctggtgcctacggggacategtgatgaccc agtctccagactccctggctgtgtctctgggcgagagggccaccatcaactgcaagtccag ccagagtgttttatacagctccaacaataagaactacttagcttggtaccagcagaaacca ggacagcctcctaagctgctcatttactgggcatctacccgggaatccggggtccctgacc gattcagtggcagcgggtctgggacagatttcactctcaccatcagcagcctgcaggctga agatgtggcagtttattactgtcagcaatattatagtactccgctcactttcggcggaggg accaaggtggagatcaaacgaactgtggctgcaccatctgtcttcatcttcccgccatctg atgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccag agaggccaaagtacagtggaaggtggataacgc 188 Light Chain Protein: DIVMTQSPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQPPKLLIYWASTRE SGVPDRFSGSGSGTDFTDTISSLQAEDVAVYYCQQYYSTPLTFGGGTKVEIK 2024204502   28 Jun 2024 Antibody UI-4 H3_13 3_1N1G1 189 Heavy Chain DNA                                         L . „„ c tgtgg t tc t tcc tcctgc tgg tggcagctcccaga tgggtcctgtcccaggtgcagctgca ggagtcgggcccaggactggtgaagccttcacagaccctgtccctcacctgcactgtctctg gtggctccatcagtagtggtgattactactggagctggatccgccagcacccagggaaggg ctggagtggattgggtacatctattacagtgggagcacctactacaacccgtccctcaagag tcgagttaccatatcagtagacacgtctaagaaccagttctccctgaagttgagctctgtga ctgccgcggacacggccgtgtattactgtgcgagagccgattacgatttttggagtggttat tttgactactggggccagggaaccctggtcaccgtctcctcagcctccaccaagggcccatc ggtcttccccctggcaccctc 190 qvqSesgpglvkpsqtlsltctvsggsissgdyywswirqhpgkglewigyiy^ MPS LKSRVTISVDTS KNQFSLKL S SVTAADTAVYY CARADYDFWSGYFDYWGQGTLVTV S S 191 Light Chain DNA gtgcccgctcagcgcctggggctcctgctgctctggttcccaggtgccaggtgtgacatcc aqatgacccagtctccatcctccctgtctgcatctgtaggagacagagtcaccatcacttg ccgggcaagtcagggcattagaaatgatttaggctggtatcagcagaaaccagggaaagcc cctaagcgcctgatctatgctgcatccagtttgcaaagtggggtcccatcaaggt cagcg gcagtggatctgggacagaattcactctcacaatcagcagcctgcagcctgaagattt g aacttattactgtctacagcataataattacccgtggacgttcggccaagggaccaaggtg gaaatcaaacgaactgtggctgcaccatctgtcttcatcttcccgccatctgatgagcagt tgaaatctggaactg SGSGSGTEFTLTISSLQPEDFATYYCLQHNNYPWTFGQGTKVEIK Antibody UI-5 193 Heavy Chain DMA: tggttcTtccttctgctggtggcagctcccagatgggtcctgtcccaggtgcagctgcagga gtcgggcccaggactggtgaagccttcacagaccctgtccctcacctgcactgtctctggtg gctccatcagcagtggtgattactactggagctggatccgccagcacccagggaagggcctg gagtggattgggtacatctattacagtgggagcacctactacaacccgtccctcaagagtcg agttaccatatcagtagacacgtctaagaaccagttctccctgaagctgagctctgtgactg ccgcggacacggccgtgtatttctgtgcgagagccgattacgatttttggagtggttatt qactactggggccagggaaccctggtcaccgtctcctcagcctccaccaagggcc SlXtXtsknqfslklssvtaadtav^ 195Light Chain DNA: atgagggtccccgctcagctcctgqqgctcctqctgctctggttcccaggtgccaggtgtga catccagatgacccagtctccatcctccctgtctgcatctgtaggagacagagtcaccatc cttgccgggcaagtcagggcattagaaatgatttaggctggtatcagcagaaaccagggaaa 2024204502   28 Jun 2024 gcccctaagcgcctgatctatgctgcatccagtttgcaaagtggggtcccatcaaggttcag cggcagtggatctgggacagaattcactctcacaatcagcagcctgcagcctgaagattttg caacttattactgtctacagcataatacttacccgtggacgttcggccaagggaocaaggtg gaaatcaaacgaactgtggctgcaccatctgtcttcatcttcccgocatctgatgagcagtt gaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaag tacagtggaaggtggataacgc 196 Light Chain Protein DiaMTQSPSSLSASVGDRVTITCRASQGIRNDLGWYQQKPGKAPKRLIYAASSLQSGVPS RFSGSGSGTEFTLTISSLQPEDFATYYCLQHNTYPWTFGQGTKVEIK - Antibody Ul-6 197 Heavy Chain DNA:                                           ■ H3 162JN1G1                                             ' u tggttcttccttctgctggtggcagctcccagatgggtcctgtcccaggtgcagctgcagga gtcgggcccaggactggtgaagccttcacagaccctgtccctcacctgcactgtctctggtg qctccatcagcagtggtgattactactggagctggatccgccagcacccagggaagggcctg qagtggattgggtacatctattacagtgggagcacctactacaacccgtccctcaagagtcg agttaccatatcagtagacacgtctaagaaccagttctccctgaagctgagctctgtgactg ccgcggacacggccgtgtatttctgtgcgagagccgattacgatttttggaatggttatttt gactactggggccagggaaccctggtcaccgtctcctcagcctccaccaagggccc 198 OVOWESGPGI>VKPSQTLSLTCTVSGGSISSGDYYWSWIRQHPGKGLEWIGYIYYSGSTY YMP SDKS RVTIS TOTS KNQF SLKLS S VTAADTAVYFCARADYDFWNGY FDYWGQGTLVTV SS 199 Light Chain DNA: H3 162. 1N1K                                                            . atgagggtccccgctcagctcctggggctcctgctgctctggttcccaggtgccaggtgtga catccagatgacccagtctccatcctccctgtctgcatctgtaggagacagagtcaccatca c ttgccgggcaagtcagggca ttagaaatgat t taggctggtatcagcagaaaccagggaaa qcccctaagcgcctgatctatgctgcttccagtttgcaaagtggggtcccatcaaggttcag cqqcagtggatctgggacagaattcactctcacaatcagcagcctgcagcctgaagattttg caacttattactgtctaoagcataatacttacccgtggacgttcggccaagggaccaaggtg gaaatcaaacgaactgtggctgcaccatctgtcttcatcttcccgccatctgatgagcagtt gaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaag tacagtggaaggtggataacgcc 20 DiQMTQSPSSLSASVGDRVTITCRASQGIRNDLGWYQQKPGKAPKRLIYAASSLQSGVPS RFSGSGSGTEFTLTISSLQPEDFATYYCLQHNTYPWTFGQGTKVEIK Antibody Ul-8 2 01 Heavy Chain DNA: H3 174_1N1G1 ttggtggcagcagctacaggcacccacgcccaggtccagctggtacagtctggggctgaggt gaagaagcctggggcctcagtgaaggtctcctgoaaggtttccggatacaccctcactgaat tatccatgtactgggtgcgacaggctcctggaaaagggcttgagtggatgggaggttttgat 2024204502   28 Jun 2024 cctgaagatggtgaaacaatctacgcacagaagttccagggcagagtcaccatgaccgagga cacatctacagacacagcctacatggagctgagcagcctgagatctgaggacacggccgtgt attactgtgcaactgggtggaactacgtctttgactactggggccagggaaccctggtcacc g tctcctcagcctccaccaagggccc 202 Heavy Chain Protein qvqlvqsgaevkkpgasvkvsckvsgytltelsmywvrqapgkglewmggfdpedgetiya QKFQGRVTMTEDTSTDTAYMELSSLRSEDTAVYYCATGWNYVFDYWGQGTLVTVSS 203 Light Chain DNA: H3„174_1N1K                                        ,       u ggatccagtggggatattgtgatgactcagtctccactctccctgcccgtcacccctggaga gccggcctccatctcctgcaggtccagtcagagcctcctgcatagtaatggatacaactatt tggattggtacctgcagaagccagggcagtctccacagctcctgatctatttggattctcat cgggcctccggggtccctgacaggttcagtggcagtggatcaggcacagattttacactgaa aatcagcagagtggaggctgaggatgttggggtttattactgcatgcaagctctacaaactc cgctcactttcggcggagggaccaaggtggagatcaaacgaactgtggctgcaccatctgtc ttcatcttcccgccat 204 Light Chain Protein. DIVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQLLIYLDSHRA SGVPDRFS G SGSGTDFTLKISRVEAEDVGVYY CMQALQTPLTFGGGTKVEIK Antibody UI-11 205 Heavy Chain DNA: H3_17a_lNlGl tggttcttccttctgctggtggcagctcccagatgggtcctgtcccaggtgcagctgcagga gtcgggcccaggactggtgaagccttcacagaccctgtccctcacctgcactgtctctggtg gctccatcagcagtggtgattactactggagctggatccgccagcacccagggaagggcctg gagtggattgggtacatctattacagtgggagcacctactacaacccgtccctcaagagtcg agttaccatatcagtagacacgtctaagaaccagttctccctgaagctgagctctgtgactg ccgcggacacggccgtgtatttctgtgcgagagccgattacgatttttggagtggttatttt gactactggggccagggaaccctggtcaccgtctcctcagcctccaccaagggcccatcgag tcttccccctgg              . 206 Heavy Chain Protein OVQWESGPGLVKPSQTLSLTCTVSGGSISSGDYYWSWIRQHPG^^^^ YNP S LKS RVTISVDT SKNQFSLKLS SVTAADTAVY FCARADYDFWSGYFDYWGQGTLVTV SS 207 Light Chain DNA: H3_178„1N1K atgagggtccccgctcagctcctggggctcctgctgctctggttcccaggtgccaggtgtg acatccagatgacccagtctccatcctccctgtctgcatctgtaggagacagagtcaccat cacttgccgggcaagtcagggcattagaaatgatttaggctggtatcagcagaaaccaggg aaagcccctaagcgcctgatctatgctgcatacagtttgcaaagtggggtcccatcaaggt tcagcggcagtggatctgggacaaaattcactctcactatcagcagcctgcagcctgaaga ttttgcaacttattactgtctacagcataatacttacccgtggacgttcggccaagggacc aaggtggaaatcagacgaactgtggctgcaccatctgtcttcatcttcccgccatctgatg agcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagaga ggccaaagtacagtggaaggtggataacgcc 2024204502   28 Jun 2024 208 srfsgsgsgtkftltisslqpedfatyyclqhntypwtfgqgtkveir Antibody Ul-16 209 Heavy Chain DNA: ac^atgaaacatctgtggttcttcctcctgctggtggcagctcacagatgggtcctgtccc aaa tacaactgcaggagtcgggcccaggactggtgaagcc ttcacagaccc tgtccc tcac ctgcactgtctctggtggctccatcagcagtggtgattactactggagctggatccgccag cacccagggaagggcctggagtggattgggtacatctattacagtgggagcacctactac acccgtccctcaagagtcgagttaccatatcagtagacacgtctaagaaccagttctccct aaaac tgagctctgtgactgccgcggacacggccgtg tat tactg tgcgagagcgga t tac gatttttggagtggttattttgactactggggccagggaatcctggtcaccgtctcctcag cctccaccaagggcccatcggtcttccccctggcaccctcctccaagaacacctctggggg cacagcggccctgggctgcctggtcaaggactacttccccgaaccggtgacggtgtcctgg aactcaggcgccctg 210 OVOLOESGPGLVKPSQTLSLTCTVSGGSISSGDYYWSWIRQHPGKGLEWIGYIYYSGSTYY 211 Light Chain DNA: atgagggtccccgctcagctcctggggctcctgctgctctggttcccaggtgccaggtgt qacatccagatgacccagtctccatcctccctgtctgcatctgtaggagacagagtcacc atcacttgccgggcaagtcagggcattagaaatgatttaggctggtatcagcagaaacca gggaaagcccctaagcgcctgatctatgctgcatccagtttgcaaagtggggtcccatca aaattcaacggcagtggatctgggacagaattcactctcacaatcagcagcctgcagcct qaagattttgcaacttattactgtctacagcataatagttacccgtggacgttcggccaa gggaccaaggtggaaatcaaacgaactgtggctgcaccatctgtcttcatcttcccgcca tctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctat 212 diqmtqspsslsasvgdrvtitcrasqgirkdlgwyqqkpgkapkrliyaasslqsgvpsr fsgsgsgteftltisslqpedfatyyclqhnsypwtfgqgtkveik Antibody UI-17 213 Heavy Chain DNA: aqtcgggcccaggactggtgaagccttcacagaccctgtccctcacctgcactgtctctgg tggctccatcagcagtggtgattactactggagctggatccgccagcacccagggaagggc . . _ i. l. _ ~ i-          r n fif1 i* 3 f11" r!        r. Cd t CCC U CadCfu tattttgactactggggccagggaacc^ catcg 2024204502   28 Jun 2024 21d Heavy Chain Protein OVOLQESGPGLVKPSQTLSL'TCTVSGGSISSGDYYWSWIRQHPGKGLEWIGYIYYSGSTYY nsslksrvtisvdtsknqfslklssvtaadtavyycaradydfwsgyfdywgq 215 Light Chain DMA: H3_224..1N1K ggtgccaggtgtgacatccagatgacccagtctccatcctccctgtctgcatctgtaggag acagagtcaccatcacttgccgggcaagtcagggcattagaaatgatttaggctggtatca gcagaaacctgggaaagcccctaagcgcctgatctatgctgcatccagtttgcaaagtggg gtcccatcaaggttcagcggcagtggatctgggacagaattcactctcacaatcagcagcc tgcagcctgaagattttgcaacttattactgtctacagcacaatagttacccgtggacgtt cggccaagggaccaaggtggaaatcaaacgaaotgtggctgcaccatctgtcttcatcttc ccgcca 216 Light Chain Protein DIQMTQSPSSLSASVGDRVTITCRASQGIRNDLGWYQQKPGKAPKRLIYAASSLQSGVPS RFSGSGSGTEFTLTISSLQPEDFATYYCLQHNSYPWTFGQGTKVEIK Antibody Hl-13 217 Heavy Chain DNA: H3_227_1N1G1 aggttcttccttctgctggtggcagctcccagatgggtcctgtcccaggtgcagctgcagg agtcgggccaaggactggtgaagccttcacagaccctgtccctcacctgcactgtctctgg tggctccatcagcagtggtgattactactggagctggatccgccagcacccagggaagggc ctggagtggattggatacatctattacagtgggagcacctactacaacccgtccctcaaga gtcgagttaccatatcagtagacacgtctaagaaccagttctccctgaagctgagctctgt gactgccgcggacacggccgtgtattactgtgcgagagccgattacgatttttggagtggt tattttgactactggggccagggaaacctggtcaccgtctcctcagcotccaccaagggcc catcggtcttccccctggcaccctcctccaagagoacctctgggggcacagcggccctggg ctgcctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgccct 218 Heavy Chain Protein 0VOWESGPGLVKPSQTLSLTCTVSGGSISSGDYYWSWIRQHPGKGLEWIGYIYYSGSTYY MPS LKS RVTISVDTSKNQF SLKLS SVTAADTAVYYCARADYCFWS GYFDYWGQGTLVTVSS 219 Light Chain DNA: H3_227_1N1K                                                 t atgagggtccccgctcagctcctggggctcctgctgctctggttcccaggtgccaggtgtga catccagatgacccagtctccatcctccctgtctgcatctgtaggagacagagtcaccatca cttgccgggcaagtcagggcattagaaatgatttaggctggtatcagcagaaaccagggaaa gcccctaagcgcctgatctatgctgcatccagtttgcaaagtggggtcccatcaaggttcag cggcagtggatctgggacagaattcactctcacaatcagcagcctgcagcctgaagattttg caacttattactgtctacagcataatagttacccgtggacgttcggccaagggaccaaggtg gaaatcaaacgaactgtggctgcaccatctgtcttcatcttcccgccatctgatgagcagtt gaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaag tacagtggaaggtggataacg 220 Licrht Chain Protein diqmtqspsslsasvgdrvtitcrasqgirndlgwyqqkpgkapkrliyaasslqsgvps RFSGSGSGTEFTLTISSLQPEDFATYYCLQHNSYPVJTFGQGTKVEIK 2024204502   28 Jun 2024 Antibody UI-33 221 Heavy Chain DNA: H4_14_1N1G4 Ctgtggttcttccttctgctggtggcagctcccagatgggtcctgtcccaggtgcagctgc aggagtcgggcccaggactggtgaagccttcacagaccctgtccctcacctgcactgtctc tggtggctccatcagcagtggtgattactactggagctggatcegccagcacccagggaag ggcctggagtggattgggtacatctattacagtgggagcacctactacaacccgtccctca agagtcgagttaccatgtcagtagacacgtctaagaaccagttctccctgaagctgagctc tgtgactgccgcggacacggccgtgtattactgtgcgagagccgattacgatttttggagt ggtcactttgactgctggggccagggaaccctggtcaccgtctcctcagcttccaccaagg gccccatccgtcttccccc 222 Heavy Chain Protein QVQLQESGPGLVKPSQTLSLTCTVSGGSISSGDYYWSWIRQHPGKGLEWIGYIYYSGSTYY npslksrvtmsvdtsknqfslklssvtaadtavyycaradydfwsghfdcwgqgtlvtvss 223 Light Chain DNA: H4_14„1N1K atgagggtccccgctcagctcctggggctcctgctgotctggttcccaggtgccaggtgtga catccagatgacccagtctccatcctccctgtctgcatctgtaggagacagagtcaccatca cttgccgggcaagtcagggcattagagatgatttaggctggtatcagcagaaaccagggaaa gcccctaagcgcctgatctatgctgaatccagtttgcaaagtggggtcccatcaaggttcag cggcagtggatctgggacagaattcactctcacaatcagcagcctgcagcctgaagattttg caacttattactgtctacagcatcatagttacccgtggacgttcggccaagggaccaaggtg gaaatcaaacgaactgtggctgcaccatctgtcttcatcttcccgcc 224 Light Chain Protein DIQMTQSPSSLSASVGDRVTITCRASQGIRDDLGWYQQKPGKAPKRLIYAESSLQSGVPSR fsgsgsgteftltisslqpedfatyyclqhhsypwtfgqgtkveik Antibody Ul-29 225 Heavy Chain DNA: H4_1O7_1N1G4 tggctgagctgggttttcctcgttgctcttttaagaggtgtccagtgtcaggtgcagctgg tggagtotgggggaggcgtggtccagcctgggaggtccctgagactctcctgtgcagcgtc tggattcaccttcaatagctatgacatgcactgggtccgccaggctccaggcaaggggctg gagtgggtggcagttatatggtatgatggaagtaataaatactatgcagactccgtgaagg gccgattcaccatctctagagacaattccaagaacacgctgtatctgcaaatgaacagcct gagagccgaggacacggctgtgtattactgtgcgagagaccgcttgtgtactaatggtgta tgctatgaagactacggtatggacgtctggggccaagggaccacggtcaccgtctcctcag cttccaccaagggcccatccgtcttccccctggcgccctgctccag^agcacctacgagag cacagccgccctgggc 226 Heavy Chain Protein QVQLVESGGGWQPGRSLRLSCAASGFTFNSYDMHWVRQAPGKGLEWVAVIWYDGSNKYYA dsvkgrftisrdnskntlylqmnslraedtavyycardrlctngvcyedyghdvwgqgttv TVSS 227 Light Chain DNA: H4_1O7_1N1K atgagggtccctgctcagotcctggggctcctgctgctctggctctcaggtgccagatgtga catccagatgacccagtctccatcctccctgtctgcatctgtaggagacagagtcaccatca 2024204502   28 Jun 2024 ct tgccaggcgagtcaggacat tagcaactatttaaattggtatcagcagaaaccagggaaa gcccctaaggtcctgatctacgatgcatccaatttggaaacaggggtcccatcaaggttcag tggaagtggatctgggacagattttactttcaecatcagcagcctgcagcctgaagatgttg caacatattactgtcaacactatgatactctcccgctcactttcggcggagggaccaaggtg gagatcaaacgaactgtggctgcaccatctgtcttcatcttcccgccatctgatgagcagtt gaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaag tacagtgg 229 Light Chain Protein DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKVLIYDASNLETGVPSR fsgsgsgtdftftisslqpedvatyycqhydtlpltfgggtkveik Antibody UI-30 229 Heavy Chain DNA: H4_116_1_1N1G4 ggactgtgcaagaacatgaaacacctgtggttcttcctcctgctggtggcagctcccagatg ggtcctgtcccaggtgcagctgcaggagtcgggcccaggactggtgaagcctttacagaccc tgtccctcacctgcactgtctctggtggctccatcagcagtggtgattactactggagctgg atccgccagcacccagggaagggcctggagtggattgggtacatctattacagtgggaccac ctactacaacccgtccctcaagagtcgagttaccatatcagtagacacgtctaagaaccagt tcgccctgaagctgaactctgtgactgccgcggacacggccgtgtattactgtgcgagagcc gattacgatttttggagtggttattttgactactggggccagggaaccctggtcaccgtctc ctcagcttccaccaagggcccatccgtcttccccctgg 230 Heavy Chain Protein qvqlqesgpglvkplqtlsltctvsggsissgdyywswirqhpgkglewigyiyysgttyy npslksrvtisvdtsknqfalklnsvtaadtavyycaradydfwsgyfdywgqgtlvtvss 231 Light Chain DNA: H4_116„1_1N1K atgagggtccctgetcagetcctggggcteetgctgctctggttcccaggtgccaggtgtg acatccagatgacccagtctccatcctccctgtctgcatctgtaggagacagagtcaccat cacttgccgggcaggtcagggca ttagaaatgatttaggctggtatcagcagaaaccaggg aaagcccctcagcgcctgatctatgctgcatccagtttgcaaagtggggtcccatcaaggt tcagcggcagtggatctgggacagaa t tctc tctcacaatc tccagcctgcagcc tgaaga ttttgcaacttattactgtctacagcataatagttacccgtggacgttcggccaagggacc aaggtggaaatcaaacgaactgtggctgcaccatctgtcttcatcttcccgccatctgatg agcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagaga ggccaaagtacagtggaaggtggataacgcccttccaatcggg 232 Light Chain Protein diqmtqspsslsasvgdrvtitcragqgirndlgwyqqkpgkapqrliyaasslqsgvpsr fsgsgsgtefsltisslqpedfatyyclqhnsypwtfgqgtkveik

