Antibody-pyrrolobenzodiazepine derivative conjugates

Abstract

The present invention provides a novel anti-DLL3 antibody-pyrrolodiazepine derivative and a novel anti-DLL3 antibody-pyrrolodiazepine derivative conjugate using the same.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 63 / 136,928, filed January 13, 2021, the disclosure of which is incorporated herein by reference in its entirety. Sequence Listing This application contains a Sequence Listing that has been submitted electronically in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy was created on January 12, 2022, is named 098065-0303_SL.txt, and is 153,716 bytes in size. Technical Field The present invention relates to an antibody-drug conjugate useful as an antitumor drug, which comprises an antibody capable of targeting tumor cells and a pyrrolobenzodiazepine derivative conjugated to each other via a linker structure moiety. [Background technology]

[0002] The following description of the background of the present technology is provided merely to aid in understanding the technology of the present invention and is not admitted to describe or constitute prior art to the technology of the present invention. Antibody-drug conjugates (ADCs) have a cytotoxic drug conjugated to an antibody that binds to an antigen expressed on the surface of cancer cells, and can internalize the antigen through binding. ADCs can effectively deliver drugs to cancer cells, thereby accumulating the drug within the cancer cells and potentially killing them. For example, the ADC Adcetris™ (brentuximab vedotin) has monomethyl auristatin E conjugated to an anti-CD30 monoclonal antibody and is approved for the treatment of Hodgkin's lymphoma and anaplastic large cell lymphoma. Kadcyla™ (trastuzumab emtansine) has emtansine conjugated to an anti-HER2 monoclonal antibody and is used to treat HER2-positive advanced and recurrent breast cancer.

[0003] A useful example of a drug conjugated for ADC is pyrrolobenzodiazepine (PBD). PBDs exhibit cytotoxicity, for example, by binding to the PuGPu sequence in the DNA minor groove. In 1965, the first naturally occurring PBD, anthramycin, was discovered, leading to the discovery of various naturally occurring PBDs and their analogues (see Mantaj, et al., Angewandte Chemie, International Edition 2016, 55, 2-29; Antonow, et al., Chemical Reviews, 2010, 111, 2815-2864; In Antibiotics III. Springer Verlag, New York, pp. 3-11; and Accounts of Chemical Research, 1986, 19, 230). The general structure of a PBD is represented by the following formula:

[0004] [ka] PBDs that differ in the number, type, and location of substituents in the A and C ring portions, as well as PBDs that differ in the degree of unsaturation in the B and C ring portions, are known.

[0005] PBDs are known to dramatically enhance cytotoxicity through the formation of dimeric structures (Journal of the American Chemical Society, 1992, 114, 4939; Journal of Organic Chemistry, 1996, 61, 8141), various ADCs with dimeric PBDs have been reported (see WO2013 / 173496, WO2014 / 130879, WO2017 / 004330, WO2017 / 004025, WO2017 / 020972, WO2016 / 036804, WO2015 / 095124, WO2015 / 052322, WO2015 / 052534, WO2016 / 115191, WO2015 / 052321, WO2015 / 031693, WO2011 / 130613 and WO2019 / 065964). Dimeric PBDs are used in some known ADCs. For example, an ADC in which a dimeric PBD is conjugated to an antibody targeting DLL3 has been reported (see WO2013 / 126746 and Saunders et al., Sci Translational Medicine 7(302): 302ra136 (2015)).

[0006] DLL3 (i.e., delta-like ligand 3 or delta-like protein 3) is a single-pass type I transmembrane protein and a Notch ligand (see Owen et al. J Hematol Oncol 12, 61 (2019)). DLL3 is selectively expressed in high-grade pulmonary neuroendocrine tumors, including SCLC and LCNEC. Increased DLL3 expression has been observed in xenograft tumors from SCLC and LCNEC patients and confirmed in primary tumors. See Saunders et al., Sci Translational Medicine 7(302):302ra136 (2015). Increased DLL3 expression has also been observed in extrapulmonary neuroendocrine carcinomas, including prostate neuroendocrine carcinoma (Puca et al., Sci Transl Med 11(484): pii: eaav0891 (2019)). DLL3 is expressed on the surface of these tumor cells, but its expression in normal adult tissues is limited. Various pharmaceutical compositions containing anti-DLL3 antibodies as active ingredients are known (see Giffin et al., Clin Cancer Res 2021;27:1526-37, WO2011 / 093097, and WO2013 / 126746). However, to date, no drugs targeting DLL3 have been approved for use as pharmaceutical agents. There is a need in the art for efficient and effective targeted therapeutics, such as ADCs, to treat various types of cancer. The present application fulfills this need, including, by way of example, ADCs that utilize PBDs. Summary of the Invention

[0007] Problems to be Solved by the Invention: The present invention provides novel anti-DLL3 antibody-pyrrolobenzodiazepine (PBD) derivative conjugates. In addition, the present invention provides pharmaceutical compositions containing the anti-DLL3 antibody-PBD derivative conjugates. Furthermore, the present invention provides methods for treating cancer by using the anti-DLL3 antibody-PBD derivative conjugates. Means for Solving the Problem: The present inventors have conducted thorough investigations and unexpectedly found that a novel anti-DLL-3 antibody-pyrrolobenzodiazepine (PBD) derivative conjugate has strong antitumor activity, thereby completing the present invention. The present invention includes the following aspects and embodiments: [1] In one aspect, the present disclosure provides an antibody-drug conjugate represented by the following formula:

[0008] [ka] During the ceremony, m 1 represents an integer between 1 and 2; D represents a drug represented by one of the following formulas:

[0009] [ka] where the asterisk represents the bond to L; L is a linker connecting the N297 glycan of Ab to D; The N297 glycan may optionally be remodeled; Ab represents an anti-DLL3 antibody that specifically binds to DLL-3.

[0010] [2] In some embodiments according to [1], the anti-DLL3 antibody comprises a heavy chain immunoglobulin variable domain (VH) and a light chain immunoglobulin variable domain (VL), (a) VH is (i) SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:5, respectively; (ii) SEQ ID NO:13, SEQ ID NO:14, and SEQ ID NO:15, respectively; (iii) SEQ ID NO:23, SEQ ID NO:24, and SEQ ID NO:25, respectively; and (iv) SEQ ID NO: 33, SEQ ID NO: 34, and SEQ ID NO: 35, respectively and / or comprising a VH-CDR1 sequence, a VH-CDR2 sequence, and a VH-CDR3 sequence selected from the group consisting of: (b)VL teeth, (i) SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:10, respectively; (ii) SEQ ID NO:18, SEQ ID NO:19, and SEQ ID NO:20, respectively; (iii) SEQ ID NO:28, SEQ ID NO:29, and SEQ ID NO:30, respectively; and (iv) SEQ ID NO: 38, SEQ ID NO: 39, and SEQ ID NO: 40, respectively V selected from the group consisting of L -CDR1 sequence, V L -CDR2 sequence, and V L -Contains CDR3 sequences.

[0011] [3] In some embodiments according to [1] or [2], the anti-DLL3 antibody comprises a heavy chain immunoglobulin variable domain (V H ) and light chain immunoglobulin variable domain (V L ), including (a) V H -CDR1 sequence, V H -CDR2 sequence, and V H - V containing the CDR3 sequence H and (b) V L -CDR1 sequence, V L -CDR2 sequence, and V L - V containing the CDR3 sequence L The combination of (i) (a) SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:5 and (b) SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:10, respectively; (ii) (a) SEQ ID NO:13, SEQ ID NO:14, and SEQ ID NO:15 and (b) SEQ ID NO:18, SEQ ID NO:19, and SEQ ID NO:20, respectively; (iii) (a) SEQ ID NO:23, SEQ ID NO:24, and SEQ ID NO:25 and (b) SEQ ID NO:28, SEQ ID NO:29, and SEQ ID NO:30, respectively; and (iv) (a) SEQ ID NO: 33, SEQ ID NO: 34, and SEQ ID NO: 35 and (b) SEQ ID NO: 38, SEQ ID NO: 39, and SEQ ID NO: 40, respectively. is selected from the group consisting of:

[0012] [4] In some embodiments according to [1] or [2], the anti-DLL3 antibody comprises one or more of the following features: (a) a light chain immunoglobulin variable domain sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to a light chain immunoglobulin variable domain sequence present in any one of SEQ ID NOs: 7, 17, 27, 37, 62, 66, or 70; and / or (b) a heavy chain immunoglobulin variable domain sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to a heavy chain immunoglobulin variable domain sequence present in any one of SEQ ID NOs: 2, 12, 22, 32, 59, 60, 61, 63, 64, 65, 67, 68, or 69.

[0013] [5] In some embodiments according to any one of [1] to [4], the anti-DLL3 antibody comprises one or more of the following characteristics: (a) a light chain immunoglobulin variable domain sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to a light chain immunoglobulin variable domain sequence present in any one of SEQ ID NOs: 17, 27, or 37; and (b) a heavy chain immunoglobulin variable domain sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to a heavy chain immunoglobulin variable domain sequence present in any one of SEQ ID NOs: 12, 22, or 32. [6] In some embodiments according to any one of [1] to [5], the anti-DLL3 antibody comprises a heavy chain immunoglobulin variable domain (V H ) and light chain immunoglobulin variable domain (V L ), including (a) V H comprises an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:12, SEQ ID NO:22, and SEQ ID NO:32; and / or (b) V L comprises an amino acid sequence selected from the group consisting of SEQ ID NO:7, SEQ ID NO:17, SEQ ID NO:27, and SEQ ID NO:37.

[0014] [7] In some embodiments according to any one of [1] to [6], the anti-DLL3 antibody comprises a heavy chain immunoglobulin variable domain (V H ) and light chain immunoglobulin variable domain (V L ), including (a) V H (b) V comprises an amino acid sequence selected from SEQ ID NO: 12, SEQ ID NO: 22, or SEQ ID NO: 32; L comprises an amino acid sequence selected from SEQ ID NO: 17, SEQ ID NO: 27 or SEQ ID NO: 37. In some embodiments according to [8][6], V H Amino acid sequence and V L The amino acid sequences are selected from the group consisting of SEQ ID NO:2 and SEQ ID NO:7 (7-I1-B), respectively; SEQ ID NO:12 and SEQ ID NO:17 (2-C8-A), respectively; SEQ ID NO:22 and SEQ ID NO:27 (10-O18-A), respectively; and SEQ ID NO:32 and SEQ ID NO:37 (6-G23-F), respectively. In some embodiments according to any one of [9][1] to [8], the antibody-drug conjugate undergoes intracellular internalization upon binding of the anti-DLL3 antibody to a DLL3 polypeptide expressed on a cell surface (e.g., a tumor cell surface).

[0015]

[10] In some embodiments according to any one of [1] to [9], the anti-DLL3 comprises an Fc domain of an isotype selected from the group consisting of IgG1 or a variant thereof, IgG2, IgG3, IgG4, IgA1, IgA2, IgM, IgD, and IgE.

[11] In some embodiments according to any one of [1]-

[10] , the anti-DLL3 comprises a heavy chain constant region of SEQ ID NO: 42, 57, or 58.

[12] In some embodiments according to any one of [1] to

[11] , the anti-DLL3 antibody is a monoclonal antibody, a chimeric antibody, a humanized antibody, a human antibody, or a bispecific antibody.

[0016]

[13] In some embodiments according to any one of [1] to

[12] , the antibody (a) a heavy chain comprising the amino acid sequence of any one of SEQ ID NOs: 59 to 61 and a light chain comprising the amino acid sequence of any one of SEQ ID NO: 62; (b) a heavy chain comprising the amino acid sequence of any one of SEQ ID NOs: 63 to 65 and a light chain comprising the amino acid sequence of any one of SEQ ID NOs: 66; and / or (c) a heavy chain comprising any one of the amino acid sequences of SEQ ID NOs: 67 to 69 and a light chain comprising any one of the amino acid sequences of SEQ ID NO: 70 Includes.

[14] In some embodiments according to any one of [1] to

[13] , the antibody (a) a heavy chain comprising the amino acid sequence of SEQ ID NO: 61 and a light chain comprising the amino acid sequence of SEQ ID NO: 62; (b) a heavy chain comprising the amino acid sequence of SEQ ID NO: 65 and a light chain comprising the amino acid sequence of SEQ ID NO: 66; or (c) a heavy chain comprising the amino acid sequence of SEQ ID NO: 69 and a light chain comprising the amino acid sequence of SEQ ID NO: 70 Includes.

[0017]

[15] In some embodiments of claim 1, the anti-DLL-3 antibody competes with the antibody of claim 9 or claim V for binding to DLL-3.

[16] In some embodiments according to any one of [1] to

[15] , the anti-DLL3 antibody binds to an epitope on DLL3 that is a conformational epitope or a non-conformational epitope.

[17] In some embodiments according to any one of [1] to

[16] , the anti-DLL3 antibody binds to a mammalian DLL3 polypeptide having an amino acid sequence comprising amino acid residues 27 to 492 of SEQ ID NO:50 or SEQ ID NO:51.

[18] In some embodiments according to any one of [1] to

[17] , the heavy or light chain of the antibody is glycosylated with or without N-linked glycosylation, O-linked glycosylation, N-terminal processing, C-terminal processing, deamidation, isomerization of aspartic acid, oxidation of methionine, addition of a methionine residue to the N-terminus, amidation of proline residues, substitution of two leucine (L) residues at positions 234 and 235 of the heavy chain with alanine (A) (EU numbering) (LALA), substitution of alanine (A) residues at positions 235 and 236 of the heavy chain with alanine (A) (EU numbering) (LALA), or substitution of alanine (A) residues at positions 236 and 237 of the heavy chain with alanine (A) (EU numbering) (LALA). the set of amino acid residues Glu (E) at position 356 and Met (M) at position 358 (EU numbering) in the heavy chain, the set of Asp (D) at position 356 and leucine (L) at position 358 (EU numbering) in the heavy chain, or any combination thereof; conversion of N-terminal glutamine or N-terminal glutamic acid to pyroglutamic acid; and deletion of one or two amino acids from the carboxyl terminus.

[0018] In some embodiments according to

[19]

[18] , one or two amino acids are deleted from the carboxyl terminus of the heavy chain. In some embodiments according to

[20]

[19] , one amino acid is deleted from each of both carboxyl termini of the heavy chain. In some embodiments according to any one of

[21]

[18] -

[20] , the proline residue at the carboxyl terminus of the heavy chain is further amidated.

[22] In some embodiments according to any one of [1] to

[21] , the anti-DLL3 antibody comprises glycosylation modifications that are adjusted to enhance antibody-dependent cellular cytotoxicity.

[23] In some embodiments according to any one of [1] to

[22] , D is

[0019] [ka] is.

[24] In some embodiments according to any one of [1] to

[22] , D is

[0020] [ka] is.

[25] In some embodiments according to any one of [1] to

[22] , D is

[0021] [ka] is.

[26] In some embodiments according to any one of [1] to

[22] , D is

[0022] [ka] is. In some embodiments according to any one of

[27]

[23] -

[26] , -OH is at the 11' position.

[28] In some embodiments according to any one of [1] to

[27] , L is -Lb-La-Lp-NH-B-CH2-O(C=O)- * where the asterisk represents the bond to D; B represents a phenyl group or a heteroaryl group; Lp represents a linker consisting of an amino acid sequence cleavable in the target cell; La is the following group: -C(=O)-(CH2CH2)n 2 -C(=O)-, -C(=O)-(CH2CH2)n 2 -C(=O)-NH-(CH2CH2)n 3 -C(=O)-, -C(=O)-(CH2CH2)n 2 -C(=O)-NH-(CH2CH2O)n 3 -CH2-C(=O)-, -C(=O)-(CH2CH2)n 2 -NH-C(=O)-(CH2CH2O)n 3 -CH2CH2-C(=O)-, and -(CH2)n 4 -OC(=O)- represents any one selected from; n 2 represents an integer of 1 to 3, and n 3 represents an integer from 1 to 5, and n 4 represents an integer between 0 and 2; Lb represents a spacer that connects La to the glycan or remodeled glycan of Ab.

[0023] In some embodiments according to

[29]

[28] , B is any one selected from a 1,4-phenyl group, a 2,5-pyridyl group, a 3,6-pyridyl group, a 2,5-pyrimidyl group, and a 2,5-thienyl group. In some embodiments according to

[30]

[29] , B is a 1,4-phenyl group. In some embodiments according to any one of

[31]

[28] to

[30] , Lp is an amino acid residue consisting of 2 to 7 amino acids. In some embodiments according to any one of

[32]

[28] to

[31] , Lp is an amino acid residue consisting of an amino acid selected from glycine, valine, alanine, phenylalanine, glutamic acid, isoleucine, proline, citrulline, leucine, serine, lysine, and aspartic acid. In some embodiments according to any one of

[33]

[28] to

[32] , Lp is selected from the following group: -GGVA- (SEQ ID NO: 85), -GG-(D-)VA-, -VA-, -GGFG- (SEQ ID NO: 86), -GGPI- (SEQ ID NO: 87), -GGVCit- (SEQ ID NO: 88), -GGVK- (SEQ ID NO: 89), -GG(D-)PI-, and -GGPL- (SEQ ID NO: 90).

[0024] In some embodiments according to any one of

[34]

[28] to

[33] , La is selected from the group: -C(=O)-CH2CH2-C(=O)-, -C(=O)-(CH2CH2)2-C(=O)-, -C(=O)-CH2CH2-C(=O)-NH-(CH2CH2)2-C(=O)-, -C(=O)-CH2CH2-C(=O)-NH-(CH2CH2O)2-CH2-C(=O)-, -C(=O)-CH2CH2-NH-C(=O)-(CH2CH2O)4-CH2CH2-C(=O)-, -CH2-OC(=O)- and -OC(=O)- is selected from. In some embodiments according to any one of

[35]

[28] to

[34] , Lb is represented by the following formula:

[0025] [ka] wherein in each structural formula of Lb shown above, Each asterisk represents binding to La, and each wavy line represents binding of Ab to a glycan or remodeled glycan.

[0026] In some embodiments according to any one of

[36]

[28] to

[35] , L is -Lb-La-Lp-NH-B-CH2-O(C=O)- * wherein: B is a 1,4-phenyl group; Lp represents any one selected from the following group: -GGVA- (SEQ ID NO: 85), -GG-(D-)VA-, -VA-, -GGFG- (SEQ ID NO: 86), -GGPI- (SEQ ID NO: 87), -GGVCit- (SEQ ID NO: 88), -GGVK- (SEQ ID NO: 89), and -GGPL- (SEQ ID NO: 90); La is the following group: -C(=O)-CH2CH2-C(=O)-, -C(=O)-(CH2CH2)2-C(=O)-, -C(=O)-CH2CH2-C(=O)-NH-(CH2CH2)2-C(=O)-, -C(=O)-CH2CH2-C(=O)-NH-(CH2CH2O)2-CH2-C(=O)-, -C(=O)-CH2CH2-NH-C(=O)-(CH2CH2O)4-CH2CH2-C(=O)-, -CH2-OC(=O)- and -OC(=O)- represents any one selected from; Lb is represented by the following formula:

[0027] [ka] wherein in each structural formula of Lb shown above, Each asterisk represents binding to La, and each wavy line represents binding of Ab to a glycan or remodeled glycan.

[0028] In some embodiments according to any one of

[37]

[28] to

[36] , L is the following group: -Z 1 —C(═O)—CHCH—C(═O)—GGVA-NH-B-CH—OC(═O)— (“GGVA” disclosed as SEQ ID NO: 85); -Z 1 -C(=O)-CH2CH2-C(=O)-GG-(D-)VA-NH-B-CH2-OC(=O)-, -Z 1 -C(=O)-CH2CH2-C(=O)-VA-NH-B-CH2-OC(=O)-, -Z 1 -C(=O)-(CH2CH2)2-C(=O)-VA-NH-B-CH2-OC(=O)-, -Z 1 —C(═O)—CHCH—C(═O)—GGPI-NH-B-CH—OC(═O)— (“GGPI” disclosed as SEQ ID NO: 87); -Z 1 —C(═O)—CH2CH2—C(═O)-GGFG-NH-B-CH2-OC(═O)— (“GGFG” disclosed as SEQ ID NO: 86), -Z 1 —C(═O)—CHCH—C(═O)-GGVCit-NH-B-CH—OC(═O)— (“GGVCit” disclosed as SEQ ID NO: 88); -Z 1—C(═O)—CHCH—C(═O)-GGVK-NH-B-CH—OC(═O)— (“GGVK” disclosed as SEQ ID NO: 89); -Z 1 —C(═O)—CHCH—C(═O)—GGPL-NH-B-CH—OC(═O)— (“GGPL” disclosed as SEQ ID NO: 90); -Z 1 -C(=O)-CH2CH2-C(=O)-NH-(CH2CH2)2-C(=O)-VA-NH-B-CH2-OC(=O)-, -Z 1 -C(=O)-CH2CH2-C(=O)-NH-(CH2CH2O)2-CH2-C(=O)-VA-NH-B-CH2-OC(=O)-, -Z 1 -C(=O)-CH2CH2-NH-C(=O)-(CH2CH2O)4-CH2CH2-C(=O)-VA-NH-B-CH2-OC(=O)-, -Z 2 -OC(=O)-GGVA-NH-B-CH-OC(=O)- ("GGVA" disclosed as SEQ ID NO: 85), and -Z 3 —CH—OC(═O)-GGVA-NH-B-CH—OC(═O)— (“GGVA” disclosed as SEQ ID NO: 85) is selected from: Z 1 has the following structural formula:

[0029] [ka] represents Z 2 has the following structural formula:

[0030] [ka] represents Z 3 has the following structural formula:

[0031] [ka] wherein Z 1 , Z 2 , and Z 3 In each structural formula, Each asterisk represents binding to La, and each wavy line represents binding of Ab to a glycan or remodeled glycan; B represents a 1,4-phenyl group.

[0032]

[38]

[37] In some embodiments, L is the following group: -Z 1 —C(═O)—CHCH—C(═O)—GGVA-NH-B-CH—OC(═O)— (“GGVA” disclosed as SEQ ID NO: 85); -Z 1 -C(=O)-CH2CH2-C(=O)-VA-NH-B-CH2-OC(=O)-, -Z 1 -C(=O)-(CH2CH2)2-C(=O)-VA-NH-B-CH2-OC(=O)-, -Z 1 -C(=O)-CH2CH2-C(=O)-GGVCit-NH-B-CH2-OC(=O)-, -Z 1 —C(═O)—CH2CH2—C(═O)—NH—(CH2CH2)2—C(═O)-VA-NH-B-CH2-OC(═O)— (“GGVCit” disclosed as SEQ ID NO: 88); -Z 1 -C(=O)-CH2CH2-C(=O)-NH-(CH2CH2O)2-CH2-C(=O)-VA-NH-B-CH2-OC(=O)-, and -Z 1 -C(=O)-CH2CH2-NH-C(=O)-(CH2CH2O)4-CH2CH2-C(=O)-VA-NH-B-CH2-OC(=O)- is selected from: B is a 1,4-phenyl group, and Z 1 has the following structural formula:

[0033] [ka] represents Z 1 In the structural formula: Each asterisk represents a Z 1 Each wavy line represents a bond to a C(=O) adjacent to the glycan, and each wavy line represents a bond of an Ab to a glycan or a remodeled glycan.

[0034]

[39] In some embodiments according to any one of [1] to

[38] , L is -Lb-La-Lp-NH-B-CH2-O(C=O)- * wherein: The asterisk represents the bond to D; B represents a 1,4-phenyl group; Lp represents -GGVA- (SEQ ID NO: 85) or -VA; La is -(CH2)n 9 -C(=O)- or -(CH2CH2)n 10 -C(=O)-NH-(CH2CH2O)n 11 represents -CH2CH2-C(=O)-, n 9 represents an integer from 2 to 7, and n 10 represents an integer of 1 to 3, and n 11 represents an integer between 6 and 10; Lb is -(succinimide-3-yl-N)-.

[0035] In some embodiments according to

[40]

[39] , L is the following group: -(succinimide-3-yl-N)-(CH2)5-C(=O)-VA-NH-B-CH2-OC(=O)-, -(succinimide-3-yl-N)-(CH)-C(=O)-GGVA-NH-B-CH-OC(=O)-)- ("GGVA" disclosed as SEQ ID NO: 85), and -(Succinimide-3-yl-N)-CH2CH2-C(=O)-NH-(CH2CH2O)8-CH2CH2-C(=O)-VA-NH-B-CH2-OC(=O)- wherein B is a 1,4-phenyl group.

[41] In some embodiments according to any one of [1] to

[40] , the antibody is an IgG.

[0036] In some embodiments according to

[42]

[41] , the antibody is an IgG1 or variant thereof, an IgG2, or an IgG4. In some embodiments according to any one of

[43] [1]-

[42] , the antibody binds to, is taken up by, and is internalized by the tumor cell. In some embodiments according to

[44]

[43] , the antibody further has anti-tumor activity. In some embodiments according to any one of

[45] [1]-

[44] , the N297 glycan is a remodeled glycan. In some embodiments according to

[46]

[45] , the N297 glycan is N297-(Fuc)MSG1, N297-(Fuc)MSG2, or a mixture thereof, or N297-(Fuc)SG, wherein N297-(Fuc)MSG1, N297-(Fuc)MSG2, and N297-(Fuc)SG have the following formula:

[0037] [ka] (In the formula, The wavy line represents antibody binding to Asn297; L(PEG) is -(CH2CH2-O)n 5 represents -CH2CH2-NH-, where the amino group at the right end is linked to the carboxylic acid via an amide bond at the 2-position of the sialic acid at the non-reducing end of the 1-3 branched chain of β-Man in the N297 glycan; The asterisk represents the bond to the linker L; n 5 represents an integer between 2 and 10)

[0038] [ka] (In the formula, The wavy line represents antibody binding to Asn297; L(PEG) is -(CH2CH2-O)n 5 represents -CH2CH2-NH-, where the amino group at the right end is linked to the carboxylic acid via an amide bond at the 2-position of the sialic acid at the non-reducing end of the 1-6 branched chain of β-Man in the N297 glycan; The asterisk represents the bond to the linker L; n 5 represents an integer from 2 to 10), and

[0039] [ka] (In the formula, The wavy line represents antibody binding to Asn297; L(PEG) is -(CH2CH2-O)n 5 represents -CH2CH2-NH-, wherein the amino group at the right end is linked to a carboxylic acid via an amide bond at the 2-position of the sialic acid at the non-reducing end of each of the 1-3 and 1-6 branched chains of β-Man in the N297 glycan; The asterisk represents the bond to the linker L; n 5 represents an integer between 2 and 10) It has a structure represented by:

[0040] In some embodiments according to

[47]

[46] , n 5 is an integer between 2 and 5.

[48] ​​In some embodiments according to any one of [1] to

[47] , m 2 represents an integer of 1 or 2; L is the following group: -Z 1 —C(═O)—CHCH—C(═O)—GGVA-NH-B-CH—OC(═O)— (“GGVA” disclosed as SEQ ID NO: 85); -Z 1 -C(=O)-CH2CH2-C(=O)-VA-NH-B-CH2-OC(=O)-, -Z 1-C(=O)-(CH2CH2)2-C(=O)-VA-NH-B-CH2-OC(=O)-, -Z 1 —C(═O)—CHCH—C(═O)-GGVCit-NH-B-CH—OC(═O)— (“GGVCit” disclosed as SEQ ID NO: 88); -Z 1 -C(=O)-CH2CH2-C(=O)-NH-(CH2CH2)2-C(=O)-VA-NH-B-CH2-OC(=O)-, -Z 1 -C(=O)-CH2CH2-C(=O)-NH-(CH2CH2O)2-CH2-C(=O)-VA-NH-B-CH2-OC(=O)-, and -Z 1 -C(=O)-CH2CH2-NH-C(=O)-(CH2CH2O)4-CH2CH2-C(=O)-VA-NH-B-CH2-OC(=O)- is selected from: B is a 1,4-phenyl group, and Z 1 has the following structural formula:

[0041] [ka] represents Z 1 In the structural formula: Each asterisk represents a Z 1 represents a bond to the C(=O) adjacent to the , and each wavy line represents a bond to the N297 glycan of an Ab; Ab represents IgG antibody; The N297 glycan of Ab represents any one of N297-(Fuc)MSG1, N297-(Fuc)MSG2, and a mixture thereof, and N297-(Fuc)SG, where N297-(Fuc)MSG1, N297-(Fuc)MSG2, and N297-(Fuc)SG have the following formula:

[0042] [ka] (In the formula, Each wavy line represents antibody binding to Asn297; L(PEG) in N297 glycan is -NH-CH2CH2-(O-CH2CH2)n 5 - * wherein: n 5 represents an integer of 2 to 5, the amino group at the left end is bonded to a carboxylic acid via an amide bond at the 2-position of the sialic acid at the non-reducing end of each or either one of the 1-3 and 1-6 branched chains of β-Man in the N297 glycan, and each asterisk represents a Z in the linker L. 1 represents a bond to the nitrogen atom at the 1- or 3-position of the triazole ring It has a structure represented by:

[49] In another embodiment, the present disclosure provides an antibody-drug conjugate selected from the group consisting of:

[0043] [ka]

[0044] [ka]

[0045] In each of the structural formulae shown above, m 2 represents an integer of 1 or 2; Ab represents an anti-DLL3 IgG antibody or a functional fragment of said antibody; The N297 glycan of Ab represents any one of N297-(Fuc)MSG1, N297-(Fuc)MSG2, and a mixture thereof, and N297-(Fuc)SG, where N297-(Fuc)MSG1, N297-(Fuc)MSG2, and N297-(Fuc)SG have the following formula:

[0046] [ka]

[0047] (In the formula, Each wavy line represents antibody binding to Asn297; L(PEG) in N297 glycan is -NH-CH2CH2-(O-CH2CH2)3- * wherein: The amino group at the left end is linked to a carboxylic acid via an amide bond at the 2-position of the sialic acid at the non-reducing end of each or either one of the 1-3 and 1-6 branched chains of the β-Man in the N297 glycan, and each asterisk represents a bond to the nitrogen atom at the 1- or 3-position of the triazole ring in the corresponding structural formula. It has a structure represented by:

[0048] In some embodiments according to

[50]

[49] , the antibody comprises a heavy chain constant region of human IgG1 or a variant thereof, human IgG2, or human IgG4. In some embodiments according to

[51]

[49] , the antibody comprises a heavy chain constant region of SEQ ID NO: 42, 57 or 58.