[0073] Light Light ______ Heavy Light Heavy         t ' Heavy          I O □' □ 15' - 1 1 (Q I 0 3 i £ Light Heavy Light Heavy Light Light ______ Heavy Heavy Light Heavy Light Heavy Light _Lig_ht__________ Heavy Light _______ Heavy Heavy J Light I 0 < £ _A. CO NJ f-k UI-10 22 c co U1-6 ---- UI-5 c — co N> —X Sequence List of OPR number: CDR1        _____-------——2 RASQGIRNDLG GGSISSGDYYWS . —.-- RS SQSLLHSNGYNYLE GGSISSGGYYWS RASQGIRNDLG ggsissgdyyws RASQGIRNDLG KASQUlKNtWJ ggsissgdyyws ggsissgdyyws RASQDIRNDLG SHAMPS SISOP RSSOSLLHSNGYNYLD gytltelsmv > co 20 o H . i r 0 1 KASUMI GGSISSGDYYWS RASQUIKNUIAj GGSISSGDYYWS 0 0 to to to Q O K 2 to rasqgirndlg GGSISSGDYYWS KSSQSVLYSSNNKNYLA RASQU1K1W1AJ_______1 GGSISSGGYYWS   | 0 X 0 > to to M XI to C to H 0 ? O 2 »< C 3 r S G to GGSINSGDYYWS I _________ AASSLQS K R io 1 to •< to r1 X to LGSNRAS YIYYSGSTYYNPSLKS AASSLQS yiyysgstyynpslks sotssw r u w v yiyysgstyynpslks SmSdNAXSSOSAAIX AASSLQS YIYYSGSTYYNPS LKS ldshras gfdpedgetiyaqkfqg £ to tn r O to yiyysgstyynpslks » k b n to n Q -» tn K Ji »< S I GO r cn yiyysgstyynpslks n K QQT.AG AASSLQS YIYYSGSTYYNPSLKS WASTRES YIYYSGSTYYNPSLKS R nw n Q -1 to 3 H n to to 6* >< > Ki n to n 0 -* to □ Jl < Ei XJ to r !X to r £ 1 I *• * i - 1 I 1 > c x c rr n £ ( 3 f c n: H k o c C S c c E 1 V G n C r iC IT 2 « 5 H- J adydfwsgyfdy IMdXNNHOT m jinoM^nirtw C a 2 w *- c X c * c 1         H 1- I r c ; r 5 < c I           5 c [ r £ 2 to « s adydfwsgyfdy L-_-......... ADYDFWNGYFDY LQHNTYPWT LQHNTYPWT t“ c IT 2 g T 5 > c c 2 u c K >2 c o c x i          T C DGYDSSGYYHGYFDY ADYDFWSGYFDY LOHNGYPWT s  § X   x 2i    U tn to £ C x IT c K o X) co । ! ■ ■ 1    .....2----------J-------------------------1------J-------—------------------ WO 2015 / 155998                                                 PCT / JP2015 / 002020 F" (O' zr <—► 1 Heavy won Heavy Light Heavy Light Heavy Heavy 1 Liaht 1 Heavy Light Heavy Liaht Heavy I [Liaht Heavy Liaht Heavy          ! Liaht                - -   ■ —            —1 Heavy          ■ Light Heavy Ir-& Heavy Heavy Light Heavy eavy c c c —k a _k G __k _k c a _k c _k cz UI- a _k U1- c: __x c c: _k CO o NJ CO -28 nj -26 -25 NJ NJ CO NJ N> NJ w^k NJ O CO co I —J. CD cn | RAGQGIRNDLG 1 GGSISSGDYYWS QASQDISNYLN GFTFNSYDMH RASQGIRNDLG GGSISSGDYYWS RASQGIRNDLG GGSISSGDYYWS RASQGIRNDLG    | GGSISSGDYYWS I RASQGIRNDLG    | GGSISSGDYYWS RASQGIRNDLG GGSISSGDYYWS 1 RASQGIRNDLG____ GGSISSGDYYWS |    DGGNaiDOSYa I GGSISSGDYYWS | oiaNHiaosvu | GGSISSGDYYWS I QASQDISNYLN__ GGSISSGGYYWS es __________ | GGSISSGDYYWS RASQGIRNDLG GGSISSGDYYWS RASQGIRNDLG H GGSISSGDYYWS I RASQGIRNDLG    | o ■ Q CQ CO o o 5 JO co <o u> tr co Q GGSVSSGGYYWS |        SOlSSW i YIYYSGTTYYNPSLKS 5 co £13 VIWYDGSNKYYADSVKG | AASSLQS yiyysgstyynpslks AASSLQS YIYYSGSTYYNPSLKS | AASSLQS YIYYSGSTYYNPSLKS | AASSLQN          1 YIYYSGSTYYNPSLKS ! AASSLQS YIYYSGSTYYNPSLKS | AASSLQS 1 YIYYSGSTYYNPSLKS | AASSLQN          1 YIYYSGSTYYNPSLKS 1        SOTMSW YIYYSGSTYYNPSLKS | VASNLET YIYDSGSTYYNPSLKS £ ft 3 a 0 § § JU & cr YIYYSGSTYYNPSLKS AASSLQS YIYYSGSTYYNPSLKS AASSLQS         I YIYYSGSTYYNSSLKS AASSLQS         | YIYYSGSTYYNPSLKS GASSRAT YIYYSGSTNYNPSLKS 1 LQHNSYPWT         1 ADYDFWSGYFDY O a a A t* tr> *3 DRLCTNGVCYEDYGMDV LQHNGYPWT ADYDFWSGYFDS LQHNGYPWT ADYDFWSGYFDF | LQHNGYPWT ADYDFWSGYFDF r o x 21 CD ADYDFWSGYFDY LQHNNYPWT ADYDFWNGYFDY LQHNSYPWT ADYDFWSGYFDY LQHNSYPWT ADYDFWSGYFDY LQHNSYPWT_______ ADYDFWSGYFDY QQCDNLPLT DQGQDGYSYGYGYYYGM DV BT | GDYDFWSGEFDY LQHNSYPWT ADYDFWSGYFDY LQHNSYPWT ADYDFWSGYFDY r* jO X co 3 s ADYDFWSGYFDY QQYDRSPLT dgdvdtamvdafdi Ui 2024204502   28 Jun 2024 WISAYDGYRNYAQKLQG DVQDYGDYDYFDY Heavy U1-31 gytftnygis Light RASQSISSYLI'l AASSLQS QQSYSTPIT Heavy UI-32 GGSISSGDYYWS YIYYSGTTYYNPSLKS ADYDFWSGYFDY Light ragqgirndlg AASSLQS LQHNSYPWT Heavy UI-33 GGSISSGDYYWS YIYYSGSTYYNFSLKS ADYDFWSGHFDC Light rasqgirddlg AESSLQS LQHHSYPWT Heavy U1-34 GYTFTNYGIS WISAYDGYRNYAQKLQG DVQDYGDYDYFDY Light RASQSISSYLN AASSLQS QQSYSTPIT Heavy U1-35 GFTFSDYYMS YISSSGNNIYHADSVKG ERYSGYDDPDGFDI Light QASQDISNYLS DASNLET QQYDNPPCS Heavy UI-36 GGSISSGYYYWS YIYYSGTTYYNPSFKS ADYDFWSGHFDY RASQGIRNDLG AASSLQS LQHNSYPWT    ___ Heavy UI-37 GYTFTSYGIS WISAYDGHTNYAQKLQG DPHDYSNYEAFDF Light RASQSISSYLN AASSLQS QQSYSTPIT______ Heavy U1-38 gfslstsgvgvg LIYWNDDKRYSPSLKS RDEVRGFDY Light RS SQSLVYSDGYTYLH KVSNWDS mqgahwpit Heavy U1-39 GFTVSSNYMS VIYSGGSTYYADSVKG GQWLDV Light RSSQSLLHSNGYNYLD LGFHRAS RQALQTPLT Heavy U1-40 GGSISSGGYYWS YIYSSGSTYYNPSLKS DRELELYYYYYGMDV Light RSSQSLLYSNGYNYLD LGSNRAS MQALQTPLT Heavy U1-41 GGSISSGGYYWS YIYYSGSTYYNPSLKS DRELEGYSNYYGVDV Light RASQAISNYLN AASSLQS QQNNSLPIT Heavy U1-42 GYSFTSYWIG IIYPGDSDTRYSPSFQG HENYGDYNY Light RASQSIRSYLN AASSLQS QQSNGSPLT Heavy U1-43 GGSISSGGYYWS YIYYSGSTYYNPSLRS DREREWDDYGDPQGMDV 2024204502   28 Jun 2024 Light rasqsissylh AASSLQS QQSYSNPLT Heavy U1-44 GYSFTSWIG 11WPGDS DTIY S ?SFQG HENYGDYWY Light RASQSIRSYLN AASSLQS QQSISSPLT Heavy UI-45 GYTFTSYDIN WMNPNSGDTGYAQVPQG FGDLPYDYSYYEWFDP Light RASQSISSYLN AASSLQS QQSYSTPLT Heavy U1-46 GDSVSSNSAAWN RTYYRSKWYNDYAVSVK S DLYDFWSGYPYYYGMDV Light sequence not available Heavy U1-47 GDSVSSNSAAWN RTYYRSKWYNDYAVSVK S DYYGSGSFYYYYGMDV Light RASQSISSYLN AASNLQS QQSYSTPRT Heavy U1 -48 GGSISSYYWS HIYTSGSTNYNPSLKS EAIFGVGPYYYYGMDV Light sequence not available           ......................... Heavy (J 1-49 GYTFTGYYMH WINPNIGGTNCAQKFQG GGRYS S SWS YYYYGMDV Light KSSQSLLLSDGGTYLY EVSNRFS MQSMQLPIT Heavy UI-50 GGSVSSGGYYWS YIYYSGSTNYNPSLKS GGD SNYEDYYYYYGMDV Light RASQSISIYLH AASSLQS QQSYTSPIT Heavy U1-51 GGSISSYYWS YIYYSGSTNYNPSLKS DSSYYDSSGYYLYYYAM DV Light KSSQSVLYSSNNKNYLA WASTRES QQYYTTPLT Heavy Light UI-52 GGSISSGGYYWS NIYYSGSTYYNPSLKS GGTGTNYYYYYGMDV RASQSVSSSYLA GASSWAT QQYGSSPLT Heavy U1-53 GFTFSIYSMN YISSSSSTIYYADSVKG DRGDFDAFDI Light QASQDITNYLN DASNLET QQCENFPIT Heavy U1-55.1 GGSVSSGGYYWN YINYSGSTNYNPSLKS DRE LELY YYYYGMDV Light Identical with U1-55__________ ..................... ——— — Heavy U1-55 Identical with U1-55.1 Light RSSQSLLYSNGYKYLD LGSNRAS MQALQTPIT Heavy          [UI-57.1__[ ......Identical with U1-57      _____________________ 2024204502   28 Jun 2024 Light RSSQSLLYSNGYKYbD LGSNRAS MQALQTPIT Heavy UI-57 GGSVSSGGYYWW YINYSGSTNYNPSLKS DRELELYYYYYGMDV Light Identical with U1-57.1 ..... ..... Heavy U1-58 GFTFSSYGMH VIWYDGSNKYYADSVKG AARLDYYYGMDV Light RASQSINSYLN GASGLQS QQSYSSPLT Heavy Ul-59 GGSFSGYYWS EINHSGSTNYNPSLKS DKWTWYFDL Light RSSQSVLYSSSNRNYLA WASTRES QQYYSTPRT Heavy UI-61.1 GVSISSGGYYWS YIYYSGSTYYNPSLKS DSESEYSSSSNYGMDV Light RASQTISSYLN AASSLQG QQSYSNPLT Heavy U1-61 GVSISSGGYYWS YIYYSGSTYYNPSLKS DSESEYSSSSNYGMDV Light Identical with U1 -61.1 Heavy UI -62 GYSFTSYWIG IIYPGDSDTRYSPSFQG QMAGNYYYGMDV Light RASQSVISIYLA GASSRAT QQYGSSPCS

[0074] When a newly produced monoclonal antibody binds to a partial peptide or a partial tertiary structure to which the Ul-49, Ul-53, Ul-59, Ul-7, or Ul-9 antibody binds, it can be determined that the antibody binds to the same epitope as the Ul-49, Ul-53, Ul-59, Ul-7, or Ul-9 antibody. Further, by confirming that the antibody competes with the Ul-49, Ul-53, Ul-59, Ul-7, or Ul-9 antibody for binding to HER3 (that is, the antibody inhibits the binding between the Ul-49, Ul-53, Ul-59, Ul-7, or Ul-9 antibody and HER3), it can be determined that the antibody binds to the same epitope as the Ul-49, Ul-53, Ul-59, Ul-7, or Ul-9 antibody even when the specific sequence or structure of an epitope is not defined. Once the epitope is confirmed to be the same, it is strongly expected that the antibody has a biological activity equivalent to that of the Ul-49, Ul-53, Ul-59, Ul-7, or Ul-9 antibody.

[0075] According to the present invention, the binding protein of the invention interacts with at least one epitope in the extracellular part of HER3. The epitopes are preferably located in domain LI (aa 19-184), which is the amino terminal domain, in domain SI (aa 185-327) and S2 (aa 500-632), which are the two Cysteine-rich domains, in domain L2 (328-499), which is flanked by the two Cysteine-rich domains or in a combination of HER3 domains. The epitopes may also be located in combinations of domains such as but not limited to an epitope comprised by parts of LI and SI. Moreover, the 2024204502   28 Jun 2024 binding protein of the invention is further characterized in that its binding to HER3 reduces HER3-mediated signal transduction. In accordance with the present invention, a reduction of HER3-mediated signal transduction may, e.g. be caused by a downregulation of HER3 resulting in an at least partial disappearance of HER3 molecules from the cell surface or by a stabilization of HER3 on the cell surface in a substantially inactive form, i.e. a form which exhibits a lower signal transduction compared to the non-stabilized form. Alternatively, a reduction of HER3-mediated signal transduction may also be caused by influencing, e.g. decreasing or inhibiting, the binding of a ligand or another member of the HER family to HER3, of GRB2 to HER-2 or of GRB2 to SHC, by inhibiting receptor tyrosine phosphorylation, AKT phosphorylation, PYK2 tyrosine phosphorylation or ERK2 phosphorylation, or by decreasing tumor invasiveness. Alternatively, a reduction of HER3 mediated signal transduction may also be caused by influencing, e.g., decreasing or inhibiting, the formation of HER3 containing dinners with other HER family members. One example among others may be the decreasing or inhibiting of the HER3-EGFR protein complex formation.

[0076] Furthermore, in accordance with the present invention, minor variations in the amino acid sequences shown in SEQ ID NOs: 1-232 are contemplated as being encompassed by the present invention, providing that even the variations in the amino acid sequence still maintain at least 75 %, more preferably at least 80 %, 90 %, 95 %, and most preferably 99 % of the sequences shown in SEQ ID NOs: 1-232. The variations may occur within the framework regions (i.e. outside the CDRs), within the CDRs, or within the framework regions and the CDRs. Preferred variations in the amino acid sequences shown in SEQ ID NOs: 1-232, i.e. deletions, insertions and / or replacements of at least one amino acid, occur near boundaries of functional domains. Structural and functional domains can be identified by comparison of the nucleotide and / or amino acid sequence data to public or proprietary sequence databases. Computerized comparison methods can be used to identify sequence motifs or predicted protein conformation domains that occur in other binding proteins of known structure and / or function. Methods to identify protein sequences that fold into a known three-dimensional structure are known. See e.g. 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), which are all incorporated herein by reference. Thus, those of skill in the art can recognize sequence motifs and structural conformations that may be used to define structural and functional domains in accordance with the invention. Among antibodies obtained by combining heavy and light chains having variations in such an amino acid sequences, an antibody equivalent to the original antibody (parent 2024204502   28 Jun 2024 antibody) or more excellent than a parent antibody may be selected. As mentioned above, the HER3-binding protein, the anti-HER3 antibody, and the like of the present invention maintain the HER3-binding activity even if having variations in their amino acid sequences. In the present invention, the term "homology" has the same meaning as the "identity. The homology between two amino acid sequences can be determined using default parameters of 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 PSLBLAST: a new generation of protein database search programs", Nucleic Acids Res. 25: 3389-3402). The Blast algorithm can be used also through the Internet by accessing the site www.ncbi.nlm.nih.gov / blast.

[0077] The chimeric antibody, humanized antibody, or human antibody obtained by the aforementioned method can be subjected to a known method for evaluating the binding property to an antigen for selecting preferable antibodies. In the anti-HER3 antibody of the present invention, MEHD-7945A (or dulig-otuzumab), RG-7116, MM-111, MM-121 (or seribantumab, MM-141, LJM-716, huHER3-8, tri-specific anti-EGFR / ErbB3 zybody, GSK-2849330, REGN-1400, KTN-3379, AV-203, monospecific surrobody (ErbB3), lumretuzumab, MP-EV-20, ZW-9, Dhnercept™ ,anti-Erb3 surrobody(SL-175 or SL-176), SYM-013, variants, active fragments, modified products thereof, and the like are also included.

[0078] As one example of another index for use in the comparison of the properties of antibodies, the stability of antibodies can be exemplified. The differential scanning calorimetry (DSC) is a device capable of quickly and accurately measuring a thermal denaturation midpoint temperature (Tm) to be used as a favorable index of the relative conformational stability of proteins. By measuring the Tm values using DSC and comparing the values, a difference in thermal stability can be compared. 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 a preferred antibody can be selected by using thermal stability as an index. Examples of other indices for selecting antibodies include the following features: the yield in an appropriate host cell is high; and the aggregability in an aqueous solution is low. For example, an antibody which shows the highest yield does not always show the highest thermal stability, and therefore, it is necessary to select an antibody most suitable for the administration to humans by making comprehensive evaluation based on the above-described indices.

[0079] The antibody of the present invention encompasses a modified product of the antibody. The modified variant refers to a variant obtained by subjecting the antibody 2024204502   28 Jun 2024 of the invention to chemical or biological modification. Examples of the chemically modified variant include variants chemically modified by linking a chemical moiety to an amino acid skeleton, variants chemically modified with an N-linked or O-linked carbohydrate chain, etc. Examples of the biologically modified variant include variants obtained by modification after translation (such as N-linked or O-linked glycosylation, N- or C-terminal processing, deamidation, isomerization of aspartic acid, or oxidation of methionine), and variants in which a methionine residue has been added to the N terminus by being expressed in a prokaryotic host cell. Further, an antibody labeled so as to enable the detection or isolation of the antibody or an antigen of the invention, for example, an enzyme-labeled antibody, a fluorescence-labeled antibody, and an affinity-labeled antibody are also included in the meaning of the modified variant. Such a modified variant of the antibody of the invention is useful for improving the stability and blood retention of the antibody, reducing the antigenicity thereof, detecting or isolating the antibody or the antigen, and so on.

[0080] Further, by regulating the modification of a glycan which is linked to the antibody of the invention (glycosylation, defucosylation, etc.), it is possible to enhance an antibody-dependent cellular cytotoxic activity. As the technique for regulating the modification of a glycan of antibodies, International Publication WO 1999 / 54342, WO 2000 / 61739, WO 2002 / 31140, etc. are known. However, the technique is not limited thereto. In the antibody of the invention, an antibody in which the modification of a glycan is regulated is also included. In the case where an antibody is produced by first isolating an antibody gene and then introducing the gene into an appropriate host, a combination of an appropriate host and an appropriate expression vector can be used. Specific examples of the antibody gene include a combination of a gene encoding a heavy chain sequence of an antibody and a gene encoding a light chain sequence thereof described in this specification. When a host cell is transformed, it is possible to insert the heavy chain sequence gene and the light chain sequence gene into the same expression vector, and also 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 the animal cells, mammalian cells, for example, simian COS cells (Gluzman, Y., Cell, (1981) 23, pp. 175-182, ATCC CRL-1650), murine fibroblasts NIH3T3 (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 ovarian cells (CHO cells; ATCC: CCL-61) can be exemplified. In the case where prokaryotic cells are used, for example, Escherichia coli and Bacillus subtilis can be exemplified. 2024204502   28 Jun 2024 By introducing a desired antibody gene into these cells through transformation, and culturing the thus transformed cells in vitro, the antibody can be obtained. In the above-described culture method, the yield may sometimes vary depending on the sequence of the antibody, and therefore, it is possible to select an antibody which is easily produced as a pharmaceutical by using the yield as an index among the antibodies having an equivalent binding activity. Therefore, in the antibody of the invention, an antibody obtained by a method of producing an antibody, characterized by including a step of culturing the transformed host cell and a step of collecting a desired antibody or a functional fragment of the antibody from a cultured product obtained in the culturing step is also included.

[0081] It is known that a lysine residue at the carboxyl terminus of the heavy chain of an antibody produced in a cultured mammalian cell could 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 terminus of the heavy chain of an antibody produced in a cultured mammalian cell could be deleted / eliminated and a proline residue newly located at the carboxyl terminus could be amidated (Analytical Biochemistry, 360: 75-83 (2007)). However, such deletion / elimination and modification of the heavy chain sequence do not affect the antigen-binding affinity and the effector function (the activation of a complement, the antibody-dependent cellular cytotoxicity, etc.) of the antibody. Therefore, in the antibody of the invention, an antibody and a functional fragment of the antibody subjected to such modification are also included, and a deletion variant in which one or two amino acids have been deleted at the carboxyl terminus of the heavy chain, a variant obtained by amidation of the deletion variant (for example, a heavy chain in which the carboxyl terminal proline residue has been amidated), and the like are also included. The type of deletion variant having a deletion at the carboxyl terminus of the heavy chain of the antibody according to the invention is not limited to the above variants as long as the antigen-binding affinity and the effector function are conserved. The two heavy chains constituting the antibody according to the invention may be of one type selected from the group consisting of a full-length heavy chain and the above-described deletion variant, or may be of two types in combination selected therefrom. The ratio of the amount of each deletion variant can be affected by the type of cultured mammalian cells which produce the antibody according to the invention and the culture conditions, however, a case where one amino acid residue at the carboxyl terminus has been deleted in both of the two heavy chains contained as main components in the antibody according to the invention can be exemplified. The scope of the whole antibody (in the present invention, also simply referred to as an "antibody") of the present invention also includes deletion variants thereof, mixtures containing one or two or more deletion 2024204502   28 Jun 2024 variants thereof, etc. The "antibody" of the present invention includes an antibody comprising a heavy or light chain in which N-terminal glutamate is in the form of pyroglutamate by cyclization and / or a heavy or light chain in which a portion of cysteine residues are in the form of cysteinyl.

[0082] In a preferred embodiment of the present invention, the anti-HER3 antibody of the invention is of the IgA, IgD-, IgEi IgG- or IgM-type, preferably of the IgG-or IgM-type including, but not limited to, the IgGI-, IgG2-, IgG3-, IgG4-, IgMI-and IgM2-type. In most preferred embodiments, the antibody is of the IgGI-, IgG2- or IgG4- type.

[0083] As the biological activity of the antibody, generally, an antigen-binding activity, activity of internalizing an antigen in cells expressing the antigen by binding with the antigen, an activity of neutralizing the activity of an antigen, an activity of enhancing the activity of an antigen, an antibody-dependent cellular cytotoxicity (ADCC) activity, a complement-dependent cytotoxicity (CDC) activity, and an antibodydependent cell-mediated phagocytosis (ADCP) can be exemplified. The function of the antibody according to the invention is a binding activity to HER3, preferably, activity of internalizing HER3 in HER3 expressing cells by binding with HER3. Further, the antibody of the invention may have an ADCC activity, a CDC activity and / or an ADCP activity in addition to the cell internalization acitivity.

[0084] In certain respects, e.g. in connection with the generation of antibodies as therapeutic candidates against HER3, it may be desirable that the anti-HER3 antibody of the invention is capable of fixing complement and participating in complement-dependent cytotoxicity (CDC). There are a number of isotypes of antibodies that are capable of the same including without limitations the following: murine IgM, murine IgG2a, murine IgG2b, murine IgG3, human IgM, human IgGI, human IgG3, and human IgA. It will be appreciated that antibodies that are generated need not initially possess such an isotype but, rather the antibody as generated can possess any isotype and the antibody can be isotype switched by appending the molecularly cloned V region genes or cDNA to molecularly cloned constant region genes or cDNAs in appropriate expression vectors using conventional molecular biological techniques that are well known in the art and then expressing the antibodies in host cells using techniques known in the art. The isotype-switched antibody may also possess an Fc region that has been molecularly engineered to possess superior CDC over naturally occurring variants (Idusogie et al., J Immunol., 166, 2571-2575) and expressed recombinantly in host cells using techniques known in the art. Such techniques include the use of direct recombinant techniques (see e.g. U.S. Patent No. 4,816,397), cell-cell fusion techniques (see e.g. U.S. Patent Nos. 5,916,771 and 6,207,418), among others. In the cell-cell fusion technique, a myeloma or other cell line such as CHO is prepared that 2024204502   28 Jun 2024 possesses a heavy chain with any desired isotype and another myeloma or other cell line such as CHO is prepared that possesses the light chain. Such cells can, thereafter, be fused and a cell line expressing an intact antibody can be isolated. By way of example, a human anti-HER3 IgG4 antibody, that possesses the desired binding to the HER3 antigen, could be readily isotype switched to generate a human IgM, human IgGI or human IgG3 isotype, while still possessing the same variable region (which defines the antibody's specificity and some of its affinity). Such molecule might then be capable of fixing complement and participating in CDC.

[0085] Moreover, it may also be desirable for the anti-HER3 antibody of the invention to be capable of binding 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 isotypes of antibodies that are capable of the same, including without limitations the following: murine IgG2a, murine IgG2b, murine IgG3, human IgGI and human IgG3. It will be appreciated that antibodies that are generated need not initially possess such an isotype but, rather the antibody as generated can possess any isotype and the antibody can be isotype switched by appending the molecularly cloned V region genes or cDNA to molecularly cloned constant region genes or cDNAs in appropriate expression vectors using conventional molecular biological techniques that are well known in the art and then expressing the antibodies in host cells using techniques known in the art. The isotype-switched antibody may also possess an Fc region that has been molecularly engineered to possess superior ADCC over naturally occurring variants (Shields et al. J Biol Chern., 276, 6591-6604) and expressed recom-binantly in host cells using techniques known in the art. Such techniques include the use of direct recombinant techniques (see e.g. U.S. Patent No. 4,816,397), cell-cell fusion techniques (see e.g. U.S. Patent Nos. 5,916,771 and 6,207,418), among others. In the cell-cell fusion technique, a myeloma or other cell line such as CHO is prepared that possesses a heavy chain with any desired isotype and another myeloma or other cell line such as CHO is prepared that possesses the light chain. Such cells can, thereafter, be fused and a cell line expressing an intact antibody can be isolated. By way of example, a human anti-HER3 IgG4 antibody, that possesses the desired binding to the HER3 antigen, could be readily isotype switched to generate a human IgGI or human IgG3 isotype, while still possessing the same variable region (which defines the antibody's specificity and some of its affinity). Such molecule might then be capable of binding to FcyR on effectors cells and participating in ADCC.

[0086] The obtained antibody can be purified to be homogeneous. The separation and purification of the antibody may be performed employing a conventional protein separation and purification method. For example, the antibody can be separated and purified by appropriately selecting and combining column chromatography, filter 2024204502   28 Jun 2024 filtration, ultrafiltration, salt precipitation, 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 thereto. Examples of such chromatography include affinity chromatography, ion exchange chromatography, hydrophobic chromatography, gel filtration chromatography, reverse phase chromatography, and adsorption chromatography. Such chromatography can be performed employing liquid chromatography such as HPLC or FPLC. As a column to be used in affinity chromatography, a Protein A column and a Protein G column can be exemplified. For example, as a column using a Protein A column, Hyper D, POROS, Sepharose FF (Pharmacia Corp.) and the like can be exemplified. Further, by using a carrier having an antigen immobilized thereon, the antibody can also be purified utilizing the binding property of the antibody to the antigen.

[0087] {Antitumor compound] The antitumor compound to be conjugated to the anti-HER3 antibody-drug conjugate of the present invention is explained. The antitumor compound used in the present invention is not particularly limited if it is a compound having an antitumor effect and a substituent or a partial structure allowing connecting to a linker structure. When a part or whole linker of the antitumor compound is cleaved in tumor cells, the antitumor compound moiety is released to exhibit the antitumor effect. As the linker is cleaved at a connecting position with a drug, the antitumor compound is released in its unmodified structure to exhibit its intrinsic antitumor effect. As an antitumor compound used in the present invention, exatecan, a camptothecin derivative ((lS,9S)-l-amino-9-ethyl-5-fluoro-2,3-dihydro-9-hydroxy-4-methyl-lH,12H-benzo[de ]pyrano[3',4':6,7]indolizino[l,2-b]quinolin-10,13(9H,15H)-dione shown in the following formula) can be preferably used.

[0088] 2024204502   28 Jun 2024 [Chem.16] Me

[0089] Although having an excellent antitumor effect, exatecan has not been commercialized as an antitumor drug. The compound can be easily obtained by a known method and the amino group at position 1 can be preferably used as a connecting position to the linker structure. Further, exatecan can be also released in tumor cells while part of the linker is still attached thereto. However, it is an excellent compound exhibiting an excellent antitumor effect even in such structure. Because exatecan has a camptothecin structure, it is known that the equilibrium shifts to a structure with a closed lactone ring (closed ring) in an acidic aqueous medium (for example, pH 3 or so) but it shifts to a structure with an open lactone ring (open ring) in a basic aqueous medium (for example, pH 10 or so). A drug conjugate being introduced with an exatecan residue corresponding to the closed ring structure and the open ring structure is also expected to have the same antitumor effect and it is needless to say that any of them is within the scope of the present invention.

[0090] Examples of other antitumor compounds include doxorubicin, daunorubicin, mitomycin C, bleomycin, cyclocytidine, vincristine, vinblastine, methotrexate, platinum-based antitumor agent (cisplatin or derivatives thereof), taxol or derivatives thereof, and other camptothecin or derivatives thereof (antitumor agent described in Japanese Patent Laid-Open No. 6-87746).

[0091] With regard to the antibody-drug conjugate, the number of conjugated drug molecules per antibody molecule is a key factor having an influence on the efficacy and safety. Production of the antibody-drug conjugate is performed by defining the reaction condition including the amounts of use of raw materials and reagents for reaction so as to have a constant number of conjugated drug molecules, a mixture containing different numbers of conjugated drug molecules is generally obtained unlike the chemical reaction of a low-molecular-weight compound. The number of drugs conjugated in an antibody molecule is expressed or specified by the average value, that is, the average number of conjugated drug molecules. Unless specifically described otherwise as a principle, the number of conjugated drug molecules means an 2024204502   28 Jun 2024 average value except in a case in which it represents an antibody-drug conjugate having a specific number of conjugated drug molecules that is included in an antibodydrug conjugate mixture having different number of conjugated drug molecules. The number of exatecan molecules conjugated to an antibody molecule is controllable, and as an average number of conjugated drug molecules per antibody, about 1 to 10 exatecans can be bound. Preferably, it is 2 to 8, and more preferably 3 to 8. Meanwhile, a person skilled in the art can design a reaction for conjugating a required number of drug molecules to an antibody molecule based on the description of the Examples of the present application and can obtain an antibody-drug conjugate with a controlled number of conjugated exatecan molecules. The antibody-drug conjugate of the present invention is unlikely to have an occurrence of aggregation, insolubility, fragmentation, or the like, 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, the linker structure for conjugating an antitumor compound to the anti-HER3 antibody is explained. The linker has the following structure: -L1-L2-Lp-NH-(CH2)n1-L“-(CH2)n2-C(=O)- or -L1-L2-Lp-, the antibody is connected to the terminal of L1 (opposite terminal to which L2 is connected), and the antitumor compound is connected to the carbonyl group of -La -(CH2)n2-C(=O)- moiety or the C terminal of Lp. n1 represents an integer of 0 to 6, preferably, an integer of 1 to 5, and more preferably 1 to 3.