[0049] In some embodiments according to any one of

[52]

[48] -

[51] , the anti-DLL3 antibody comprises a heavy chain immunoglobulin variable domain (V H ) and light chain immunoglobulin variable domain (V L ), (a)V H teeth, (i) SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:5, respectively; (ii) SEQ ID NO:13, SEQ ID NO:14, and SEQ ID NO:15, respectively; (iii) SEQ ID NO:23, SEQ ID NO:24, and SEQ ID NO:25, respectively; and (iv) SEQ ID NO: 33, SEQ ID NO: 34, and SEQ ID NO: 35, respectively V selected from the group consisting of H -CDR1 sequence, V H -CDR2 sequence, and V H - comprising a CDR3 sequence; and / or (b)V L teeth, (i) SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:10, respectively; (ii) SEQ ID NO:18, SEQ ID NO:19, and SEQ ID NO:20, respectively; (iii) SEQ ID NO:28, SEQ ID NO:29, and SEQ ID NO:30, respectively; and (iv) SEQ ID NO: 38, SEQ ID NO: 39, and SEQ ID NO: 40, respectively V selected from the group consisting of L -CDR1 sequence, V L -CDR2 sequence, and V L -Contains CDR3 sequences.

[0050] In some embodiments according to

[53]

[52] , the anti-DLL3 antibody comprises a heavy chain immunoglobulin variable domain (V H ) and light chain immunoglobulin variable domain (V L ), including (a) V H -CDR1 sequence, V H -CDR2 sequence, and V H - V containing the CDR3 sequence H and (b) V L -CDR1 sequence, V L -CDR2 sequence, and V L - V containing the CDR3 sequence L The combination of (i) (a) SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:5 and (b) SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:10, respectively; (ii) (a) SEQ ID NO:13, SEQ ID NO:14, and SEQ ID NO:15 and (b) SEQ ID NO:18, SEQ ID NO:19, and SEQ ID NO:20, respectively; (iii) (a) SEQ ID NO:23, SEQ ID NO:24, and SEQ ID NO:25 and (b) SEQ ID NO:28, SEQ ID NO:29, and SEQ ID NO:30, respectively; and (iv) (a) SEQ ID NO: 33, SEQ ID NO: 34, and SEQ ID NO: 35 and (b) SEQ ID NO: 38, SEQ ID NO: 39, and SEQ ID NO: 40, respectively. is selected from the group consisting of:

[0051] In some embodiments according to any one of

[54]

[49] to

[53] , the antibody is selected from the group consisting of the following combinations (1) to (4): (1) a light chain comprising a variable domain sequence of SEQ ID NO: 7 and a heavy chain comprising a variable domain sequence of SEQ ID NO: 2; (2) a light chain comprising a variable domain sequence of SEQ ID NO: 17 and a heavy chain comprising a variable domain sequence of SEQ ID NO: 12; (3) a light chain comprising a variable domain sequence of SEQ ID NO: 27 and a heavy chain comprising a variable domain sequence of SEQ ID NO: 22; and (4) a light chain comprising a variable domain sequence of SEQ ID NO: 37 and a heavy chain comprising a variable domain sequence of SEQ ID NO: 32 or a functional fragment of said antibody.

[0052] In some embodiments according to

[55]

[54] , the antibody comprises a light chain comprising the variable domain sequence of SEQ ID NO: 17 and a heavy chain comprising the variable domain sequence of SEQ ID NO: 12. In some embodiments according to

[56]

[54] , the antibody comprises a light chain comprising the variable domain sequence of SEQ ID NO:27 and a heavy chain comprising the variable domain sequence of SEQ ID NO:22. In some embodiments according to

[57]

[54] , the antibody comprises a light chain comprising the variable domain sequence of SEQ ID NO: 37 and a heavy chain comprising the variable domain sequence of SEQ ID NO: 32. In some embodiments according to

[58]

[54] , the antibody comprises a light chain comprising the variable domain sequence of SEQ ID NO:7 and a heavy chain comprising the variable domain sequence of SEQ ID NO:2.

[59] In some embodiments according to any one of

[49] to

[58] , the antibody (a) a heavy chain comprising the amino acid sequence of any one of SEQ ID NOs: 59 to 61 and a light chain comprising the amino acid sequence of SEQ ID NO: 62; (b) a heavy chain comprising the amino acid sequence of any one of SEQ ID NOs: 63 to 65 and a light chain comprising the amino acid sequence of SEQ ID NO: 66; or (c) a heavy chain comprising any one of the amino acid sequences of SEQ ID NOs: 67 to 69 and a light chain comprising the amino acid sequence of SEQ ID NO: 70 Includes.

[0053] In some embodiments according to any one of

[60] ,

[49] , and

[59] , the antibody (a) a heavy chain comprising the amino acid sequence of SEQ ID NO: 61 and a light chain comprising the amino acid sequence of SEQ ID NO: 62; (b) a heavy chain comprising the amino acid sequence of SEQ ID NO: 65 and a light chain comprising the amino acid sequence of SEQ ID NO: 66; or (c) a heavy chain comprising the amino acid sequence of SEQ ID NO: 69 and a light chain comprising the amino acid sequence of SEQ ID NO: 70 Includes.

[0054] In some embodiments according to any one of

[61] ,

[49] , or

[60] , the heavy chain or light chain may be modified by N-linked glycosylation, O-linked glycosylation, N-terminal processing, C-terminal processing, deamidation, isomerization of aspartic acid, oxidation of methionine, addition of a methionine residue to the N-terminus, amidation of a proline residue, substitution of two leucine (L) residues at positions 234 and 235 of the heavy chain with alanine (A) (according to the EU index) (LALA), or substitution of the amino acid residues at positions 235 and 236 of the heavy chain with alanine (A) (according to the EU index) (LALA). The antibody has one or two or more amino acid residue modifications or sets selected from the group consisting of Glu (E) at position 356 and Met (M) at position 358 (according to the EU index), Asp (D) at position 356 and leucine (L) at position 358 in the heavy chain (according to the EU index), or any combination thereof; conversion of an N-terminal glutamine or N-terminal glutamic acid to pyroglutamic acid; and deletion of one or two amino acids from the carboxyl terminus.

[0010] In another embodiment, the present disclosure provides an antibody-drug conjugate represented by the following formula:

[0055] [ka] During the ceremony, m 2 represents an integer of 1 or 2; Ab represents an anti-DLL3 IgG antibody comprising a heavy chain having the amino acid sequence of SEQ ID NO: 61 and a light chain having the amino acid sequence of SEQ ID NO: 62; The N297 glycan of Ab represents any one of N297-(Fuc)MSG1, N297-(Fuc)MSG2, and a mixture thereof, and N297-(Fuc)SG, where N297-(Fuc)MSG1, N297-(Fuc)MSG2, and N297-(Fuc)SG have the following formula:

[0056] [ka]

[0057] (In the formula, Each wavy line represents antibody binding to Asn297; L(PEG) in N297 glycan is -NH-CH2CH2-(O-CH2CH2)3- * wherein: The amino group at the left end is linked to a carboxylic acid via an amide bond at the 2-position of the sialic acid at the non-reducing end of each or either one of the 1-3 and 1-6 branched chains of the β-Man in the N297 glycan, and each asterisk represents a bond to the nitrogen atom at the 1- or 3-position of the triazole ring in the corresponding structural formula. It has a structure represented by:

[0010] In another embodiment, the present disclosure provides an antibody-drug conjugate represented by the following formula:

[0058] [ka] (In the formula, m 2 represents an integer of 1 or 2; Ab represents an anti-DLL3 IgG antibody comprising a heavy chain having the amino acid sequence of SEQ ID NO: 65 and a light chain having the amino acid sequence of SEQ ID NO: 66; The N297 glycan of Ab represents any one of N297-(Fuc)MSG1, N297-(Fuc)MSG2, and a mixture thereof, and N297-(Fuc)SG, where N297-(Fuc)MSG1, N297-(Fuc)MSG2, and N297-(Fuc)SG have the following formula:

[0059] [ka]

[0060] (In the formula, Each wavy line represents antibody binding to Asn297; L(PEG) in N297 glycan is -NH-CH2CH2-(O-CH2CH2)3- * wherein: The amino group at the left end is linked to a carboxylic acid via an amide bond at the 2-position of the sialic acid at the non-reducing end of each or either one of the 1-3 and 1-6 branched chains of the β-Man in the N297 glycan, and each asterisk represents a bond to the nitrogen atom at the 1- or 3-position of the triazole ring in the corresponding structural formula. It has a structure represented by:

[0013] In another embodiment, the present disclosure provides an antibody-drug conjugate represented by the following formula:

[0061] [ka] (In the formula, m 2 represents an integer of 1 or 2; Ab represents an anti-DLL3 IgG antibody comprising a heavy chain having the amino acid sequence of SEQ ID NO: 69 and a light chain having the amino acid sequence of SEQ ID NO: 70; The N297 glycan of Ab represents any one of N297-(Fuc)MSG1, N297-(Fuc)MSG2, and a mixture thereof, and N297-(Fuc)SG, where N297-(Fuc)MSG1, N297-(Fuc)MSG2, and N297-(Fuc)SG have the following formula:

[0062] [ka] (In the formula, Each wavy line represents antibody binding to Asn297; L(PEG) in N297 glycan is -NH-CH2CH2-(O-CH2CH2)3- * wherein: The amino group at the left end is linked to a carboxylic acid via an amide bond at the 2-position of the sialic acid at the non-reducing end of each or either one of the 1-3 and 1-6 branched chains of the β-Man in the N297 glycan, and each asterisk represents a bond to the nitrogen atom at the 1- or 3-position of the triazole ring in the corresponding structural formula. It has a structure represented by:

[0063]

[65] In another aspect, the present disclosure provides a pharmaceutical composition comprising the antibody-drug conjugate according to any one of [1] to

[64] , a salt thereof, or a hydrate of the conjugate or salt. In some embodiments according to

[66]

[65] , the pharmaceutical composition is an anti-tumor drug. In some embodiments according to

[67]

[65] or

[66] , the pharmaceutical composition is for use in treating a tumor, wherein the tumor is a DLL3-expressing tumor. In some embodiments according to

[68]

[66] or

[67] , the tumor is small cell lung cancer (SCLC); large cell neuroendocrine carcinoma (LCNEC); neuroendocrine tumors of various tissues, including kidney, genitourinary tract (bladder, prostate, ovary, cervix, and endometrium), gastrointestinal tract (stomach, colon), thyroid (medullary thyroid carcinoma), pancreas, and lung; glioma; or pseudoneuronocrine tumor (pNET).

[0064]

[69] In another aspect, the present disclosure provides a method for making a glycan-remodeled antibody, comprising: i) culturing host cells containing nucleic acid encoding an anti-DLL3 antibody and collecting the targeted antibody from the resulting culture; ii) treating the antibody obtained in step i) with a hydrolase to produce a (Fucα1,6)GlcNAc-antibody; and iii)-1 A step of reacting a glycan donor molecule with a (Fucα1,6)GlcNAc-antibody in the presence of transglycosidase, wherein the glycan donor molecule is obtained by introducing an azide group-containing PEG linker into the carbonyl group of the carboxylic acid at the 2-position of the sialic acid in MSG(9) or SG(10) and oxazolinating the reducing end; or iii)-2 A step of reacting a (Fucα1,6)GlcNAc-antibody with a glycan donor molecule in the presence of transglycosidase, in which the glycan donor molecule is obtained by introducing an azide group-containing PEG linker into the carbonyl group of the carboxylic acid at the 2-position of the sialic acid in (MSG-)Asn or (SG-)Asn, whose α-amino group may be protected, and the carbonyl group of the carboxylic acid in Asn, inducing the action of a hydrolase, and then oxazolinating the reducing end. The present invention provides a method comprising:

[0065] In some embodiments according to

[70]

[69] , the method further comprises purifying the (Fucα1,6)GlcNAc-antibody by purifying the reaction solution in step ii) on a hydroxyapatite column.

[71] In another aspect, the present disclosure provides a method for producing an antibody-drug conjugate form according to any one of [1] to

[64] , comprising: i) producing a glycan-remodeled antibody by using the method described in

[69] or

[70] ; and ii) reacting the drug linker with DBCO and the azide group in the glycan of the glycan-remodeled antibody produced in step i). The present invention provides a method comprising:

[72] In another aspect, the present disclosure provides a glycan-remodeled antibody obtained by using the method of

[69] or

[70] .

[0066]

[73] In another aspect, the present disclosure provides an antibody-drug conjugate obtained by using the method described in

[71] .

[74] In another aspect, the present disclosure provides a method for treating a tumor, the method comprising administering to an individual having a tumor the antibody-drug conjugate according to any one of [1] to

[64] , a salt of the antibody-drug conjugate, or a hydrate of the antibody-drug conjugate or salt.

[75] In another aspect, the present disclosure provides a method for treating a tumor, comprising simultaneously, separately, or sequentially administering to an individual a pharmaceutical composition comprising at least one component selected from the antibody-drug conjugate according to any one of [1] to

[64] , a salt thereof, and a hydrate of the conjugate or salt thereof, and at least one anti-tumor agent.

[0067] In some embodiments according to

[76]

[74] or

[75] , the tumor is a DLL3-expressing tumor. In some embodiments according to any one of

[77]

[74] -

[76] , the tumor is a small cell lung cancer (SCLC), a large cell neuroendocrine carcinoma (LCNEC), a neuroendocrine tumor of various tissues, including the kidney, genitourinary tract (bladder, prostate, ovary, cervix, and endometrium), gastrointestinal tract (stomach, colon), thyroid (medullary thyroid carcinoma), pancreas, and lung, a glioma, or a pseudoneuroendocrine tumor (pNET). Advantageous Effects of the Invention: The novel anti-DLL-3 antibody-pyrrolobenzodiazepine (PBD) derivative conjugate provided by the present invention has excellent antitumor activity and safety, and is therefore useful as an antitumor agent. [Brief explanation of the drawings]

[0068] [Figure 1]Schematic diagram of a drug-conjugate form (molecule (I)) of the present invention. (a) shows drug D, (b) shows linker L, (c) shows N3-L(PEG)-, and (d) shows N297 glycan (open oval: NeuAc(Sia), open hexagon: human, closed hexagon: GlcNAc, open diamond: Gal, open inverted triangle: Fuc). (b) and (c) are combined together to form a triazole ring by reaction of the azide group (closed teardrop shape) of (c) with the spacer (open semicircle) of (b). The Y-shaped figure represents an antibody Ab. For convenience, in this schematic diagram, the N297 glycan is shown as N297-(Fuc)MSG, and this diagram illustrates an embodiment in which one of the two branches of each N297 glycan has a sialic acid attached to an azide-containing PEG linker (N3-L(PEG)-) and the other branch does not have a sialic acid at its non-reducing end (i.e., N297-(Fuc)MSG), although an alternative embodiment in which each of the two branches of the N297 glycan has a sialic acid attached to an azide-containing PEG linker at its non-reducing end (i.e., N297-(Fuc)SG) is also acceptable. Unless otherwise specified, this diagrammatic scheme applies throughout this specification. [Figure 2] 2A-2C are schematic diagrams illustrating the structures of (Fucα1,6)GlcNAc-antibody (molecule A in (II) of FIG. 2), which is an intermediate in the production of the drug-conjugate form of the present invention, and an antibody with MSG-type glycan remodeling (molecule (III) in (B) of FIG. 2). In each diagram, the Y-shaped figure represents the antibody Ab, as in FIG. 1. In FIG. 2A, (e) shows the N297 glycan consisting solely of GlcNAc at position 6 connected to position 1 of Fuc via an α-glycosidic bond. In FIG. 2B, (d) shows the same N297 glycan as in FIG. 1, and (f) shows the structure of a PEG linker moiety bearing an azide group, specifically, an azide group for binding to the terminal linker L. The binding mode of the PEG linker bearing an azide group is as described with reference to FIG. 1. [Figure 3]This is a schematic diagram of the process for producing an antibody with remodeled MSG-type glycans from an antibody produced in animal cells. As in Figure 2, molecules (II) and (III) in this diagram represent a (Fucα1,6)GlcNAc-antibody and an antibody with remodeled MSG-type glycans, respectively. Molecule (IV) is an antibody produced in animal cells and is a mixture of molecules with heterogeneous N297 glycan moieties. Figure 3A illustrates the process for producing a homogeneous (Fucα1,6)GlcNAc-antibody (II) by treating the heterogeneous N297 glycan moiety of (IV) with a hydrolase such as EndoS. Figure 3B illustrates the process for producing an MSG-type glycan-remodeled antibody (III) by exposing the GlcNAc of the N297 glycan in antibody (II) to a transglycosidase such as the EndoS D233Q / Q303L variant to transglycosylate the glycan of an MSG-type glycan donor molecule. The MSG-type glycan donor molecule used here has a sialic acid at the non-reducing end of MSG modified with an azide-containing PEG linker. Therefore, the resulting MSG-type N297 glycan-remodeled antibody also has a sialic acid at the non-reducing end modified in the same manner as described in Figure 2B. For convenience, Figure 3B shows MSG as the donor molecule. However, a glycan-remodeled antibody in which an azide-containing linker molecule is attached to each non-reducing end of the N297 glycan can also be synthesized as a remodeled antibody (III) by using SG(10) as the glycan donor. [Figure 4A] FIG. 1 shows the nucleotide and amino acid sequences of human (Homo sapiens) Delta-like canonical Notch ligand 3 (DLL3) isoform 1, represented as SEQ ID NO: 55 and SEQ ID NO: 50, respectively. [Figure 4B] FIG. 1 shows the nucleotide and amino acid sequences of human Delta-like canonical Notch ligand 3 (DLL3) isoform 2, represented as SEQ ID NO: 56 and SEQ ID NO: 51, respectively. [Figure 5A]Figure 1 shows the nucleotide and amino acid sequences of the VH domain of antibody 7-I1-B, represented as SEQ ID NO: 1 and SEQ ID NO: 2, respectively. VH CDR1 (SEQ ID NO: 3) is shown in bold font, VH CDR2 (SEQ ID NO: 4) is underlined, and VH CDR3 (SEQ ID NO: 5) is shown in italic underlined font. [Figure 5B] 1 shows the nucleotide and amino acid sequences of the VL domain of antibody 7-I1-B, represented as SEQ ID NO: 6 and SEQ ID NO: 7, respectively. VL CDR1 (SEQ ID NO: 8) is shown in bold font, VL CDR2 (SEQ ID NO: 9) is underlined, and VL CDR3 (SEQ ID NO: 10) is shown in italic underlined font. [Figure 6A] Figure 1 shows the nucleotide and amino acid sequences of the VH domain of antibody 2-C8-A, represented as SEQ ID NO: 11 and SEQ ID NO: 12, respectively. VH CDR1 (SEQ ID NO: 13) is shown in bold font, VH CDR2 (SEQ ID NO: 14) is underlined, and VH CDR3 (SEQ ID NO: 15) is shown in italic underlined font. [Figure 6B] 1 shows the nucleotide and amino acid sequences of the VL domain of antibody 2-C8-A, represented as SEQ ID NO: 16 and SEQ ID NO: 17, respectively. VL CDR1 (SEQ ID NO: 18) is shown in bold font, VL CDR2 (SEQ ID NO: 19) is underlined, and VL CDR3 (SEQ ID NO: 20) is shown in italic underlined font. [Figure 7A] Figure 1 shows the nucleotide and amino acid sequences of the VH domain of antibody 10-O18-A, represented as SEQ ID NO: 21 and SEQ ID NO: 22, respectively. VH CDR1 (SEQ ID NO: 23) is shown in bold font, VH CDR2 (SEQ ID NO: 24) is underlined, and VH CDR3 (SEQ ID NO: 25) is shown in italic underlined font. [Figure 7B]1 shows the nucleotide and amino acid sequences of the VL domain of antibody 10-O18-A, represented as SEQ ID NO: 26 and SEQ ID NO: 27, respectively. VL CDR1 (SEQ ID NO: 28) is shown in bold font, VL CDR2 (SEQ ID NO: 29) is underlined, and VL CDR3 (SEQ ID NO: 30) is shown in italic underlined font. [Figure 8A] Figure 1 shows the nucleotide and amino acid sequences of the VH domain of antibody 6-G23-F, represented as SEQ ID NO: 31 and SEQ ID NO: 32, respectively. VH CDR1 (SEQ ID NO: 33) is shown in bold font, VH CDR2 (SEQ ID NO: 34) is underlined, and VH CDR3 (SEQ ID NO: 35) is shown in italic underlined font. [Figure 8B] 1 shows the nucleotide and amino acid sequences of the VL domain of antibody 6-G23-F, represented as SEQ ID NO: 36 and SEQ ID NO: 37, respectively. VL CDR1 (SEQ ID NO: 38) is shown in bold font, VL CDR2 (SEQ ID NO: 39) is underlined, and VL CDR3 (SEQ ID NO: 40) is shown in italic underlined font. [Figure 9] Figure 1 shows the results of a Fab ZAP assay, a cytotoxicity-based internalization assay performed to assess DLL3 internalization by the indicated antibodies. Reference DLL3 monoclonal antibody SC16 was used as a positive control. See WO2015127407. All tested anti-DLL3 antibodies exhibited killing activity comparable to the reference monoclonal antibody. A hook effect was observed at higher concentrations of anti-DLL3 antibody, as free anti-DLL3 competed with cell-bound anti-DLL3 for Fab ZAP. [Figure 10]Figures 10A-10D show binding curves for antibodies 7-I1-B (Figure 10A), 6-G23-F (Figure 10B), 10-O18-A (Figure 10C), and 2-C8-A (Figure 10D) measured via Octet HTX at 25 °C using PBS 0.1% BSA 0.02% Tween 20 as the binding buffer and 10 mM glycine pH 1.7 as the regeneration buffer. Monoclonal antibodies (5 μg / mL each) were loaded onto anti-mouse Fc sensors, and the loaded sensors were immersed in the indicated dilutions of recombinant human DLL3 protein (amino acids Ala27 to Ala479, catalog number 9749-DL, R&D Systems) at a starting concentration of 200 nM in seven serial 1:3 dilutions. Actual measurements and curve fits are shown for each DLL3 dilution. Figure 10E shows the dissociation constant (KD) values ​​for the four monoclonal antibodies described herein (6-G23-F, 2-C8-A, 7-I1-B, and 10-O18-A), which were calculated using the binding curves shown in Figures 2A-2D and applying a monovalent (1:1) binding model. [Figure 11] This figure shows that monoclonal antibodies (mAbs) 6-G23-F, 10-O18-A, and 2-C8-A selectively bind to DLL3 but not to DLL1 or DLL4. 7-I1-B mAb binds to both DLL3 and DLL4 but not to DLL1. [Figure 12] FIG. 1 shows the reaction scheme for "Scheme R: Preparation of Antibodies" described in Section VI(E) below. [Figure 13] Figure 13 shows the glycan remodeling scheme of H2-C8-A. [Figure 14] FIG. 1 shows the glycan remodeling scheme of H6-G23-F. [Figure 15] FIG. 1 shows the glycan remodeling scheme of H10-O18-A. [Figure 16] FIG. 1 shows the synthetic steps for preparing H2-C8-AADC. [Figure 17] FIG. 1 shows the synthetic steps for preparing H6-G23-FADC. [Figure 18] FIG. 1 shows the synthetic steps for preparing H10-O18-AADC. [Figure 19] Figure 1 shows the in vivo antitumor activity of three human anti-DLL3-drug antibody-drug conjugates (H2-C8-A-conjugate, H6-G23-F-conjugate, and H10-O18-A-conjugate) or an anti-LPS antibody-conjugate. Evaluation was performed using an animal model in which immunodeficient mice were inoculated with the DLL3-positive human small cell lung cancer cell line NCI-H209. The abscissa indicates the number of days after inoculation, and the ordinate indicates the estimated tumor volume. Error bars indicate standard error (SE) values. Arrows indicate the date of administration. [Figure 20] Figure 1 shows the in vivo antitumor activity of three human anti-DLL3 antibody-drug conjugates (H2-C8-A-conjugate, H6-G23-F-conjugate, and H10-O18-A-conjugate) or an anti-LPS antibody-conjugate. Evaluation was performed using an animal model in which immunodeficient mice were inoculated with the DLL3-positive human small cell lung cancer cell line NCI-H524. The abscissa indicates the number of days after inoculation, and the ordinate indicates the estimated tumor volume. Error bars indicate standard error (SE) values. Arrows indicate the date of administration. [Figure 21] Figure 1 shows the in vivo antitumor activity of three human anti-DLL3 antibody-drug conjugates (H2-C8-A-conjugate, H6-G23-F-conjugate, and H10-O18-A-conjugate) or an anti-DLL3 antibody-drug conjugate (SC16LD6.5). Evaluation was performed using an animal model in which immunodeficient mice were inoculated with the DLL3-positive human small cell lung cancer cell line NCI-H510A. The abscissa indicates the number of days after inoculation, and the ordinate indicates the estimated tumor volume. Error bars indicate standard error (SE) values. Arrows indicate the date of administration. [Figure 22] FIG. 1 shows the glycan remodeling scheme of H2-C8-A-3. [Figure 23] FIG. 1 shows the glycan remodeling scheme of H10-O18-A-3. [Figure 24]FIG. 1 shows the synthetic steps for preparing H2-C8-A-3ADC. [Figure 25] FIG. 1 shows the synthetic steps for preparing H10-O18-A-3ADC. [Figure 26] This figure shows the in vivo antitumor activity of two human anti-DLL3 antibody-drug conjugates (H2-C8-A-3-conjugate, H10-O18-A-3-conjugate), an anti-DLL3 antibody-drug conjugate (SC16LD6.5), or an anti-LPS antibody-conjugate. Evaluation was performed using an animal model in which immunodeficient mice were inoculated with the DLL3-positive human small cell lung cancer cell line NCI-H510A. The abscissa indicates the number of days after administration, and the ordinate indicates the estimated tumor volume. Error bars indicate standard error (SE) values. [Figure 27] This figure shows the in vivo antitumor activity of two human anti-DLL3 antibody-drug conjugates (H2-C8-A-3-conjugate, H10-O18-A-3-conjugate). Evaluation was performed using an animal model in which immunodeficient mice were inoculated with the DLL3-positive human small cell lung cancer cell line NCI-H209. The abscissa indicates the number of days after administration, and the ordinate indicates the estimated tumor volume. Error bars indicate standard error (SE) values. [Figure 28] This figure shows the in vivo antitumor activity of two human anti-DLL3 antibody-drug conjugates (H2-C8-A-3-conjugate, H10-O18-A-3-conjugate), an anti-DLL3 antibody-drug conjugate (SC16LD6.5), or an anti-LPS antibody-conjugate. Evaluation was performed using an animal model in which immunodeficient mice were inoculated with the DLL3-positive human small cell lung cancer cell line NCI-H82. The abscissa indicates the number of days after administration, and the ordinate indicates the estimated tumor volume. Error bars indicate standard error (SE) values. DETAILED DESCRIPTION OF THE INVENTION

[0069] The antibody-drug conjugate of the present invention is an antitumor drug in which an antitumor compound is conjugated via a linker structural moiety to an antibody capable of recognizing or binding to tumor cells.

[0070] It will be appreciated that certain aspects, modes, embodiments, variations and features of the inventive technology are described below at varying levels of detail in order to provide a substantial understanding of the inventive technology.

[0071] In practicing the techniques of the present invention, many conventional techniques in molecular biology, protein biochemistry, cell biology, immunology, microbiology and recombinant DNA are used. For example, Sambrook and Russell eds. (2001) Molecular Cloning: A Laboratory Manual, 3rd edition; the series Ausubel et al. eds. (2007) Current Protocols in Molecular Biology; the series Methods in Enzymology (Academic Press, Inc., NY); MacPherson et al. (1991) PCR 1: A Practical Approach (IRL Press at Oxford University Press); MacPherson et al. (1995) PCR 2: A Practical Approach; Harlow and Lane eds. (1999) Antibodies, A Laboratory Manual; Freshney (2005) Culture of Animal Cells: A Manual of Basic Technique, 5th edition; Gait ed. (1984) Oligonucleotide Synthesis; U.S. Patent No. 4,683,195; Hames and Higgins eds. (1984) Nucleic Acid Hybridization; Anderson (1999) Nucleic Acid Hybridization; Hames and Higgins eds. (1984) Transcription and Translation; Immobilized Cells and Enzymes (IRL Press (1986)); Perbal (1984) A Practical Guide to Molecular Cloning; Miller and Calos eds.See, for example, (1987) Gene Transfer Vectors for Mammalian Cells (Cold Spring Harbor Laboratory); Makrides ed. (2003) Gene Transfer and Expression in Mammalian Cells; Mayer and Walker eds. (1987) Immunochemical Methods in Cell and Molecular Biology (Academic Press, London); and Herzenberg et al. eds. (1996) Weir's Handbook of Experimental Immunology. Methods for detecting and measuring levels of polypeptide gene expression products (i.e., gene translation levels) are well known in the art and include the use of polypeptide detection methods such as antibody detection and quantification techniques. (See also Strachan & Read, Human Molecular Genetics, Second Edition (John Wiley and Sons, Inc., NY, 1999)). Hereinafter, preferred embodiments for carrying out the present invention will be described with reference to the drawings. It should be noted that the embodiments described below are merely exemplary embodiments of the present invention, and the scope of the present invention should not be construed narrowly by these examples.

[0072] I. Definition In the description of the present invention, the term "cancer" is used synonymously with the term "tumor". In describing the present invention, the term "gene" is used to include not only DNA but also its mRNA and cDNA, as well as their cRNA. In describing the present invention, the terms "polynucleotide" or "nucleotide" are used synonymously with the meaning of nucleic acid, and include DNA, RNA, probes, oligonucleotides, and primers. In describing the present invention, the terms "polynucleotide" and "nucleotide" can be used synonymously with each other unless otherwise specified. In describing the present invention, the terms "polypeptide" and "protein" may be used interchangeably. In describing the present invention, the term "cell" includes cells of an individual animal and cells in culture.

[0073] In describing the present invention, the term "DLL3" can be used synonymously with the DLL3 protein. In describing the present invention, human DLL3 is also referred to as "hDLL3." In the description of the present invention, the term "cytotoxic activity" is used to mean that a pathological change is caused in a cell in some way. This term refers not only to direct trauma but also to any type of structural or functional damage caused to a cell, such as DNA breaks, base dimer formation, chromosome breaks, damage to the cell division apparatus, and reduction in the activity of various types of enzymes.

[0074] In the description of the present invention, the phrase "exerting toxicity in cells" is used to mean that toxicity is exhibited in cells in some given way. This term not only refers to direct trauma, but also to all types of structural, functional, or metabolic effects on cells, such as DNA breaks, base dimer formation, chromosome breaks, damage to the cell division apparatus, reduction in the activity of various types of enzymes, and inhibition of the action of cell growth factors.

[0075] In the description of the present invention, the term "functional antibody fragment," also referred to as "antigen-binding fragment of antibody," is used to mean a partial fragment of an antibody that retains antigen-binding activity, and includes Fab, F(ab'), scFv, diabodies, linear antibodies, and multispecific antibodies formed from antibody fragments. Fab' is a monovalent fragment of the antibody variable region obtained by treating F(ab') under reducing conditions, and is also included in the antigen-binding fragment of an antibody. However, antigen-binding fragments of antibodies are not limited to these molecules, so long as they retain the ability to bind to an antigen. These antigen-binding fragments include not only those obtained by treating a full-length antibody protein molecule with an appropriate enzyme, but also proteins produced in a suitable host cell using genetically engineered antibody genes.

[0076] In the present description, the term "epitope" refers to a partial peptide or partial three-dimensional structure of DLL3 to which a specific anti-DLL3 antibody binds. Such epitopes are the partial peptides of DLL3 described above and can be determined by methods well known to those skilled in the art, such as immunoassays. First, various partial structures of the antigen are prepared. Known oligopeptide synthesis techniques can be applied to prepare such partial structures. For example, a series of polypeptides in which DLL3 is successively truncated to an appropriate length from its C-terminus or N-terminus can be prepared by recombinant DNA techniques well known to those skilled in the art. The reactivity of antibodies against such polypeptides is then studied to roughly determine the recognition site. Further shorter peptides can then be synthesized, and their reactivity against these peptides can then be studied to determine the epitope. When an antibody that binds to a membrane protein with multiple extracellular domains is directed against a three-dimensional structure composed of multiple domains as an epitope, the domain to which the antibody binds can be determined by modifying the amino acid sequence of a specific extracellular domain, thereby altering the three-dimensional structure. An epitope is the partial three-dimensional structure of an antigen that binds to a specific antibody, which can also be determined by identifying amino acid residues of the antigen that flank the antibody by X-ray structural analysis.

[0077] In the context of this invention, a "humanized antibody" refers to an antibody that comprises at least one chain comprising variable region framework residues from a human antibody chain and at least one complementarity-determining region (CDR) from a non-human antibody (e.g., murine). The term "human antibody," as used herein, is intended to include antibodies having variable and constant regions derived from human immunoglobulin sequences. However, the term "human antibody," as used herein, is not intended to include antibodies in which CDR sequences from another mammalian species, such as a mouse, have been grafted onto human framework sequences.

[0078] In describing the present invention, the phrase "antibodies that bind to the same epitope" is used to mean antibodies that bind to a common epitope. A first antibody and a second antibody can be determined to bind to the same epitope if the second antibody binds to a partial peptide or partial three-dimensional structure to which the first antibody binds. Alternatively, by confirming that the second antibody competes with the first antibody for binding to the antigen of the first antibody (i.e., the second antibody interferes with the binding of the first antibody to the antigen), it can be determined that the first antibody and the second antibody bind to the same epitope, even if the specific sequence or structure of the epitope has not been determined. In describing the present invention, the phrase "binding to the same epitope" refers to the case where the first antibody and the second antibody are determined to bind to a common epitope by either or both of these determination methods. When a first antibody and a second antibody bind to the same epitope and the first antibody has a special activity such as anti-tumor activity or internalization activity, the second antibody can be expected to have the same activity as the first antibody.

[0079] In the description of the present invention, the term "CDR" is used to mean a complementarity-determining region. It is known that the heavy and light chains of an antibody molecule each have three CDRs. Such CDRs, also called hypervariable regions, are located in the variable regions of the heavy and light chains of an antibody. These regions have particularly highly variable primary structures and are divided into three regions in the primary structure of the polypeptide chain in each of the heavy and light chains. In the description of the present invention, with respect to the CDRs of an antibody, the CDRs of the heavy chain are referred to as CDRH1, CDRH2, and CDRH3, respectively, from the amino-terminus of the heavy chain amino acid sequence, while the CDRs of the light chain are referred to as CDRL1, CDRL2, and CDRL3, respectively, from the amino-terminus of the light chain amino acid sequence. These regions are located close to each other in the three-dimensional structure and determine the specificity of the antibody for the antigen to which it binds.

[0080] The term "CDR-grafted antibody," as used herein, means an antibody in which at least one CDR of an "acceptor" antibody has been replaced with a CDR "graft" from a "donor" antibody having the desired antigen specificity. The terms "individual," "patient," or "subject," as used herein, can refer to an individual organism, a vertebrate, a mammal, or a human. In some embodiments, the individual, patient, or subject is a human. The term "pharmaceutically acceptable carrier," as used herein, is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal compounds, isotonic and absorption delaying compounds, and the like, that are compatible with pharmaceutical administration. Pharmaceutically acceptable carriers and their formulations are known to those skilled in the art and are described, for example, in Remington's Pharmaceutical Sciences (20 th edition, ed. A. Gennaro, 2000, Lippincott, Williams & Wilkins, Philadelphia, PA.

[0081] "Treating" or "treatment," as used herein, encompasses treatment of a disease or disorder described herein in a subject, e.g., a human, including, for example, (i) inhibiting the disease or disorder, i.e., halting its development; (ii) relieving the disease or disorder, i.e., causing regression of the disorder; (iii) slowing the progression of the disorder; and / or (iv) inhibiting, alleviating, or slowing the progression of one or more symptoms of the disease or disorder. In some embodiments, treating means that the symptoms associated with the disease are, for example, alleviated, reduced, cured, or placed in remission. "Specifically binds to," as used herein, refers to a molecule (e.g., an antibody or antigen-binding fragment thereof) that recognizes and binds to one molecule (e.g., an antigen) but does not substantially recognize and bind to another molecule. The terms "specific binding," "specifically binds to," or "specific for" a particular molecule (e.g., a polypeptide, or epitope on a polypeptide), as used herein, refer to, for example, a molecule that recognizes and binds to the molecule to which it binds with approximately 10 -4 M, 10 -5 M, 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M or 10 -12 K of M D The term "specifically binds to" can also refer to binding such that a molecule (e.g., an antibody or antigen-binding fragment thereof) binds to a particular polypeptide (e.g., a DLL3 polypeptide) or an epitope on a particular polypeptide, but does not substantially bind to any other polypeptides or polypeptide epitopes.

[0082] In describing the present invention, the term "one or more" is used to mean 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 or 2. In the present invention, examples of a "halogen atom" include, but are not limited to, a fluorine atom, a chlorine atom, a bromine atom, and an iodine atom.

[0083] In the present invention, the term "C1-C6 alkyl group" refers to a straight-chain or branched-chain alkyl group having 1 to 6 carbon atoms. Examples of the "C1-C6 alkyl group" include, but are not limited to, methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, s-butyl, t-butyl, n-pentyl, and n-hexyl. In the present invention, the term "C1-C6 alkoxy group" refers to an alkoxy group having a linear or branched alkyl group having 1 to 6 carbon atoms. Examples of the "C1-C6 alkoxy group" include, but are not limited to, a methoxy group, an ethoxy group, an n-propoxy group, an i-propoxy group, an n-butoxy group, an i-butoxy group, an s-butoxy group, an n-pentyloxy group, and an n-hexyloxy group. In the present invention, the term "C1-C6 alkylthio group" refers to an alkylthio group having a straight-chain or branched-chain alkyl group having 1 to 6 carbon atoms. Examples of the "C1-C6 alkylthio group" include, but are not limited to, methylthio, ethylthio, n-propylthio, i-propylthio, n-butylthio, i-butylthio, s-butylthio, t-butylthio, n-pentylthio, and n-hexylthio.

[0084] In the present invention, the term "3- to 5-membered saturated hydrocarbon ring" refers to a saturated cyclic hydrocarbon group having 3 to 5 carbon atoms. Examples of the "3- to 5-membered saturated hydrocarbon ring" include, but are not limited to, a cyclopropyl group, a cyclobutyl group, and a cyclopentyl group. In the present invention, the term "C3-C5 cycloalkoxy group" refers to a cycloalkoxy group having a saturated cyclic hydrocarbon group having 3 to 5 carbon atoms. Examples of the "C3-C5 cycloalkoxy group" include, but are not limited to, a cyclopropoxy group, a cyclobutoxy group, and a cyclopentyloxy group. In the present invention, examples of the "3- to 5-membered saturated heterocycle" include, but are not limited to, 1,3-propylene oxide, azacyclobutane, trimethylene sulfide, tetrahydrofuran, and pyrrolidine.

[0085] In the present invention, examples of the "aryl group" include, but are not limited to, a phenyl group, a benzyl group, an indenyl group, a naphthyl group, a fluorenyl group, an anthranyl group, and a phenanthrenyl group. In the present invention, examples of "heteroaryl groups" include, but are not limited to, thienyl, pyrrolyl, pyrazolyl, triazolyl, oxazolyl, oxadiazolyl, thiazolyl, pyridyl, pyrimidyl, pyridazyl, pyrazinyl, quinolyl, quinoxalyl, benzothiophenyl, benzimidazolyl, benzotriazolyl, and benzofuranyl groups. In the present invention, examples of the "6-membered heterocycle" include, but are not limited to, a pyridine ring, a pyrimidine ring, and a pyridazine ring. In the present invention, "spiro-linked" refers to a situation in which a pyrrolidine ring to which A and A are bonded, or a pyrrolidine ring to which E and E are bonded, forms a spiro ring, as illustrated in the examples.

[0086] II. Delta-like ligand 3 (DLL3) DLL3 (i.e., Delta-like ligand 3 or Delta-like protein 3) is selectively expressed in high-grade pulmonary neuroendocrine tumors, such as SCLC and LCNEC. Increased expression of DLL3 was observed in xenograft tumors from SCLC and LCNEC patients and was also confirmed in primary tumors. See Saunders et al., Sci Translational Medicine 7(302): 302ra136 (2015). Increased expression of DLL3 has also been observed in extrapulmonary neuroendocrine cancers, such as prostate neuroendocrine carcinoma (Puca et al., Sci Transl Med 11(484): pii: eaav0891 (2019)). DLL3 is expressed on the surface of such tumor cells, but not in normal tissues. The present disclosure provides immunoglobulin-related compositions (e.g., antibodies or antigen-binding fragments thereof) that are internalized upon binding to DLL3 on tumor cells and are therefore useful for delivering toxic payloads to these tumor cells. The immunoglobulin-related compositions of the present technology are useful in methods for detecting or treating DLL3-associated cancers in a subject in need thereof. Accordingly, various aspects of the methods of the present invention relate to the preparation, characterization, and manipulation of anti-DLL3 antibodies. The immunoglobulin-related compositions of the present technology are useful alone or in combination with additional therapeutic agents for treating cancer. In some embodiments, the immunoglobulin-related composition is a humanized antibody, a chimeric antibody, or a bispecific antibody.

[0087] In Drosophila melanogaster, Notch signaling is primarily mediated by Notch receptors. Delta is one of the Drosophila ligands for Notch, which activates signaling in neighboring cells. Humans have four known Notch receptors (NOTCH1-NOTCH4) and three homologs of Delta, called Delta-like ligands: DLL1, DLL3, and DLL4. DLL3, unlike DLL1 and DLL4, has been reported to inhibit, rather than activate, Notch signaling.

[0088] DLL3 (also known as Delta-like 3 or SCDO1) is a member of the Delta-like family of Notch DSL ligands. Representative DLL3 protein orthologs include, but are not limited to: Human (accession number NP_058637: (SEQ ID NO: 50) and NP_982353: (SEQ ID NO:51)); Chimpanzee (accession number XP_003316395: (SEQ ID NO:52)); Mouse (accession number NP_031892: (SEQ ID NO: 53) and rats (accession number NP_446118: (SEQ ID NO: 54)

[0089] In humans, the DLL3 gene consists of eight exons spanning 9.5 kb located on chromosome 19q13. Alternative splicing within the last exon generates a 2389 bp transcript (accession number NM_016941 (SEQ ID NO: 55)) and a 2052 bp transcript (accession number NM_203486 (SEQ ID NO: 56)). The former transcript encodes a 618 amino acid long protein (accession number NP_058637 (SEQ ID NO: 50)), whereas the latter transcript encodes a 587 amino acid long protein (accession number NP_982353 (SEQ ID NO: 51)). See Figures 4A-4B. These two protein isoforms of DLL3 share 100% overall identity across their extracellular and transmembrane domains, differing only in that the longer isoform contains an extended cytoplasmic tail containing 32 additional residues at the carboxy terminus of the protein.

[0090] Both isoforms can be detected in tumor cells. Indeed, aberrant DLL3 expression (genotypic and / or phenotypic) is associated with various tumorigenic cell subpopulations, such as cancer stem cells and tumor-initiating cells. Accordingly, the present disclosure provides DLL3 antibodies that may be particularly useful for targeting such cells (e.g., cancer stem cells, tumor-initiating cells, and cancers, such as small cell lung cancer, large cell neuroendocrine carcinoma, pulmonary neuroendocrine carcinoma, extrapulmonary neuroendocrine carcinoma, and melanoma), thereby facilitating the treatment, management, or prevention of neoplastic disorders.

[0091] The DLL3 protein used in the present invention can be directly purified from cells expressing DLL3 from human or non-human mammals (e.g., rats, mice, or monkeys) and then used, or a cell membrane fraction of the aforementioned cells can be prepared and used as the DLL3 protein. Alternatively, DLL3 can also be obtained by synthesizing it in vitro or by genetically engineering host cells to produce DLL3. Through such genetic engineering, the DLL3 protein can be obtained, specifically, by incorporating DLL3 cDNA into a vector capable of expressing the DLL3 cDNA and then synthesizing DLL3 in a solution containing enzymes, substrates, and energy materials necessary for transcription and translation, or by transforming other prokaryotic or eukaryotic host cells so that they can express DLL3. Cells expressing DLL3 based on the above-mentioned genetic engineering or cell lines expressing DLL3 can also be used to present the DLL3 protein. Alternatively, an expression vector incorporating DLL3 cDNA can be directly administered to an animal to be immunized, and DLL3 can be expressed in the body of the immunized animal in this manner. Furthermore, the term "DLL3" also includes proteins consisting of an amino acid sequence that contains one or more amino acid substitutions, deletions, and / or additions in the amino acid sequence of the above-mentioned DLL3 and has biological activity equivalent to that of the DLL3 protein.

[0092] III. Anti-DLL3 antibody The present technology describes methods and compositions for the production and use of anti-DLL3 immunoglobulin-related compositions (e.g., anti-DLL3 antibodies or antigen-binding fragments thereof). The anti-DLL3 immunoglobulin-related compositions of the present disclosure may be useful in the diagnosis or treatment of DLL3-related cancers (e.g., small cell lung cancer, large cell neuroendocrine carcinoma, pulmonary neuroendocrine carcinoma, extrapulmonary neuroendocrine carcinoma, and melanoma). Anti-DLL3 immunoglobulin-related compositions within the scope of the present technology include, but are not limited to, monoclonal, chimeric, humanized, human, bispecific antibodies, and diabodies that specifically bind to a target polypeptide, a homolog, derivative, or fragment thereof. The present disclosure also provides an antigen-binding fragment of any of the anti-DLL3 antibodies disclosed herein, selected from the group consisting of Fab, F(ab)'2, Fab', scFv, and Fv.

[0093] The present technology discloses anti-DLL3 antibodies that can promote the internalization of DLL3-antibody complexes and are therefore useful for delivering toxic payloads to tumor cells. Figures 5-8 provide the nucleotide and amino acid sequences of the VH and VL, as well as the CDR sequences of the antibodies disclosed herein (SEQ ID NOS: 1-40). The following table also lists the VH and VL sequences. H and V L The amino acid sequences of the antibodies are also provided, in addition to the CDR sequences of the antibodies disclosed herein.

[0094] [Table 1] TIFF2024503657000029.tif249156 TIFF2024503657000030.tif249156 TIFF2024503657000031.tif249160

[0095] In one aspect, the present disclosure provides an antibody or antigen-binding fragment thereof comprising a heavy chain immunoglobulin variable domain (VH) and a light chain immunoglobulin variable domain (VL), wherein (a) the VH has a VH-CDR1 sequence selected from the group consisting of: (i) SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:5, respectively; (ii) SEQ ID NO:13, SEQ ID NO:14, and SEQ ID NO:15, respectively; (iii) SEQ ID NO:23, SEQ ID NO:24, and SEQ ID NO:25, respectively; and (iv) SEQ ID NO:33, SEQ ID NO:34, and SEQ ID NO:35, respectively. , VH-CDR2 sequence, and VH-CDR3 sequence; and / or (b) VL comprises a VL-CDR1 sequence, a VL-CDR2 sequence, and a VL-CDR3 sequence selected from the group consisting of: (i) SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:10, respectively; (ii) SEQ ID NO:18, SEQ ID NO:19, and SEQ ID NO:20, respectively; (iii) SEQ ID NO:28, SEQ ID NO:29, and SEQ ID NO:30, respectively; and (iv) SEQ ID NO:38, SEQ ID NO:39, and SEQ ID NO:40, respectively. In some embodiments, the present disclosure provides an antibody or antigen-binding fragment thereof comprising a heavy chain immunoglobulin variable domain (VH) and a light chain immunoglobulin variable domain (VL), wherein (a) V H -CDR1 sequence, V H -CDR2 sequence, and V H - V containing the CDR3 sequence H and (b) V L -CDR1 sequence, V L -CDR2 sequence, and V L - V containing the CDR3 sequence LThe combination provides an antibody or antigen-binding fragment thereof selected from the group consisting of: (i) (a) SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:5, respectively, and (b) SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:10; (ii) (a) SEQ ID NO:13, SEQ ID NO:14, and SEQ ID NO:15, respectively, and (b) SEQ ID NO:18, SEQ ID NO:19, and SEQ ID NO:20; (iii) (a) SEQ ID NO:23, SEQ ID NO:24, and SEQ ID NO:25, respectively, and (b) SEQ ID NO:28, SEQ ID NO:29, and SEQ ID NO:30; and (iv) (a) SEQ ID NO:33, SEQ ID NO:34, and SEQ ID NO:35, respectively, and (b) SEQ ID NO:38, SEQ ID NO:39, and SEQ ID NO:40. In some embodiments, the antibody further comprises an Fc domain of any isotype, including, but not limited to, IgG (including IgG1 and variants (SEQ ID NOS: 42, 57, and 58), IgG2, IgG3, and IgG4), IgA (including IgA1 and IgA2), IgD, IgE, or IgM, and IgY. In some embodiments, the antibody comprises a heavy chain constant region of SEQ ID NOS: 42, 57, or 58, preferably SEQ ID NOS: 57 or 58, more preferably SEQ ID NO: 58. Non-limiting examples of constant region sequences include: Human IgD constant region, Uniprot:P01880 (SEQ ID NO: 41) APTKAPDVFPIISGCRHPKDNSPVVLACLITGYHPTSVTVTWYMGTQSQPQRTFPEIQRRDSYYMTSSQLSTPLQQWRQGEYKCVVQHTASKSKKEIFRWPESPKAQASSVPTAQPQAEGSLAKATTAPATTRNTGRGGEEKKKEKEKEEQEERETKTPECPSHTQPLGVYLLTPAVQDLWLRDKATFTCFV VGSDLKDAHLTWEVAGKVPTGGVEEGLLERHSNGSQSQHSRLTLPRSLWNAGTSVTCTLNHPSLPPQRLMALREPAAQAPVKLSLNLLASSDPPEAASWLLCEVSGFSPPNILLMWLEDQREVNTSGFAPARPPPQPGSTTFWAWSVLRVPAPPSPQPATYTCVVSHEDSRTLLNASRSLEVSYVTDHGPMK Human IgG1 constant region, Uniprot:P01857 (SEQ ID NO: 42) ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Human IgG1 variant constant region (SEQ ID NO: 57) ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Human IgG1 variant constant region (SEQ ID NO: 58) ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Human IgG2 constant region, Uniprot:P01859 (SEQ ID NO: 43) ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKDTLMISRTPEVTCVVDVSHEDPEVQFNWYVDGVEV HNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDISVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Human IgG3 constant region, Uniprot:P01860 (SEQ ID NO: 44) ASTKGPSVFPLAPCSRSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYTCNVNHKPSNTKVDKRVELKTPLGDTTHTCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPAPELLGGPSVFLFPPKPKDTLMISRTPE VTCVVVDVSHEDPEVQFKWYVDGVEVHNAKTKPREEQYNSTFRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKTKGQPREPQVYTLP PSREEMTKNQVSLTCLVKGFYPSDIAVEWESSGQPENNYNTTPPMLDSDGSFFLYSKLTVDKSRWQQGNIFSCSVMHEALHNRFTQKSLSLSPGK Human IgM constant region, Uniprot:P01871 (SEQ ID NO: 45) GSASAPTLFPLVSCENSPSDTSSVAVGCLAQDFLPDSITLSWKYKNNSDISSTRGFPSVLRGGKYAATSQVLLPSKDVMQGTDEHVVCKVQHPNGNKEKNVPLPVIAELPPKV SVFVPPRDGFFGNPRKSKLICQATGFSPRQIQVSWLREGKQVGSGVTTDQVQAEAKESGPTTYKVTSTLTIKESDWLGQSMFTCRVDHRGLTFQQNASSMCVPDQDTAIRVFA IPPSFASIFLTKSTKLTCLVTDLTTYDSVTISWTRQNGEAVKTHTNISESHPNATFSAVGEASICEDDWNSGERFTCTVTHTDLPSPLKQTISRPKGVALHRPDVYLLPPARE QLNLRESATITCLVTGFSPADVFVQWMQRGQPLSPEKYVTSAPMPEPQAPGRYFAHSILTVSEEEWNTGETYTCVAHEALPNRVTERTVDKSTGKPTLYNVSLVMSDTAGTCY Human IgG4 constant region, Uniprot:P01861 (SEQ ID NO: 46) ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVE VHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK Human IgA1 constant region, Uniprot:P01876 (SEQ ID NO: 47) ASPTSPKVFPLSLCSTQPDGNVVIACLVQGFFPQEPLSVTWSESGQGVTARNFPPSQDASGDLYTTSSQLTLPATQCLAGKSVTCHVKHYTNPSQDVTVPCPVPSTPPTPSPSTPPTPSPSCCHPRLSLHRPALEDLLLGSEANLTCTLTGLRDASGVTFTWTPSSGKSAVQGPPE RDLCGCYSVSSVLPGCAEPWNHGKTFTCTAAYPESKTPLTATLSKSGNTFRPEVHLLPPPSEELALNELVTLTCLARGFSPKDVLVRWLQGSQELPREKYLTWASRQEPSQGTTTFAVTSILRVAAEDWKKGDTFSCMVGHEALPLAFTQKTIDRLAGKPTHVNVSVVMAEVDGTCY Human IgA2 constant region, Uniprot:P01877 (SEQ ID NO: 48) ASPTSPKVFPLSLDSTPQDGNVVVACLVQGFFPQEPLSVTWSESGQNVTARNFPPSQDASGDLYTTSSQLTLPATQCPDGKSVTCHVKHYTNPSQDVTVPCPVPPPPPCCHPRLSLHRPALEDLLLGSEANLTCTLTGLRDASGATFTWTPSSGKSAVQGPPERDLCGCY SVSSVLPGCAQPWNHGETFTCTAAHPELKTPLTANITKSGNTFRPEVHLLPPPSEELALNELVTLTCLARGFSPKDVLVRWLQGSQELPREKYLTWASRQEPSQGTTTFAVTSILRVAAEDWKKGDTFSCMVGHEALPLAFTQKTIDRMAGKPTHVNVSVVMAEVDGTCY Human Ig kappa constant region, Uniprot: P01834 (SEQ ID NO: 49) TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0096] In some embodiments, the immunoglobulin-related compositions of the present technology comprise a heavy chain constant region at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NOs: 41-48, 57, and 58. Additionally, or alternatively, in some embodiments, the immunoglobulin-related compositions of the present technology comprise a light chain constant region at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NO: 49. In some embodiments, the immunoglobulin-related compositions of the present technology bind to the extracellular domain of DLL3. In some embodiments, the epitope is a conformational epitope.

[0097] In another aspect, the present disclosure provides an isolated immunoglobulin-related composition (e.g., an antibody or antigen-binding fragment thereof) comprising a heavy chain immunoglobulin variable domain (VH) amino acid sequence comprising SEQ ID NO:2, SEQ ID NO:12, SEQ ID NO:22, or SEQ ID NO:32, or a variant thereof having one or more conservative amino acid substitutions, or a heavy chain amino acid sequence comprising SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:68, or SEQ ID NO:69, or a variant thereof having one or more conservative amino acid substitutions. Additionally, or alternatively, in some embodiments, the immunoglobulin-related compositions of the present technology comprise a light chain immunoglobulin variable domain (VL) amino acid sequence comprising SEQ ID NO:7, SEQ ID NO:17, SEQ ID NO:27, or SEQ ID NO:37, or a variant thereof with one or more conservative amino acid substitutions, or a light chain amino acid sequence comprising SEQ ID NO:62, SEQ ID NO:66, or SEQ ID NO:70, or a variant thereof with one or more conservative amino acid substitutions.

[0098] In some embodiments, the immunoglobulin-related compositions of the present technology comprise a heavy chain immunoglobulin variable domain (VH) or heavy chain amino acid sequence and a light chain immunoglobulin variable domain (VL) or light chain amino acid sequence selected from the group consisting of SEQ ID NO:2 and SEQ ID NO:7, respectively (7-I1-B); SEQ ID NO:12 and SEQ ID NO:17, respectively (2-C8-A); SEQ ID NO:59 and SEQ ID NO:62, respectively (H2-C8-A); SEQ ID NO:60 and SEQ ID NO:62, respectively (H2-C8-A-2); SEQ ID NO:61 and SEQ ID NO:62, respectively (H2-C8-A-3); SEQ ID NO:22 and SEQ ID NO:27, respectively (10-O18-A); SEQ ID NO:67 and SEQ ID NO:70, respectively (H10-O18-A); SEQ ID NO:68 and SEQ ID NO:70, respectively (H10-O18-A-2); SEQ ID NO:69 and SEQ ID NO:70 (H10-O18-A-3); SEQ ID NO:32 and SEQ ID NO:37, respectively (6-G23-F).

[0099] In any of the above embodiments of the immunoglobulin-related composition, the HC and LC immunoglobulin variable domain sequences constitute an antigen-binding site that binds to the extracellular domain of DLL3. In any of the above embodiments of the immunoglobulin-related composition, the HC and LC immunoglobulin variable domain sequences constitute an antigen-binding site that binds to DLL3 and promotes internalization of the immunoglobulin-related composition. In some embodiments, the epitope is a conformational epitope. In some embodiments, the HC and LC immunoglobulin variable domain sequences are components of the same polypeptide chain. In other embodiments, the HC and LC immunoglobulin variable domain sequences are components of different polypeptide chains. In certain embodiments, the antibody is a full-length antibody.

[0100] In some embodiments, the immunoglobulin-related compositions of the present technology specifically bind to at least one DLL3 polypeptide. In some embodiments, the immunoglobulin-related compositions of the present technology specifically bind to at least one DLL3 polypeptide in a proportion of about 10 -3 M, 10 -4 M, 10 -5 M, 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M or 10 -12 It binds with a dissociation constant (KD) of M. In certain embodiments, the immunoglobulin-related composition is a monoclonal antibody, a chimeric antibody, a humanized antibody, a human antibody, or a bispecific antibody. In some embodiments, the antibody comprises a human antibody framework region. In certain embodiments, the immunoglobulin-related compositions comprise one or more of the following characteristics: (a) a light chain immunoglobulin variable domain sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to a light chain immunoglobulin variable domain sequence present in any one of SEQ ID NOs: 7, 17, 27, 37, 62, 66, or 70; and / or (b) a heavy chain immunoglobulin variable domain sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to a heavy chain immunoglobulin variable domain sequence present in any one of SEQ ID NOs: 2, 12, 22, 32, 59, 60, 61, 63, 64, 65, 67, 68, or 69. In another aspect, one or more amino acid residues in the immunoglobulin-related compositions provided herein are substituted with another amino acid. The substitution may be a "conservative substitution" as defined herein.