[0093] 1. L1 L1 is represented by a structure shown below: -(Succinimid-3-yl-N)-(CH2)n3-C(=O)- In the above, n3 is an integer of 2 to 8, and "-(Succinimid-3-yl-N)-" has a structure represented by the following formula:

[0094] [Chern. 17] 0 0

[0095] Position 3 of the above partial structure is the connecting position to the anti-HER3 antibody. The connection to the antibody at position 3 is characterized by forming a thioether bond. The nitrogen atom at position 1 of the structure moiety is connected to 2024204502   28 Jun 2024 the carbon atom of methylene which is present within the linker including the structure. Specifically, -(Succinimid-3-yl-N)-(CH2)n3-C(=O)-L2- is a structure represented by the following formula (herein, "antibody -S-" is derived from an antibody).

[0096] [Chern. 18] O Antibody — L\n- (CH2)n3-C(=O)-L2- O

[0097] In the formula, n3 is an integer of 2 to 8, and preferably 2 to 5.

[0098] Specific examples of L1 include the followings. -(Succinimid-3-yl-N)-CH2CH2-C(=O)-, -(Succinimid-3-yl-N)-CH2CH2CH2-C(=O)-, -(Succinimid-3-yl-N)-CH2CH2CH2CH2-C(=O)-, -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-

[0099] 2. L2 L2 has a structure represented by the following formula: -NH-(CH2CH2-O)n4-CH2CH2-C(=O)- L2 may not be present, and in such a case, L2 is a single bond. In the drug-linker structure of the present invention, in particular, Lp may be directly connected to a drug, and in such a case, L2 is particularly preferably a single bond, n4 is an integer of 1 to 6, and preferably 2 to 4. L2 is connected to L1 at its terminal amino group and is connected to Lp at the carbonyl group of the opposite terminal.

[0100] Specific examples of L2 include the followings. -NH-CH2CH2-O-CH2CH2-C(=O)-, -NH-CH2CH2-O-CH2CH2-O-CH2CH2-C(=O)-, -NH-CH2CH2-O-CH2CH2-O-CH2CH2-O-CH2CH2-C(=O)-, -NH-CH2CH2-O-CH2CH2-O-CH2CH2-O-CH2CH2-O-CH2CH2-C(=O)-, -NH-CH2CH2-O-CH2CH2-O-CH2CH2-O-CH2CH2-O-CH2CH2-O-CH2CH2-C(=O)-, -NH-CH2CH2-O-CH2CH2-O-CH2CH2-O-CH2CH2-O-CH2CH2-O-CH2CH2-O-CH2CH2-C(=O)-

[0101] 3. Lp Lp is a peptide residue consisting of 2 to 7 amino acids. Specifically, it consists of an oligopeptide residue in which 2 to 7 amino acids are linked by a peptide bond. Lp is connected to L2 at N terminal and it is connected to the amino group of -NH-(CH2)n1-L a-(CH2)n2-C(=O)- moiety of the linker at C terminal.

[0102] The amino acid constituting Lp is not particularly limited, and the examples thereof include an L- or a D-amino acid, preferably an L-amino acid. Further, it can be an amino acid having a structure such as beta-alanine, epsilon-aminocaproic acid, or 2024204502   28 Jun 2024 gamma-aminobutyric acid in addition to an alpha-amino acid, further, it can be a nonnatural type amino acid such as N-methylated amino acid. Sequence of the amino acid of Lp is not particularly limited, but examples of the constituting amino acid include phenylalanine (Phe; F), tyrosine (Tyr; Y), leucine (Leu; L), glycine (Gly; G), alanine (Ala; A), valine (Vai; V), lysine (Lys; K), citrulline (Cit), serine (Ser; S), glutamic acid (Glu; E), and aspartic acid (Asp; D). Among them, preferred examples include phenylalanine, glycine, valine, lysine, citrulline, serine, glutamic acid, and aspartic acid. Depending on the type of the amino acid, drug release pattern can be controlled. The number of the amino acid can be between 2 to 7.

[0103] Specific examples of Lp include the followings. -GGF-, -DGGF-, -(D-)D-GGF-, -EGGF-, -GGFG-, -SGGF-, -KGGF-, -DGGFG-, -GGFGG-, -DDGGFG-, -KDGGFG-, -GGFGGGF- The "(D-)D" described above means D-aspartic acid. Examples of the particularly preferred Lp of the antibody-drug conjugate of the present invention include -GGFG-and -DGGFG- peptide residue. Further, in the drug-linker structure of the present invention, Lp may be directly connected to the drug, and for such a case, preferred examples of Lp include a pentapeptide residue of -DGGFG-.

[0104] 4. La-(CH2)n2-C(=O)- La in La-(CH2)n2-C(=O)- is a structure of -O- or a single bond, n2 is an integer of 0 to 5, preferably, 0 to 3, and more preferably 0 or 1. Examples of La-(CH2)n2-C(=O)- include the followings. -O-CH2-C(=O)-, -O-CH2CH2-C(=O)-, -O-CH2CH2CH2-C(=O)-, -O-CH2CH2CH2CH2-C(=O)-, -O-CH2CH2CH2CH2CH2-C(=O)-, -CH2-C(=O)-, -CH2CH2-C(=O)-, 2024204502   28 Jun 2024 -CH2CH2CH2-C(=O)-, -CH2CH2CH2CH2-C(=O)-, -CH2CH2CH2CH2CH2-C(=O)-. Among them, those with -O-CH2-C(=O)-, -O-CH2CH2-C(=O)- or those in which La is a single bond and n2 is 0 are preferable.

[0105] Specific examples of the linker structure represented by -NH-(CH2)n1-La-(CH2)n2 - C(=O)- include the followings. -NH-CH2-C(=O)-, -NH-CH2CH2-C(=O)-, -NH-CH2-O-CH2-C(=O)-, -NH-CH2CH2-O-C(=O)-, -NH-CH2CH2-O-CH2-C(=O)-, -NH-CH2CH2CH2-C(=O)-, -NH-CH2CH2CH2CH2-C(=O)-, -NH-CH2CH2CH2CH2CH2-C(=O)-

[0106] Among them, the examples are more preferably the followings. -NH-CH2CH2CH2-C(=O)-, -NH-CH2-O-CH2-C(=O)-, -NH-CH2CH2-O-C(=O)-

[0107] As for the linker -NH-(CH2)n'-La-(CH2)n2-C(=O)-, the chain length of 4 to 7 atoms is preferable, and more preferably, are those having the chain length of 5 or 6 atoms.

[0108] With regard to the anti-HER3 antibody-drug conjugate of the present invention, when it is transferred to the inside of tumor cells, it is thought that the linker moiety is cleaved and the drug derivative having a structure represented by NH2-(CH2)n'-La-(CH 2)n2-C(=O)-(NH-DX) is released to express an antitumor action. Examples of the antitumor derivative exhibiting an antitumor effect by releasing from the antibody-drug conjugate of the present invention include an antitumor derivative having a structure moiety in which the terminal of the structure represented by -NH-(CH2)n1-La-(CH2)n2 -C(=O)- of the linker is an amino group, and the particularly preferred include the followings. NH2-CH2CH2-C(=O)-(NH-DX), NH2-CH2CH2CH2-C(=O)-(NH-DX), NH2-CH2-O-CH2-C(=O)-(NH-DX), NH2-CH2CH2-O-CH2-C(=O)-(NH-DX). Meanwhile, in case of NH2-CH2-O-CH2-C(=O)-(NH-DX), it was confirmed that, as the aminal structure in the molecule is unstable, it again undergoes a self- degradation 2024204502   28 Jun 2024 to release the following HO-CH2-C(=O)-(NH-DX). Those compounds can be also preferably used as a production intermediate of the antibody-drug conjugate of the present invention. Further, in the drug-linker structure of the present invention, there arises a case in which Lp may be directly connected to the drug. In such a case, when the C terminal of Lp is glycine, the antitumor drug to be released is exatecan itself or a compound having glycine bonded to the amino group of exatecan.

[0109] For the antibody-drug conjugate of the present invention in which exatecan is used as a drug, the drug-linker structure moiety having the following structure -L1-L2-Lp-NH-(CH2)n1-La-(CH2)n2-C(=O)-(NH-DX) or -L*-L2-LP-(NH-DX) to which the antibody is connected is preferable. The conjugated number of these drug-linker structure moiety may be from 1 to 10 as the average conjugated number per antibody, preferably, 2 to 8, and more preferably 3 to 8. -(Succinimid-3-yl-N)-CH2CH2-C(=O)-GGFG-NH-CH2CH2-C(=O)-(NH-DX), -(Succinimid-3-yl-N)-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-(NH-DX), -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2-C(=O)-(NH- DX), -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-( NH-DX), -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2CH2CH2- C(=O)-(NH-DX), -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-DGGFG-NH-CH2CH2-C(=O)-(N H-DX), -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-DGGFG-NH-CH2CH2CH2-C(=O) -(NH-DX), -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-DGGFG-NH-CH2CH2CH2CH2CH 2-C(=O)-(NH-DX), -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2-O-CH2-C(=O)-( NH-DX), -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2-O-CH2-C(= O)-(NH-DX), -(Succinimid-3-yl-N)-CH2CH2-C(=O)-NH-CH2CH2-O-CH2CH2-O-CH2CH2-C(=O)-G GFG-NH-CH2CH2-C(=O)-(NH-DX), -(Succinimid-3-yl-N)-CH2CH2-C(=O)-NH-CH2CH2-O-CH2CH2-O-CH2CH2-C(=O)-G GFG-NH-CH2CH2CH2-C(=O)-(NH-DX), -(Succinimid-3-yl-N)-CH2CH2-C(=O)-NH-CH2CH2-O-CH2CH2-O-CH2CH2-O-CH2C H2-O-CH2CH2-C(=O)-GGFG-NH-CH2CH2-C(=O)-(NH-DX), 2024204502   28 Jun 2024 -(Succinimid-3-yl-N)-CH2CH2-C(=O)-NH-CH2CH2-O-CH2CH2-O-CH2CH2O-CH2CH2- O-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-(NH-DX), -(Succinimid-3-yl-N)-CH2CH2-C(=O)-GGFG-(NH-DX), -(Succinimid-3-yl-N)-CH2CH2-C(=O)-DGGFG-(NH-DX), -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-(NH-DX), -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-DGGFG-(NH-DX). Among them, the more preferred are the followings. -(Succinimid-3-yl-N)-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-(NH-DX), -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-(N H-DX), -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-DGGFG-NH-CH2CH2CH2-C(=O)-( NH-DX), -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2-O-CH2-C(=O)-(N H-DX), -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2-O-CH2-C(=O) -(NH-DX), -(Succinimid-3-yl-N)-CH2CH2-C(=O)-NH-CH2CH2-O-CH2CH2-O-CH2CH2-C(=O)-GG FG-NH-CH2CH2CH2-C(=O)-(NH-DX), -(Succinimid-3-yl-N)-CH2CH2-C(=O)-NH-CH2CH2-O-CH2CH2-O-CH2CH2-O-CH2CH2 -O-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-(NH-DX), -(Succinimid-3-yl-N)-CH2CH2-C(=O)-DGGFG-(NH-DX), -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-DGGFG-(NH-DX). The still more preferred are the followings. -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2-O-CH2-C(=O)-(N H-DX), -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2-O-CH2-C(=O) -(NH-DX), -(Succinimid-3-yl-N)-CH2CH2-C(=O)-NH-CH2CH2-O-CH2CH2-O-CH2CH2-C(=O)-GG FG-NH-CH2CH2CH2-C(=O)-(NH-DX), -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-DGGFG-(NH-DX). The particularly preferred are the followings. -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2-O-CH2-C(=O)-(N H-DX), -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2-O-CH2-C(=O) -(NH-DX), -(Succinimid-3-yl-N)-CH2CH2-C(=O)-NH-CH2CH2-O-CH2CH2-O-CH2CH2-C(=O)-GG FG-NH-CH2CH2CH2-C(=O)-(NH-DX).

[0110] With regard to the linker structure for conjugating the anti-HER3 antibody and a 2024204502   28 Jun 2024 drug in the antibody-drug conjugate of the present application, the preferred linker can be constructed by connecting preferred structures shown for each part of the linker explained above. As for the linker structure, those with the following structure can be preferably used. Meanwhile, the left terminal of the structure is a connecting position to the antibody and the right terminal is a connecting position to the drug. -(Succinimid-3-yl-N)-CH2CH2-C(=O)-GGFG-NH-CH2CH2-C(=O)-, -(Succinimid-3-yl-N)-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-, -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2-C(=O)-, -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-, -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2CH2CH2-C (=0)-, -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-DGGFG-NH-CH2CH2-C(=O)-, -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-DGGFG-NH-CH2CH2CH2-C(=O)-, -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-DGGFG-NH-CH2CH2CH2CH2CH2-C(=O)-, -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2-O-CH2-C(=O)-, -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2-O-CH2-C(=O) -(Succinimid-3-yl-N)-CH2CH2-C(=O)-NH-CH2CH2-O-CH2CH2-O-CH2CH2-C(=O)-GG FG-NH-CH2CH2-C(=O)-, -(Succinimid-3-yl-N)-CH2CH2-C(=O)-NH-CH2CH2-O-CH2CH2-O-CH2CH2-C(=O)-GG FG-NH-CH2CH2CH2-C(=O)-, -(Succinimid-3-yl-N)-CH2CH2-C(=O)-NH-CH2CH2-O-CH2CH2-O-CH2CH2-O-CH2CH2 -O-CH2CH2-C(=O)-GGFG-NH-CH2CH2-C(=O)-, -(Succinimid-3-yl-N)-CH2CH2-C(=O)-NH-CH2CH2-O-CH2CH2-O-CH2CH2-O-CH2CH2 -O-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-, -(Succinimid-3-yl-N)-CH2CH2-C(=O)-GGFG-, -(Succinimid-3-yl-N)-CH2CH2-C(=O)-DGGFG-, -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-, -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-DGGFG-. Among them, more preferred are the followings. -(Succinimid-3-yl-N)-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-, -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-, -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-DGGFG-NH-CH2CH2CH2-C(=O)-, -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2-O-CH2-C(=O)-, -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2-O-CH2-C(=O) -(Succinimid-3-yl-N)-CH2CH2-C(=O)-NH-CH2CH2-O-CH2CH2-O-CH2CH2-C(=O)-GG 2024204502   28 Jun 2024 FG-NH-CH2CH2CH2-C(=O)-, -(Succinimid-3-yl-N)-CH2CH2-C(=O)-NH-CH2CH2-O-CH2CH2-O-CH2CH2-O-CH2CH2 -O-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-, -(Succinimid-3-yl-N)-CH2CH2-C(=O)-DGGFG-, -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-DGGFG-. Still more preferred are the followings. -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2-O-CH2-C(=O)-, -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2-O-CH2-C(=O) -(Succinimid-3-yl-N)-CH2CH2-C(=O)-NH-CH2CH2-O-CH2CH2-O-CH2CH2-C(=O)-GG FG-NH-CH2CH2CH2-C(=O)-, -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-DGGFG-. Particularly preferred are the followings. -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2-O-CH2-C(=O)-, -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2-O-CH2-C(=O) -(Succinimid-3-yl-N)-CH2CH2-C(=O)-NH-CH2CH2-O-CH2CH2-O-CH2CH2-C(=O)-GG FG-NH-CH2CH2CH2-C(=O)-.

[0111] {Production method} Next, explanations are given for the representative method for producing the antibody-drug conjugate of the present invention or a production intermediate thereof. Meanwhile, the compounds are hereinbelow described with the number shown in each reaction formula. Specifically, they are referred to as a "compound of the formula (1)", a "compound (1)", or the like. The compounds with numbers other than those are also described similarly.

[0112] 1. Production method 1 The antibody-drug conjugate represented by the formula (1) in which the antibody is conjugated to the drug-linker structure via thioether can be produced by the following method, for example.

[0113] [Chern. 19] AB Lr-L2-Lp-NH-(CH2)n1^La-(CH2)n2-C(=O)<NH-DX)         3a AB-L1-L2-Lp-NH-(CH2)n1-La-(CH2)n2-C(=O)-(NH-DX) °r                                   *                          or L"-I2I.MNH-DX)                                             AB-L1-L2-Lp-(NH-DX) 2                                                                                 1

[0114] [in the formula, AB represents an antibody with a sulfhydryl group and L1’ corresponds to L1 having a structure in which the linker terminal is converted to a maleimidyl group (formula shown below). 2024204502   28 Jun 2024

[0115] [Chem.20]

[0116] (in the formula, the nitrogen atom is the connecting position) Specifically, it represents a linker having a structure which, within the structure of L1 represented as -(Succinimid-3-yl-N)-(CH2)n2-C(=O)-, said -(Succinimid-3-yl-N)-moiety is converted into a maleimidyl group. Further, the -(NH-DX) represents a structure represented by the following formula:

[0117] [Chem.21] Me

[0118] and it represents a group that is derived by removing one hydrogen atom of the amino group at position 1 of exatecan.]