[0101] In certain embodiments, the immunoglobulin-related composition contains an IgG1 constant region comprising one or more amino acid substitutions or a set of amino acid residues selected from the group consisting of N297A and K322A, two amino acid substitutions (LALA) of two leucine (L) residues at positions 234 and 235 (according to the EU index) of the heavy chain with alanine (A), a set of amino acid residues: Glu (E) at position 356 and Met (M) at position 358 (according to the EU index) of the heavy chain, or a set of Asp (D) at position 356 and leucine (L) at position 358 (according to the EU index) of the heavy chain, or any combination thereof. Additionally or alternatively, in some embodiments, the immunoglobulin-related composition contains an IgG4 constant region comprising a S228P mutation. Engineered antibodies containing the above LALA substitutions exhibit antitumor activity without any of the undesirable effects of toxicity, PK profile, and loss of stability caused by Fc-mediated effector immune function (Pharmacol Ther. 2019; 200: 110-125).

[0102] In some embodiments, the anti-DLL3 immunoglobulin-related compositions described herein contain structural modifications to facilitate rapid binding and cellular uptake and / or delayed release. In some embodiments, the anti-DLL3 immunoglobulin-related compositions (e.g., antibodies) of the present technology may contain deletions in the CH2 constant heavy chain region to facilitate rapid binding and cellular uptake and / or delayed release. In some embodiments, Fab fragments are used to facilitate rapid binding and cellular uptake and / or delayed release. In some embodiments, F(ab)'2 fragments are used to facilitate rapid binding and cellular uptake and / or delayed release.

[0103] Amino acid sequence modifications of the anti-DLL3 antibodies described herein are contemplated. For example, it may be desirable to improve the binding affinity and / or other biological properties of the antibody. Amino acid sequence variants of anti-DLL3 antibodies are prepared by introducing appropriate nucleotide changes into the antibody nucleic acid or by peptide synthesis. Such modifications include, for example, deletion of, and / or insertion into, and / or substitution of, residues within the amino acid sequence of the antibody. Any combination of deletion, insertion, and substitution can be made to obtain the desired antibody, provided that the resulting antibody possesses the desired properties. Modifications also include altering the glycosylation pattern of the protein. Sites of greatest interest for mutagenesis include hypervariable regions, although FR alterations are also contemplated. "Conservative substitutions" are shown in the table below.

[0104] [Table 2]

[0105] One type of substitutional variant involves substituting one or more hypervariable region residues of a parent antibody. A convenient method for generating such substitutional variants involves affinity maturation using phage display. Specifically, several hypervariable region sites (e.g., 6-7 sites) are mutated to generate any possible amino acid substitution at each site. The antibody variants thus generated are displayed in a monovalent manner from filamentous phage particles as fusions to the M13 gene III product packaged within each particle. The phage-displayed variants are then screened for biological activity (e.g., binding affinity) as disclosed herein. To identify candidate hypervariable region sites for modification, alanine scanning mutagenesis may be performed to identify hypervariable region residues significantly involved in antigen binding. Alternatively, or in addition, it may be beneficial to analyze a crystal structure of an antigen-antibody complex to identify contact points between the antibody and antigen. Such contact residues and neighboring residues are candidates for substitution using the techniques detailed herein. Once such variants are generated, the panel of variants can be subjected to screening as described herein, and antibodies with similar or superior properties can be selected in one or more relevant assays for further development.

[0106] In one aspect, the present technology provides nucleic acid sequences encoding any of the immunoglobulin-related compositions described herein. Also disclosed herein are recombinant nucleic acid sequences encoding any of the antibodies described herein. In some embodiments, the nucleic acid sequence is selected from the group consisting of SEQ ID NOs: 1, 6, 11, 16, 21, 26, 31, and 36. In another aspect, the present technology provides host cells or expression vectors that express any nucleic acid sequence encoding any of the immunoglobulin-related compositions described herein.

[0107] The immunoglobulin-related compositions (e.g., anti-DLL3 antibodies) of the present technology may be monospecific, bispecific, trispecific, or of greater multispecificity. Multispecific antibodies may be specific for different epitopes of one or more DLL3 polypeptides, or may be specific for both a DLL3 polypeptide and a heterologous composition, such as a heterologous polypeptide or solid support material. See, for example, WO93 / 17715; WO92 / 08802; WO91 / 00360; WO92 / 05793; Tutt et al., J. Immunol. 147: 60-69 (1991); U.S. Patent Nos. 5,573,920, 4,474,893, 5,601,819, 4,714,681, 4,925,648; 6,106,835; Kostelny et al., J. Immunol. 148: 1547-1553 (1992). In some embodiments, the immunoglobulin-related composition is chimeric. In certain embodiments, the immunoglobulin-related composition is humanized.

[0108] The immunoglobulin-related compositions of the present technology may further be recombinantly fused at the N- or C-terminus to heterologous polypeptides or chemically conjugated to polypeptides or other compositions (including covalent and non-covalent conjugation). For example, the immunoglobulin-related compositions of the present technology may be recombinantly fused or conjugated to molecules useful as labels in detection assays, and effector molecules such as heterologous polypeptides, drugs, or toxins. See, e.g., WO92 / 08495; WO91 / 14438; WO89 / 12624; U.S. Pat. No. 5,314,995; and EP0396387. In any of the above embodiments of the immunoglobulin-related compositions of the present technology, the antibody or antigen-binding fragment may optionally be conjugated to an agent selected from the group consisting of an isotope, a dye, a chromagen, an imaging agent, a drug, a toxin, a cytokine, an enzyme, an enzyme inhibitor, a hormone, a hormone antagonist, a growth factor, a radionuclide, a metal, a liposome, a nanoparticle, RNA, DNA, or any combination thereof. In some embodiments, the antibody or antigen-binding fragment of the present technology may be combined with a pharmaceutically acceptable carrier. In the case of chemical or physical binding, a functional group on the immunoglobulin-related composition typically associates with a functional group on the agent. Alternatively, a functional group on the agent associates with a functional group on the immunoglobulin-related composition.

[0109] The functional groups on the drug and the immunoglobulin-related composition may be directly associated. For example, a functional group (e.g., a sulfhydryl group) on the drug may associate with a functional group (e.g., a sulfhydryl group) on the immunoglobulin-related composition to form a disulfide. Alternatively, the functional groups may be associated via a crosslinker (i.e., a linker). Some examples of crosslinkers are described below. The crosslinker may be attached to either the drug or the immunoglobulin-related composition. The number of drugs or immunoglobulin-related compositions in the conjugate is also limited by the number of functional groups present on the other. For example, the maximum number of drugs associated with the conjugate is determined by the number of functional groups present on the immunoglobulin-related composition. Alternatively, the maximum number of immunoglobulin-related compositions associated with the drug is determined by the number of functional groups present on the drug. In yet another embodiment, the conjugate comprises an immunoglobulin-related composition associated with an agent. In one embodiment, the conjugate comprises at least one agent chemically bound (e.g., conjugated) to at least one immunoglobulin-related composition. The agent may be chemically bound to the immunoglobulin-related composition by any method known to those skilled in the art. For example, a functional group on the agent may be directly attached to a functional group on the immunoglobulin-related composition. Some examples of suitable functional groups include, for example, amino, carboxyl, sulfhydryl, maleimide, isocyanato, isothiocyanate, and hydroxyl.

[0110] The agent may also be chemically bound to the immunoglobulin-related composition by a cross-linking agent, such as a dialdehyde, carbodiimide, dimaleimide, or the like. Cross-linking agents can be obtained, for example, from Pierce Biotechnology, Inc., Rockford, Ill. The Pierce Biotechnology, Inc. website can provide assistance. Additional cross-linking agents include platinum cross-linkers described in U.S. Patent Nos. 5,580,990; 5,985,566; and 6,133,038 to Kreatech Biotechnology, BV, Amsterdam, The Netherlands. Alternatively, the functional groups on the drug and the immunoglobulin-related composition may be the same. To crosslink identical functional groups, a homobifunctional crosslinker is typically used. Examples of homobifunctional crosslinkers include EGS (i.e., ethylene glycol bis[succinimidyl succinate]), DSS (i.e., disuccinimidyl suberate), DMA (i.e., dimethyl adipimidate.2HCl), DTSSP (i.e., 3,3'-dithiobis[sulfosuccinimidyl propionate]), DPDPB (i.e., 1,4-di-[3'-(2'-pyridyldithio)-propionamido]butane), and BMH (i.e., bismaleimidohexane). Such homobifunctional crosslinkers are also available from Pierce Biotechnology, Inc.

[0111] In other instances, it may be beneficial to cleave the agent from the immunoglobulin-related composition. The Pierce Biotechnology, Inc. website mentioned above can also provide assistance to those skilled in the art in selecting a suitable crosslinker that is cleavable in a cell, e.g., by an enzyme. Thus, the agent may be separated from the immunoglobulin-related composition. Examples of cleavable linkers include SMPT (i.e., 4-succinimidyloxycarbonyl-methyl-a-[2-pyridyldithio]toluene), sulfo-LC-SPDP (i.e., sulfosuccinimidyl 6-(3-[2-pyridyldithio]-propionamido)hexanoate), LC-SPDP (i.e., succinimidyl 6-(3-[2-pyridyldithio]-propionamido)hexanoate), sulfo-LC-SPDP (i.e., sulfosuccinimidyl 6-(3-[2-pyridyldithio]-propionamido)hexanoate), SPDP (i.e., N-succinimidyl 3-[2-pyridyldithio]-propionamidohexanoate), and AEDP (i.e., 3-[(2-aminoethyl)dithio]propionic acid HCl).

[0112] In another embodiment, the conjugate comprises at least one agent physically associated with at least one immunoglobulin-related composition. Any method known to those skilled in the art can be used to physically associate the agent with the immunoglobulin-related composition. For example, the immunoglobulin-related composition and the agent can be mixed together by any method known to those skilled in the art. The order of mixing is not important. For example, the agent can be physically mixed with the immunoglobulin-related composition by any method known to those skilled in the art. For example, the immunoglobulin-related composition and the agent can be placed in a container and agitated, for example, by shaking the container, to mix the immunoglobulin-related composition and the agent. The immunoglobulin-related compositions can be modified by any method known to those skilled in the art, for example, the immunoglobulin-related compositions can be modified with crosslinking agents or functional groups as described above.

[0113] The antibodies of the present invention also include modified antibodies. The term "modification" is used to refer to chemical or biological modifications of the antibodies of the present invention. Examples of such chemical modifications include the attachment of chemical moieties to the amino acid backbone and chemical modifications of N- or O-linked carbohydrate chains. Examples of such biological modifications include antibodies that have undergone post-translational modifications (e.g., N- or O-linked glycosylation, N- or C-terminal processing, deamidation, aspartic acid isomerization, methionine oxidation, and conversion of N-terminal glutamine or N-terminal glutamic acid to pyroglutamic acid) and antibodies that have an additional methionine residue at their N-terminus as a result of expression in a prokaryotic host cell. In addition, such modifications also include labeled antibodies, such as enzyme-labeled, fluorescent-labeled, and affinity-labeled antibodies, that enable detection or isolation of the antibodies or antigens of the present invention. Such modifications of the antibodies of the present invention are useful for improving the stability and retention of the antibodies in blood; reducing antigenicity; detecting or isolating the antibodies or antigens, and the like. Furthermore, antibody-dependent cellular cytotoxicity can be enhanced by adjusting the sugar chain modifications (glycosylation, defucosylation, etc.) attached to the antibodies of the present invention. Techniques for adjusting the sugar chain modifications of antibodies are known, including those described in International Publications WO1999 / 54342, WO2000 / 61739, WO2002 / 31140, and WO2007 / 133855, but the techniques are not limited thereto. The antibodies of the present invention also include antibodies in which the aforementioned sugar chain modifications have been adjusted.

[0114] Once the antibody genes have been isolated, they can be introduced into a suitable host to produce antibodies using a suitable combination of host and expression vector. A specific example of an antibody gene can be a combination of a gene encoding the heavy chain sequence of an antibody described in the present description and a gene encoding the light chain sequence of an antibody described therein. When transforming a host cell, such heavy chain sequence gene and light chain sequence gene can be inserted into a single expression vector, or alternatively, these genes can be inserted into different expression vectors. When eukaryotic cells are used as hosts, animal cells, plant cells, or eukaryotic microorganisms may be used. Particularly, examples of animal cells include mammalian cells, such as monkey COS cells (Gluzman, Y., Cell (1981) 23, pp. 175-182, ATCC CRL-1650), mouse fibroblast NIH3T3 (ATCC No. CRL-1658), dihydrofolate reductase-deficient Chinese hamster ovary cell line (CHO cells, ATCC CCL-61) (Urlaub, G. and Chasin, LA Proc. Natl. Acad. Sci. USA (1980) 77, pp. 4126-4220), and FreeStyle 293F cells (Invitrogen Corp.). When prokaryotic cells are used as hosts, for example, Escherichia coli or Bacillus subtilis may be used.

[0115] The desired antibody gene is introduced into these cells for transformation, and the transformed cells are then cultured in vitro to obtain the antibody. Since the yield during the aforementioned culture may vary depending on the antibody sequence, it is possible to use the yield as an indicator to select antibodies that can be easily produced as pharmaceuticals from antibodies with equivalent binding activity. Therefore, the antibody of the present invention also includes antibodies obtained by the above-mentioned method for producing an antibody, which includes the steps of culturing transformed host cells and collecting the desired antibody or a functional fragment of the antibody from the culture obtained in the aforementioned step.

[0116] It is known that the lysine residue at the carboxyl terminus of the heavy chain of an antibody produced in cultured mammalian cells is deleted (Journal of Chromatography A, 705: 129-134 (1995)). It is also known that two amino acid residues, glycine and lysine, are deleted at the carboxyl terminus of the heavy chain, and that the newly added proline residue at the carboxyl terminus is amidated (Analytical Biochemistry, 360: 75-83 (2007)). However, such deletions and modifications of these heavy chain sequences do not affect the antigen-binding activity and effector functions (complement activation, antibody-dependent cellular cytotoxicity, etc.) of the antibody. Therefore, the antibodies of the present invention also include antibodies that have undergone the above-mentioned modifications and functional fragments of such antibodies. Specific examples of such antibodies include deletion mutants containing one or two amino acid deletions at the carboxyl terminus of the heavy chain, and deletion mutants formed by amidating the above-mentioned deletion mutants (e.g., heavy chains in which the proline residue at the carboxyl terminus is amidated). However, deletion mutants containing deletions at the carboxyl termini of the heavy chains of the antibodies of the present invention are not limited to the deletion mutants described above, as long as they retain antigen-binding activity and effector function. The two heavy chains constituting the antibodies of the present invention may be either a heavy chain selected from the group consisting of full-length antibodies or one of the deletion mutants described above, or a combination of any two types selected from the aforementioned group. The ratio of the individual deletion mutants may be affected by the type and culture conditions of the cultured mammalian cells used to produce the antibodies of the present invention. An example of a major component of the antibodies of the present invention is an antibody in which one amino acid residue is deleted at the carboxyl termini of each of the two heavy chains. Examples of the biological activities of antibodies generally include antigen-binding activity, the activity of internalizing into cells expressing the antigen by binding to the antigen, the activity of neutralizing antigen activity, the activity of enhancing antigen activity, antibody-dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), and antibody-dependent cellular phagocytosis (ADCP). The function of the antibodies according to the present invention is their binding activity to DLL3, and preferably their activity of internalizing into cells expressing DLL3 by binding to DLL3. Furthermore, the antibodies of the present invention may have ADCC, CDC, and / or ADCP activity, as well as cell-internalizing activity.

[0117] IV. Generation of anti-DLL3 antibodies The anti-DLL3 antibodies of the present invention may be derived from any species. Preferred species include human, monkey, rat, mouse, and rabbit. When the anti-DLL3 antibodies of the present invention are derived from a species other than human, the anti-DLL3 antibodies are preferably chimerized or humanized by well-known techniques. The antibodies of the present invention may be polyclonal or monoclonal, with monoclonal antibodies being preferred. The anti-DLL3 antibodies of the present invention are antibodies capable of targeting tumor cells. Specifically, the anti-DLL3 antibodies of the present invention have the properties of being able to recognize tumor cells, being able to bind to tumor cells, and / or being internalized into tumor cells through cellular uptake. Therefore, the anti-DLL3 antibodies of the present invention may be conjugated to a compound having anti-tumor activity via a linker to prepare an antibody-drug conjugate.

[0118] Antibody binding activity to tumor cells can be confirmed by flow cytometry. Antibody uptake into tumor cells can be confirmed by (1) an assay using a fluorescently labeled secondary antibody that binds to the antibody to visualize the antibody uptake by the cells under a fluorescence microscope (Cell Death and Differentiation, 2008, 15, 751-761), (2) an assay using a fluorescently labeled secondary antibody that binds to the antibody to measure the amount of fluorescence uptake by the cells (Molecular Biology of the Cell, Vol. 15, 5268-5282, December 2004), or (3) the Mab-ZAP assay, which uses an immunotoxin that binds to the antibody and is released upon cellular uptake to inhibit cell growth (BioTechniques, 28: 162-165, January 2000). A recombinant conjugate protein of the catalytic domain of diphtheria toxin and protein G may be used as the immunotoxin.

[0119] In describing the present invention, the term "high internalization ability" is used to mean that the viability of DLL3-expressing cells administered with the above-mentioned antibody and saporin-labeled anti-rat IgG antibody (as expressed as a ratio to the cell viability in the absence of antibody addition, which is defined as 100%) is preferably 70% or less, more preferably 60% or less. The anti-tumor antibody-drug conjugate of the present invention comprises a conjugate compound that exerts an anti-tumor effect. Therefore, it is preferable, but not necessarily, that the antibody itself has an anti-tumor effect. For the purpose of specifically and / or selectively exerting the cytotoxicity of the anti-tumor compound in tumor cells, it is important and preferable that the antibody has the property of being internalized and translocated into tumor cells. Anti-DLL3 antibodies can be obtained by immunizing animals with a polypeptide that acts as an antigen, followed by collecting and purifying the antibodies produced in vivo, using methods commonly used in the art. It is preferable to use DLL3 that retains its three-dimensional structure as the antigen. An example of such a method is DNA immunization.

[0120] The origin of the antigen is not limited to humans, and therefore animals can also be immunized with antigens derived from non-human animals such as mice or rats. In this case, antibodies applicable to human diseases can be selected by testing the cross-reactivity of the obtained antibodies that bind to the heterologous antigen with human antigens. Furthermore, antibody-producing cells that produce antibodies against an antigen may be fused with myeloma cells by known methods to establish hybridomas to obtain monoclonal antibodies (e.g., Kohler and Milstein, Nature (1975) 256, 495-497; and Kennet, R. ed., Monoclonal Antibodies, 365-367, Plenum Press, NY (1980)). A method for obtaining an antibody against DLL3 will be specifically described below.

[0121] General Overview: First, a target polypeptide is selected that can raise antibodies of the present technology. For example, antibodies can be raised against the full-length DLL3 protein or against a portion of the extracellular domain of the DLL3 protein. Techniques for generating antibodies against such target polypeptides are well known to those skilled in the art. Examples of such techniques include, but are not limited to, display libraries, xeno- or human-mice, and hybridomas. Target polypeptides within the scope of the present technology include any polypeptide derived from the DLL3 protein that contains the extracellular domain and that is capable of eliciting an immune response. It is to be understood that recombinantly engineered antibodies and antibody fragments, including antibody-related polypeptides and fragments thereof, directed against DLL3 proteins are suitable for use in accordance with the present disclosure. Anti-DLL3 antibodies that can be subjected to the techniques described herein include monoclonal and polyclonal antibodies, as well as antibody fragments, such as Fab, Fab', F(ab')2, Fd, scFv, diabodies, antibody light chains, antibody heavy chains, and / or antibody fragments. Methods useful for the high-yield production of antibody Fv-containing polypeptides, such as Fab' and F(ab')2 antibody fragments, have been described. See U.S. Patent No. 5,648,237.

[0122] Generally, antibodies are obtained from a source species. More particularly, the nucleic acid or amino acid sequence of the variable portion of the light chain, heavy chain, or both, of an antibody of the source species having specificity for a target polypeptide antigen is obtained. The source species can be any species that has been useful for generating antibodies or antibody libraries of the present technology, such as rat, mouse, rabbit, chicken, monkey, human, etc. Phage or phagemid display technology is a useful technique for obtaining antibodies of the present technology. Techniques for generating and cloning monoclonal antibodies are well known to those skilled in the art. Expression of sequences encoding antibodies of the present technology can be carried out in E. coli.

[0123] Due to the degeneracy of nucleic acid coding sequences, other sequences that encode substantially the same amino acid sequence as that of the naturally occurring protein may be used in the practice of the present technology. Examples include, but are not limited to, nucleic acid sequences comprising all or part of the nucleic acid sequences encoding the above-described polypeptides, where such nucleic acid sequences have been altered by the substitution of different codons that encode functionally equivalent amino acid residues within the sequence, thus resulting in silent changes. Nucleotide sequences of immunoglobulins according to the present technology can be sequenced using standard methods (see, for example, "Current Methods in Sequence Comparison and Analysis," Macromolecule Sequencing and Synthesis, Selected Methods and Applications, pp. 127-149, 1998, Alan R. Liss, It is understood that variations in sequence homology of up to 25% are permitted, as calculated by the International Association of Clinical Oncology, Inc., as long as such variants form effective antibodies that recognize the DLL3 protein. For example, one or more amino acid residues within a polypeptide sequence may be substituted by another amino acid of a similar polarity, which acts as a functional equivalent, resulting in a silent alteration. Substitutes for an amino acid within the sequence may be selected from other members of the class to which the amino acid belongs. For example, nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, and the like. The polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine. The positively charged (basic) amino acids include arginine, lysine, and histidine. The negatively charged (acidic) amino acids include aspartic acid and glutamic acid. Also included within the scope of the present technology are proteins or fragments or derivatives thereof that are differentially modified during or after translation, for example, by glycosylation, proteolytic cleavage, linkage to antibody molecules or other cellular ligands, etc.In addition, nucleic acid sequences encoding immunoglobulins may be mutated in vitro or in vivo to create and / or destroy translation, initiation, and / or termination sequences, or to create variations in the coding region and / or to create new or destroy existing restriction endonuclease sites, or to facilitate further in vitro modifications. Any technique for mutagenesis known in the art can be used, including, but not limited to, in vitro site-directed mutagenesis, J. Biol. Chem. 253:6551, the use of Tab linkers (Pharmacia), etc.

[0124] Preparation of Polyclonal Antisera and Immunogens. Methods for generating antibodies or antibody fragments of the present technology typically involve immunizing a subject (generally a non-human subject, e.g., a mouse or rabbit) with purified DLL3 protein or a fragment thereof or cells expressing DLL3 protein or a fragment thereof. An appropriate immunogenic preparation may contain, for example, recombinantly expressed DLL3 protein or a chemically synthesized DLL3 peptide. The extracellular domain of the DLL3 protein, or a portion or fragment thereof, can be used as an immunogen to generate anti-DLL3 antibodies that bind to the DLL3 protein, or a portion or fragment thereof, using standard techniques for polyclonal and monoclonal antibody preparation.

[0125] The full-length DLL3 protein or a fragment thereof is useful as the fragment is an immunogen. In some embodiments, the DLL3 fragment comprises the extracellular domain of DLL3, such that antibodies raised against the peptide form specific immune complexes with the DLL3 protein. The extracellular domain of DLL3 is 466 amino acids long, spanning amino acids 27 to 492 of the full-length DLL3 protein. In some embodiments, antigenic DLL3 peptides contain at least 5, 8, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, or 450 amino acid residues. Depending on the use, and by methods well known to those skilled in the art, longer antigenic peptides are sometimes preferable over shorter antigenic peptides. Multimers of a given epitope are sometimes more effective than monomers.

[0126] If necessary, the immunogenicity of the DLL3 protein (or a fragment thereof) can be increased by fusion or conjugation to a carrier protein, such as keyhole limpet hemocyanin (KLH) or ovalbumin (ovum). Many such carrier proteins are known in the art. The DLL3 protein can also be combined with a conventional adjuvant, such as Freund's complete or incomplete adjuvant, to increase the subject's immune response to the polypeptide. Various adjuvants used to increase the immune response include, but are not limited to, Freund's (complete and incomplete), mineral gels (e.g., aluminum hydroxide), surfactants (e.g., lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, dinitrophenol, etc.), human adjuvants such as Bacille Calmette-Guerin and Corynebacterium parvum, or similar immunostimulatory compounds. These techniques are standard in the industry.

[0127] In describing the technology of the present invention, an immune response can be described as either a "primary" or a "secondary" immune response. A primary immune response, also described as a "protective" immune response, refers to an immune response generated in an individual as a result of some initial exposure to a particular antigen, such as a DLL3 protein (e.g., an initial "immunization"). In some embodiments, immunization can occur as a result of administering an antigen-containing vaccine to an individual. For example, the vaccine can be a DLL3 vaccine containing antigens derived from one or more DLL3 proteins. A primary immune response can weaken or decay over time, and may even disappear or at least become undetectably attenuated. Thus, the technology of the present invention also relates to a "secondary" immune response, also described herein as a "memory immune response." The term secondary immune response refers to an immune response elicited in an individual after a primary immune response has already occurred.

[0128] Thus, a secondary immune response can be elicited, for example, to strengthen a pre-existing immune response that has become weakened or attenuated, or to revive a previous immune response that has either disappeared or is no longer detectable. A secondary or memory immune response can be either a humoral (antibody) response or a cellular response. A secondary or memory humoral response occurs with the stimulation of memory B cells generated upon initial antigen presentation. A delayed-type hypersensitivity (DTH) response is a response that activates the CD4 + It is a type of cell-mediated secondary or memory immune response mediated by T cells. Initial antigen exposure stimulates the immune system, and subsequent exposures result in DTH. After appropriate immunization, anti-DLL3 antibodies can be prepared from the serum of the subject. If desired, antibody molecules against the DLL3 protein can be isolated from the mammal (e.g., from the blood) and further purified by well-known techniques, such as polypeptide A chromatography, to obtain the IgG fraction.

[0129] Monoclonal Antibodies. In one embodiment of the present technology, the antibody is an anti-DLL3 monoclonal antibody. For example, in some embodiments, the anti-DLL3 monoclonal antibody can be a human or murine anti-DLL3 monoclonal antibody. For preparation of monoclonal antibodies directed against a DLL3 protein, or derivatives, fragments, analogs, or homologs thereof, any technique that provides for the production of antibody molecules by continuous cell line culture can be utilized. Such techniques include, but are not limited to, hybridoma techniques (see, e.g., Kohler & Milstein, 1975. Nature 256: 495-497); trioma techniques; human B-cell hybridoma techniques (see, e.g., Kozbor, et al., 1983. Immunol. Today 4: 72) and EBV hybridoma techniques for producing human monoclonal antibodies (see, e.g., Cole, et al., 1985. In: MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp. 77-96). Human monoclonal antibodies can be utilized in the practice of the present technology and can be produced by using human hybridomas (see, e.g., Cote, et al., 1983. Proc. Natl. Acad. Sci. USA 80: 2026-2030) or by transforming human B cells in vitro with Epstein-Barr virus (see, e.g., Cole, et al., 1985. In: MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp. 77-96). For example, a population of nucleic acids encoding regions of an antibody can be isolated. PCR using primers derived from sequences encoding conserved regions of the antibody can be used to amplify sequences encoding portions of the antibody from the population, and DNA encoding the antibody or a fragment thereof, e.g., the variable domain, can then be reconstituted from the amplified sequences.Such amplified sequences may also be fused to DNA encoding other proteins, such as bacteriophage coat or bacterial cell surface proteins, for expression and display of the fusion polypeptide in phage or bacteria. The amplified sequences can then be expressed and further selected or isolated based on, for example, the affinity of the expressed antibodies or fragments thereof for antigens or epitopes present on the DLL3 protein. Alternatively, hybridomas expressing anti-DLL3 monoclonal antibodies can be prepared by immunizing a subject and then isolating hybridomas from the subject's spleen using conventional methods. See, for example, Milstein et al. (Galfre and Milstein, Methods Enzymol (1981) 73: 3-46). Screening the hybridomas using standard methods is expected to produce monoclonal antibodies with diverse specificities (i.e., for different epitopes) and affinities. Selected monoclonal antibodies exhibiting desired properties, such as DLL3 binding, can be used as expressed by hybridomas, conjugated to molecules such as polyethylene glycol (PEG) to alter their properties, or the cDNA encoding them can be isolated, sequenced, and manipulated in various ways. Synthetic dendrimeric trees can be added to reactive amino acid side chains, such as lysine, to enhance the immunogenic properties of the DLL3 protein. CPG-dinucleotide technology can also be used to enhance the immunogenic properties of the DLL3 protein. Other manipulations include substituting or deleting specific aminoacyl residues that contribute to antibody instability during storage or after administration to a subject, and affinity maturation techniques to improve the affinity of the antibody for the DLL3 protein.

[0130] Hybridoma Technology. In some embodiments, the antibody of the present technology is an anti-DLL3 monoclonal antibody produced by a hybridoma comprising B cells obtained from a transgenic non-human animal, e.g., a transgenic mouse having a genome comprising a human heavy chain transgene and a human light chain transgene fused to an immortalized cell. Hybridoma technology includes those known in the art and taught in Harlow et al., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 349 (1988); Hammerling et al., Monoclonal Antibodies And T-Cell Hybridomas, 563-681 (1981). Other methods for producing hybridomas and monoclonal antibodies are well known to those skilled in the art.

[0131] Phage Display Technology. As described above, the antibodies of the present technology can be generated through the application of recombinant DNA and phage display technology. For example, anti-DLL3 antibodies can be prepared using various phage display methods known in the art. In phage display methods, functional antibody domains are displayed on the surface of phage particles that carry the polynucleotide sequences encoding them. Phage with desired binding properties are selected from repertoire or combinatorial antibody libraries (e.g., human or murine) by direct selection using antigen, typically antigen bound or captured to a solid surface or bead. The phage used in these methods are typically filamentous phage such as fd and M13, which have Fab, Fv, or disulfide-stabilized Fv antibody domains recombinantly fused to either the phage gene III or gene VIII protein. In addition, the method can be adapted for the construction of Fab expression libraries (see, e.g., Huse, et al., Science 246: 1275-1281, 1989), thereby allowing the rapid and efficient identification of monoclonal Fab fragments with a desired specificity for a DLL3 polypeptide, e.g., a polypeptide or derivative, fragment, analog, or homolog thereof.Other examples of phage display methods that can be used to generate antibodies of the present technology include those described in Huston et al., Proc. Natl. Acad. Sci USA, 85: 5879-5883, 1988; Chaudhary et al., Proc. Natl. Acad. Sci USA, 87: 1066-1070, 1990; Brinkman et al., J. Immunol. Methods 182: 41-50, 1995; Ames et al., J. Immunol. Methods 184: 177-186, 1995; Kettleborough et al., Eur. J. Immunol. 24: 952-958, 1994; Persic et al., Gene 187: 9-18, 1997; Burton et al., Advances in Immunology 57: 191-280, 1994;PCT / GB91 / 01134;WO90 / 02809;WO91 / 10737;WO92 / 01047;WO92 / 18619;WO93 / 11236;WO95 / 15982;WO95 / 20401;WO96 / 06213;WO92 / 01047(Medical Research Council et al. al.); WO97 / 08320 (Morphosys); WO92 / 01047 (CAT / MRC); WO91 / 17271 (Affymax); and those disclosed in U.S. Patent Nos. 5,698,426, 5,223,409, 5,403,484, 5,580,717, 5,427,908, 5,750,753, 5,821,047, 5,571,698, 5,427,908, 5,516,637, 5,780,225, 5,658,727, and 5,733,743. A method useful for displaying polypeptides on the surface of bacteriophage particles by attaching the polypeptides via disulfide bonds is described by Lohning in US Pat. No. 6,753,136.After phage selection, as described in the above references, the antibody coding region can be isolated from the phage and used to generate whole antibodies, including human antibodies, or any other desired antigen-binding fragment, which can be expressed in any desired host, such as mammalian cells, insect cells, plant cells, yeast, and bacteria. For example, techniques for recombinantly producing Fab, Fab', and F(ab')2 fragments can also be employed using methods known in the art, such as those disclosed in WO92 / 22324; Mullinax et al., BioTechniques 12:864-869, 1992; and Sawai et al., AJRI 34:26-34, 1995; and Better et al., Science 240:1041-1043, 1988.

[0132] Generally, antibodies or antibody fragments thereof are expected to be presented on the surface of phage or phagemid particles, and hybrid antibodies or hybrid antibody fragments cloned into display vectors can be selected against appropriate antigens to identify variants that retain superior binding activity. See, e.g., Barbas III et al., Phage Display, A Laboratory Manual (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 2001). However, other vector formats can be used in this process, such as cloning antibody fragment libraries into lytic phage vectors (modified T7 or Lambda Zap systems) for selection and / or screening.

[0133] Expression of Recombinant Anti-DLL3 Antibodies. As described above, the antibodies of the present technology can be produced through the application of recombinant DNA technology. Recombinant polynucleotide constructs encoding the anti-DLL3 antibodies of the present technology typically include an expression control sequence operably linked to the coding sequence of the anti-DLL3 antibody chain, such as a naturally associated or heterologous promoter region. Accordingly, another aspect of the present technology includes vectors containing one or more nucleic acid sequences encoding the anti-DLL3 antibodies of the present technology. For recombinant expression of one or more of the polypeptides of the present technology, a nucleic acid containing all or part of the nucleotide sequence encoding the anti-DLL3 antibody is inserted into an appropriate cloning vector or expression vector (i.e., a vector containing the necessary elements for transcription and translation of the inserted polypeptide coding sequence) by recombinant DNA techniques well known in the art, as further detailed below. Methods for producing a diverse collection of vectors are described by Lerner et al. in U.S. Patent Nos. 6,291,160 and 6,680,192.

[0134] In general, expression vectors useful in recombinant DNA techniques are often in the form of plasmids. In this disclosure, the terms "plasmid" and "vector" can be used interchangeably, as plasmids are the most commonly used form of vector. However, the present technology is intended to include other forms of expression vectors that are not technically plasmids, such as viral vectors (e.g., replication-deficient retroviruses, adenoviruses, and adeno-associated viruses), which serve equivalent functions. Such viral vectors allow infection of a subject and expression of the construct in the subject. In some embodiments, the expression control sequences are eukaryotic promoter systems in vectors capable of transforming or transfecting eukaryotic host cells. Once the vector is incorporated into an appropriate host, the host is maintained under conditions suitable for high-level expression of nucleotide sequences encoding anti-DLL3 antibodies, and the collection and purification of anti-DLL3 antibodies, e.g., cross-reactive anti-DLL3 antibodies. See generally US 2002 / 0199213. These expression vectors are typically replicable in the host organism either as episomes or as an integral part of the host chromosomal DNA. Generally, expression vectors contain a selectable marker, such as ampicillin resistance or hygromycin resistance, to permit detection of cells transformed with the desired DNA sequences. The vector may also encode a signal peptide, such as a pectate lyase, useful for directing the secretion of extracellular antibody fragments. See U.S. Patent No. 5,576,195.

[0135] The recombinant expression vector of the present technology contains a nucleic acid encoding a protein having DLL3-binding properties in a form suitable for expression of the nucleic acid in a host cell, meaning that the recombinant expression vector contains one or more regulatory sequences operably linked to the nucleic acid sequence to be expressed, selected based on the host cell to be used for expression. Within a recombinant expression vector, "operably linked" is intended to mean that the nucleotide sequence of interest is linked to a regulatory sequence in a manner that allows expression of the nucleotide sequence (e.g., in an in vitro transcription / translation system or in a host cell when the vector is introduced into the host cell). The term "regulatory sequence" is intended to include promoters, enhancers, and other expression control elements (e.g., polyadenylation signals). Such regulatory sequences are described, for example, in Goeddel, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990). Regulatory sequences include those that direct constitutive expression of a nucleotide sequence in many types of host cell and those that direct expression of the nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences). Those skilled in the art will understand that the design of an expression vector may depend on factors such as the choice of host cell to be transformed, the desired expression level of the polypeptide, and the like. Exemplary regulatory sequences useful as promoters for recombinant polypeptide expression (e.g., anti-DLL3 antibodies) include, but are not limited to, promoters of 3-phosphoglycerate kinase and other glycolytic enzymes. Inducible yeast promoters include, among others, promoters from alcohol dehydrogenase, isocytochrome C, and enzymes involved in maltose and galactose utilization. In one embodiment, a polynucleotide encoding an anti-DLL3 antibody of the present technology is operably linked to the ara B promoter and expressible in a host cell. See U.S. Patent No. 5,028,530.By introducing the expression vectors of the present technology into host cells, polypeptides or peptides (e.g., anti-DLL3 antibodies) comprising the fusion polypeptides encoded by the nucleic acids described herein can be produced.

[0136] Another aspect of the present technology relates to host cells expressing anti-DLL3 antibodies, containing nucleic acids encoding one or more anti-DLL3 antibodies. The recombinant expression vectors of the present technology can be designed for expression of anti-DLL3 antibodies in prokaryotic or eukaryotic cells. For example, anti-DLL3 antibodies can be expressed in bacterial cells, such as Escherichia coli, insect cells (using baculovirus expression vectors), fungal cells, such as yeast, yeast cells, or mammalian cells. Suitable host cells are further discussed in Goeddel, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990). Alternatively, the recombinant expression vector can be transcribed and translated in vitro, for example, using T7 promoter regulatory sequences and T7 polymerase. Previously, methods useful for preparing and screening polypeptides with predetermined properties, such as anti-DLL3 antibodies, via the expression of stochastically generated polynucleotide sequences have been described. See U.S. Patent Nos. 5,763,192; 5,723,323; 5,814,476; 5,817,483; 5,824,514; 5,976,862; 6,492,107; and 6,569,641.

[0137] Expression of polypeptides in prokaryotes is most often carried out in Escherichia coli using vectors containing constitutive or inducible promoters directing the expression of either fusion or non-fusion polypeptides. Fusion vectors add a predetermined number of amino acids to the polypeptide encoded therein, usually to the amino terminus of the recombinant polypeptide. Such fusion vectors typically serve three purposes: (i) increasing the expression of the recombinant polypeptide; (ii) increasing the solubility of the recombinant polypeptide; and (iii) aiding in the purification of the recombinant polypeptide by acting as a ligand in affinity purification. Often, a proteolytic cleavage site is introduced into fusion expression vectors at the junction between the fusion moiety and the recombinant polypeptide, allowing separation of the recombinant polypeptide from the fusion moiety following purification of the fusion polypeptide. Such enzymes, and their cognate recognition sequences, include factor Xa, thrombin, and enterokinase. Exemplary fusion expression vectors include pGEX (Pharmacia Biotech Inc; Smith and Johnson, 1988. Gene 67:31-40), pMAL (New England Biolabs, Beverly, Mass.), and pRIT5 (Pharmacia, Piscataway, NJ), which fuse glutathione S-transferase (GST), maltose E-binding polypeptide, or polypeptide A, respectively, to the target recombinant polypeptide.

[0138] Examples of suitable inducible non-fusion E. coli expression vectors include pTrc (Amrann et al., (1988) Gene 69: 301-315) and pET 11d (Studier et al., GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990) 60-89). Methods for the targeted assembly of separate active peptide or protein domains to obtain multifunctional polypeptides via polypeptide fusion have been described by Pack et al. in U.S. Pat. Nos. 6,294,353 and 6,692,935. One strategy for maximizing recombinant polypeptide expression in E. coli, e.g., anti-DLL3 antibodies, is to express the polypeptide in a host bacterium with an impaired ability to proteolytically cleave the recombinant polypeptide. See, for example, Gottesman, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990) 119-128. Another strategy is to modify the nucleic acid sequence of the nucleic acid to be inserted into the expression vector so that the individual codons for each amino acid are those preferentially utilized in the expression host, e.g., E. coli (see, for example, Wada, et al., 1992. Nucl. Acids Res. 20: 2111-2118). Such modification of the nucleic acid sequence of the technology of the present invention can be carried out by standard DNA synthesis techniques.

[0139] In another embodiment, the anti-DLL3 antibody expression vector is a yeast expression vector. Examples of vectors for expression in the yeast Saccharomyces cerevisiae include pYepSec1 (Baldari, et al., 1987, EMBO J. 6: 229-234), pMFa (Kurjan and Herskowitz, Cell 30: 933-943, 1982), pJRY88 (Schultz et al., Gene 54: 113-123, 1987), pYES2 (Invitrogen, San Diego, Calif.), and picZ (Invitrogen, San Diego, Calif.). Alternatively, anti-DLL3 antibodies can be expressed in insect cells using baculovirus expression vectors. Baculovirus vectors that can be used to express polypeptides, such as anti-DLL3 antibodies, in cultured insect cells (e.g., SF9 cells) include the pAc series (Smith, et al., Mol. Cell. Biol. 3: 2156-2165, 1983) and the pVL series (Lucklow and Summers, 1989. Virology 170: 31-39).

[0140] In yet another embodiment, a nucleic acid encoding an anti-DLL3 antibody of the present technology is expressed in mammalian cells using a mammalian expression vector. Examples of mammalian expression vectors include, but are not limited to, pCDM8 (Nature 329: 840, 1987) and pMT2PC (Kaufman, et al., EMBO J. 6: 187-195, 1987). When used in mammalian cells, the expression vector's control functions are often provided by viral regulatory elements. For example, commonly used promoters are derived from polyoma, adenovirus 2, cytomegalovirus, and simian virus 40. For other suitable expression systems for both prokaryotic and eukaryotic cells useful for expressing the anti-DLL3 antibodies of the present technology, see, e.g., Chapters 16 and 17 of Sambrook, et al., MOLECULAR CLONING: A LABORATORY MANUAL. 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989.

[0141] In another embodiment, the recombinant mammalian expression vector is capable of directing expression of the nucleic acid in a particular cell type (e.g., tissue-specific regulatory elements). Tissue-specific regulatory elements are known in the art. Non-limiting examples of suitable tissue-specific promoters include the albumin promoter (liver-specific; Pinkert, et al., Genes Dev. 1: 268-277, 1987), lymphocyte-specific promoters (Calame and Eaton, Adv. Immunol. 43: 235-275, 1988), T-cell receptor promoters (Winoto and Baltimore, EMBO J. 8: 729-733, 1989) and immunoglobulin promoters (Banerji, et al., 1983. Cell 33: 729-740; Queen and Baltimore, Cell 33: 741-748, 1983), neuron-specific promoters (e.g., the neurofilament promoter; Byrne and Ruddle, Proc. Natl. Acad. Sci. USA 86: 5473-5477, 1988), and the like. 1989), pancreatic-specific promoters (Edlund, et al., 1985. Science 230: 912-916), and mammary gland-specific promoters (e.g., milk whey promoters; U.S. Pat. No. 4,873,316 and European Patent Application Publication No. 264,166). Developmentally regulated promoters are also included, such as the mouse hox promoters (Kessel and Gruss, Science 249: 374-379, 1990) and the alpha-fetoprotein promoter (Campes and Tilghman, Genes Dev. 3: 537-546, 1989).

[0142] Another aspect of the methods of the present invention relates to host cells into which a recombinant expression vector of the present technology has been introduced. The terms "host cell" and "recombinant host cell" are used interchangeably herein. It is understood that such terms refer not only to the particular subject cell, but also to the progeny or potential progeny of such a cell. Because certain modifications may occur in subsequent generations, either due to mutation or environmental influences, such progeny may not actually be identical to the parent cell, but are still included within the scope of the terms used herein.

[0143] Host cells can be any prokaryotic or eukaryotic cell. For example, anti-DLL3 antibodies can be expressed in bacterial cells, such as E. coli, insect cells, yeast, or mammalian cells. Mammalian cells are suitable hosts for expressing nucleotide segments encoding immunoglobulins or fragments thereof. See Winnacker, From Genes To Clones (VCH Publishers, NY, 1987). Numerous suitable host cell lines capable of secreting intact heterologous proteins have been developed in the art, including Chinese hamster ovary (CHO) cell lines, various COS cell lines, HeLa cells, L cells, and myeloma cell lines. In some embodiments, the cells are non-human. Expression vectors for these cells may contain expression control sequences, such as an origin of replication, a promoter, an enhancer, and necessary processing information sites, such as ribosome binding sites, RNA splice sites, polyadenylation sites, and transcription terminator sequences. Queen et al., Immunol. Rev. 89: 49, 1986. Exemplary expression control sequences include promoters derived from endogenous genes, cytomegalovirus, SV40, adenovirus, bovine papillomavirus, etc. Co et al., J Immunol. 148: 1149, 1992. Other suitable host cells are known to those skilled in the art.

[0144] Vector DNA can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques. The terms "transformation" and "transfection," as used herein, are intended to refer to various art-recognized techniques for introducing foreign nucleic acid (e.g., DNA) into host cells, including calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection, electroporation, gene guns, or viral-based transfection. Other methods used to transform mammalian cells include the use of polybrene, protoplast fusion, liposomes, electroporation, and microinjection (see generally, Sambrook et al., Molecular Cloning). Suitable methods for transforming or transfecting host cells can be found in Sambrook, et al. (MOLECULAR CLONING: A LABORATORY MANUAL. 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989) and other laboratory manuals. The vectors containing the DNA segments of interest can be transferred into the host cell by well-known methods, depending on the type of cellular host.

[0145] For stable transfection of mammalian cells, it is known that, depending on the expression vector and transfection technique used, only a small percentage of cells may integrate the foreign DNA into their genome. To identify and select these integrants, a gene encoding a selectable marker (e.g., antibiotic resistance) is generally introduced into the host cells along with the gene of interest. Various selectable markers include those that confer resistance to drugs, such as G418, hygromycin, and methotrexate. The nucleic acid encoding the selectable marker may be introduced into the host cells on the same vector as that encoding the anti-DLL3 antibody, or may be introduced on a separate vector. Cells stably transfected with the introduced nucleic acid can be identified by drug selection (e.g., cells that have incorporated the selectable marker gene are expected to survive, while other cells die).

[0146] Host cells, e.g., prokaryotic or eukaryotic host cells, containing the anti-DLL3 antibody of the present technology in culture can be used to produce (i.e., express) a recombinant anti-DLL3 antibody. In one embodiment, the method includes culturing host cells (introduced with a recombinant expression vector encoding the anti-DLL3 antibody) in a suitable medium to produce the anti-DLL3 antibody. In another embodiment, the method further includes isolating the anti-DLL3 antibody from the medium or host cells. Once expressed, the anti-DLL3 antibody, e.g., a harvest of the anti-DLL3 antibody or anti-DLL3 antibody-related polypeptide, is purified from the culture medium and host cells. The anti-DLL3 antibody can be purified according to standard procedures in the art, including HPLC purification, column chromatography, gel electrophoresis, and the like. In one embodiment, the anti-DLL3 antibody is produced in a host organism by the method of Boss et al., U.S. Pat. No. 4,816,397. Typically, the anti-DLL3 antibody chain is expressed with a signal sequence, thus releasing it into the culture medium. However, if the anti-DLL3 antibody chains are not naturally secreted by the host cells, they can be released by treatment with mild detergent. Purification of recombinant polypeptides is well known in the art and includes, for example, ammonium sulfate precipitation, affinity chromatography purification techniques, column chromatography, ion exchange purification techniques, gel electrophoresis, and the like (see generally, Scopes, Protein Purification (Springer-Verlag, NY, 1982)).

[0147] A polynucleotide encoding an anti-DLL3 antibody, e.g., an anti-DLL3 antibody coding sequence, may be incorporated into a transgene for introduction into the genome of a transgenic animal and subsequent expression in the milk of the transgenic animal. See, e.g., U.S. Patent Nos. 5,741,957, 5,304,489, and 5,849,992. Suitable transgenes include light and / or heavy chain coding sequences operably linked to a promoter and enhancer from a mammary gland-specific gene, such as casein or β-lactoglobulin. To generate transgenic animals, the transgene may be microinjected into fertilized oocytes or incorporated into the genome of embryonic stem cells, the nucleus of which is transferred into an enucleated oocyte.

[0148] Single-chain antibodies. In one embodiment, the anti-DLL3 antibody of the present technology is a single-chain anti-DLL3 antibody. Techniques can be adapted to produce single-chain antibodies specific to DLL3 protein in accordance with the present technology (see, for example, U.S. Pat. No. 4,946,778). Examples of techniques that can be used to produce single-chain Fvs and antibodies of the present technology include those described in U.S. Pat. Nos. 4,946,778 and 5,258,498; Huston et al., Methods in Enzymology, 203: 46-88, 1991; Shu, L. et al., Proc. Natl. Acad. Sci. USA, 90: 7995-7999, 1993; and Skerra et al., Science, 240: 1038-1040, 1988.

[0149] Chimeric and humanized antibodies. In one embodiment, the anti-DLL3 antibody of the present technology is a chimeric anti-DLL3 antibody. In one embodiment, the anti-DLL3 antibody of the present technology is a humanized anti-DLL3 antibody. In one embodiment of the present technology, the donor and acceptor antibodies are monoclonal antibodies from different species. For example, the acceptor antibody is a human antibody (to minimize its antigenicity in humans), in which case the resulting CDR-grafted antibody is called a "humanized" antibody.

[0150] Recombinant anti-DLL3 antibodies, such as chimeric and humanized monoclonal antibodies containing both human and non-human portions, can be produced using standard recombinant DNA techniques and are within the scope of the present technology. For some uses, such as the use of the anti-DLL3 antibodies of the present technology in humans in vivo, as well as the use of these agents in in vitro detection assays, chimeric or humanized anti-DLL3 antibodies can be used. Such chimeric and humanized monoclonal antibodies can be produced by recombinant DNA techniques known in the art.Such useful methods include, but are not limited to, those described in International Patent Application No. PCT / US86 / 02269; U.S. Patent No. 5,225,539; European Patent No. 184187; European Patent No. 171496; European Patent No. 173494; PCT International Publication No. WO86 / 01533; U.S. Patent Nos. 4,816,567; 5,225,539; European Patent No. 125023; Better, et al., 1988. Science 240: 1041-1043; Liu, et al., 1987. Proc. Natl. Acad. Sci. USA 84: 3439-3443; Liu, et al., 1987. J. Immunol. 139: 3521-3526; Sun, et al., 1987. Proc. Natl. Acad. Sci. USA 84: 214-218; Nishimura, et al., 1987. Cancer Res. 47: 999-1005; Wood, et al., 1985. Nature 314: 446-449; Shaw, et al., 1988. J. Natl. Cancer Inst. 80: 1553-1559; Morrison (1985) Science 229: 1202-1207; Oi, et al. (1986) BioTechniques 4: 214; Jones, et al., 1986. Nature 321: 552-525; Verhoeyan, et al., 1988. Science 239: 1534; Morrison, Science 229: 1202, 1985; et al., BioTechniques 4: 214, 1986; Gillies et al., J. Immunol. Methods, 125: 191-202, 1989; U.S. Patent No. 5,807,715; and Beidler, et al., 1988. J. Immunol. 141: 4053-4060.For example, antibodies can be humanized using a variety of techniques, such as CDR grafting (EP 0239400; WO 91 / 09967; U.S. Patent Nos. 5,530,101; 5,585,089; 5,859,205; 6,248,516; EP 460167), veneering or resurfacing (EP 0592106; EP 0519596; Padlan EA, Molecular Immunology, 28: 489-498, 1991; Studnicka et al., Protein Engineering 7: 805-814, 1994; Roguska et al., PNAS 91: 969-973, 1994), and chain shuffling (U.S. Patent No. 5,565,332).In one embodiment, cDNA encoding a murine anti-DLL3 monoclonal antibody is digested with restriction enzymes specifically selected to remove sequences encoding the Fc constant region, and the equivalent portion of cDNA encoding a human Fc constant region is substituted (Robinson et al., PCT / US86 / 02269; Akira et al., European Patent Application No. 184,187; Taniguchi, European Patent Application No. 171,496; Morrison et al., European Patent Application No. 173,494; Neuberger et al., WO 86 / 01533; Cabilly et al., U.S. Pat. No. 4,816,567; Cabilly et al., European Patent Application No. 125,023; Better et al. (1988) Science 240: 1041-1043; Liu et al. (1987) Proc. Natl. Acad. Sci. USA 84: 3439-3443; Liu et al. (1987) J Immunol 139: 3521-3526; Sun et al. (1987) Proc. Natl. Acad. Sci. USA 84: 214-218; Nishimura et al. (1987) Cancer Res 47: 999-1005; Wood et al. (1985) Nature 314: 446-449; and Shaw et al. (1988) J. Natl. Cancer Inst. 80: 1553-1559; U.S. Patent No. 6,180,370; U.S. Patent No. 6,300,064;

[0151] In one embodiment, the technology of the present invention provides for the construction of humanized anti-DLL3 antibodies that are less likely to induce a human anti-mouse antibody (hereinafter referred to as "HAMA") response, yet still possess effective antibody effector functions. The terms "human" and "humanized," as used herein with respect to antibodies, refer to any antibody that is predicted to elicit a therapeutically acceptable, weak immunogenic response in human subjects. In one embodiment, the technology of the present invention provides humanized anti-DLL3 antibodies, heavy and light chain immunoglobulins.

[0152] CDR-grafted antibodies. In some embodiments, the anti-DLL3 antibodies of the present technology are anti-DLL3 CDR-grafted antibodies. Generally, the donor and acceptor antibodies used to generate anti-DLL3 CDR antibodies are monoclonal antibodies from different species; typically, the acceptor antibody is a human antibody (to minimize its antigenicity in humans), in which case the resulting CDR-grafted antibody is called a "humanized" antibody. Specifically, a "humanized antibody" refers to an antibody comprising at least one chain comprising variable region framework residues from a human antibody chain and at least one complementarity-determining region (CDR) from a non-human antibody (e.g., mouse). The term "human antibody," as used herein, is intended to include antibodies having variable and constant regions derived from human immunoglobulin sequences. However, the term "human antibody," as used herein, is not intended to include antibodies in which CDR sequences from another mammalian species, such as a mouse, have been grafted onto human framework sequences. Grafting occurs within a single V region of the acceptor antibody. H or V L It may be a graft of a single CDR (or part of a single CDR) within V H and V LThe grafting may involve multiple CDRs (or portions thereof) within one or both of the variable domains of the acceptor antibody. Often, all three CDRs in all variable domains of the acceptor antibody will be replaced with the corresponding donor CDRs, but only to the extent necessary to allow sufficient binding of the resulting CDR-grafted antibody to the DLL3 protein. Methods for producing CDR-grafted and humanized antibodies are taught by Queen et al., U.S. Patent Nos. 5,585,089; 5,693,761; 5,693,762; and Winter, U.S. Patent No. 5,225,539; and EP 0682040. H and V L Methods useful for preparing polypeptides are taught by Winter et al., U.S. Patent Nos. 4,816,397; 6,291,158; 6,291,159; 6,291,161; 6,545,142; EP 0368684; EP 0451216; and EP 0120694.

[0153] After selecting suitable framework region candidates from the same family and / or same family members, either or both of the heavy and light chain variable regions are created by grafting CDRs from the originating species into the hybrid framework regions. Assembly of hybrid antibodies or hybrid antibody fragments having hybrid variable chain regions according to any of the above aspects can be achieved using conventional methods known to those skilled in the art. For example, DNA sequences encoding the hybrid variable domains described herein (i.e., frameworks based on the target species and CDRs from the originating species) can be generated by oligonucleotide synthesis and / or PCR. Nucleic acids encoding CDR regions can also be isolated from the originating species antibody using suitable restriction enzymes and ligated to the target species framework by ligation with a suitable ligation enzyme. Alternatively, the framework regions of the variable chains of the originating species antibody can be altered by site-directed mutagenesis. Because hybrids are composed of a selection of multiple candidates corresponding to each framework region, there are many combinations of sequences suitable for construction according to the principles described herein. Thus, libraries of hybrids can be assembled having members with different combinations of individual framework regions. Such libraries can be electronic database collections of sequences or physical collections of hybrids.

[0154] This process typically does not alter the FRs of the acceptor antibody adjacent to the grafted CDRs. However, one skilled in the art may sometimes improve the antigen-binding affinity of the resulting anti-DLL3 CDR-grafted antibody by replacing specific residues in a given FR to make the FR more similar to the corresponding FR of the donor antibody. Suitable locations for substitution include amino acid residues adjacent to the CDR or capable of interacting with the CDR (see, e.g., U.S. Pat. No. 5,585,089, especially columns 12-16). Alternatively, one skilled in the art may start with the donor FR and modify it to more closely resemble the acceptor FR or human consensus FR. Techniques for making these modifications are known in the art. In particular, if the resulting FR matches the human consensus FR for that position or is at least 90% or more identical to such consensus FR, doing so may not significantly increase the antigenicity of the resulting modified anti-DLL3 CDR-grafted antibody compared to the same antibody with fully human FRs.

[0155] Bispecific antibodies (BsAbs). Bispecific antibodies are antibodies that can simultaneously bind to two targets with distinct structures, e.g., two different target antigens or two different epitopes on the same target antigen. BsAbs can be generated, for example, by combining heavy and / or light chains that recognize different epitopes of the same or different antigens. In some embodiments, by molecular function, a bispecific binding agent binds to one antigen (or epitope) on one of its two binding arms (one VH / VL pair) and to a different antigen (or epitope) on its second arm (a different VH / VL pair). By this definition, a bispecific binding agent has two distinct antigen-binding arms (both in specificity and CDR sequences) and is monovalent for each antigen to which it binds. Bispecific antibodies (BsAbs) and bispecific antibody fragments (BsFabs) of the present technology have, for example, at least one arm that specifically binds to DLL3 and at least one other arm that specifically binds to a second target antigen. In certain embodiments, the BsAbs are capable of binding to tumor cells that express the DLL3 antigen on their cell surface.

[0156] Various bispecific fusion proteins can be generated using molecular engineering. For example, BsAbs have been constructed that utilize either a complete immunoglobulin framework (e.g., IgG), a single-chain variable fragment (scFv), or a combination thereof. In some embodiments, the bispecific fusion protein is bivalent, e.g., comprising an scFv with a single binding site for one antigen and a Fab fragment with a single binding site for a second antigen. In some embodiments, the bispecific fusion protein is bivalent, e.g., comprising an scFv with a single binding site for one antigen and another scFv fragment with a single binding site for a second antigen. In other embodiments, the bispecific fusion protein is tetravalent, e.g., comprising an immunoglobulin (e.g., IgG) with two binding sites for one antigen and two identical scFvs for the second antigen. BsAbs, composed of two scFv units in tandem, have been shown to be a clinically successful bispecific antibody format. In some embodiments, the BsAb is designed to contain two single-chain variable fragments (scFvs) in tandem, where an scFv that binds a tumor antigen (e.g., DLL3) is linked to an scFv that binds a different target antigen.

[0157] Recent methods for generating BsAbs include engineered recombinant monoclonal antibodies with additional cysteine ​​residues so that they cross-link more strongly than more common immunoglobulin isotypes. See, e.g., FitzGerald et al., Protein Eng. 10(10):1221-1225 (1997). Another approach is to engineer recombinant fusion proteins linking two or more different single-chain antibody or antibody fragment segments with the required bispecificity. See, e.g., Coloma et al., Nature Biotech. 15:159-163 (1997). A variety of bispecific fusion proteins can be generated using molecular engineering.