[0119] Further, the compound of the formula (1) in the above reaction formula can be interpreted as a structure in which one structure moiety from drug to the linker terminal is connected to one antibody. However, it is only the description given for the sake of convenience, and there are actually many cases in which a plurality of said structure moieties is connected to one antibody molecule. The same applies to the explanation of the production method described below.

[0120] Specifically, the antibody-drug conjugate (1) can be produced by reacting the compound (2), which is obtainable by the method described below, with the antibody (3a) having a sulfhydryl group. The antibody (3a) having a sulfhydryl group can be obtained by a method well known in the art (Hermanson, G.T, Bioconjugate Techniques, pp. 56-136, pp. 456-493, Academic Press (1996)). Examples include: Traufs reagent is reacted with the amino group of the antibody; N-succinimidyl S-acetylthioalkanoates are reacted with the 2024204502   28 Jun 2024 amino group of the antibody followed by reaction with hydroxylamine; after reacting with N-succinimidyl 3-(pyridyldithio)propionate, it is reacted 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 disulfide bond at a hinge part in the antibody to form a sulfhydryl group, but it is not limited thereto. Specifically, using 0.3 to 3 molar equivalents of TCEP as a reducing agent per disulfide bonds at hinge part in the antibody and reacting with the antibody in a buffer solution containing a chelating agent, the antibody which the disulfide bonds at hinge part in the antibody is partially or completely reduced can be obtained. Examples of the chelating agent include ethylenediamine tetraacetic acid (EDTA) and diethylenetriamine pentaacetic acid (DTPA). It can be used at concentration of 1 mM to 20 mM. Examples of the buffer solution which may be used include a solution of sodium phosphate, sodium borate, or sodium acetate. Specifically, by reacting the antibody with TCEP at 4C to 37C for 1 to 4 hours, the antibody (3a) having partially or completely reduced sulfhydryl groups can be obtained. Meanwhile, by performing an addition reaction of a sulfhydryl group to a drug-linker moiety, the drug-linker moiety can be conjugated by a thioether bond. Using 2 to 20 molar equivalents of the compound (2) per the antibody (3a) having a sulfhydryl group, the antibody-drug conjugate (1) in which 2 to 8 drug molecules are conjugated per antibody can be produced. Specifically, it is sufficient that the solution containing the compound (2) dissolved therein is added to a buffer solution containing the antibody (3a) having a sulfhydryl group for the reaction. Herein, examples of the buffer solution which may be used include sodium acetate solution, sodium phosphate, and sodium borate. pH for the reaction is 5 to 9, and more preferably the reaction is performed near 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-pyrrolidone (NMP). The reaction may be carried out by adding the organic solvent solution containing the compound (2) dissolved therein at 1 to 20% v / v to a buffer solution containing the antibody (3a) having a sulfhydryl group. The reaction temperature is 0 to 37C, more preferably 10 to 25C, and the reaction time is 0.5 to 2 hours. The reaction can be terminated by deactivating the reactivity of unreacted compound (2) with a thiol-containing reagent. Examples of the thiol-containing reagent include cysteine and N-acetyl-L-cysteine (NAC). More specifically, by adding 1 to 2 molar equivalents of NAC to the compound (2) used and, by incubating at room temperature for 10 to 30 minutes, the reaction can be terminated. The produced antibody-drug conjugate (1) can be subjected to, after concentration, buffer exchange, purification, and measurement of antibody concentration and average 2024204502   28 Jun 2024 number of conjugated drug molecules per antibody molecule according to common procedures described below, to make an identification of the antibody-drug conjugate (1).

[0121] Common procedure A: Concentration of aqueous solution of antibody or antibodydrug conjugate To a Amicon Ultra (50,000 MWCO, Millipore Corporation) container, a solution of antibody or antibody-drug conjugate was added and the solution of the antibody or antibody-drug conjugate was concentrated by centrifugation (centrifuge for 5 to 20 minutes at 2000 G to 3800 G) using a centrifuge (Allegra X-15R, Beckman Coulter, Inc.)

[0122] Common procedure B: Measurement of antibody concentration Using a UV detector (Nanodrop 1000, Thermo Fisher Scientific Inc.), measurement of the antibody concentration was performed according to the method defined by the manufacturer. Here, 280 nm absorption coefficient can be estimated from the amino acid sequence of an antibody using a known calculation method (Protein Science, 1995, vol. 4, 2411-2423), and 280 nm absorption coefficient different for each antibody was used (1.3 mLmg 'em1 to 1.8 mLmg 'em'). In the case of Ul-59, 280 nm absorption coefficient of 1.768 mLmg 'em 1 was used as an estimated value according to its amino acid sequence.

[0123] Common procedure C: Buffer Exchange for antibody NAP-25 column (Cat. No. 17-0852-02, GE Healthcare Japan Corporation) using Sephadex G-25 carrier was equilibrated with phosphate buffer (10 mM, pH 6.0; it is referred to as PBS6.0 / EDTA in the specification) containing sodium chloride (137 mM) and ethylene diamine tetraacetic acid (EDTA, 5 mM) according to the method defined by the manufacturer. Aqueous solution of the antibody was applied in an amount of 2.5 mL to single NAP-25 column, and then the fraction (3.5 mL) eluted with 3.5 mL of PBS6.0 / EDTA was collected. The resulting fraction was concentrated by the Common procedure A. After measuring the concentration of the antibody using the Common procedure B, the antibody concentration was adjusted to 10 mg / mL using PBS6.0 / EDTA.

[0124] Common procedure D: Purification of antibody-drug conjugate NAP-25 column was equilibrated with acetate buffer containing sorbitol (5%) (10 mM, pH 5.5; it is referred to as ABS in the specification). Aqueous reaction solution of the antibody-drug conjugate (about 2.5 mL) was applied to the NAP-25 column, and then eluted with the buffer in an amount as defined by the manufacturer to collect the antibody fraction. By conducting a gel filtration purification process, in which said collected fraction was again applied to the NAP-25 column and eluted with buffer, was repeated 2 to 3 times in total, the antibody-drug conjugate excluding non-conjugated 2024204502   28 Jun 2024 drug linker and a low-molecular-weight compound (tris(2-carboxyethyl)phosphine hydrochloride (TCEP), N-acetyl-L-cysteine (NAC), and dimethyl sulfoxide) was obtained.

[0125] Common procedure E: Measurement of antibody concentration in antibody-drug conjugate and average number of conjugated drug molecules per antibody molecule (1) The conjugated drug concentration in the antibody-drug conjugate can be calculated by measuring UV absorbance of an aqueous solution of the antibody-drug conjugate at two wavelengths of 280 nm and 370 nm, followed by performing the calculation shown below. Because the total absorbance at any wavelength is equal to the sum of the absorbance of every light-absorbing chemical species that are present in a system [additivity of absorbance], when the molar absorption coefficients of the antibody and the drug remain the same before and after conjugation between the antibody and the drug, the antibody concentration and the drug concentration in the antibody-drug conjugate are expressed with the following equations. A280 = ADj28o + Aa,28o = EDj28oCd + Ea,28oCa Equation (1) A370 = ADj37o+Aa,37o = EDj37oCd+Ea,37oCa Equation (2) In the above, A28o represents the absorbance of an aqueous solution of the antibodydrug conjugate at 280 nm, A370 represents the absorbance of an aqueous solution of the antibody-drug conjugate at 370 nm, Aa,28o represents the absorbance of an antibody at 280 nm, AAj37o represents the absorbance of an antibody at 370 nm, AD 28o represents the absorbance of a conjugate precursor at 280 nm, ADj370 represents the absorbance of a conjugate precursor at 370 nm, EAj28o represents the molar absorption coefficient of an antibody at 280 nm, EAj37o represents the molar absorption coefficient of an antibody at 370 nm, ED 28o represents the molar absorption coefficient of a conjugate precursor at 280 nm, ED 370 represents the molar absorption coefficient of a conjugate precursor at 370 nm, CA represents the antibody concentration in an antibody-drug conjugate, and C D represent the drug concentration in an antibody-drug conjugate. As for EAj28o, EAj37o, Ed 28o? and EDj370 in the above, previously prepared values (estimated value based on calculation or measurement value obtained by UV measurement of the compound) are used. For example, EAj28o can be estimated from the amino acid sequence of an antibody using a known calculation method (Protein Science, 1995, vol. 4, 2411-2423). EAj37o is generally zero. In the case of Ul-59, EAj28o of 259400 was used as an estimated value according to its amino acid sequence. ED 28o and Ed 37o can be obtained based on Lambert-Beer's law (Absorbance = molar concentration " molar absorption coefficient" cell path length) by measuring the absorbance of a solution in which the conjugate precursor to be used is dissolved at a certain molar concentration. By measuring A28o and A370 of an aqueous solution of the 2024204502   28 Jun 2024 antibody-drug conjugate and solving the simultaneous equations (1) and (2) using the values, CA and CD can be obtained. Further, by diving CD by CA, the average drug binding number per antibody can be obtained. In the present invention, the method for determining the average number of conjugated drug molecules per antibody as described above is referred to as a "UV method".

[0126] Common procedure F: Measurement of average number of conjugated drug molecules per antibody molecule in 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) analysis using the following method, in addition to the aforementioned Common procedure E. {F-l. Preparation of sample for HPLC analysis (Reduction of antibody-drug conjugate)} An antibody-drug conjugate solution (about 1 mg / mL, 60 u ("u" represents "micro")L) is mixed with an aqueous solution of dithiothreitol (DTT) (100 rnM, 15 uL). By incubating the mixture at 37C for 30 minutes, the disulfide bond between the L and H chains of the antibody-drug conjugate is cleaved. The resulting sample is used in HPLC analysis. {F-2. HPLC analysis} The HPLC analysis is carried out under the following measurement conditions. HPLC system: Agilent 1290 HPLC system (Agilent Technologies) Detector: Ultraviolet absorption spectrometer (measurement wavelength: 280 nm) Column: PLRP-S (2.1'50 mm, 8 um, 1000 angstroms; Agilent Technologies, P / N PL1912-1802) Column temperature: 80C Mobile phase A: 0.04% aqueous trifluoroacetic acid (TFA) solution 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.) Sample injection: 15 uL {F-3. Data analysis} [F-3-1] Compared with non-conjugated antibody L (Lo) and H (Ho) chains, drug-conjugated L (L chain bound to one drug molecule: LJ and H (H chain bound to one drug molecule: Hi, H chain bound to two drug molecule: H2, H chain bound to three drug molecules: H3) chains exhibit higher hydrophobicity in proportion to the number of conjugated drug molecules and thus have a larger retention time. These chains are therefore eluted in the order of Lo and Li or Ho, Hi, H2, and H3. Detection peaks can be assigned to any of Lo, Lb Ho, H h H2, and H3 by the comparison of retention times with 2024204502   28 Jun 2024 Lo and Ho. [F-3-2] Since the drug linker has UV absorption, peak area values are corrected in response to the number of conjugated drug linker molecules according to the following expression using the molar absorption coefficients of the L or H chain and the drug linker.

[0127] [Math.l] Corrected val ue of the peak area of the L chain (Li) = Peak area Molar absorption coefficient of the L chain x-------------------------------------------------------- Molar absorption coefficient of the L chain + The number of conjugated drug molecules x Molar absorption coefficient of the drug linker

[0128] [Math.2] Corrected value of the peak area of the H chain (Hi) = Peak area Molar absorption coefficient of the H chain x-------------------------------------------------------- Molar absorption coefficient of the H chain + The number of conjugated drug molecules x Molar absorption coefficient of the drug linker

[0129] Here, a value estimated from the amino acid sequence of the L or H chain of each antibody using a known calculation method (Protein Science, 1995, vol. 4, 2411-2423) can be used as the molar absorption coefficient (280 nm) of the L or H chain of each antibody. In the case of UI-59, a molar absorption coefficient of 34690 and a molar absorption coefficient of 95000 were used as estimated values for the L and H chains, respectively, according to its amino acid sequence. The actually measured molar absorption coefficient (280 nm) of a compound in which the maleimide group has been converted to succinimide thioether by the reaction of each drug linker with mercaptoethanol or N-acetylcysteine was used as the molar absorption coefficient (280 nm) of the drug linker. [F-3-3] The peak area ratio (%) of each chain is calculated for the total of the corrected values of peak areas.

[0130] [Math.3] Peak area ratio of the L chain =------x 100 J Peak area ratio of the H chain =----------—---------x 100 •^0 +   +    +  3 Corrected values of respective peak areas of ALi, AHiiLi, Hi

[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 expression. 2024204502   28 Jun 2024 Average number of conjugated drug molecules = (Lo peak area ratio x 0 + Lo peak area ratio x 1 + Ho peak area ratio x 0 + Hi peak area ratio x 1 + H2 peak area ratio x 2 + H3 peak area ratio x 3) / 100 x 2

[0132] Hereinbelow, production intermediate compounds used in Production method 1 are described. The compound represented by the formula (2) in the production method 1 is a compound represented by the following formula:

[0133] (maleimid-N-yl)-(CH2)n3-C(=O)-L2-Lp-NH-(CH2)n1-L“-(CH2)n2-C(=O)-(NH-DX) or (maleimid-N-yl)-(CH2)n3-C(=O)-L2-Lp-(NH-DX). In the formula, n3 represents an integer of 2 to 8, L2 represents -NH-(CH2CH2-O)n4-CH2CH2-C(=O)- or a single bond, wherein n4 represents an integer of 1 to 6, Lp represents a peptide residue consisting of 2 to 7 amino acids selected from phenylalanine, glycine, valine, lysine, citrulline, serine, glutamic acid, and aspartic acid, n1 represents an integer of 0 to 6, n2 represents an integer of 0 to 5, La represents -O- or a single bond, (maleimid-N-yl)- is a maleimidyl group (2,5-dioxo-2,5-dihydro-lH-pyrrol-l-yl group) represented by the following formula: [Chern. 22]

[0134] wherein the nitrogen atom is the connecting position, and -(NH-DX) is a group represented by the following formula: 2024204502   28 Jun 2024 [Chem.23] Me

[0135] wherein the nitrogen atom of the amino group at position 1 is the connecting position.

[0136] As for the peptide residue Lp, those consisting of an amino acid selected from phenylalanine, glycine, valine, lysine, citrulline, serine, glutamic acid, and aspartic acid is preferred as a production intermediate. Among the peptide residue Lp, those consisting of 4 or 5 amino acids is preferred as a production intermediate. More specifically, those in which Lp is a tetrapeptide residue of -GGFG- or a pentapeptide of -DGGFG- is preferred as a production intermediate, more preferably, -GGFG-.

[0137] Further, as for the -NH-(CH2)n1-L“-(CH2)n2-, those having -NH-CH2CH2-, -NH-CH2 CH2CH2-, -NH-CH2CH2CH2CH2-, -NH-CH2CH2CH2CH2CH2-, -NH-CH2-O-CH2-, or -NH-CH2CH2-O-CH2- is preferred as a production intermediate. A compound of -NH-CH2CH2CH2-, -NH-CH2-O-CH2-, or -NH-CH2CH2-O-CH2 is more preferred. As for n3, those in which it is an integer of 2 to 8 is preferred as a production intermediate. As for L2, those in which it is a single bond or -NH-(CH2CH2-O)n4-CH2CH2-C(=O)-and n4 is an integer of 2 to 4 is preferred as a production intermediate.

[0138] Further, those in which n3 is an integer of 2 to 5, L2 is a single bond, and -NH-(CH2)n ‘-La-(CH2)n2- is -NH-CH2CH2-, -NH-CH2CH2CH2-, -NH-CH2CH2CH2CH2-, -NH-CH2 CH2CH2CH2CH2-, -NH-CH2-O-CH2-, or -NH-CH2CH2-O-CH2- is preferred as a production intermediate. Further, more preferred among them is those in which -NH-(CH2)n1-L“-(CH2)n2- is -NH-CH2CH2-, -NH-CH2CH2CH2-, -NH-CH2-O-CH2-, or -NH-CH2CH2-O-CH2-. Further, those in which n3 is an integer of 2 or 5 is preferred.

[0139] Further, those in which n3 is an integer of 2 to 5, L2 is -NH-(CH2CH2-O)n4-CH2CH2 -C(=O)-, n4 is an integer of 2 to 4, and -NH-(CH2)n'-La-(CH2)n2- is -NH-CH2CH2-, -nh-ch2ch2ch2-, -nh-ch2ch2ch2ch2-, -nh-ch2ch2ch2ch2ch2-, -nh-ch2 -O-CH2-, or -NH-CH2CH2-O-CH2- is preferred as a production intermediate. More preferred among them is those in which n4 is an integer of 2 or 4. Further, those in 2024204502   28 Jun 2024 which -NH-(CH2)n'-La- is -NH-CH2CH2CH2-, -NH-CH2-O-CH2-, or -NH-CH2CH2 -O-CH2- is preferred.

[0140] Preferred examples of the intermediate that are useful for production of the compound of the present invention include those exemplified below: (maleimid-N-yl)-CH2CH2-C(=O)-GGFG-NH-CH2CH2-C(=O)-(NH-DX), (maleimid-N-yl)-CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2-C(=O)-(NH-DX), (maleimid-N-yl)-CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2-C(=O)-(NH-DX), (maleimid-N-yl)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2-C(=O)-(NH-DX), (maleimid-N-yl)-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-(NH-DX), (maleimid-N-yl)-CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-(NH-DX), (maleimid-N-yl)-CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-(NH-DX), (maleimid-N-yl)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-(NH- DX), (maleimid-N-yl)-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2CH2-C(=O)-(NH-DX), (maleimid-N-yl)-CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2CH2-C(=O)-(NH-DX), (maleimid-N-yl)-CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2CH2-C(=O)-(NH- DX), (maleimid-N-yl)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2CH2-C(=O)-( NH-DX), (maleimid-N-yl)-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2CH2CH2-C(=O)-(NH-DX), (maleimid-N-yl)-CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2CH2CH2-C(=O)-(NH- DX), (maleimid-N-yl)-CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2CH2CH2-C(=O)-( NH-DX), (maleimid-N-yl)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2CH2CH2-C(= O)-(NH-DX), (maleimid-N-yl)-CH2CH2CH2CH2CH2-C(=O)-DGGFG-NH-CH2CH2CH2-C(=O)-(NH -DX), (maleimid-N-yl)-CH2CH2CH2CH2CH2-C(=O)-DGGFG-NH-CH2CH2CH2CH2-C(=O)-(NH-DX), (maleimid-N-yl)-CH2CH2CH2CH2CH2-C(=O)-DGGFG-NH-CH2CH2CH2CH2CH2-C( =O)-(NH-DX), (maleimid-N-yl)-CH2CH2-C(=O)-GGFG-NH-CH2-O-CH2-C(=O)-(NH-DX), (maleimid-N-yl)-CH2CH2CH2-C(=O)-GGFG-NH-CH2-O-CH2-C(=O)-(NH-DX), (maleimid-N-yl)-CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2-O-CH2-C(=O)-(NH-DX), (maleimid-N-yl)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2-O-CH2-C(=O)-(NH-DX), (maleimid-N-yl)-CH2CH2-C(=O)-GGFG-NH-CH2CH2-O-CH2-C(=O)-(NH-DX), 2024204502   28 Jun 2024 (maleimid-N-yl)-CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2-O-CH2-C(=O)-(NH-DX), (maleimid-N-yl)-CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2-O-CH2-C(=O)-(NH-D X), (maleimid-N-yl)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2-O-CH2-C(=O)-(NH -DX), (maleimid-N-yl)-CH2CH2-C(=O)-NH-CH2CH2-O-CH2CH2-O-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-(NH-DX), (maleimid-N-yl)-CH2CH2-C(=O)-NH-CH2CH2-O-CH2CH2-O-CH2CH2-O-CH2CH2-C(= O)-GGFG-NH-CH2CH2CH2-C(=O)-(NH-DX), (maleimid-N-yl)-CH2CH2-C(=O)-NH-CH2CH2-O-CH2CH2-O-CH2CH2-O-CH2CH2-O-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-(NH-DX), (maleimid-N-yl)-CH2CH2-C(=O)-GGFG-(NH-DX), (maleimid-N-yl)-CH2CH2CH2-C(=O)-GGFG-(NH-DX), (maleimid-N-yl)-CH2CH2CH2CH2-C(=O)-GGFG-(NH-DX), (maleimid-N-yl)-CH2CH2CH2CH2CH2-C(=O)-GGFG-(NH-DX), (maleimid-N-yl)-CH2CH2-C(=O)-DGGFG-(NH-DX), (maleimid-N-yl)-CH2CH2CH2-C(=O)-DGGFG-(NH-DX), (maleimid-N-yl)-CH2CH2CH2CH2-C(=O)-DGGFG-(NH-DX), or (maleimid-N-yl)-CH2CH2CH2CH2CH2-C(=O)-DGGFG-(NH-DX).

[0141] By the reaction of the drug-linker compound selected from the aforementioned group of intermediate compounds with an anti-Her3 antibody or a reactive derivative thereof, a thioether bond can be formed at a disulfide bond moiety present in a hinge part of the anti-Her3 antibody, and as a result, the anti-Her3 antibody-drug conjugate of the present invention can be produced. In this case, it is preferable to use a reactive derivative of an anti-Her3 antibody. A reactive derivative obtained by reducing an anti-Her3 antibody is particularly preferred.

[0142] The followings are a compound which is more preferred as a production intermediate. (maleimid-N-yl)-CH2CH2-C(=O)-GGFG-NH-CH2CH2-C(=O)-(NH-DX), (maleimid-N-yl)-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-(NH-DX), (maleimid-N-yl)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2-C(=O)-(NH-DX), (maleimid-N-yl)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-(NH-DX), (maleimid-N-yl)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2CH2CH2-C(= O)-(NH-DX), (maleimid-N-yl)-CH2CH2CH2CH2CH2-C(=O)-DGGFG-NH-CH2CH2CH2-C(=O)-(NH -DX), (maleimid-N-yl)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2-O-CH2-C(=O)-(NH- 2024204502   28 Jun 2024 DX), (maleimid-N-yl)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2-O-CH2-C(=O)-(NH -DX), (maleimid-N-yl)-CH2CH2-C(=O)-NH-CH2CH2-O-CH2CH2-O-CH2CH2-C(=O)-GGFG- NH-CH2CH2-C(=O)-(NH-DX), (maleimid-N-yl)-CH2CH2-C(=O)-NH-CH2CH2-O-CH2CH2-O-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-(NH-DX), (maleimid-N-yl)-CH2CH2-C(=O)-NH-CH2CH2-O-CH2CH2-O-CH2CH2-O-CH2CH2-O-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-(NH-DX), (maleimid-N-yl)-CH2CH2-C(=O)-DGGFG-(NH-DX), (maleimid-N-yl)-CH2CH2CH2-C(=O)-DGGFG-(NH-DX), (maleimid-N-yl)-CH2CH2CH2CH2-C(=O)-DGGFG-(NH-DX), (maleimid-N-yl)-CH2CH2CH2CH2CH2-C(=O)-DGGFG-(NH-DX).

[0143] Further, among the aforementioned intermediate compound group, the intermediates represented by the following formula are a more preferred compound: (maleimid-N-yl)-CH2CH2-C(=O)-NH-CH2CH2-O-CH2CH2-O-CH2CH2-C(=O)-GGFG -NH-CH2CH2CH2-C(=O)-(NH-DX), (maleimid-N-yl)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2-O-CH2-C(=O)-(NH-DX), (maleimid-N-yl)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2-O-CH2-C(=O)-(N H-DX), (maleimid-N-yl)-CH2CH2CH2CH2CH2-C(=O)-DGGFG-NH-CH2CH2CH2-C(=O)-(NH -DX), or (maleimid-N-yl)-CH2CH2CH2CH2CH2-C(=O)-DGGFG-(NH-DX). Particularly preferred are the compounds that are represented by the following formula: (maleimid-N-yl)-CH2CH2-C(=O)-NH-CH2CH2-O-CH2CH2-O-CH2CH2-C(=O)-GGFG -NH-CH2CH2CH2-C(=O)-(NH-DX), (maleimid-N-yl)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2-O-CH2-C(=O)-(NH-DX), or (maleimid-N-yl)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2-O-CH2-C(=O)-(N H-DX).

[0144] 2. Production method 2 The compound represented by the formula (2) or a pharmacologically acceptable salt thereof used as an intermediate in the previous production method can be produced by the following method, for example.

[0145] 2024204502   28 Jun 2024 [Chem.24] nh2-dx 4 P1-NH-(CH2)n<L“-(CH2)n?-C(=O)-OH 5 T P1-NH-(CH2)nl-La-{CH2)n2-C(=O)-(NH-DX) 6 NH2-<CH2)n1-L’-(CH2)n2-C(=O)-OP3 12 NH2-(CH2)n,-L>-(CH2)n2-C(=O)-(NH-DX) 7 P2-L’-OH 8                            NH2-DX ’                                                                    4 P2-LP-NH-(CH2)n’-La-(CH2)n2-C(=O)-(NH-DX) *-------- 9 H-Lp-NH-(CH2)n1-La-(CH2)n2-C(=O)-(NH"DX) 10 L1-L2-OH 11                             NH2-DX '                                                                4 L '-L2-Lp XH iCH2^' !CH2kr C^CX         -------- 2 P2-LP-OH S v pS-LP-NH-fCH^n’-P-lCHjJrf-Ct^-OP3 P2-Lp-NH-(CH2)nXLa-(CH2)r^C(=O)-OH 14                    ' -I Ls NH (CHjn1 U (CHjn2 15 L’'-L2-OH 11 U'-L^LP-NH-tCHJn’-L^CH^-C;^ L1XL2<p4MH-(CH2)nXLa-(CH2}n2x:^ 17

[0146] [in the formula, Lr corresponds to L1 having a structure in which the terminal is converted to a maleimidyl group and P1, P2, and P3 represent a protecting group].

[0147] The compound (6) can be produced by derivatizing the carboxylic acid (5) into an active ester, mixed acid anhydride, acid halide, or the like and, in the presense of base, reacting it with NH2-DX [indicating exatecan; chemical name: (lS,9S)-l-amino-9-ethyl-5-fluoro-2,3-dihydro-9-hydroxy-4-methyl-lH,12H-benzo[de] pyrano[3',4':6,7]indolizino[l,2-b]quinolin-10,13(9H,15H)-dione] (4) ora pharmacologically acceptable salt thereof. Reaction reagents and conditions that are commonly used for peptide synthesis can be employed for the reaction. There are various kinds of active ester,for example, it can be produced by reacting phenols such as p-nitrophenol, N-hydroxy benzotriazole, N-hydroxy succinimide, or the like, with the carboxylic acid (5) using a condensing agent such as N,N'-dicyclohexylcarbodiimide or l-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride; further, the active ester can be also produced by a reaction of the carboxylic acid (5) with pentafluorophenyl trifluoroacetate or the like; a reaction of the carboxylic acid (5) with 1-benzotriazolyl oxytripyrrolidinophosphonium hexafluorophosphite; a reaction of the carboxylic acid (5) with diethyl cyanophosphonate (Shioiri method); a reaction of the carboxylic acid (5) with triphenylphosphine and 2,2'-dipyridyl disulfide (Mukaiyama method); a reaction of the carboxylic acid (5) with a triazine derivative such as 4-(4,6-dimethoxy-l,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMTMM); or the like. Further, the reaction can be also performed by, e.g., an acid halide method by which the carboxylic acid (5) is treated with acid halide such as thionyl chloride and 2024204502   28 Jun 2024 oxalyl chloride in the presence of a base. By reacting the active ester, mixed acid anhydride, or acid halide of the carboxylic acid (5) obtained as above with the compound (4) in the presence of a suitable base in an inert solvent at a reaction temperature of -78C to 150C, the compound (6) can be produced. Meanwhile, "inert solvent" indicates a solvent which does not inhibit a desired reaction for which the solvent is used.

[0148] Specific examples of the base used for each step described above include a carbonate, an alkoxide, a hydroxide or a hydride of an alkali metal or an alkali earth metal such as sodium carbonate, potassium carbonate, sodium ethoxide, potassium butoxide, sodium hydroxide, potassium hydroxide, sodium hydride, or potassium hydride; organometallic base represented by an alkyl lithium such as n-butyl lithium, or dialkylamino lithium such as lithium diisopropylamide; organometallic base such as bissilylamine including lithium bis(trimethylsilyl)amide; and organic base such as pyridine, 2,6-lutidine, collidine, 4-dimethylaminopyridine, triethylamine, N-methyl morpholine, diisopropylethylamine, and diazabicyclo[5.4.0]undec-7-ene (DBU).

[0149] Examples of the inert solvent which 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; an 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 them, 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 may be used in some case. Alternatively, these solvents may be used as a mixed solvent.

[0150] As for the protecting group P1 for the terminal amino group of the compound (6), a protecting group for an amino group which is generally used for peptide synthesis, for example, tert-butyloxy carbonyl group, 9-fluorenylmethyloxy carbonyl group, and benzyloxy carbonyl group, can be used. Examples of the other protecting group for an amino group include an alkanoyl group such as acetyl group; an alkoxycarbonyl group such as methoxy carbonyl group and ethoxy carbonyl group; an arylmethoxy carbonyl group such as paramethoxybenzyloxy carbonyl group, and para (or ortho)nitroybenzyloxy carbonyl group; an arylmethyl group such as benzyl group and triphenyl methyl group; an aroyl group such as benzoyl group; and an aryl sulfonyl group such as 2,4-dinitrobenzene sulfonyl group and orthonitrobenzene sulfonyl group. The protecting group P1 can be selected depending on, e.g., properties of a compound having an amino group to be protected. By deprotecting the protecting group P1 for the terminal amino group of the compound (6) obtained, the compound (7) can be produced. In this deprotection, 2024204502   28 Jun 2024 reagents and conditions can be selected depending on the protecting group. The compound (9) can be produced by derivatizing the peptide carboxylic acid (8) having the N terminal protected with P2 into an active ester, mixed acid anhydride, or the like and reacting it with the compound (7) obtained. The reaction conditions, reagents, base, and inert solvent used for a peptide bond formation between the peptide carboxylic acid (8) and the compound (7) can be suitably selected from those described for the synthesis of the compound (6). The protecting group P2 can be suitably selected from those described for the protecting group of the compound (6), and the selection can be made based on, e.g., the properties of the compound having an amino group to be protected. As it is generally used for peptide synthesis, by repeating sequentially the reaction and deprotection of the amino acid or peptide constituting the peptide carboxylic acid (8) for elongation, the compound (9) can be also produced. By deprotecting P2 as the protecting group for the amino group of the compound (9) obtained, the compound (10) can be produced. In this deprptection, reagents and conditions can be selected depending on the protecting group. It is possible to produce the compound (2) by derivatizing the carboxylic acid (11) into an active ester, mixed acid anhydride, acid halide, or the like and reacting it with the compound (10) obtained. The reaction conditions, reagents, base, and inert solvent used for forming a peptide bond between the carboxylic acid (11) and the compound (10) can be suitably selected from those described for the synthesis of the compound (6).

[0151] The compound (9) can be also produced by the following method, for example. The compound (13) can be produced by derivatizing the peptide carboxylic acid (8) having the N terminal protected with P2 into active ester, mixed acid anhydride, or the like and reacting it with the amine compound (12) having the carboxy group protected with P3 in the presence of a base. The reaction conditions, reagents, base, and inert solvent used for forming a peptide bond between the peptide carboxylic acid (8) and the compound (12) can be suitably selected from those described for the synthesis of the compound (6). The protecting group P2 for the amino group of the compound (13) is not particularly limited if it is a protecting group which is commonly used. Specifically, examples of the protecting group for a hydroxyl group include an alkoxymethyl group such as methoxymethyl group; an arylmethyl group such as benzyl group, 4-methoxybenzyl group, and triphenylmethyl group; an alkanoyl group such as acetyl group; an aroyl group such as benzoyl group; and a silyl group such as tert-butyl diphenylsilyl group. Carboxy group can be protected by an ester with an alkyl group such as methyl group, ethyl group, and tert-butyl group, an allyl group, or an arylmethyl group such as benzyl group. As for the amino group, an alkyloxy carbonyl group such as tert-butyloxy 2024204502   28 Jun 2024 carbonyl group, methoxycarbonyl group, and ethoxycarbonyl group; an arylmethoxy carbonyl group such as allyloxycarbonyl group, 9-fluorenylmethyloxy carbonyl group, benzyloxy carbonyl group, paramethoxybenzyloxy carbonyl group, and para (or ortho)nitroybenzyloxy carbonyl group; an alkanoyl group such as acetyl group; an arylmethyl group such as benzyl group and triphenyl methyl group; an aroyl group such as benzoyl group; and an aryl sulfonyl group such as 2,4-dinitrobenzene sulfonyl group or orthonitrobenzene sulfonyl group can be mentioned. As for the protecting group P3 for a carboxy group, a protecting group commonly used as a protecting group for a carboxy group in organic synthetic chemistry, in particular, peptide synthesis can be used. A carboxyl group can be protected as an ester with an alkyl group such as a methyl group, an ethyl group, or a tert-butyl, an allyl group, and an arylmethyl group such as a benzyl group. In such case, it is preferable that the protecting group for an amino group and the protecting group for a carboxy group can be removed by a different method or different conditions. For example, a representative example includes a combination in which P2 is a tert-butyloxy carbonyl group and P3 is a benzyl group. The protecting groups can be selected from the aforementioned ones depending on, e.g., the properties of a compound having an amino group and a carboxy group to be protected. For removal of the protecting groups, reagents and conditions can be selected depending on the protecting group. By deprotecting the protecting group P3 for the carboxy group of the compound (13) obtained, the compound (14) can be produced. In this deprotection, reagents and conditions are selected depending on the protecting group. The compound (9) can be produced by derivatizing the compound (14) obtained into active ester, mixed acid anhydride, acid halide, or the like and reacting with the compound (4) in the presence of a base. For the reaction, reaction reagents and conditions that are generally used for peptide synthesis can be also used, and the reaction conditions, reagents, base, and inert solvent used for the reaction can be suitably selected from those described for the synthesis of the compound (6).

[0152] The compound (2) can be also produced by the following method, for example. By deprotecting the protecting group P2 for the amino group of the compound (13), the compound (15) can be produced. In this deprotection, reagents and conditions can be selected depending on the protecting group. The compound (16) can be produced by derivatizing the carboxylic acid derivative (11) into active ester, mixed acid anhydride, acid halide, or the like and reacting it with the compound (15) obtained in the presence of a base. The reaction conditions, reagents, base, and inert solvent used for forming an amide bond between the peptide carboxylic acid (11) and the compound (15) can be suitably selected from those 2024204502   28 Jun 2024 described for the synthesis of the compound (6). By deprotecting the protecting group for the carboxy group of the compound (16) obtained, the compound (17) can be produced. In this deprotection, it can be carried out similar to deprotecting carboxy group for producing the compound (14). The compound (2) can be produced by derivatizing the compound (17) into active ester, mixed acid anhydride, acid halide, or the like and reacting it with the compound (4) in the presence of a base. For the reaction, reaction reagents and conditions that are generally used for peptide synthesis can be also used, and the reaction conditions, reagents, base, and inert solvent used for the reaction can be suitably selected from those described for the synthesis of the compound (6).

[0153] 3. Production method 3 The compound represented by the formula (2) used as an intermediate can be also produced by the following method.

[0154] [Chem.25] H-LP-OP4 18 L1'-L2-OH 11 L1-L2-LP-OP4 19 L1-L2-Lp-OH 20 NH2-(CH2)n1-La-(CH2)n2-C(=O)-(NH-DX) 7 L1'-L2-LP-NH-(CH2)n1-La-(CH2)n2-C(=O)-(NH-DX) 2

[0155] [in the formula, Lr corresponds to L1 having a structure in which the terminal is converted to a maleimidyl group and P4 represents a protecting group].