[0158] Bispecific fusion proteins linking two or more different single-chain antibodies or antibody fragments are produced in a similar manner. Recombinant methods can be used to produce a variety of fusion proteins. In certain embodiments, a BsAb according to the technology of the present invention comprises an immunoglobulin, which comprises a heavy chain and a light chain, and an scFv. In certain embodiments, the scFv is linked to the C-terminus of the heavy chain of any DLL3 immunoglobulin disclosed herein. In certain embodiments, the scFv is linked to the C-terminus of the light chain of any DLL3 immunoglobulin disclosed herein. In various embodiments, the scFv is linked to the heavy or light chain via a linker sequence. The appropriate linker sequence required for in-frame connection of the heavy chain Fd to the scFv can be synthesized via a PCR reaction. L and V kappa The DNA fragment encoding the scFv is then ligated into a staging vector containing a DNA sequence encoding the CH1 domain. The resulting scFv-CH1 construct is excised and transformed into the V domain of the DLL3 antibody. H The resulting vector can be used to transfect suitable host cells, such as mammalian cells, for expression of the bispecific fusion protein.

[0159] In some embodiments, the linker is at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more amino acids in length. In some embodiments, the linker is characterized by not adopting a rigid three-dimensional structure but rather tending to provide flexibility to the polypeptide (e.g., the first and / or second antigen-binding site). In some embodiments, linkers are employed in the BsAbs described herein based on specific properties they confer to the BsAb, such as increased stability. In some embodiments, the BsAbs of the present technology comprise a G4S linker (SEQ ID NO: 82). In certain embodiments, the BsAb of the present technology is (G4S) n and a linker, wherein n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or greater (SEQ ID NO: 83).

[0160] Fc Modifications. In some embodiments, the anti-DLL3 antibodies of the present technology comprise a variant Fc region, which comprises at least one amino acid modification relative to a wild-type Fc region (or parent Fc region) such that the molecule has altered affinity for an Fc receptor (e.g., FcγR), provided that the variant Fc region does not have substitutions at positions that make direct contact with an Fc receptor based on crystallographic and structural analyses of Fc-Fc receptor interactions, such as those disclosed by Sondermann et al., Nature, 406:267-273 (2000). Examples of positions within the Fc region that make direct contact with an Fc receptor, such as FcγR, include amino acids 234-239 (hinge region), amino acids 265-269 (B / C loop), amino acids 297-299 (C7E loop), and amino acids 327-332 (F / G loop).

[0161] In some embodiments, the anti-DLL3 antibodies of the present technology have altered affinity for activating and / or inhibitory receptors and have a variant Fc region with one or more amino acid modifications, wherein the one or more amino acid modifications are substitution of N297 with alanine or substitution of K322 with alanine. Glycosylation modifications. In some embodiments, the anti-DLL3 antibodies of the present technology have an Fc region with variant glycosylation compared to the parent Fc region. In some embodiments, the variant glycosylation comprises the absence of fucose; in some embodiments, the variant glycosylation results from expression in GnT1-deficient CHO cells. In some embodiments, antibodies of the present technology may have altered glycosylation sites compared to a suitable reference antibody that binds to an antigen of interest (e.g., DLL3) without altering the functionality of the antibody, e.g., its binding activity to the antigen. "Glycosylation site," as used herein, includes any specific amino acid sequence in an antibody to which an oligosaccharide (i.e., a carbohydrate containing two or more monosaccharides linked together) is predicted to be specifically and covalently attached.

[0162] Oligosaccharide side chains are typically linked to the antibody backbone via either N- or O-linkages. N-linked glycosylation refers to the attachment of an oligosaccharide moiety to the side chain of an asparagine residue. O-linked glycosylation refers to the attachment of an oligosaccharide moiety to a hydroxyamino acid, e.g., serine or threonine. For example, an Fc-glycoform (hDLL3-IgGln) lacking certain oligosaccharides, such as fucose and terminal N-acetylglucosamine, can be produced in specialized CHO cells and exhibit enhanced ADCC effector function.

[0163] In some embodiments, the carbohydrate content of the immunoglobulin-related compositions disclosed herein is modified by adding or deleting glycosylation sites. Methods for modifying the carbohydrate content of antibodies are well known in the art and are included within the technology of the present invention, see, e.g., U.S. Patent No. 6,218,149; EP 0359096 B1; U.S. Patent Publication No. US 2002 / 0028486; International Patent Application Publication No. WO 03 / 035835; U.S. Patent Publication No. 2003 / 0115614; U.S. Patent No. 6,218,149; and U.S. Patent No. 6,472,511, all of which are incorporated herein by reference in their entireties. In some embodiments, the carbohydrate content of an antibody (or a relevant portion or component thereof) is modified by deleting one or more endogenous carbohydrate moieties of the antibody. In certain embodiments, the technology of the present invention involves deleting a glycosylation site in the Fc region of an antibody by modifying position 297 from asparagine to alanine.

[0164] Engineered glycoforms can be useful for a variety of purposes, including, but not limited to, enhancing or reducing effector function. Engineered glycoforms can be produced by any method known to those of skill in the art, for example, by using engineered or variant expression strains, by co-expression with one or more enzymes, such as N-acetylglucosaminyltransferase III (GnTIII), by expressing an Fc region-containing molecule in a different organism or cell line from a different organism, or by modifying the carbohydrate after the Fc region-containing molecule has been expressed. Methods for producing engineered glycoforms are known in the art, examples of which include, but are not limited to, Umana et al., 1999, Nat. Biotechnol. 17: 176-180; Davies et al., 2001, Biotechnol. Bioeng. 74:288-294; Shields et al., 2002, J. Biol. Chem. 277:26733-26740; Shinkawa et al., 2003, J. Biol. Chem., each of which is incorporated by reference in its entirety. 278:3466-3473; U.S. Patent No. 6,602,684; U.S. Patent Application No. 10 / 277,370; U.S. Patent Application No. 10 / 113,929; International Patent Application Publication Nos. WO 00 / 61739 A1; WO 01 / 292246 A1; WO 02 / 311140 A1; WO 02 / 30954 A1; POTILLEGENT™ technology (Biowa, Inc., Princeton, NJ); GLYCOMAB™ glycosylation engineering technology (GLYCART biotechnology AG, Zurich, Switzerland). See, for example, International Patent Application Publication No. WO 00 / 061739; U.S. Patent Publication No. 2003 / 0115614; Okazaki et al., 2004, JMB, 336: 1239-49.

[0165] Fusion Protein. In one embodiment, the anti-DLL3 antibody of the present technology is a fusion protein. When fused to a second protein, the anti-DLL3 antibody of the present technology can be used as an antigenic tag. Examples of domains that can be fused to a polypeptide include heterologous signal sequences as well as other heterologous functional regions. Fusion does not necessarily have to be direct fusion, but can occur via a linker sequence. Furthermore, the fusion protein of the present technology can also be engineered to improve the characteristics of the anti-DLL3 antibody. For example, additional amino acids, particularly a region of charged amino acids, can be added to the N-terminus of the anti-DLL3 antibody to improve stability and persistence during purification from host cells or subsequent handling and storage. Peptide moieties can also be added to the anti-DLL3 antibody to facilitate purification. Such regions can be removed before the final preparation of the anti-DLL3 antibody. The addition of peptide moieties to facilitate handling of a polypeptide is a routine technique well known in the art. The anti-DLL3 antibody of the present technology can also be fused to a marker sequence, such as a peptide that facilitates purification of the fused polypeptide. In selected embodiments, the amino acid sequence of the marker is a hexahistidine peptide (SEQ ID NO: 84), such as the tag provided in the pQE vector (QIAGEN, Inc., Chatsworth, Calif.), among others, many of which are commercially available. For example, hexahistidine (SEQ ID NO: 84) allows for convenient purification of the fusion protein, as described by Gentz ​​et al., Proc. Natl. Acad. Sci. USA 86: 821-824, 1989. Another peptide tag useful for purification, the "HA" tag, corresponds to an epitope derived from the influenza hemagglutinin protein. Wilson et al., Cell 37: 767, 1984.

[0166] Thus, any of these above fusion proteins can be engineered using the polynucleotides or polypeptides of the present technology. In some embodiments, the fusion proteins described herein also exhibit increased half-life in vivo. Fusion proteins with disulfide-bonded dimeric structures (e.g., IgG) can be more efficient at binding and neutralizing other molecules than monomeric secreted proteins or protein fragments alone. Fountoulakis et al., J. Biochem. 270: 3958-3964, 1995.

[0167] Similarly, EP-A-0464533 (Canadian counterpart 2045869) discloses fusion proteins comprising various portions of the constant region of an immunoglobulin molecule together with another human protein or fragment thereof. In many cases, the Fc portion in a fusion protein is beneficial in therapy and diagnosis, and can therefore result in, for example, improved pharmacokinetic properties. See EP-A-0232262. Alternatively, it may be desirable to delete or modify the Fc portion after the fusion protein has been expressed, detected, and purified. For example, if the fusion protein is used as an antigen for immunization, the Fc portion may interfere with therapy and diagnosis. In drug discovery, for example, human proteins, such as hIL-5, have been fused to an Fc portion for the purpose of high-throughput screening assays to identify hIL-5 antagonists. Bennett et al., J. Molecular Recognition 8: 52-58, 1995; Johanson et al., J. Biol. Chem., 270: 9459-9471, 1995.

[0168] Antigen preparation: DLL3 antigens can be obtained by genetically engineering host cells to produce genes encoding antigen proteins. Specifically, vectors capable of expressing the antigen gene are constructed, and the vectors are then introduced into host cells so that the genes are expressed therein, after which the expressed antigen can be purified. Antibodies can also be obtained by immunizing animals with cells expressing the antigen based on the above-mentioned genetic engineering or with cell lines expressing the antigen. Alternatively, antibodies can also be obtained without using the antigen protein by incorporating the cDNA of the antigen protein into an expression vector, then administering the expression vector to an animal to be immunized, and expressing the antigen protein in the body of the animal thus immunized, so that antibodies against the antigen protein are produced therein.

[0169] The anti-DLL3 antibody used in the present invention is not particularly limited. For example, an antibody identified by the amino acid sequence shown in the sequence listing of the present application can be preferably used. The anti-DLL3 antibody used in the present invention preferably has the following properties: (1) An antibody having the following properties: (a) specifically binds to DLL3; and (b) having the activity of being internalized into cells expressing DLL3 by binding to DLL3; or (2) The antibody according to (1) above, wherein the DLL3 is human DLL3. The method for obtaining the antibody against DLL3 of the present invention is not particularly limited, as long as an anti-DLL3 antibody can be obtained. It is preferable to use DLL3 that retains its conformation as the antigen.

[0170] An example of a method for obtaining antibodies is DNA immunization. DNA immunization involves transfecting an animal (e.g., a mouse or rat) with an antigen-expressing plasmid, then expressing the antigen in the animal to induce immunity against the antigen. Transfection approaches include direct injection of a plasmid into muscle, intravenous injection of a transfection reagent such as liposomes or polyethyleneimine, an approach using a viral vector, an approach using a gene gun to inject gold particles with a plasmid attached, and a hydrodynamic method in which a large amount of a plasmid solution is rapidly injected into a vein. Regarding the transfection method of intramuscular injection of an expression plasmid, a technique called in vivo electroporation involves applying electroporation to the site of intramuscular injection of the plasmid, which is known as an approach to improve expression levels (Aihara H, Miyazaki J. Nat Biotechnol. 1998 Sep; 16 (9): 867-70 or Mir LM, Bureau MF, Gehl J, Rangara R, Rouy D, Caillaud JM, Delaere P, Branellec D, Schwartz B, Scherman D. Proc Natl Acad Sci U S A. 1999 Apr 13; 96 (8): 4262-7. This approach further improves expression levels by treating the muscle with hyaluronidase before intramuscular injection of the plasmid (McMahon JM1, Signori E, Wells KE, Fazio VM, Wells DJ., Gene Ther. 2001 Aug; 8 (16): 1264-70). Furthermore, hybridoma production may be carried out by known methods, or may be carried out using, for example, the Hybrimune Hybridoma Production System (Cyto Pulse Sciences, Inc.).

[0171] A specific example of obtaining a monoclonal antibody may include the following steps: (a) An immune response can be induced by incorporating DLL3 cDNA into an expression vector (e.g., pcDNA3.1; Thermo Fisher Scientific Inc.) and directly administering the vector to an animal to be immunized (e.g., a rat or mouse) by methods such as electroporation or a gene gun so as to express DLL3 in the animal's body. Administration of the vector, such as by electroporation, may be carried out once or more, preferably multiple times, as necessary to enhance antibody titers; (b) collecting tissues (e.g., lymph nodes) containing antibody-producing cells from the aforementioned animals in which an immune response has been induced; (c) preparation of myeloma cells (hereinafter referred to as "myeloma") (e.g., mouse myeloma SP2 / 0-ag14 cells); (d) cell fusion between antibody-producing cells and myeloma; (e) selection of a group of hybridomas producing the desired antibody; (f) division into single-cell clones (cloning); (g) optionally, culturing hybridomas or breeding animals inoculated with hybridomas for the large-scale production of monoclonal antibodies; and / or (h) Studying the physiological activity (internalization activity) and binding specificity of the monoclonal antibodies thus produced, or examining the properties of the antibodies as labeling reagents.

[0172] Examples of methods for measuring antibody titers used herein include, but are not limited to, flow cytometry and Cell-ELISA. The antibodies of the present invention also include genetically engineered antibodies, such as chimeric antibodies, humanized antibodies, and human antibodies, which have been artificially modified to reduce the antigenicity of heterologous genotypes to humans, as well as the above-mentioned monoclonal antibodies against DLL3. These antibodies can be produced by known methods. Examples of chimeric antibodies include antibodies whose variable and constant regions are heterologous to each other, such as chimeric antibodies formed by conjugating the variable regions of a mouse- or rat-derived antibody to a human-derived constant region (see Proc. Natl. Acad. Sci. USA, 81, 6851-6855, (1984)).

[0173] Examples of humanized antibodies include antibodies formed by incorporating only the complementarity-determining regions (CDRs) into a human-derived antibody (see Nature (1986) 321, pp. 522-525), antibodies formed by incorporating amino acid residues from some framework and CDR sequences into a human antibody using the CDR grafting method (International Publication No. WO90 / 07861), and antibodies formed by modifying the amino acid sequences of some CDRs while maintaining the ability to bind to an antigen.

[0174] Further examples of the antibodies of the present invention include human antibodies that bind to DLL3. An anti-DLL3 human antibody refers to a human antibody that has only the gene sequence of the antibody derived from a human chromosome. Human anti-DLL3 antibodies can be obtained by a method using a human antibody-producing mouse carrying a human chromosomal fragment containing heavy and light chain genes of a human antibody (see, for example, 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., Matsuda, T. and Iijima, S. eds.), Kluwer Academic Publishers, 1999; Tomizuka, K. et al., Proc. Natl. Acad. Sci. USA (2000) 97, pp. 722-727).

[0175] Such human antibody-producing mice may be specifically produced by using genetically modified animals in which their endogenous immunoglobulin heavy and light chain loci have been disrupted and then human immunoglobulin heavy and light chain loci have been introduced in their place, such as using yeast artificial chromosome (YAC) vectors, and then producing knockout and transgenic animals from such genetically modified animals, and then breeding such animals with each other. Alternatively, anti-DLL3 human antibodies can also be obtained by transforming eukaryotic cells with cDNAs encoding the heavy and light chains of such human antibodies, or preferably with vectors containing the cDNAs, using genetic recombination techniques, and then culturing the transformed cells to produce genetically modified human monoclonal antibodies, such that the antibodies are obtained from the culture supernatant.

[0176] In this context, eukaryotic cells, preferably mammalian cells, such as CHO cells, lymphocytes or myeloma cells, for example, can be used as hosts. Furthermore, methods for obtaining phage-display-derived human antibodies selected from human antibody libraries are also known (see, for example, Wormstone, I. M. et al., Investigative Ophthalmology & Visual Science. (2002) 43 (7), pp. 2301-2308; Carmen, S. et al., Briefings in Functional Genomics and Proteomics (2002), 1 (2), pp. 189-203; Siriwardena, D. et al., Ophthalmology (2002) 109 (3), pp. 427-431).

[0177] For example, a phage display method can be applied, which involves expressing the variable region of a human antibody on the surface of a phage as a single-chain antibody (scFv), and then selecting phages that bind to the antigen (Nature Biotechnology (2005), 23, (9), pp. 1105-1116). By analyzing the genes of selected phage for their ability to bind to the antigen, the DNA sequence encoding the variable region of the human antibody that binds to the antigen can be determined. Once the DNA sequence of an scFv that binds to an antigen has been determined, an expression vector carrying the aforementioned sequence is constructed, and the constructed expression vector is then introduced into an appropriate host and expressed therein, thereby yielding a human antibody (International Publication Nos. WO92 / 01047, WO92 / 20791, WO93 / 06213, WO93 / 11236, WO93 / 19172, WO95 / 01438, and WO95 / 15388, Annu. Rev. Immunol (1994) 12, pp. 433-455, Nature Biotechnology (2005) 23 (9), pp. 1105-1116).

[0178] The amino acid substitutions in the present invention are preferably conservative amino acid substitutions. Conservative amino acid substitutions are substitutions that occur within amino acid groups related to specific amino acid side chains. Preferred amino acid groups are as follows: acidic group = aspartic acid and glutamic acid; basic group = lysine, arginine, and histidine; nonpolar group = alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan; and uncharged polar group = glycine, asparagine, glutamine, cysteine, serine, threonine, and tyrosine. Other preferred amino acid groups are as follows: aliphatic hydroxy group = serine and threonine; amide-containing group = asparagine and glutamine; aliphatic group = alanine, valine, leucine, and isoleucine; and aromatic group = phenylalanine, tryptophan, and tyrosine. Such amino acid substitutions are preferably made without impairing the properties of the substance having the original amino acid sequence.

[0179] V. Identification and Characterization of Anti-DLL3 Antibodies Methods for Identifying and / or Screening Anti-DLL3 Antibodies of the Invention. Methods useful for identifying and screening antibodies to DLL3 polypeptides for those with the desired specificity for the DLL3 protein (e.g., those that bind to the extracellular domain of DLL3) include any immunologically mediated technique known in the art. Components of the immune response can be detected in vitro by a variety of methods well known to those skilled in the art. For example, (1) cytotoxic T lymphocytes can be incubated with radiolabeled target cells, and the lysates of these target cells can be detected by the release of radioactivity; (2) helper T lymphocytes can be incubated with antigen and antigen-presenting cells, and cytokine synthesis and secretion can be measured by standard methods (Windhagen A et al., Immunity, 2: 373-80, 1995); (3) antigen-presenting cells can be incubated with whole protein antigen, and presentation of the antigen on MHC can be detected by either T lymphocyte activation assays or biophysical methods (Harding et al., Proc. Natl. Acad. Sci., 86: 4230-4, 1989); (4) mast cells can be incubated with a reagent that crosslinks their Fc-epsilon receptors, and histamine release can be measured by enzyme immunoassay (Siraganian et al., TIPS, 4: 432-437, 1983); and (5) enzyme-linked immunosorbent assay (ELISA).

[0180] Similarly, the products of an immune response in either a model organism (eg, a mouse) or a human subject can be detected by a variety of methods well known to those skilled in the art. For example, (1) the production of antibodies in response to vaccination can be easily detected by standard methods currently used in clinical laboratories, such as ELISA; (2) the migration of immune cells to sites of inflammation can be detected by scratching the surface of the skin, placing it in a sterile container, and capturing cells that migrate across the scratch site (Peters et al., Blood, 72: 1310-5, 1988); (3) the proliferation of peripheral blood mononuclear cells (PBMCs) in response to mitogens or mixed lymphocyte reactions can be measured using 3H-thymidine; (4) the phagocytic ability of granulocytes, macrophages, and other phagocytes in PBMCs can be measured by placing PBMCs in wells with labeled particles (Peters et al., Blood, 72: 1310-5, 1988); and (5) the differentiation of immune system cells can be measured by labeling PBMCs with antibodies against CD molecules, such as CD4 and CD8, and measuring the percentage of PBMCs that express these markers.

[0181] In one embodiment, the anti-DLL3 antibody of the technology of the present invention is selected using the display of DLL3 peptide on the surface of replicable genetic packaging.See, for example, U.S. Patent Nos. 5,514,548; 5,837,500; 5,871,907; 5,885,793; 5,969,108; 6,225,447; 6,291,650; 6,492,160; EP585287; EP605522; EP616640; EP1024191; EP589877; EP774511; EP844306.A method useful for producing / selecting filamentous bacteriophage particles containing phagemid genomes encoding binding molecules with desired specificity is described. See, for example, EP774511; US5871907; US5969108; US6225447; US6291650; US6492160.

[0182] In some embodiments, the anti-DLL3 antibodies of the present technology are selected using display of a DLL3 peptide on the surface of a yeast host cell. A method useful for isolating scFv polypeptides by yeast surface display is described by Kieke et al., Protein Eng. 1997 Nov; 10(11): 1303-10.

[0183] In some embodiments, the anti-DLL3 antibodies of the present technology are selected using ribosome display. Methods useful for identifying ligands in peptide libraries using ribosome display are described by Mattheakis et al., Proc. Natl. Acad. Sci. USA 91: 9022-26, 1994; and Hanes et al., Proc. Natl. Acad. Sci. USA 94: 4937-42, 1997. In certain embodiments, the anti-DLL3 antibodies of the present technology are selected using tRNA display of a DLL3 peptide. A method useful for selecting ligands using in vitro tRNA display is described by Merryman et al., Chem. Biol., 9: 741-46, 2002. In one embodiment, the anti-DLL3 antibodies of the present technology are selected using RNA display. Methods useful for selecting peptides and proteins using RNA display libraries are described by Roberts et al. Proc. Natl. Acad. Sci. USA, 94: 12297-302, 1997; and Nemoto et al., FEBS Lett., 414: 405-8, 1997. Methods useful for selecting peptides and proteins using non-natural RNA display libraries are described by Frankel et al., Curr. Opin. Struct. Biol., 13: 506-12, 2003.

[0184] In some embodiments, the anti-DLL3 antibodies of the present technology are expressed in the periplasm of Gram-negative bacteria and mixed with labeled DLL3 protein. See WO02 / 34886. In clones expressing recombinant polypeptides with affinity for DLL3 protein, as described in Harvey et al., Proc. Natl. Acad. Sci. 22: 9193-98 2004 and U.S. Patent Publication No. 2004 / 0058403, the concentration of labeled DLL3 protein bound to the anti-DLL3 antibody increases, allowing the cells to be isolated from the rest of the library. After selection of a desired anti-DLL3 antibody, it is expected that the antibody can be produced in large quantities by any technique known to those of skill in the art, such as, for example, by prokaryotic or eukaryotic expression. Anti-DLL3 antibodies, including, but not limited to, anti-DLL3 hybrid antibodies or fragments, can be produced by using conventional techniques to construct expression vectors encoding antibody heavy chains, in which the CDRs necessary to retain the original species antibody binding specificity, and optionally a minimal portion of the variable region framework (such as engineered by the techniques described herein), are derived from the source species antibody, and the remainder of the antibody is derived from a target species immunoglobulin, which can be engineered as described herein, thereby creating a vector for expression of the hybrid antibody heavy chain.

[0185] Measuring DLL3 Binding. In some embodiments, a DLL3 binding assay refers to an assay format in which a DLL3 protein and an anti-DLL3 antibody are mixed under conditions suitable for binding between the DLL3 protein and the anti-DLL3 antibody, and the amount of binding between the DLL3 protein and the anti-DLL3 antibody is assessed. The amount of binding is compared to a suitable control, which can be the amount of binding in the absence of the DLL3 protein, the amount of binding in the presence of a nonspecific immunoglobulin composition, or both. The amount of binding can be assessed by any suitable method. Binding assay methods include, for example, ELISA, radioimmunoassay, scintillation proximity assay, fluorescence energy transfer assay, liquid chromatography, membrane filtration assay, etc. Biophysical assays for directly measuring DLL3 protein binding to an anti-DLL3 antibody include, for example, nuclear magnetic resonance, fluorescence, fluorescence polarization, surface plasmon resonance (BIACORE chip), biolayer interferometry, etc. Specific binding is determined by standard assays known in the art, such as radioligand binding assays, ELISA, FRET, immunoprecipitation, SPR, NMR (2D-NMR), mass spectrometry, etc. If the specific binding of the candidate anti-DLL3 antibody is at least 1 percent greater than the binding observed in the absence of the candidate anti-DLL3 antibody, then the candidate anti-DLL3 antibody is useful as an anti-DLL3 antibody in the present technology.

[0186] By combining sequences that show high identity to the above-mentioned heavy chain and light chain amino acid sequences, it is possible to select antibodies with biological activity equivalent to that of each of the above-mentioned antibodies. Such identity is generally 80% or more, preferably 90% or more, more preferably 95% or more, and most preferably 99% or more. Furthermore, by combining heavy and light chain amino acid sequences that contain substitutions, deletions, or additions of one or several amino acid residues compared to the heavy or light chain amino acid sequences, it is also possible to select antibodies with biological activity equivalent to that of each of the above-mentioned antibodies. Identity between two types of amino acid sequences can be determined by aligning the sequences using the default parameters of Clustal W version 2 (Larkin MA, Blackshields G, Brown NP, Chenna R, McGettigan PA, McWilliam H, Valentin F, Wallace IM, Wilm A, Lopez R, Thompson JD, Gibson TJ and Higgins DG (2007), "Clustal W and Clustal X version 2.0", Bioinformatics. 23 (21): 2947-2948).

[0187] If a newly generated human antibody binds to the partial peptide or partial three-dimensional structure bound by any one of the rat anti-human DLL3 antibodies described in the present description, it can be determined that the human antibody binds to the same epitope as the rat anti-human DLL3 antibody, chimeric anti-human DLL3 antibody, or humanized anti-human DLL3 antibody. Alternatively, by confirming that the human antibody competes with the rat anti-human DLL3 antibody, chimeric anti-human DLL3 antibody, or humanized anti-human DLL3 antibody described in the present description for antibody binding to DLL3, it can be determined that the human antibody binds to the same epitope as the rat anti-human DLL3 antibody, chimeric anti-human DLL3 antibody, or humanized anti-human DLL3 antibody described in the present description, even if the specific sequence or structure of the epitope has not been determined. In the present description, if a human antibody is determined to "bind to the same epitope" by at least one of these determination methods, as with the rat anti-human DLL3 antibody, chimeric anti-human DLL3 antibody, or humanized anti-human DLL3 antibody described in the present description, it is concluded that the newly prepared human antibody "binds to the same epitope." If it is confirmed that a human antibody binds to the same epitope, it is predicted that the human antibody will have biological activity equivalent to that of the rat anti-human DLL3 antibody, chimeric anti-human DLL3 antibody, or humanized anti-human DLL3 antibody.

[0188] The chimeric, humanized, or human antibodies obtained by the above-described methods are evaluated for their binding activity to antigens by known methods, etc., so that preferred antibodies can be selected. Another example of an indicator for comparing antibody properties is antibody stability. Differential scanning calorimetry (DSC) is an instrument capable of rapidly and accurately measuring the thermal denaturation midpoint (Tm), which serves as an excellent indicator for the relative structural stability of proteins. Differences in thermal stability can be compared by measuring Tm values ​​using DSC and comparing the obtained values. It is known that the storage stability of antibodies has a specific correlation with their thermal stability (Lori Burton, et al., Pharmaceutical Development and Technology (2007) 12, pp. 265-273). Therefore, preferred antibodies can be selected using thermal stability as an indicator. Other examples of indicators for antibody selection include high yield in suitable host cells and low aggregation in aqueous solutions. For example, because the antibody with the highest yield does not necessarily exhibit the highest thermal stability, it is necessary to comprehensively determine the thermal stability based on the above-mentioned indicators to select the most suitable antibody for human administration.

[0189] The obtained antibody may be purified to a homogeneous state. Separation and purification methods commonly used for proteins can be used to separate and purify antibodies. For example, column chromatography, filtration, ultrafiltration, salting out, dialysis, preparative polyacrylamide gel electrophoresis, and isoelectric focusing are appropriately selected and combined to separate and purify antibodies (Strategies for Protein Purification and Characterization: A Laboratory Course Manual, Daniel R. Marshak et al. eds., Cold Spring Harbor Laboratory Press (1996); and Antibodies: A Laboratory Manual. Ed Harlow and David Lane, Cold Spring Harbor Laboratory (1988)). Examples of separation and purification methods include, but are not limited to, column chromatography, filtration, ultrafiltration, salting out, dialysis, preparative polyacrylamide gel electrophoresis, and isoelectric focusing.

[0190] Examples of chromatography include affinity chromatography, ion exchange chromatography, hydrophobic chromatography, gel filtration chromatography, reversed phase chromatography, and absorption chromatography. These chromatographic techniques can be performed using liquid chromatography, such as HPLC or FPLC. Examples of columns used in affinity chromatography include Protein A and Protein G. Examples of columns that use Protein A include Hyper D, POROS, and Sepharose FF (Pharmacia). Antibodies can also be purified by using a carrier on which an antigen is immobilized and utilizing the binding activity of the antibody to the antigen.

[0191] VI. Anti-DLL3 Antibody-Drug Conjugates (ADCs) The anti-DLL3 antibodies described herein (e.g., 2-C8-A, 6-G23-F, and 10-O18-A or human antibodies: H2-C8-A, H2-C8-A-2, H2-C8-A-3, H6-G23-F, H6-G23-F-2, H6-G23-F-3, H10-O18-A, H10-O18-A-2, and H10-O18-A-3) can be conjugated to a drug via a linker structure moiety to prepare an anti-DLL3 antibody-drug conjugate (ADC). The drug is not particularly limited as long as it has a substituent or moiety that can be connected to the linker structure. Anti-DLL3 antibody-drug conjugates (ADCs) can be used for various purposes depending on the conjugated drug. Examples of such drugs include substances with antitumor activity, substances effective in treating blood disorders, substances effective in treating autoimmune diseases, anti-inflammatory substances, antimicrobial substances, antifungal substances, antiparasitic substances, antiviral substances, and anti-anesthetic substances. The antibody-drug conjugates of the present invention have the following formula:

[0192] [ka] where m1 represents the number of drug molecules conjugated per antibody molecule in the antibody-drug conjugate, Ab represents an antibody or a functional fragment of the antibody, L represents a linker connecting Ab and D, and D represents a drug.