[0156] The compound (19) can be produced by derivatizing the compound (11) into active ester, mixed acid anhydride, or the like and reacting it with the peptide carboxylic acid (18) having the C terminal protected with P4 in the presence of a base. The reaction conditions, reagents, base, and inert solvent used for forming a peptide bond between the peptide carboxylic acid (18) and the compound (11) can be suitably selected from 2024204502   28 Jun 2024 those described for the synthesis of the compound (6). The protecting group P4 for the carboxy group of the compound (18) can be suitably selected from the aforementioned protective groups. By deprotecting the protecting group for the carboxy group of the compound (19) obtained, the compound (20) can be produced. In this deprotecion, it can be performed similar to the deprotection of the carboxy group for producing the compound (14). The compound (2) can be produced by derivatizing the compound (20) obtained into active ester, mixed acid anhydride, or the like and reacting it with the compound (7). For the reaction, reaction reagents and conditions that are generally used for peptide synthesis can be also used, and the reaction conditions, reagents, base, and inert solvent used for the reaction can be suitably selected from those described for the synthesis of the compound (6).

[0157] 4. Production method 4 Hereinbelow, within the production intermediate (10) described in production method 2, the method for producing the compound (10b) having n1 = 1 and La = 0 is described in detail. The compound represented by the formula (10b), a salt or a solvate thereof can be produced according to the following method, for example.

[0158] [Chem.26] HO-CH2-C(=O)-OPs ps.X-NH-CH -O-L _______22         P5-X-NH-CH2-O-CH2-C(=O)-OPe ___________P5-X-NH-CH2-O-CH,-C(=O)-OH 2i 2                      *             23                        *             24 h2n-dx                                                                                p7-y-oh ‘ 4      P5-X-NH-CH2-O-CH2-C(=O)-(NH-DX)               H-X-NH-CHq-O-CHq-C(=O)-(NH-DX) 27 ------*                25               ------------ ‘   26                        " P<L^NH-CK-O-CH,-C(=Or(NH-DX)  H-LP-NH-CH2-O-CH,-C(=O)-(NH-DX) 9b                                           10b

[0159] [in the formula, Lp is as defined above, L represents an acyl group including an alkanoyl group such as acetyl group or an aroyl group such as benzoyl group, or represents a hydrogen atom or the like, X and Y represent an oligopeptide consisting of 1 to 3 amino acids, P5 and P7 represent a protecting group for an amino group, and P6 represents a protecting group for a carboxy group].

[0160] A compound represented by the formula (21) can be produced by using or applying the method described in Japanese Patent Laid-Open No. 2002-60351 or the literature (J. Org. Chern., Vol. 51, page 3196, 1986), and if necessary, by removing the protecting groups or modifying the functional groups. Alternatively, it can be also obtained by treating an amino acid with a protected terminal amino group or acid amide of oligopeptide with protected amino group with aldehyde or ketone. By reacting the compound (21) with the compound (22) having a hydroxyl group 2024204502   28 Jun 2024 under temperature conditions ranging from under cooling to room temperature in an inert solvent in the presence of an acid or a base, the compound (23) can be produced. Here, examples of the acid which may be used can include 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, paratoluene sulfonic acid, and methane sulfonic acid; and a Lewis acid such as tetrafluoroborate, zinc chloride, tin chloride, aluminum chloride, and iron chloride. Among them, sulfonic acids are preferable, and paratoluene sulfonic acid is particularly preferable. As for the base, any one of the aforementioned base can be suitably selected and used. Preferred examples thereof include an alkali metal alkoxide such as potassium tert-butoxide, an alkali metal or alkaline earth metal hydroxide such as sodium hydroxide and potassium hydroxide; alkali metal hydride such as sodium hydride and potassium hydride; organometallic base represented by dialkylamino lithium such as lithium diisopropylamide; and organometallic base of bissilylamine such as lithium bis(trimethylsilyl)amide. Examples of the solvent to 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. Those solvents can be prepared as a mixture with water. Further, the protecting group for an amino group as represented as P5 is not particularly limited if it is a group commonly used for protection of an amino group. Representative examples can include the protecting groups for an amino group that are described in Production method 2. However, the protecting group for an amino group as drepresented as P5 may be cleaved off within the course of the reaction. In such case, a protecting group can be re-introduced by appropriately performing a reaction with a suitable reagent for protecting an amino group as required. The compound (24) can be derived by removing the protecting group P6 of the compound (23). Herein, although the representative examples of the protecting group for a carboxy group as represented as P6 are described in Production method 2, it can be appropriately selected from these examples. In the compound (23), it is desirable that the protecting group P5 for an amino group and the protecting group P6 for a carboxy group are the protecting groups that can be removed by a different method or different conditions. For example, a representative example can include a combination in which P5 is a 9-fluorenylmethyloxy carbonyl group and P6 is a benzyl group. The protecting groups can be selected depending on, e.g., the properties of a compound having an amino group and a carboxy group to be protected. For removal of the protecting groups, reagents and conditions are selected depending on the protecting group. The compound (26) can be produced by derivatizing the compound (24) into active 2024204502   28 Jun 2024 ester, mixed acid anhydride, acid halide, or the like and reacting it with the compound (4) or a pharmacologically acceptable salt thereof in the presence of a base to produce the compound (25) followed by removing the protecting group P5 of the compound (25) obtained. For the reaction between the compound (4) and the carboxylic acid (24) and the reaction for removing the protecting group P6, the same reagents and reaction conditions as those described for Production method 2 can be used. The compound (10b) can be produced by reacting the compound (26) with an amino acid with protected terminal amino group or the oligopeptide (27) with protected amino group to produce the compound (9b) and removing the protective group P7 of the compound (9b) obtained. The protective group for an amino group as represented as P7 is not particularly limited if it is generally used for protection of an amino group. Representative examples thereof include the protecting groups for an amino group that are described in Production method 2. For removing the protective group, reagents and conditions are selected depending on the protecting group. For the reaction between the compound (26) and the compound (27), reaction reagents and conditions that are commonly used for peptide synthesis can be employed. The compound (10b) produced by the aforementioned method can be derivatized into the compound (1) of the present invention according to the method described above.

[0161] 5. Production method 5 The compound represented by the formula (2) as an intermediate can be also produced by the method shown below.

[0162] 2024204502   28 Jun 2024 [Chem.27] H2N-(CH2)n’-L^(CH2)n2-C(=O)-OP3 12 H2N-(CH2)n'-L<(CH2)p2-C(=O)-(NH-DX) 7 । pe_Lp2 qh (     28 P3-Lp2-NH-(CH2)n1-La-(CH2)n2-C(=O)-(NH-DX) 29 t H-Lp2-NH-(CH2)n1-L“-(CH2)n2-C(<>)-(NH^^ 30 |P9-Lp1(pi°)-OH 1      31 pS-LfP-NH-fCH^n’-L’-lCH^rf-C^OJ-OP3 35 P8-Lp2-NH-(CH2)n'-La-(CH2)n2-C(=O)-OH 36 4 H-Lp2-NH-(CH2)n’-La-(CH2)n2-C(=O)-OP: ■    37 31 ( P^LP^P^j-L^-NH-tC^Jn'-L’-tCHjJn’-C^OJ-OP3 38 Ps-Lpl(Pw)-Lp2.NH-(CH2)n,-La-(CH2)n2-C(=O)-OH P3-Lp\P':,:!)-Lp2-NH-(CH2)n1-La-(CH2)n2-C(=O)-(NH-DX) 32 H-Lp1(PK)-Lp2-NH-(CH2)nUE-(CH2)n2<(=O)-(NH-DX) 33 L1-L2-OH 1 / -12.^ (F' 0)-L-t2-N H-(C H2)n'-La-(C H2)n2-C(=O)-(NH-DX) 34 L1-L2-Lp'(P,0)-Lp2-OH 48 39 4 I 30 '(P^-LPXOP'2 47 46 H-Lp1(P’°)-Lp2-OP12 50 H-L^P^-L^-NH-CCH^n’-LMCH^^-C^CO-OP3 40 r-L^LM^-LP^NH-pH^nXL’-pHJr^ 41 — P^L^P^-L^-OP' 49 | 24 H-Lp2-OP12 46 J 42 L1'-L2-Lp1(P1,3)-OH 45 L1-L2-Lp1(P10)-OP 44 I 4 4 H-Lp1(P10)-OP’ 43 L1-L2-Lp-NH-(CH2)nl-L’-(CH2)n2-C(=O)-(NH-DX) 2

[0163] [in the formula, Lr corresponds to L1 having a structure in which the terminal is converted to a maleimidyl group, Lp represents a structure consisting of -Lp'-Lp2-, and ps ps p9 pio, pii, anj pn represent a protecting group].

[0164] Because Lp is formed by connecting Lp1 to Lp2, the hydrophilic amino acid at N terminal of Lp is derived from Lpl, and thus, those that having a hydrophilic amino acid at the N terminal are suitably employed as Lpl. Meanwhile, plural hydrophilic amino acids may be present therein. Further, when Lp2 with hydrophilic amino acid is employed, Lp having plural hydrophilic amino acids at the N terminal of Lp or at the N terminal and at other positions can be produced depending on the location of the hydrophilic amino acid.

[0165] The compound (29) can be produced by derivatizing the peptide or amino acid (28) having the N terminal protected with P2 into active ester, mixed acid anhydride, or the like and reacting it with the compound (7) obtained. The reaction conditions, reagents, base, and solvent used for forming an amide bond between the peptide or amino acid (28) and the compound (7) can be suitably selected from those described for the synthesis of the compound (6). The protecting group P8 for an amino group can be 2024204502   28 Jun 2024 suitably selected from those described for the protecting group of the compound (6), and the selection can be made based on the properties of the compound or the like. As it is generally used for peptide synthesis, by repeating sequentially the reaction and deprotection of the amino acid or peptide constituting the peptide or amino acid (28) for elongation, the compound (29) can be also produced. By deprotection of P8 as a protecting group of the amino group of the compound (29) obtained, the compound (23) can be produced. In this deprotection, reagents and conditions can be selected depending on the protecting group. The compound (32) can be produced by derivatizing the amino acid or peptide (31) having the N terminal protected with P8 and the protected carboxy group, hydroxy group, or amino group in side chain protected into active ester, mixed acid anhydride, or the like and reacting it with the compound (30) obtained. The reaction conditions, reagents, base, and inert solvent used for forming a peptide bond between the amino acid or peptide (31) and the compound (30) can be suitably selected from those described for the synthesis of the compound (6). As for the protecting groups P8 and P9, the protecting groups can be suitably selected from those described as protecting group for an amino group, carboxy group, or hydroxy group of the compound (6). However, in such case, it is necessary that the protecting group P9 for an amino group and the protecting group P10for a functional group in side chain can be removed by a different method or different conditions. For example, a representative example includes a combination in case P9 is a 9-fluorenylmethyloxy carbonyl group and P10 is a tert-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 tert-butyloxycarbonyl group or the like as a protecting group for an amino group. The protective group P10 for a functional group in a side chain is preferably a protecting group which can be deprotected by a treatment under acidic conditions. However, it is not limited thereto, and it can be selected from the aforementioned ones depending on, e.g., the properties of amino group, carboxy group, or a hydroxy group of a compound to be protected. For removal of the protecting groups, reagents and conditions are selected depending on the protecting group. As it is generally used for peptide synthesis, by repeating sequentially the reaction and deprotection of the constituting amino acid or peptide for elongation, the compound (32) can be also produced. By deprotection of P9 as a protecting group of the terminal amino group of the compound (32) obtained, the compound (33) can be produced. In this deprotection, reagents and conditions can be selected depending on the protecting group. It is possible to produce the compound (34) by derivatizing the carboxylic acid derivative (11) into active ester, mixed acid anhydride, acid halide, or the like and reacting it with the compound (33) obtained. Herein, the carboxylic acid derivative 2024204502   28 Jun 2024 (11) is a compound with a structure in which the linker terminal of Lr has a maleimidyl group. The reaction conditions, reagents, base, and solvent used for forming a peptide bond between the carboxylic acid derivative (11) and the compound (33) can be suitably selected from those described for the synthesis of the compound (6). By deprotecting the protecting group P10for the carboxy group, hydroxy group, or amino group in the amino acid side chain of the peptide moiety of the compound (34) obtained, the compound (2) can be produced. Reagents and conditions can be selected depending on the protecting group.

[0166] The compound (29) can be also produced by the following method, for example. The compound (35) can be produced by derivatizing the peptide or amino acid (28) having the N terminal protected with P8 into active ester, mixed acid anhydride, or the like and reacting it with the amine compound (12) having the terminal carboxy group protected with P3 in the presence of a base. The reaction conditions, reagents, base, and solvent used for forming a peptide bond between the peptide or amino acid (28) and the compound (12) can be suitably selected from those described for the synthesis of the compound (6). The protecting group P8 for an amino group of the compound (35) can be suitably selected and used from those described as a protecting group for the compound (6). As for the protecting group P3 for a carboxy group, a protecting group commonly used as a protecting group for a carboxy group in organic synthetic chemistry, in particular, peptide synthesis can be used. Specific examples include alkyl ester such as methyl group, ethyl group, and tert-butyl, allyl ester, and benzyl ester, and it can be suitably selected and used from the protecting groups that are described for the compound (6). In such case, it is necessary that the protecting group P8 for an amino group and the protecting group P3 for a carboxy group can be removed by a different method or different conditions. For example, a representative example includes a combination in which P8 is a tert-butyloxy carbonyl group and P3 is a benzyl group. The protecting groups can be selected from the aforementioned ones depending on, e.g., the properties of a compound having an amino group and a carboxy group to be protected. For removal of the protecting groups, reagents and conditions are selected depending on the protecting group. By deprotecting the protecting group P3 for the carboxy group of the compound (35) obtained, the compound (36) can be produced. In this deprotection, reagents and conditions are selected depending on the protecting group. The compound (29) can be produced by derivatizing the compound (36) obtained into active ester, mixed acid anhydride, acid halide, or the like and reacting it with the compound (4) in the presence of a base. For the reaction, reaction reagents and conditions that are generally used for peptide synthesis can be also used, and the 2024204502   28 Jun 2024 reaction conditions, reagents, base, and solvent used for the reaction can be suitably selected from those described for the synthesis of the compound (6).

[0167] The compound (32) can be also produced by the following method, for example. By deprotecting the protecting group P8 for the amino group of the compound (35), the compound (37) can be produced. In this deprotection, reagents and conditions can be selected depending on the protecting group. The compound (38) can be produced by derivatizing the amino acid or peptide (31) into active ester, mixed acid anhydride, acid halide, or the like and reacting it with the compound (37) obtained in the presence of a base. The reaction conditions, reagents, base, and solvent used for forming an amide bond between the amino acid or peptide (31) and the compound (37) can be suitably selected from those described for the synthesis of the compound (6). In such case, it is necessary that the protecting group P9 and P10for the amino acid or peptide (31) and the protecting group P3for the compound (37) can be removed by a different method or different conditions. For example, a representative example includes a combination in which P9 is a 9-fluorenylmethyloxy carbonyl group, P10 is a tert-butyloxy carbonyl group, tert-butyl group, or a methoxymethyl group, and P3 is a benzyl group. Further, the protective group P10 for a functional group in a side chain is preferably a protecting group which can be deprotected by a treatment under acidic conditions as described above. However, it is not limited thereto, and it can be selected from the aforementioned ones depending on, e.g., the properties of amino group, carboxy group, or a hydroxy group of a compound to be protected. For removal of the protecting groups, reagents and conditions are selected depending on the protecting group. By deprotecting the protecting group P3for the carboxy group of the compound (38) obtained, the compound (39) can be produced. In this deprotection, reagents and conditions can be selected depending on the protecting group. The compound (32) can be produced by derivatizing the compound (39) into active ester, mixed acid anhydride, acid halide, or the like and reacting it with the compound (4) in the presence of a base. For the reaction, reaction reagents and conditions that are generally used for peptide synthesis can be also used, and the reaction conditions, reagents, base, and solvent used for the reaction can be suitably selected from those described for the synthesis of the compound (6).

[0168] The compound (34) can be also produced by the following method, for example. By deprotecting the protecting group P9 for the amino group of the compound (38), the compound (40) can be produced. In this deprotection, reagents and conditions can be selected depending on the protecting group. The compound (41) can be produced by derivatizing the carboxylic acid derivative (11) into active ester, mixed acid anhydride, acid halide, or the like and reacting it with 2024204502   28 Jun 2024 the compound (40) obtained in the presence of a base. The reaction conditions, reagents, base, and solvent used for forming an amide bond between the carboxylic acid derivative (11) and the compound (40) can be suitably selected from those described for the synthesis of the compound (6). By deprotecting the protecting group P3for the carboxy group of the compound (41) obtained, the compound (42) can be produced. In this deprotection, reagents and conditions can be selected depending on the protecting group. The compound (34) can be produced by derivatizing the compound (42) into active ester, mixed acid anhydride, acid halide, or the like and reacting it with the compound (4) in the presence of a base. For the reaction, reaction reagents and conditions that are generally used for peptide synthesis can be also used, and the reaction conditions, reagents, base, and solvent used for the reaction can be suitably selected from those described for the synthesis of the compound (6).

[0169] The compound (34) can be also produced by the following method, for example. The compound (44) can be produced by derivatizing the carboxylic acid derivative (11) into active ester, mixed acid anhydride, acid halide, or the like and reacting it with the amino acid or peptide (43) having the carboxy group protected with P11 and the carboxy group, hydroxy group, or amino group in side chain protected with P10in the presence of a base. The reaction conditions, reagents, base, and solvent used for forming an amide bond between the carboxylic acid derivative (11) and the compound (43) can be suitably selected from those described for the synthesis of the compound (6). As for the protecting groups P10 and P11 of the compound (44), the protecting groups can be suitably selected from those described as protecting group for a carboxy group, hydroxy group, or amino group of the compound (6). Meanwhile, in such case, it is necessary that the protecting group P11 for a carboxy group and the protecting group P10for a functional group in side chain can be removed by a different method or different conditions. For example, a representative example includes a combination in which P11 is a benzyl group and P10 is a tert-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 tert-butyloxycarbonyl group or the like as a protecting group for an amino group. The protective group P10 for a functional group in a side chain is preferably a protecting group which can be deprotected by a treatment under acidic conditions. However, it is not limited thereto, and it can be selected from the aforementioned ones depending on, e.g., the properties of amino group, carboxy group, or a hydroxy group of a compound to be protected. For removing the protecting group, the reagents and conditions can be selected depending on the protecting group. By deprotecting the protecting group P11 for the carboxy group of the compound (44) obtained, the compound (45) can be produced. In this deprotection, reagents and 2024204502   28 Jun 2024 conditions can be selected depending on the protecting group. The compound (34) can be produced by derivatizing the compound (45) into active ester, mixed acid anhydride, acid halide, or the like and reacting it with the compound (30) in the presence of a base. For the reaction, reaction reagents and conditions that are generally used for peptide synthesis can be also used, and the reaction conditions, reagents, base, and solvent used for the reaction can be suitably selected from those described for the synthesis of the compound (6). The compound (47) can be produced by derivatizing the compound (45) into active ester, mixed acid anhydride, acid halide or the like and reacting it with the amino acid or peptide (46) having the carboxy group protected with P12in the presence of a base. For the reaction, the reaction reagents and 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 the compound (6). As for the protecting groups P10 and P12 of the compound (47), the protecting groups can be suitably selected and used from those described as protecting group for a carboxy group, hydroxy group, or amino group of the compound (6). Meanwhile, in such case, it is necessary that the protecting group P12for a carboxy group and the protecting group P10for a functional group in side chain can be removed by a different method or different conditions. For example, a representative example includes a combination in which P12 is a benzyl group and P10 is a tert-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 tert-butyloxycarbonyl group or the like as a protecting group for an amino group. The protective group P10 for a functional group in a side chain is preferably a protecting group which can be deprotected by a treatment under acidic conditions. However, it is not limited thereto, and it can be selected from the aforementioned ones depending on, e.g., the properties of amino group, carboxy group, or a hydroxy group of a compound to be protected. For removing the protecting group, the reagents and conditions can be selected depending on the protecting group. Further, the compound (47) can be also produced by repeating sequentially the reaction and deprotection of constituting amino acid or peptide for elongation. By deprotecting the protecting group P12 for the carboxy group of the compound (47) obtained, the compound (48) can be produced. Reagents and conditions can be selected depending on the protecting group. The compound (34) can be produced by derivatizing the compound (48) into active ester, mixed acid anhydride, acid halide, or the like and reacting it with the compound (7) in the presence of a base. For the reaction, reaction reagents and conditions that are generally used for peptide synthesis can be also used, and the reaction conditions, reagents, base, and solvent used for the reaction can be suitably selected from those 2024204502   28 Jun 2024 described for the synthesis of the compound (6). The compound (47) can be also produced by the following method, for example. The peptide (49) can be produced by derivatizing the amino acid or peptide (46) into active ester, mixed acid anhydride, acid halide, or the like and reacting it with the amino acid or peptide (31) having the N terminal protected with P9 and the carboxy group, hydroxy group, or amino group in side chain protected with P10 in the presence of a base. The reaction conditions, reagents, base, and solvent used for forming 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 the compound (6). Meanwhile, in this case, it is necessary that the protecting group P12for a carboxy group of the amino acid or peptide (46) and the protecting group P9 and P10for the amino acid or peptide (31) can be removed in the same manner as described above but by a different method or different conditions. For example, a representative example includes a combination in which P9 is a 9-fluorenylmethyloxy carbonyl group, P10 is a tert-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 tert-butyloxycarbonyl group as a protecting group or the like for an amino group, and P12 is a benzyl group. The protective group P10 for a functional group in a side chain is preferably a protecting group which can be deprotected by a treatment under acidic conditions. However, it is not limited thereto, and it can be selected from the aforementioned ones depending on, e.g., the properties of amino group, carboxy group, or a hydroxy group of a compound to be protected. For removing the protecting group, the reagents and conditions can be selected depending on the protecting group. By deprotecting the protecting group P9for the N terminal of the peptide (49) obtained, the compound (50) can be produced. Reagents and conditions can be selected depending on the protecting group. The compound (47) can be produced by derivatizing the carboxylic acid derivative (11) into active ester, mixed acid anhydride, acid halide, or the like and reacting it with the peptide (50) obtained in the presence of a base. The reaction conditions, reagents, base, and solvent used for forming an amide bond between the carboxylic acid derivative (11) and the peptide (50) can be suitably selected from those described for the synthesis of the compound (6).

[0170] 6. Production method 6 Within the production intermediate (2), those in which the linker has a structure represented by -L'-L2-^-, and said Lp is the peptide residue containing a hydrophilic amino acid at the N terminal and said hydrophilic amino acid located at the N terminal is other than glycine, can be also produced by the following method.

[0171] 2024204502   28 Jun 2024 [Chem.28] nh2-dx 4 P”-L"'2-OH 28 u P8-LP2-(NH-DX) 51 H-LP2-(NH-DX) 52 p9_Lpi^piO)_oH 9      31 P9-LP1(P1°)-LP2-(NH-DX) 53 H_Lpi(piO)_Lp2-(NH-DX) 54 L1'-L2-OH 11 ♦ L1'-L2-Lp1(P10)-Lp2-(NH-DX) 55 o L1'-L2.|_P-(NH-DX) 2 p9_Lp1(p10)_Lp2_Opi2 49 p9_|_p1^p10)_|_p2_OH 56 4           Lr-L2-LP1(P10)-LP2-OH ।--------------- 48 1                   L1’-L2-LP1(P10)-OH H-Lp2-(NH-DX)

[0172] [in the formula, Lr corresponds to L1 having a structure in which the terminal is modified to maleimidyl group, Lp represents a structure consisting of -Lp'-Lp2-, and P8, P9, P10, and P12 represent a protecting group].

[0173] Because Lp is formed by connecting Lpl to Lp2, the hydrophilic amino acid at N terminal of Lp is derived from Lpl, and thus, those that having a hydrophilic amino acid at the N terminal are suitably employed as Lpl. Meanwhile, plural hydrophilic amino acids may present therein. Further, when Lp2 with hydrophilic amino acid is employed, Lp having plural hydrophilic amino acids at the N terminal of Lp or at the N terminal and at other positions can be produced depending on its location of hydrophilic amino acid. The compound (51) can be produced by derivatizing the peptide or amino acid (28) 2024204502   28 Jun 2024 described in Production method 5, which has the N terminal protected with P8, into active ester, mixed acid anhydride, or the like, and reacting with the compound (4) and a salt thereof. The reaction conditions, reagents, base, and solvent used for forming a peptide bond between the peptide or amino acid (28) and the compound (4) can be suitably selected from those described for the synthesis of the compound (6). The protective group P8 can be suitably selected and used from those described as the protecting group for the compound (6), and it can be selected depending on, e.g., a property of the compound having an amino group to be protected. Further, as it is generally used for peptide synthesis, by repeating sequentially the reaction and deprotection of the amino acid or peptide constituting the peptide or amino acid (28) for elongation, the compound (51) can be also produced. By deprotecting the protecting group P8for the amino group of the compound (51) obtained, the compound (52) can be produced. In this deprotection, reagents and conditions can be selected depending on the protecting group. The compound (53) can be produced by derivatizing the amino acid or peptide (31) having the N terminal protected with P9 and the carboxy group, hydroxy group, or amino group in side chain protected with P10 as described in Production method 4 into active ester, mixed acid anhydride, or the like and reacting it with the compound (52) obtained. The reaction conditions, reagents, base, and solvent used for forming a peptide bond between the amino acid or peptide (31) and the compound (52) can be suitably selected from those described for the synthesis of the compound (6). The protecting group P9 and P10are the same as those described in Production method 5. Further, as it is generally used for peptide synthesis, by repeating sequentially the reaction and deprotection of the constituting amino acid or peptide for elongation, the compound (53) can be also produced. By deprotection of P9 as the protecting group of the amino group of the compound (53) obtained, the compound (54) can be produced. In this deprotection, reagents and conditions can be selected depending on the protecting group. It is possible to produce the compound (55) by derivatizing the carboxylic acid derivative (11) into active ester, mixed acid anhydride, acid halide, or the like and reacting it with the compound (54) obtained. The reaction conditions, reagents, base, and solvent used for forming a peptide bond between the carboxylic acid derivative (11) and the compound (54) can be suitably selected from those described for the synthesis of the compound (6). By deprotecting the protecting group P10for the carboxy group, hydroxy group, or amino group of the compound (55) obtained, the compound (2) can be produced. In this deprotection, reagents and conditions can be selected depending on the protecting group. 2024204502   28 Jun 2024

[0174] The compound (53) can be also produced by the following method, for example. By deprotecting the protecting group P12for the carboxy group of the compound (49) described in Production method 5, the peptide (56) can be produced. In this deprotection, reagents and conditions can be selected depending on the protecting group. The compound (53) can be produced by derivatizing the peptide (56) obtained into active ester, mixed acid anhydride, acid halide, or the like and reacting it with the compound (4) or a salt thereof. The reaction conditions, reagents, base, and solvent used for forming a peptide bond between the compound (56) and the compound (4) can be suitably selected from those described for the synthesis of the compound (6).

[0175] The compound (55) can be also produced by the following method, for example. The compound (55) can be produced by derivatizing the compound (48) described in Production method 5 into active ester, mixed acid anhydride, or the like, and reacting it with the compound (4) in the presence of a base, or derivatizing the amino acid or peptide (45) described in Production method 5 into active ester, mixed acid anhydride, or the like, and reacting it with the compound (52) in the presence of a base. The reaction conditions, reagents, base, and solvent used for forming each peptide bond can be suitably selected from those described for the synthesis of the compound (6).

[0176] 7. Production method 7 Within the production intermediate represented by the formula (2), those having the linker structure of -L1-L2-Lp-NH-(CH2)n1-La-NH-(CH2)n2-C(=O)-, and said Lp is the peptide residue having a hydrophilic amino acid at the N terminal, and said hydrophilic amino acid located at N terminal is other than glycine can be also produced by the following method.

[0177] [Chem.29] P9-LP1(P13)-Lp2-NH-(CH2)n1-La-(CH2)n2-C(=O)-(NH-DX) 57 I H-Lp-NH-(CH2)n1-La-(CH2)n2-C(=O)-(NH-DX) 58 l1'-l2-oh 11 L1 -L2-Lp-NH-(CH2)n1-La-(CH2)n2-C(=O)-( NH-DX) 2

[0178] [in the formula, Lr corresponds to L1 having a structure in which the terminal is modified to maleimidyl group, Lp represents a structure consisting of -Lp'-Lp2-, and P9 and P13 represent a protecting group]. p9.LPl(p13).LP2.(NH.DX) 59 I H-LP-(NH-DX) 60 U'-L2-OH F 11 L1'-L2-Lp-(NH-DX) 2 2024204502   28 Jun 2024

[0179] The production intermediate represented by the formula (2) includes the following two modes, that is, a structure in which the linker is represented by -L1-L2-LP-NH-(CH2 )n'-La-NH-(CH2)n2-C(=O)- and a structure in which the linker is represented by -L'-L2 -Lp-. The compound (2) with a structure in which the linker is represented by -L'-L2-Lp -NH-(CH2)n'-La-NH-(CH2)n2-C(=O)- can be produced as follows. The compound (57) can be synthesized in the same manner as the compound (32) described in Production method 5. However, unlike the compound (32), it is not necessary that the protecting group P9 for the amino group and the protecting group P13 for the functional group in side chain can be removed by a different method or different conditions. The functional group in 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 hydroxy group in side chain can be simultaneously deprotected. For example, a representative example includes a combination in which P9 is a tert-butyloxy carbonyl group and P13 is a tert-butyl group or a trityl group, or P3 is a benzyloxy carbonyl group and P13 is a benzyl group. The protecting groups can be suitably selected from the aforementioned ones with regard to the protecting groups for the compound (6) depending on, e.g., the properties of an amino group, a carboxy group, or a hydroxy group of the compound to be protected. For removal of the protecting groups, reagents and conditions are selected depending on the protecting group. By using the protected amino acid or peptide satisfying above properties, the compound (57) can be synthesized in the same manner as Production method 5. By sequential or simultaneous deprotection of the protecting group P9 and P13 of the compound (57), the compound (51) can be produced. Reagents and conditions can be selected depending on the protecting group. A functional group in hydrophilic side chain of Lpin the compound (58) is not particularly protected, however, by reaction with the compound (11) derivatized into active ester, mixed acid anhydride, or the like in the presence of a base, the compound (2) can be produced. The reaction conditions, reagents, base, and solvent used for forming each peptide bond can be suitably selected from those described for the synthesis of the compound (6). The compound (2) with a structure in which the linker is represented by -L'-L2-Lp-can be produced as follows. The compound (59) can be also synthesized in the same manner as the compound (53) described in Production method 6. However, unlike the compound (53), it may not be necessary that the protecting group P3 for the amino group and the protecting group P8 for the functional group in side chain can be removed by a different method or different conditions. The functional group in side chain is a carboxy group or a 2024204502   28 Jun 2024 hydroxy group, and the protecting group P9 for the amino group and the protecting group P13 for the carboxy group or hydroxy group in side chain can be simultaneously deprotected. For example, a representative example includes a combination in which P9 is a tert-butyloxy carbonyl group and P13 is a tert-butyl group or a trityl group, or P3 is a benzyloxy carbonyl group and P13 is a benzyl group. The protecting groups can be suitably selected from the aforementioned ones with regard to the protecting groups for the compound (6) depending on, e.g., the properties of an amino group, a carboxy group, or a hydroxy group of the compound to be protected. For removal of the protecting groups, reagents and conditions are selected depending on the protecting group. By using the protected amino acid or peptide satisfying above properties, the compound (59) can be synthesized in the same manner as Production method 6. By sequential or simultaneous deprotection of the protecting group P9 and P13 of the compound (59), the compound (53) can be produced. Reagents and conditions can be selected depending on the protecting group. A functional...

Claims

1. A method for producing a pharmaceutical composition comprising an anti-HER3 antibody-drug conjugate, a salt thereof or a hydrate thereof as an active ingredient, and a pharmaceutically acceptable formulation ingredient, wherein the anti-HER3 antibody-drug conjugate has a drug-linker structure represented by the following formula:-(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-(NH-DX), andthe method comprises a step of treating an anti-HER3 antibody in a reducing condition and thereafterreacting the anti-HER3 antibody with a compound represented by the following formula: (maleimid-N-yl)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-(NH-DX), wherein-(Succinimid-3-yl-N)- has a structure represented by the following formula:0which is connected to the anti-HER3 antibody at position 3 thereof via thioether bond which is formed at a disulfide bond moiety present in a hinge part of the anti-HER3 antibody and is connected to a methylene group in the linker structure containing this structure on the nitrogen atom at position 1,-(NH-DX) represents a group represented by the following formula:2024204502   28 Jun 2024wherein the nitrogen atom of the amino group at position 1 is a connecting position, (maleimid-N-yl)- is a group represented by the following formula:wherein the nitrogen atom is a connecting position, and-GGFG- represents a tetrapeptide residue of -Gly-Gly-Phe-Gly-.

2. A method for producing a pharmaceutical composition comprising an anti-HER3 antibody-drug conjugate as an active ingredient, and a pharmaceutically acceptable formulation ingredient, wherein the anti-HER3 antibody-drug conjugate has a drug-linker structure represented by the following formula:-(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-(NH-DX), andthe method comprises a step of treating an anti-HER3 antibody in a reducing condition and thereafterreacting the anti-HER3 antibody with a compound represented by the following formula: (maleimid-N-yl)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-(NH-DX), wherein-(Succinimid-3-yl-N)- has a structure represented by the following formula:2024204502   28 Jun 2024which is connected to the anti-HER3 antibody at position 3 thereof via thioether bond which is formed at a disulfide bond moiety present in a hinge part of the anti-HER3 antibody and is connected to a methylene group in the linker structure containing this structure on the nitrogen atom at position 1,-(NH-DX) represents a group represented by the following formula:wherein the nitrogen atom of the amino group at position 1 is a connecting position, (maleimid-N-yl)- is a group represented by the following formula:wherein the nitrogen atom is a connecting position, and-GGFG- represents a tetrapeptide residue of -Gly-Gly-Phe-Gly-.

3. The method according to claim 1 or claim 2, wherein the anti-HER3 antibody comprises the CDRH1 to CDRH3 and CDRL1 to CDRL3 of U1-49, U1-53, U1-59, U1-7 orU1-9 in the heavy and light chains, respectively.2024204502   28 Jun 2024

4. The method according to claim 1 or claim 2, wherein the anti-HER3 antibody comprises the heavy chain variable region and the light chain variable region of U1-49, U1-53, U1-59, U1-7 or U1-9 on the heavy and light chains, respectively.

5. The method according to claim 1 or claim 2, wherein the anti-HER3 antibody comprises the amino acid sequences of SEQ ID No: 42 and 44, SEQ ID No: 54 and 56, SEQ ID No: 70 and 72, SEQ ID No: 92 and 94, or, SEQ ID No: 96 and 98, in the heavy and light chains, respectively.

6. The method according to claim 1 or claim 2, wherein the anti-HER3 antibody comprises the amino acid sequences of SEQ ID No: 583 and 584 in the heavy and light chains, respectively.

7. The method according to claim 6, wherein the anti-HER3 antibody lacks a lysine residue at the carboxyl terminus of the heavy chain.

8. The method according to any one of claims 1 to 7, wherein the average number of units of the drug-linker structure conjugated per antibody is in a range of from 2 to 8.

9. The method according to any one of claims 1 to 7, wherein the average number of units of the drug-linker structure conjugated per antibody is in a range of from 3 to 8.

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