[0193] A. Drugs Drug D conjugated to the antibody-drug conjugate of the present invention is described herein. Drug D of the present invention is preferably an anti-tumor compound. The anti-tumor compound exerts its anti-tumor effect when the linker is partially or entirely cleaved in tumor cells, releasing the anti-tumor compound moiety. When the linker and drug are cleaved at the binding moiety, the anti-tumor compound in its original structure is released, thereby exerting its original anti-tumor effect. The antitumor compound in the antibody-drug conjugate of the present invention is a pyrrolobenzodiazepine derivative (PBD derivative) represented by the general formula (V):

[0194] [ka] The variables of the formula are described in more detail below.

[0195] The asterisk represents the bond to the linker L. n 1 represents an integer of 2 to 8, preferably an integer of 2 to 6, and more preferably an integer of 3 to 5. The alkyl chain is denoted by the subscript n 1 is an integer of 2 to 8, preferably an integer of 2 to 6, more preferably an integer of 3 to 5, and may contain a double bond. A represents a spiro-bonded 3- to 5-membered saturated hydrocarbon ring or 3- to 5-membered saturated heterocycle, preferably a 3- to 5-membered saturated hydrocarbon ring (cyclopropane, cyclobutane, or cyclopentane), more preferably cyclopropane or cyclobutane, and most preferably cyclopropane. The spiro-bonded 3- to 5-membered saturated hydrocarbon ring may be substituted with 1 to 4 halogen atoms, preferably 1 or 2 fluorine atoms (e.g., 2,2-difluorocyclopropane). R 1 and R 2 each independently represents a C1-C6 alkoxy group, a C1-C6 alkyl group, a hydrogen atom, a hydroxy group, a thiol group, a C1-C6 alkylthio group, a halogen atom, or -NR'R''; each is preferably a C1-C6 alkoxy group, a C1-C6 alkyl group, or a hydroxy group, more preferably a C1-C3 alkoxy group, and most preferably a methoxy group. R 3 , R 4 , and R 5 is as described in any one of (i) to (iii) below. i. Compound Embodiment 1 R 3 and R 4 However, as shown below, R 3 and R 4When combined with the carbon atom to which it is attached to form a double bond:

[0196] [ka] R 5 represents an aryl group or heteroaryl group optionally having one or more substituents selected from Group 1, or a C1-C6 alkyl group optionally having one or more substituents selected from Group 2, preferably an aryl group optionally having one or more substituents selected from Group 1.

[0197] R 5 The "aryl group" in the "aryl group or heteroaryl group optionally having one or more substituents selected from Group 1" is preferably a phenyl group or a naphthyl group, and more preferably a phenyl group. R 5 The "heteroaryl group" in the "aryl group or heteroaryl group optionally having one or more substituents selected from Group 1" is preferably a thienyl group, a pyridyl group, a pyrimidyl group, a quinolyl group, a quinoxalyl group, or a benzothiophenyl group, more preferably a 2-thienyl group, a 3-thienyl group, a 2-pyridyl group, a 3-pyridyl group, or a 4-pyridyl group, and even more preferably a 3-pyridyl group or a 3-thienyl group.

[0198] R 5 Examples of substituents on the aryl or heteroaryl group for the above include, but are not limited to, the following a) to j): a) a C1-C6 alkoxy group optionally substituted with 1 to 3 halogen atoms; b) a C1-C6 alkyl group optionally substituted with 1 to 3 halogen atoms, a hydroxy group, -OCOR', -NR'R'', -C(=NR')-NR''R''', and -NHC(=NR')-NR''R''', c) a halogen atom, d) a C3-C5 cycloalkoxy group, e) a C1-C6 alkylthio group, f)-NR'R'', g)-C(=NR')-NR''R'''', h)-NHC(=NR')-NR''R'''', i) -NHCOR', and j) Hydroxy groups. Here, R', R'', and R''' in b) and f) to i) each independently represent a hydrogen atom or a C1-C6 alkyl group, and preferably each independently represent a hydrogen atom or a C1-C3 alkyl group.

[0199] a) to j) are preferably as follows: a) a C1-C3 alkoxy group optionally substituted with 1 to 3 halogen atoms, more preferably a methoxy group, an ethoxy group, an n-propoxy group, an i-propoxy group, or a trifluoromethoxy group, even more preferably a methoxy group, an ethoxy group, or a trifluoromethoxy group, and most preferably a methoxy group; b) a C1-C3 alkyl group, a hydroxy group, -OCOR', -C(=NR')-NR''R''', or -NHC(=NR')-NR''R''' (wherein R', R'', and R''' are each independently a hydrogen atom or a C1-C3 alkyl group, and more preferably, a C1-C3 alkyl group, optionally substituted with 1 to 3 halogen atoms, a hydroxy group, -OCOR', -C(=NR')-NR''R'''', or -NHC(=NR')-NR''R'' ... R"R"', wherein R', R", and R"' are each independently a hydrogen atom or a methyl group, and more preferably a methyl group, an ethyl group, an n-propyl group, an i-propyl group, a fluoromethyl group, a difluoromethyl group, a trifluoromethyl group, a hydroxymethyl group, -CH2OCOMe, -CH2-NHC(=NH)-NH2, or -CH2-NHC(=NMe)-NH2; c) a halogen atom, preferably a fluorine atom or a chlorine atom; d) a C3-C5 cycloalkoxy group, more preferably a cyclopropoxy group; e) a C1-C3 alkylthio group, more preferably a methylthio group or an ethylthio group; f) -NR'R'' (wherein R' and R'' are each independently a hydrogen atom or a C1-C3 alkyl group, more preferably -NH2, -NHMe, -NMe2, -NHEt, or -NEt2); g) —C(═NR′)—NR″R′″, wherein R′, R″, and R′″ are each independently a hydrogen atom or a C1-C3 alkyl group, more preferably —C(═NH)—NH2 or —C(═NMe)—NH2; h) -NHC(=NR')-NR''R''' [wherein R', R'', and R''' each independently represent a hydrogen atom or a C1-C3 alkyl group, more preferably -NHC(=NH)-NH2 or -NHC(=NMe)-NH2]; i) -NHCOR', where R' is a hydrogen atom or a C1-C3 alkyl group, more preferably -NHCOMe or -NHCOEt; and j) Hydroxy groups.

[0200] R 5 The aryl group (preferably a phenyl group) or heteroaryl group (preferably a pyridyl group) in the formula (I) may have at least one substituent at any position. When multiple substituents are present, the substituents may be the same or different. R 5 When is an aryl group, each substituent is preferably a), b), d), g), h), or j), more preferably a), b), d), or j). R 5 is a phenyl group, R 5 R may have a substituent at any position, or may have multiple substituents, preferably one or two substituents at the 3-position and / or 4-position, more preferably one substituent at the 4-position. 5 is a naphthyl group, R 5may have a substituent at any position, and may have multiple substituents, preferably one substituent at the 6-position.

[0201] R 5 is a phenyl group, R 5 is more preferably a phenyl group, a 4-methoxyphenyl group, a 3-methoxyphenyl group, a 4-ethoxyphenyl group, a 4-(n-propoxy)-phenyl group, a 4-(i-propoxy)-phenyl group, a 4-cyclopropoxy-phenyl group, a 4-trifluoromethylphenyl group, a 4-hydroxymethyl-phenyl group, a 4-acetoxymethyl-phenyl group, or a 4-carbamimidamidomethyl-phenyl group, and even more preferably a phenyl group, a 4-methoxyphenyl group, a 3-methoxyphenyl group, a 4-cyclopropoxy-phenyl group, a 4-hydroxymethyl-phenyl group, a 4-acetoxymethyl-phenyl group, a 4-carbamimidamidomethyl-phenyl group, or a 4-trifluoromethylphenyl group.

[0202] R 5 is a naphthyl group, R 5 is more preferably a naphthyl group or a 6-methoxy-2-naphthyl group. Most preferably, it is a 4-methoxyphenyl group. R 5 When is a heteroaryl group, each substituent is preferably a), b), d), g), h), or j), more preferably a) or b). R 5 When R is a heteroaryl group, 5 R may have at least one substituent at any position. 5 When R is a 3-pyridyl group, the substituents are preferably located at the 6- and / or 5-positions. 5 When R is 2-pyridyl, the substituents are preferably at the 5- and / or 4-positions, or at the 5- and / or 6-positions. 5 When is 4-pyridyl, the substituents are preferably at the 2- and / or 6-positions.

[0203] R 5 When R is a heteroaryl group, 5 may have multiple substituents, preferably has one or two substituents, and more preferably has one substituent. R 5 is a pyridyl group, R 5 is preferably a 6-methoxy-3-pyridyl group or a 6-methyl-3-pyridyl group. R 5 is a 3-thienyl group or a 6-quinoxalyl group, R 5 is preferably unsubstituted. R 5 The "C1-C6 alkyl group" in the "C1-C6 alkyl group optionally having one or more substituents selected from Group 2" is preferably a C1-C3 alkyl group, more preferably a methyl group or an ethyl group.

[0204] R 5 The substituents in the "C1-C6 alkyl group optionally having one or more substituents selected from Group 2" are each a halogen atom, a hydroxy group, or a C1-C6 alkoxy group (preferably a C1-C3 alkoxy group), preferably a hydroxy group, a methoxy group, or an ethoxy group, more preferably a hydroxy group. ii. Compound embodiment 2 R 3 If represents a hydrogen atom, R 4 and R 5 is R 4 and R 5 is combined with the carbon atom to which it is attached to form a 3- to 5-membered saturated hydrocarbon ring or a 3- to 5-membered saturated heterocyclic ring, as shown below, or CH2=:

[0205] [ka] The 3- to 5-membered saturated hydrocarbon ring may be substituted with 1 to 4 halogen atoms, preferably 1 or 2 fluorine atoms.

[0206] R 4 and R 5 are preferably combined to form a 3- to 5-membered saturated hydrocarbon ring or CH2=, more preferably to form cyclopropane, cyclobutane, or CH2= (exomethylene group), even more preferably to form cyclopropane. R 4 and R 5 are combined to form a 3- to 5-membered saturated hydrocarbon ring or a 3- to 5-membered saturated heterocyclic ring, the 3- to 5-membered saturated hydrocarbon ring or the 3- to 5-membered saturated heterocyclic ring is preferably the same as A. More preferably, A is a 3- to 5-membered saturated hydrocarbon ring, and R 4 and R 5 are combined to form a 3- to 5-membered saturated hydrocarbon ring, and even more preferably, A is a cyclopropane ring and R 4 and R 5 are combined to form a cyclopropane ring.

[0207] iii. Compound embodiment 3 R 3 , R 4 , and R 5 is R 3 and the carbon atom to which R is bonded 4 and R 5 are combined together with the carbon atom to which they are attached to form a benzene ring or a 6-membered heterocycle, optionally bearing one or more substituents selected from Group 3. The following formula is R 3 and R 4 are combined to form a benzene ring optionally bearing one or more substituents:

[0208] [ka] The benzene ring or heterocycle may have at least one substituent at any position. When multiple substituents are present, the substituents may be the same or different. Each substituent on the benzene ring or heterocycle is a halogen atom, a C1-C6 alkyl group optionally substituted with 1 to 3 halogen atoms, or a C1-C6 alkoxy group, preferably a halogen atom, a C1-C3 alkyl group optionally substituted with 1 to 3 halogen atoms, or a C1-C3 alkoxy group, more preferably a halogen atom, a methyl group, or a methoxy group. The "benzene ring or 6-membered heterocyclic ring optionally having one or more substituents" is preferably an unsubstituted benzene ring.

[0209] R 3 , R 4 and R 5 most preferably satisfies subsection (i) "Compound embodiment 1" above. R 6 and R 7 Each represents a hydrogen atom, or R 6 and R 7 taken together represent an imine bond (C=N). R 8 is a hydroxy group or a C1-C3 alkoxy group, preferably a hydroxy group or a methoxy group, more preferably a hydroxy group. 8 may be a bisulfite adduct (OSO3M, where M is a metal cation). R 8 is attached to an asymmetric carbon atom, providing the configuration represented by the following partial structure (Va) or (Vb): In general formula (V), each wavy line represents a bond to Y, and each asterisk represents a bond to L.

[0210] [ka] X and Y each independently represent an oxygen atom, a nitrogen atom, or a sulfur atom, and are preferably an oxygen atom. The drug D of the present invention is preferably any one compound selected from the following group:

[0211] [ka] In the formula, each asterisk represents where a drug is attached to the linker structure (L).

[0212] In some embodiments, the -OH is at the 11' position.

[0213] B. Linker Structure The linker structure for binding an antitumor drug to an antibody in the antibody-drug conjugate of the present invention is described below. The linker L has the following formula: -Lb-La-Lp-NH-B-CH2-O(C=O)- * where the asterisk represents the bond to the nitrogen atom at the N10' position of drug D, and Lb represents a spacer connecting La to the glycan or remodeled glycan of Ab.

[0214] B represents a phenyl group or a heteroaryl group, preferably a 1,4-phenyl group, a 2,5-pyridyl group, a 3,6-pyridyl group, a 2,5-pyrimidyl group, or a 2,5-thienyl group, and more preferably a 1,4-phenyl group. Lp represents a linker consisting of an amino acid sequence that is cleavable in vivo or in target cells, for example, by the action of an enzyme such as an esterase or peptidase. Lp is a peptide residue composed of 2 to 7 (preferably 2 to 4) amino acids, that is, Lp is composed of an oligopeptide residue in which 2 to 7 amino acids are connected via peptide bonds. The N-terminus of Lp is linked to the carbonyl group of La in the form of Lb-La-, and the C-terminus forms an amide bond with the amino group (-NH-) of the -NH-B-CH2-O(C=O)- moiety of the linker. The bond between the C-terminus of Lp and -NH- is cleaved by enzymes such as esterases.

[0215] The amino acids constituting Lp are not limited to any particular amino acid and may be, for example, L- or D-amino acids, preferably L-amino acids. The amino acids may include not only α-amino acids but also amino acids having the structure of, for example, β-alanine, ε-aminocaproic acid, or γ-aminobutyric acid, and may further include unnatural amino acids such as N-methylated amino acids. The amino acid sequence of Lp is not limited to a specific amino acid sequence, and examples of amino acids constituting Lp include, but are not limited to, glycine (Gly; G), valine (Val; V), alanine (Ala; A), phenylalanine (Phe; F), glutamic acid (Glu; E), isoleucine (Ile; I), proline (Pro; P), citrulline (Cit), leucine (Leu; L), serine (Ser; S), lysine (Lys; K), and aspartic acid (Asp; D). Among these, glycine (Gly; G), valine (Val; V), alanine (Ala; A), and citrulline (Cit) are preferred.

[0216] Any of these amino acids may occur multiple times, and Lp has an amino acid sequence containing any selected amino acid. Drug release patterns can be controlled through the type of amino acid. Specific sequences of the linker Lp include, but are not limited to, -GGVA- (SEQ ID NO: 85), -GG-(D-)VA-, -VA-, -GGFG- (SEQ ID NO: 86), -GGPI- (SEQ ID NO: 87), -GGVCit- (SEQ ID NO: 88), -GGVK- (SEQ ID NO: 89), -GG(D-)PI-, -GGPL- (SEQ ID NO: 90), -EGGVA (SEQ ID NO: 91), -PI-, -GGF-, DGGF- (SEQ ID NO: 92), (D-)D-GGF-, -EGGF- (SEQ ID NO: 93), -SGGF- (SEQ ID NO: 94), -KGGF- (SEQ ID NO: 95), -DGGFG- (SEQ ID NO: 96), -GGFGG- (SEQ ID NO: 97), -DDGGFG- (SEQ ID NO: 98), -KDGGFG- (SEQ ID NO: 99), and -GGFGGGF- (SEQ ID NO: 100). Here, "(D-)V" indicates D-valine, "(D)-P" indicates D-proline, and "(D-)D" indicates D-aspartic acid.

[0217] The linker Lp is preferably one of the following: -GGVA- (SEQ ID NO: 85), -GG-(D-)VA-, -VA-, -GGFG- (SEQ ID NO: 86), -GGPI- (SEQ ID NO: 87), -GGVCit- (SEQ ID NO: 88), -GGVK- (SEQ ID NO: 89), -GG(D-)PI-, and -GGPL- (SEQ ID NO: 90). The linker Lp is more preferably one of the following: -GGVA- (SEQ ID NO: 85), -GGVCit- (SEQ ID NO: 88), and -VA-. Lb represents a spacer connecting La to the glycan or remodeled glycan of Ab. In some embodiments, Lb is selected from the group: -C(=O)-(CH2CH2)n 2 -C(=O)-, -C(=O)-(CH2CH2)n 2 -C(=O)-NH-(CH2CH2)n 3 -C(=O)-, -C(=O)-(CH2CH2)n 2 -C(=O)-NH-(CH2CH2O)n 3 -CH2-C(=O)-, -C(=O)-(CH2CH2)n 2 -NH-C(=O)-(CH2CH2O)n 3 -CH2CH2-C(=O)-, -(CH2)n 4 -OC(=O)- represents one selected from In the formula, n 2 represents an integer of 1 to 3 (preferably 1 or 2), and n 3 represents an integer of 1 to 5 (preferably an integer of 2 to 4, more preferably 2 or 4), and n 4 represents an integer of 0 to 2 (preferably 0 or 1).

[0218] In some embodiments, La is preferably selected from the group: -C(=O)-CH2CH2-C(=O)-, -C(=O)-(CH2CH2)2-C(=O)-, -C(=O)-CH2CH2-C(=O)-NH-(CH2CH2)2-C(=O)- -C(=O)-CH2CH2-C(=O)-NH-(CH2CH2O)2-CH2-C(=O)-, -C(=O)-CH2CH2-NH-C(=O)-(CH2CH2O)4-CH2CH2-C(=O)-, -CH2-OC(=O)- and -OC(=O)- and La is more preferably —C(═O)—CH 2 CH 2 —C(═O)— or —C(═O)—(CH 2 CH 2 ) 2 —C(═O)—. The spacer Lb is not limited to a particular spacer, examples of which include, but are not limited to, spacers represented by the following formulas:

[0219] [ka]

[0220] In the structural formula of Lb shown above, each asterisk represents a -(C=O) or -(CH2)n at the left end of La. 4Each wavy line represents Ab binding to a glycan or remodeled glycan.

[0221] In each structural formula of Lb (Lb-1, Lb-2, or Lb-3) shown above, the triazole ring moiety formed through the click reaction of the azide group and DBCO provides a geometric isomeric structure, and the Lb molecule exists as either one of the two structures or as a mixture of both. m per molecule of the antibody-drug conjugate of the present invention 1 There are m "-LD" portions, 1 In each L of the "-LD" moiety, either one of the two structures is present as Lb (Lb-1, Lb-2, or Lb-3), or both of them are present simultaneously.

[0222] In some embodiments, L is preferably -Lb-La-Lp-NH-B-CH2-O(C=O)- * wherein: B is a 1,4-phenyl group; Lp is the following group: -GGVA- (SEQ ID NO: 85), -GG-(D-)VA-, -VA-, -GGFG- (SEQ ID NO: 86), -GGPI- (SEQ ID NO: 87), -GGVCit- (SEQ ID NO: 88), -GGVK- (SEQ ID NO: 89), -GG(D-)PI-, and -GGPL- (SEQ ID NO: 90) represents one selected from La is the following group: -C(=O)-CH2CH2-C(=O)-, -C(=O)-(CH2CH2)2-C(=O)-, -C(=O)-CH2CH2-C(=O)-NH-(CH2CH2)2-C(=O)-, -C(=O)-CH2CH2-C(=O)-NH-(CH2CH2O)2-CH2-C(=O)-, -C(=O)-CH2CH2-NH-C(=O)-(CH2CH2O)4-CH2CH2-C(=O)-, -CH2-OC(=O)-, -OC(=O)- represents one selected from Lb represents any of the structural formulae above for Lb.

[0223] In some embodiments, L is more preferably selected from the group: -Z 1 —C(═O)—CHCH—C(═O)—GGVA-NH-B-CH—OC(═O)— (“GGVA” disclosed as SEQ ID NO: 85); -Z 1 -C(=O)-CH2CH2-C(=O)-GG-(D-)VA-NH-B-CH2-OC(=O)-, -Z 1 -C(=O)-CH2CH2-C(=O)-VA-NH-B-CH2-OC(=O)-, -Z 1 -C(=O)-(CH2CH2)2-C(=O)-VA-NH-B-CH2-OC(=O)-, -Z 1 —C(═O)—CHCH—C(═O)—GGPI-NH-B-CH—OC(═O)— (“GGPI” disclosed as SEQ ID NO: 87); -Z 1 —C(═O)—CH2CH2—C(═O)-GGFG-NH-B-CH2-OC(═O)— (“GGFG” disclosed as SEQ ID NO: 86), -Z 1 —C(═O)—CHCH—C(═O)-GGVCit-NH-B-CH—OC(═O)— (“GGVCit” disclosed as SEQ ID NO: 88); -Z 1 —C(═O)—CHCH—C(═O)-GGVK-NH-B-CH—OC(═O)— (“GGVK” disclosed as SEQ ID NO: 89); -Z 1 —C(═O)—CHCH—C(═O)—GGPL-NH-B-CH—OC(═O)— (“GGPL” disclosed as SEQ ID NO: 90); -Z 1 -C(=O)-CH2CH2-C(=O)-NH-(CH2CH2)2-C(=O)-VA-NH-B-CH2-OC(=O)-, -Z 1-C(=O)-CH2CH2-C(=O)-NH-(CH2CH2O)2-CH2-C(=O)-VA-NH-B-CH2-OC(=O)-, -Z 1 -C(=O)-CH2CH2-NH-C(=O)-(CH2CH2O)4-CH2CH2-C(=O)-VA-NH-B-CH2-OC(=O)-, -Z 2 —OC(═O)-GGVA-NH-B-CH—OC(═O)- (“GGVA” disclosed as SEQ ID NO: 85), -Z 3 —CH—OC(═O)-GGVA-NH-B-CH—OC(═O)— (“GGVA” disclosed as SEQ ID NO: 85); wherein Z is any one selected from 1 has the following structural formula as described for Lb:

[0224] [ka] represents Z 2 has the following structural formula as described for Lb:

[0225] [ka] represents Z 3 has the following structural formula as described for Lb:

[0226] [ka] and B is a 1,4-phenyl group.

[0227] L is most preferably: -Z 1 —C(═O)—CHCH—C(═O)—GGVA-NH-B-CH—OC(═O)— (“GGVA” disclosed as SEQ ID NO: 85); -Z 1 -C(=O)-CH2CH2-C(=O)-VA-NH-B-CH2-OC(=O)-, -Z 1 -C(=O)-(CH2CH2)2-C(=O)-VA-NH-B-CH2-OC(=O)-, -Z 1 —C(═O)—CHCH—C(═O)-GGVCit-NH-B-CH—OC(═O)— (“GGVCit” disclosed as SEQ ID NO: 88); -Z 1 -C(=O)-CH2CH2-C(=O)-NH-(CH2CH2)2-C(=O)-VA-NH-B-CH2-OC(=O)-, -Z 1 -C(=O)-CH2CH2-C(=O)-NH-(CH2CH2O)2-CH2-C(=O)-VA-NH-B-CH2-OC(=O)-, and -Z 1 -C(=O)-CH2CH2-NH-C(=O)-(CH2CH2O)4-CH2CH2-C(=O)-VA-NH-B-CH2-OC(=O)-, wherein B is a 1,4-phenyl group; Z 1 has the following structural formula as described for Lb:

[0228] [ka] Represents. The antibody-drug conjugates of the present invention are presumed to exhibit antitumor activity through a process in which most of the antibody-drug conjugate molecules are transported to tumor cells, and then a portion of the linker (e.g., Lp) is cleaved by an enzyme or the like to activate the antibody-drug conjugate, thereby releasing a portion of drug D (hereinafter referred to as free drug (described below)). Therefore, it is preferred that the antibody-drug conjugates of the present invention are stable outside tumor cells.

[0229] C.Antibodies The antibodies used in the disclosed antibody-drug conjugates (ADCs) are anti-DLL3 antibodies (e.g., 2-C8-A, 6-G23-F, and 10-O18-A, or human antibodies H2-C8-A, H6-G23-F, H10-O18-A, H2-C8-A-3, and H10-O18-A-3). The disclosed antibodies that can be incorporated into the disclosed ADCs are described in detail above in Section III, "Anti-DLL3 Antibodies."

[0230] Briefly, in some embodiments, the anti-DLL3 antibody of the ADC comprises a heavy chain immunoglobulin variable domain (V H ) and light chain immunoglobulin variable domain (V L ), and (a) V H are selected from the group consisting of: (i) SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:5, respectively; (ii) SEQ ID NO:13, SEQ ID NO:14, and SEQ ID NO:15, respectively; (iii) SEQ ID NO:23, SEQ ID NO:24, and SEQ ID NO:25, respectively; and (iv) SEQ ID NO:33, SEQ ID NO:34, and SEQ ID NO:35, respectively. H -CDR1 sequence, V H -CDR2 sequence, and V H - comprising a CDR3 sequence; and / or (b) V L are selected from the group consisting of: (i) SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:10, respectively; (ii) SEQ ID NO:18, SEQ ID NO:19, and SEQ ID NO:20, respectively; (iii) SEQ ID NO:28, SEQ ID NO:29, and SEQ ID NO:30, respectively; and (iv) SEQ ID NO:38, SEQ ID NO:39, and SEQ ID NO:40, respectively. L -CDR1 sequence, V L -CDR2 sequence, and V L -Contains CDR3 sequences.

[0231] In some embodiments, the anti-DLL3 antibody of the ADC may comprise one or more of the following characteristics: (a) a light chain immunoglobulin variable domain sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to a light chain immunoglobulin variable domain sequence present in any one of SEQ ID NOs: 7, 17, 27, 37, 62, 66, or 70; and / or (b) a heavy chain immunoglobulin variable domain sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to a heavy chain immunoglobulin variable domain sequence present in any one of SEQ ID NOs: 2, 12, 22, 32, 59, 60, 61, 63, 64, 65, 67, 68, or 69.

[0232] In some embodiments, the anti-DLL3 antibody of the ADC comprises a heavy chain immunoglobulin variable domain (V H ) or heavy and light chain immunoglobulin variable domains (V L ) or light chain, and (a) V H or the heavy chain comprises an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:12, SEQ ID NO:22, SEQ ID NO:32, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:68, and SEQ ID NO:69; and / or (b) V L Or the light chain comprises an amino acid sequence selected from the group consisting of SEQ ID NO:7, SEQ ID NO:17, SEQ ID NO:27, SEQ ID NO:37, SEQ ID NO:62, SEQ ID NO:66, and SEQ ID NO:70. H or heavy chain amino acid sequence and V LAlternatively, the light chain amino acid sequences are SEQ ID NO:2 and SEQ ID NO:7, respectively (7-I1-B); SEQ ID NO:12 and SEQ ID NO:17, respectively (2-C8-A); SEQ ID NO:59 and SEQ ID NO:62, respectively (H2-C8-A); SEQ ID NO:60 and SEQ ID NO:62, respectively (H2-C8-A-2); SEQ ID NO:61 and SEQ ID NO:62, respectively (H2-C8-A-3); SEQ ID NO:22 and SEQ ID NO:27, respectively (10-O18-A); SEQ ID NO:67 and SEQ ID NO:70, respectively (H1 H10-O18-A; SEQ ID NO: 68 and SEQ ID NO: 70, respectively (H10-O18-A-2); SEQ ID NO: 69 and SEQ ID NO: 70 (H10-O18-A-3); SEQ ID NO: 32 and SEQ ID NO: 37, respectively (H6-G23-F); SEQ ID NO: 63 and SEQ ID NO: 66, respectively (H6-G23-F); SEQ ID NO: 64 and SEQ ID NO: 66, respectively (H6-G23-F-2); and SEQ ID NO: 65 and SEQ ID NO: 66, respectively (H6-G23-F-3).

[0233] In some embodiments, the anti-DLL3 antibodies of the ADCs may have remodeled or modified glycans, as discussed in more detail below. Functional fragments of the present invention include those that have a sufficiently conserved asparagine (Asn297) and the amino acids surrounding Asn297 to be modified with N-linked glycans in the Fc region of an IgG heavy chain, while retaining antigen-binding activity.

[0234] i. Glycan remodeling of the disclosed anti-DLL3 antibodies: Recently, methods for uniformly introducing functionalized glycans into antibody glycoproteins by enzymatic remodeling have been reported (ACS Chemical Biology 2012, 7, 110, ACS Medicinal Chemistry Letters 2016, 7, 1005). Attempts have been made to use this glycan remodeling technology to synthesize uniform ADCs by site-specific drug introduction (Bioconjugate Chemistry 2015, 26, 2233, Angew. Chem. Int. Ed. 2016, 55, 2361-2367, US2016361436). In the glycan remodeling method of the present invention, heterogeneous glycans attached to a protein (e.g., an antibody) are cleaved using a hydrolase, leaving only GlcNAc at each end, thereby generating a homogeneous protein moiety (hereinafter referred to as the "acceptor") bearing GlcNAc. Subsequently, a separately prepared arbitrary glycan (hereinafter referred to as the "donor") is provided, and the acceptor and donor are linked to each other using a transglycosidase. This allows the synthesis of homogeneous glycoproteins with arbitrary glycan structures.

[0235] As used herein, "glycan" refers to a structural unit of two or more monosaccharides linked together via a glycosidic bond. Specific monosaccharides and glycans are sometimes abbreviated, for example, as "GlcNAc-," "MSG-," etc. When any of these abbreviations is used in a structural formula, it is intended that the oxygen or nitrogen atom involved in the glycosidic bond at the reducing end to another structural unit is not included in the abbreviation for glycan, unless specifically defined.

[0236] In the present invention, unless otherwise specified, monosaccharides as basic units of glycans are shown for convenience in such a way that the positions of the carbon atom bonded to the oxygen atom constituting the ring and the carbon atom directly bonded to the hydroxyl group (or the oxygen atom involved in the glycosidic bond) in the ring structure are defined as position 1 (position 2 only in the case of sialic acid). The names of the compounds in the examples are provided taking into account the chemical structure as a whole, and the rules are not necessarily applied. When a glycan is represented herein as a symbol (e.g., GLY, SG, MSG, GlcNAc), the symbol is intended to include the carbon atoms extending to the reducing end, unless otherwise specified, and is not intended to include the N or O involved in the N- or O-glycosidic bond. In the present invention, unless specifically stated otherwise, a partial structure in which a glycan is linked to the side chain of an amino acid is represented in such a manner that a portion of the side chain is shown in parentheses, e.g., "(SG-)Asn".

[0237] D. Antibody-Drug Conjugates (ADCs) In some embodiments, the anti-DLL3 antibody-drug conjugate of the invention has the following formula: [ka] wherein the antibody Ab or a functional fragment of the antibody may be directly bound to L from a side chain of an amino acid residue thereof (e.g., cysteine, lysine), or may be bound to L via a glycan or a remodeled glycan of the Ab, preferably via a glycan or a remodeled glycan of the Ab, more preferably via a remodeled glycan of the Ab. The glycans in the Abs of the present invention are N-linked or O-linked glycans, preferably N-linked glycans. N-linked and O-linked glycans are attached to amino acid side chains of the antibody via N-glycosidic and O-glycosidic bonds, respectively.

[0238] In some embodiments, the antibody (Ab) of the present invention is an IgG, preferably an IgG1, IgG2, or IgG4. In some embodiments, the antibody (Ab) is 2-C8-A, 6-G23-F, 10-O18-A, H2-C8-A, H2-C8-A-2, H2-C8-A-3, H6-G23-F, H6-G23-F-2, H6-G23-F-3, H10-O18-A, H10-O18-A-2, or H10-O18-A-3. IgG has a well-conserved N-linked glycan on the asparagine residue at position 297 in the Fc region of its heavy chain (hereinafter referred to as "Asn297 or N297"), and N-linked glycans are known to contribute to the activity and kinetics of antibody molecules (Biotechnol. Prog., 2012, 28, 608-622, Sanglier-Cianferani, S., Anal. Chem., 2013, 85, 715-736).

[0239] The amino acid sequence in the constant region of IgG is highly conserved, and each amino acid is identified by the Eu index numbering system in Edelman et al. (Proc. Natl. Acad. Sci. USA, Vol. 63, No. 1 (May 15, 1969), pp. 78-85). For example, Asn297, to which an N-linked glycan is attached in the Fc region, corresponds to position 297 in the Eu index numbering system. Each amino acid is uniquely identified by the Eu index numbering system, even if its actual position changes due to fragmentation of the molecule or deletion of a region.

[0240] In some embodiments of the antibody-drug conjugate of the present invention, the antibody or a functional fragment of the antibody can be bound to L via a glycan attached to the side chain of Asn297 (hereinafter referred to as "N297 glycan"), and even more preferably, the antibody or a functional fragment of the antibody is bound to L via the N297 glycan, and the N297 glycan is a remodeled glycan. The following formula illustrates the situation where an antibody-drug conjugate of the invention or a functional fragment of the antibody is attached to L via the N297 glycan.

[0241] [ka] Antibodies with remodeled glycans are referred to as glycan-remodeled antibodies. SGP, an abbreviation for sialylglycopeptide, is a typical N-linked complex glycan. SGP can be isolated / purified from hen's egg yolk, for example, by using the method described in WO2011 / 0278681. Purified SGP products are commercially available (Tokyo Chemical Industry Co., Ltd., Fushimi Pharmaceutical Co., Ltd.) and may be purchased. For example, disialooctasaccharide (Tokyo Chemical Industry Co., Ltd.) is a glycan formed by deleting one GlcNAc at the reducing end of the glycan moiety of SG (hereinafter referred to as "SG(10)") and is commercially available. In the present invention, the glycan structure formed by deleting sialic acid at only one of the non-reducing ends of the β-Man branches in SG(10) is referred to as MSG(9), the structure having sialic acid only in the 1-3 branched chain is referred to as MSG1, and the structure having sialic acid only in the 1-6 branched chain is referred to as MSG2.

[0242] The remodeled glycans of the present invention are N297-(Fuc)MSG1, N297-(Fuc)MSG2, or a mixture of N297-(Fuc)MSG1 and N297-(Fuc)MSG2, or N297-(Fuc)SG, preferably N297-(Fuc)MSG1, N297-(Fuc)MSG2, or N297-(Fuc)SG, more preferably N297-(Fuc)MSG1 or N297-(Fuc)MSG2. N297-(Fuc)MSG1 is represented by the following structural or sequence formula:

[0243] [ka]

[0244] In the above formula: Each wavy line represents antibody binding to Asn297; L(PEG) is -(CH2CH2-O)n 5 -CH2CH2-NH-, where the amino group at the right end represents an amide bond to the carboxylic acid at the 2-position of the sialic acid at the non-reducing end of the 1-3 branched chain of β-Man in the N297 glycan; each asterisk represents a bond to the linker L, in particular a nitrogen atom at position 1 or 3 of the 1,2,3-triazole ring of L in the linker L; n 5 is an integer of 2 to 10, preferably an integer of 2 to 5. N297-(Fuc)MSG2 is represented by the following structural or sequence formula:

[0245] [ka]

[0246] In the above formula: Each wavy line represents antibody binding to Asn297; L(PEG) is -(CH2CH2-O)n 5 -CH2CH2-NH-, where the amino group at the right end represents an amide bond to the carboxylic acid at the 2-position of the sialic acid at the non-reducing end in the 1-6 branched chain of β-Man in the N297 glycan; each asterisk represents a bond to the linker L, in particular a nitrogen atom at position 1 or 3 of the 1,2,3-triazole ring of L in the linker L; n 5 is an integer of 2 to 10, preferably an integer of 2 to 5. N297-(Fuc)SG is represented by the following structural or sequence formula:

[0247] [ka]

[0248] In the above formula: Each wavy line represents antibody binding to Asn297; L(PEG) is -(CH2CH2-O)n 5 -CH2CH2-NH-, wherein the amino group at the right end represents an amide bond to the carboxylic acid at the 2-position of the sialic acid at the non-reducing end in each of the 1-3 and 1-6 branched chains of β-Man in the N297 glycan; each asterisk represents a bond to the linker L, in particular a nitrogen atom at position 1 or 3 of the 1,2,3-triazole ring of L in the linker L; n 5 is an integer of 2 to 10, preferably an integer of 2 to 5.

[0249] When the N297 glycan of the antibody in the antibody-drug conjugate of the present invention is N297-(Fuc)MSG1, N297-(Fuc)MSG2, or a mixture thereof, the antibody-drug conjugate is a molecule (m) in which two molecules of the linker L and two molecules of the drug D are conjugated, since the antibody is a dimer. 2 =1) (see Figure 1).

[0250] When the N297 glycan of the antibody in the antibody-drug conjugate of the present invention is N297-(Fuc)SG, the antibody-drug conjugate is a molecule in which four molecules of the linker L and four molecules of the drug D are conjugated (m 2 =2). The N297 glycan is preferably N297-(Fuc)MSG1, N297-(Fuc)MSG2, or N297-(Fuc)SG, more preferably N297-(Fuc)MSG1 or N297-(Fuc)MSG2.

[0251] When the N297 glycan of the antibody in the antibody-drug conjugate of the present invention is N297-(Fuc)MSG1, N297-(Fuc)MSG2, or N297-(Fuc)SG, a homogeneous ADC can be obtained. The antibody-drug conjugate of the present invention is most preferably one antibody-drug conjugate selected from the following group:

[0252] [ka]

[0253] [ka]

[0254] [ka]

[0255] [ka]

[0256] [ka]

[0257] [ka]

[0258] In each of the above structural formulas: m 2 represents 1 or 2 (preferably, m 2 is 1), the antibody Ab is an anti-DLL3 IgG antibody (preferably IgG1, IgG2, or IgG4, more preferably IgG1), or a functional fragment of such an antibody; N297 glycan represents any one of N297-(Fuc)MSG1, N297-(Fuc)MSG2, and a mixture thereof, and N297-(Fuc)SG (preferably N297-(Fuc)MSG1); L(PEG) is -NH-CH2CH2-(O-CH2CH2)3- * wherein the amino group at the left end represents an amide bond to the carboxylic acid at the 2-position of the sialic acid at the non-reducing end of each or either one of the 1-3 and 1-6 branched chains (preferably the 1-3 branched chain) of the β-Man in the N297 glycan, and the asterisk represents a bond to the nitrogen atom at the 1- or 3-position of the triazole ring of Lb in the linker L. In some embodiments, the anti-DLL3 antibody is, for example, 2-C8-A, 6-G23-F, 10-O18-A, H2-C8-A, H2-C8-A-2, H2-C8-A-3, H6-G23-F, H6-G23-F-2, H6-G23-F-3, H10-O18-A, H10-O18-A-2, or H10-O18-A-3.

[0259] For convenience, the most preferred antibody-drug conjugates are those having two or four units of the structure "-(N297 glycan)-LD" in which the N297 glycan is attached to the nitrogen atom at position 1 of the triazole ring of Lb in one conjugate molecule (m 2 = 1 or 2) ("(N297 glycan)-(N1Lb)LD"), or a structure having two or four units of "-(N297 glycan)-LD" in which the N297 glycan is bound to the nitrogen atom at position 3 of the triazole ring of Lb in one conjugate molecule of L (m 2 = 1 or 2) ("(N297 glycan)-(N3Lb)LD") is exemplified, but "(N297 glycan)-(N1Lb)LD" (m 2 = 1 means one unit, m 2 = 2, 1, 2, or 3 units) and "(N297 glycan)-(N3Lb)LD" (m 2 = 1 means one unit, m 2In other words, either "(N297 glycan)-(N1Lb)LD" or "(N297 glycan)-(N3Lb)LD" or both of them may be present simultaneously in one conjugate molecule.

[0260] The antibody-drug conjugates of the present invention and the anti-DLL3 antibody-drug conjugates of the present invention exhibit strong antitumor activity (in vivo antitumor activity, in vitro anticellular activity) and satisfactory in vivo kinetic and physical properties, and are highly safe, and therefore are useful as pharmaceuticals.

[0261] The antibody-drug conjugates of the present invention may exist as stereoisomers, optical isomers due to asymmetric carbon atoms, geometric isomers, tautomers, or optical isomers such as d-isomers and l-isomers, and atropisomers. The present invention encompasses the free drug or intermediate products of the antibody-drug conjugates, as well as these isomers, optical isomers, and mixtures thereof. The PBD derivatives (V) and (VI) of the present invention have an asymmetric carbon at the 11'-position, and thus exist as optical isomers. In the present specification, these isomers and mixtures of these isomers are all represented by a single formula, i.e., general formula (V) or (VI). Therefore, (V) or (VI) encompasses all optical isomers and mixtures of optical isomers in any ratio. The absolute configuration at the 11'-position of (V) or (VI) can be determined by X-ray crystallography or NMR, e.g., the Mosher method, of the crystalline product or intermediate, or a derivative thereof. Absolute configuration may then be determined by using crystalline products or intermediates that are derivatized with a reagent containing an asymmetric center of known configuration. If desired, stereoisomers of the compounds synthesized according to the present invention may be obtained by isolation using conventional optical resolution or separation methods.

[0262] The number of drug molecules conjugated per antibody molecule is an important factor affecting the efficacy and safety of the antibody-drug conjugates of the present invention. Antibody-drug conjugates are prepared using reaction conditions, such as the amounts of reacted raw materials and reagents, specified to obtain a consistent number of conjugated drug molecules. However, in contrast to chemical reactions of low-molecular-weight compounds, a mixture containing a large number of different conjugated drug molecules is typically obtained. The number of drug molecules conjugated per antibody molecule is specified as an average value, i.e., the average number of conjugated drug molecules (DAR: drug-to-antibody ratio). The number of pyrrolobenzodiazepine derivative molecules conjugated to an antibody molecule is controllable, and the average number of drug molecules conjugated per antibody molecule (DAR) can be 1 to 10, but preferably the number is 1 to 8, and more preferably 1 to 5.

[0263] When an antibody binds to L in an antibody-drug conjugate of the present invention via a remodeled glycan of the antibody, m is the number of drug molecules conjugated per antibody molecule in the antibody-drug conjugate. 2 is an integer of 1 or 2. When the glycan is an N297 glycan, and the glycan is N297-(Fuc)MSG1, N297-(Fuc)MSG2, or a mixture of N297-(Fuc)MSG1 and N297-(Fuc)MSG2, m 2 is 1, and the DAR is in the range of 1 to 3 (preferably in the range of 1.0 to 2.5, more preferably in the range of 1.2 to 2.2). When the N297 glycan is N297-(Fuc)SG, m 2 is 2, and the DAR is in the range of 3 to 5 (preferably in the range of 3.2 to 4.8, more preferably in the range of 3.5 to 4.2).

[0264] In any of the foregoing embodiments, the anti-DLL3 antibody (i.e., "antibody" or "Ab") can be selected from 2-C8-A, 6-G23-F, 10-O18-A, H2-C8-A, H2-C8-A-2, H2-C8-A-3, H6-G23-F, H6-G23-F-2, H6-G23-F-3, H10-O18-A, H10-O18-A-2, or H10-O18-A-3. In some embodiments, the anti-DLL3 antibody of the ADC comprises a heavy chain immunoglobulin variable domain (V H ) and light chain immunoglobulin variable domain (V L ), and (a) V H are selected from the group consisting of: (i) SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:5, respectively; (ii) SEQ ID NO:13, SEQ ID NO:14, and SEQ ID NO:15, respectively; (iii) SEQ ID NO:23, SEQ ID NO:24, and SEQ ID NO:25, respectively; and (iv) SEQ ID NO:33, SEQ ID NO:34, and SEQ ID NO:35, respectively. H -CDR1 sequence, V H -CDR2 sequence, and V H - comprising a CDR3 sequence; and / or (b) V L are selected from the group consisting of: (i) SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:10, respectively; (ii) SEQ ID NO:18, SEQ ID NO:19, and SEQ ID NO:20, respectively; (iii) SEQ ID NO:28, SEQ ID NO:29, and SEQ ID NO:30, respectively; and (iv) SEQ ID NO:38, SEQ ID NO:39, and SEQ ID NO:40, respectively. L -CDR1 sequence, V L -CDR2 sequence, and V L-CDR3 sequences. In some embodiments, the anti-DLL3 antibodies of the ADCs may comprise one or more of the following features: (a) a light chain immunoglobulin variable domain sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to a light chain immunoglobulin variable domain sequence or light chain sequence present in any one of SEQ ID NOs: 7, 17, 27, 37, 62, 66, or 70; and / or (b) a heavy chain immunoglobulin variable domain sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to a heavy chain immunoglobulin variable domain sequence or heavy chain sequence present in any one of SEQ ID NOs: 2, 12, 22. In some embodiments, the anti-DLL3 antibodies of the ADCs may comprise a heavy chain immunoglobulin variable domain (V H ) or heavy and light chain immunoglobulin variable domains (V L ) or light chain, and (a) V H or the heavy chain comprises an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:12, SEQ ID NO:22, SEQ ID NO:32, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:68, and SEQ ID NO:69; and / or (b) V L comprises an amino acid sequence selected from the group consisting of SEQ ID NO:7, SEQ ID NO:17, SEQ ID NO:27, SEQ ID NO:37, SEQ ID NO:62, SEQ ID NO:66, and SEQ ID NO:70. H or heavy chain amino acid sequence and V LAlternatively, the light chain amino acid sequences are SEQ ID NO:2 and SEQ ID NO:7, respectively (7-I1-B); SEQ ID NO:12 and SEQ ID NO:17, respectively (2-C8-A); SEQ ID NO:59 and SEQ ID NO:62, respectively (H2-C8-A); SEQ ID NO:60 and SEQ ID NO:62, respectively (H2-C8-A-2); SEQ ID NO:61 and SEQ ID NO:62, respectively (H2-C8-A-3); SEQ ID NO:22 and SEQ ID NO:27, respectively (10-O18-A); SEQ ID NO:67 and SEQ ID NO:70, respectively (H1 H10-O18-A; SEQ ID NO: 68 and SEQ ID NO: 70, respectively (H10-O18-A-2); SEQ ID NO: 69 and SEQ ID NO: 70 (H10-O18-A-3); SEQ ID NO: 32 and SEQ ID NO: 37, respectively (H6-G23-F); SEQ ID NO: 63 and SEQ ID NO: 66, respectively (H6-G23-F); SEQ ID NO: 64 and SEQ ID NO: 66, respectively (H6-G23-F-2); and SEQ ID NO: 65 and SEQ ID NO: 66, respectively (H6-G23-F-3).

[0265] Those skilled in the art can conjugate a required number of drug molecules to each antibody molecule based on the explanations in the Examples herein, and design a reaction method to obtain an antibody having a controlled number of conjugated pyrrolobenzodiazepine derivative molecules. The antibody-drug conjugates of the present invention, free drugs, or production intermediates thereof may absorb moisture and allow the adsorbed water to adhere, or may become hydrated or recrystallize when left in the atmosphere, and such water-containing compounds and salts are also included in the present invention.

[0266] When the antibody-drug conjugates of the present invention, free drugs, or product intermediates have a basic group such as an amino group, they may be converted into pharmaceutically acceptable salts as required. Examples of such salts include, but are not limited to, hydrohalide salts such as hydrochloride and hydroiodide; inorganic acid salts such as nitrate, perchlorate, sulfate, and phosphate; lower alkane sulfonates such as methanesulfonate, trifluoromethanesulfonate, and ethanesulfonate; arylsulfonates such as benzenesulfonate and p-toluenesulfonate; organic acid salts such as formate, acetate, malate, fumarate, succinate, citrate, tartrate, oxalate, and maleate; and amino acid salts such as ornithinate, glutamate, and aspartate.

[0267] When the antibody-drug conjugate of the present invention, the free drug, or a product intermediate has an acidic group such as a carboxy group, a base addition salt can generally be formed. Examples of pharmaceutically acceptable salts include, but are not limited to, alkali metal salts such as sodium salt, potassium salt, and lithium salt; alkaline earth metal salts such as calcium salt and magnesium salt; inorganic salts such as ammonium salt; and organic amine salts such as dibenzylamine salt, morpholine salt, phenylglycine alkyl ester salt, ethylenediamine salt, N-methylglucamate, diethylamine salt, triethylamine salt, cyclohexylamine salt, dicyclohexylamine salt, N,N'-dibenzylethylenediamine salt, diethanolamine salt, N-benzyl-N-(2-phenylethoxy)amine salt, piperazine salt, tetramethylammonium salt, and tris(hydroxymethyl)aminomethane salt.

[0268] The antibody-drug conjugates, free drugs, or product intermediates of the present invention may exist as hydrates, for example, by absorbing moisture from the air. The solvates of the present invention are not limited to specific solvates and may be any pharmaceutically acceptable solvates, and specifically, hydrates, ethanol solvates, 2-propanol solvates, etc. are preferred. When a nitrogen atom is present in the antibody-drug conjugates, free drugs, or product intermediates of the present invention, they may be in the form of their N-oxides, and these solvates and N-oxide forms are included within the scope of the present invention.

[0269] The present invention includes compounds labeled with various radioactive or non-radioactive isotopes. The antibody-drug conjugates, free drugs, or product intermediates of the present invention may contain one or more component atoms with unnatural proportions of atomic isotopes. Examples of atomic isotopes include, but are not limited to, deuterium ( 2 H), tritium ( 3 H), iodine-125( 125 I), and carbon-14 ( 14 The compounds of the present invention can be substituted with tritium ( 3 H), iodine-125( 125 I), and carbon-14 ( 14 The antibody-drug conjugates of the present invention may be radiolabeled with a radioactive isotope, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 110, 111, 112, 113, 114, 115, 116, 117, 118, 120, 122, 123, 124, 125, 130, 131, 132,

[0270] E. Preparation method Representative methods for producing the antibody-drug conjugates of the present invention and their free drugs or product intermediates are disclosed and described herein. Hereinafter, the compound numbers shown in the reaction schemes are used to distinguish the compounds from one another. Specifically, references will be made in the form of "compound of formula (1)," "compound (1)," etc. Compounds with other numbers will be referred to in the same manner. Scheme R: Antibody preparation Glycan-remodeled antibodies can be made, for example, according to the methods described in WO2013 / 120066 and by using the method illustrated in Figure 3. The following description of synthetic steps R1 and R2 is given in relation to Figure 12 and the following scheme.

[0271] [ka] Step R-1: Hydrolysis of the glycosidic bond at GlcNAcβ1-4GlcNAc of the chitobiose structure at the reducing end This process involves preparing glycan-truncated antibodies by cleaving the N-linked glycan bond to the asparagine at position 297 of the amino acid sequence of the targeted antibody (N297-linked glycan) using known enzymatic reactions.

[0272] The targeted antibody (20 mg / mL) in a buffer solution (e.g., 50 mM phosphate buffer solution) is subjected to a hydrolysis reaction of the glycosidic bond between GlcNAcβ1 and 4GlcNAc in the chitobiose structure at the reducing end using a hydrolase such as the enzyme EndoS at 0°C to 40°C. The reaction time is 10 minutes to 72 hours, preferably 1 hour to 6 hours. The amount of wild-type enzyme EndoS used is 0.1 to 10 mg, preferably 0.1 to 3 mg, per 100 mg of antibody. After the reaction is complete, the antibody is purified by affinity chromatography and / or hydroxyapatite column, as described below, to produce a (Fucα1,6)GlcNAc antibody in which the glycan has been hydrolyzed between GlcNAcβ1 and 4GlcNAc.

[0273] Step R-2: Glycosyl transfer reaction This process involves using an enzymatic reaction to conjugate a (Fucα1,6)GlcNAc antibody to an MSG (MSG1, MSG2) or SG glycan oxazoline form (hereinafter referred to as "azidoglycan oxazoline form") having an azide group-containing PEG linker, thereby producing an antibody with a remodeled glycan. The glycan-truncated antibody in a buffer solution (e.g., phosphate buffer solution) is subjected to a glycosyl transfer reaction by reacting with an azidoglycan oxazoline form in the presence of a catalytic amount of a transglycosidase, such as EndoS(D233Q / Q303L), at 0°C to 40°C. The reaction time is 10 minutes to 72 hours, preferably 1 to 6 hours. The amount of the enzyme EndoS(D233Q / Q303L) used is 1 to 10 mg, preferably 1 to 3 mg, per 100 mg of antibody. The amount of the azidoglycan oxazoline form used is 2 equivalents to an excess of 2 equivalents, preferably 2 to 20 equivalents.

[0274] After the reaction is complete, the glycan-remodeled antibody is purified by affinity chromatography and purification on a hydroxyapatite column. Azidoglycan oxazoline (e.g., [N3-PEG(3)]-MSG1-Ox, Example 56 of WO19065964) forms may be prepared according to the methods described in Examples 55-57 of WO19065964. N3-(CH2CH2-O)n may be prepared by using reactions known in the field of synthetic organic chemistry (e.g., condensation reactions). 5 A PEG linker containing an azide group (N3-L(PEG)) may be introduced into MSG (MSG1, MSG2) or disialooctasaccharide (Tokyo Chemical Industry Co., Ltd.). Specifically, the carboxylate at the 2-position of the sialic acid and the N3-(CH2CH2-O)n 5 The amino group at the right end of -CH2CH2-NH2 undergoes a condensation reaction to form an amide bond.

[0275] Examples of condensing agents used in the condensation reaction include, but are not limited to, N,N'-dicyclohexylcarbodiimide (DCC), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI), carbonyldiimidazole (CDI), 2-(2H-benzotriazol-2-yl)-4-(1,1,3,3-tetramethylbutyl)phenol (BOP), 1H-benzotriazol-1-yloxytripyrrolidinophosphonium hexafluorophosphate (PyBOP), and O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU). Examples of solvents for the reaction include, but are not limited to, dichloromethane, DMF, THF, ethyl acetate, and mixtures thereof.

[0276] The reaction temperature is typically −20° C. to 100° C. or the boiling point of the solvent, preferably within the range of −5° C. to 50° C. If necessary, an organic base such as triethylamine, diisopropylethylamine, N-methylmorpholine, or 4-dimethylaminopyridine, or an inorganic base such as potassium carbonate, sodium carbonate, potassium bicarbonate, or sodium bicarbonate may be added. Furthermore, for example, 1-hydroxybenzotriazole or N-hydroxysuccinimide may be added as a reaction promoter. MSG, MSG1, or MSG2 can be obtained by hydrolysis of (MSG-)Asn or isolated / purified (MSG1-)Asn or (MSG2-)Asn using a hydrolase such as EndoM. Oxazolination may be performed from GlcNAc at the reducing end of MSG-type (MSG1, MSG2) or SG-type glycans according to known literature (J. Org Chem., 2009, 74(5), 2210-2212. Helv. Chim. Acta, 2012, 95, 1928-1936.).

[0277] Step R-3: Glycosyl transfer reaction Instead of step R-2, the following steps may be applied to the production of the above-mentioned glycan-remodeled antibody using the two enzymes described in WO 2018 / 003983 in conjunction with this method. The (Fucα1,6)GlcNAc antibody obtained in step R-1 is reacted with a glycan donor molecule, such as (SG-)Asn or (MSG-)Asn, in which an azide group has been introduced into the sialic acid, in the presence of two glycosyltransferases (enzyme A and enzyme B) to synthesize an Fc-containing molecule in which an SG-type glycan has been remodeled and in which an azide group has been introduced into the sialic acid. Examples of enzyme A include EndoM, EndoOm, and EndoCC, as well as EndoM mutants, EndoOm mutants, and EndoCC mutants exhibiting reduced hydrolysis activity. Enzyme A is preferably EndoMN175Q, EndoCC N180H, or EndoOm N194Q. Examples of enzyme B include EndoS and EndoS2 (EndoS49), as well as EndoS mutants and EndoS2 (EndoS49) mutants exhibiting reduced hydrolytic activity. Enzyme B is preferably EndoS D233Q, EndoS D233Q / Q303L, EndoS D233Q / E350A, EndoS D233Q / E350Q, EndoS D233Q / E350D, EndoS D233Q / E350N, EndoS D233Q / D405A, EndoS2D184M, EndoS2T138Q, etc. In preparing a glycan-remodeled antibody, concentration of an aqueous antibody solution, concentration determination, and buffer exchange may be performed according to the following general operations A to C.

[0278] i. General Operation A: Concentration of Aqueous Antibody Solution The antibody or antibody-drug conjugate solution was placed in an Amicon Ultra (30,000 to 50,000 MWCO, Millipore) container, and the antibody or antibody-drug conjugate solution described below was concentrated by centrifugation (2000 to 4000 G for 5 to 20 minutes) using a centrifuge (Allegra X-15R, Beckman Coulter, Inc.). ii. General Operation B: Measurement of Antibody Concentration Antibody concentrations were measured using a UV measurement device (Nanodrop 1000, Thermo Fisher Scientific Inc.) according to the method specified by the manufacturer. The absorbance coefficient at 280 nm (1.3 mL mg) was then calculated, which differs between antibodies. -1 ·cm -1 ~1.8 mL mg -1 ·cm -1 ) was used. iii. General Operation C: Buffer Exchange for Each Antibody A buffer solution (e.g., phosphate buffered saline (pH 6.0), phosphate buffer (pH 6.0)) was added to an aqueous solution of antibody, which was then concentrated according to General Operation A. This operation was repeated several times, and then the antibody concentration was measured using General Operation B and adjusted to 10-20 mg / mL with a buffer solution (e.g., phosphate buffered saline (pH 6.0), phosphate buffer (pH 6.0)).

[0279] Scheme S: Conjugation The preparation method is a method for preparing an antibody-drug conjugate by conjugating the above-mentioned glycan-remodeled antibody to the product intermediate (2) via the SPAAC (Strain-Promoted Alkyne Azide Cycloaddition: J. AM. CHEM. SOC. 2004, 126, 15046-15047) reaction, where Ab represents the glycan-remodeled antibody.

[0280] [ka] The SPAAC reaction proceeds by mixing a buffer solution of antibody Ab (sodium acetate solution, sodium phosphate solution, sodium borate solution, etc., or a mixture thereof) and a solution of compound (2) dissolved in an appropriate solvent (dimethyl sulfoxide (DMSO), dimethylformamide (DMF), dimethylacetamide (DMA), N-methyl-2-pyridone (NMP), propylene glycol (PG), etc., or a mixture thereof).

[0281] The molar amount of compound (2) used is 2 moles to an excess molar amount per mole of antibody, preferably 1 mole to 30 moles, and the ratio of the organic solvent to the antibody buffer is preferably 1 to 200% v / v. The reaction temperature is 0°C to 37°C, preferably 10°C to 25°C, and the reaction time is 1 to 150 hours, preferably 6 to 100 hours. The pH during the reaction is preferably 5 to 9. Antibody-drug conjugate compounds (ADCs) can be identified from one another via buffer exchange, purification, and measurement of antibody concentration and the average number of drug molecules conjugated per antibody molecule according to general operations A through C described above and general operations D through F described below.

[0282] iv. General Operation D: Purification of Antibody-Drug Conjugates A NAP-25 column (GE Healthcare) was equilibrated with commercially available acetate buffer solution (10 mM, pH 5.5; referred to herein as ABS) containing sorbitol (5%). The aqueous antibody-drug conjugate reaction solution (approximately 1.5–2.5 mL) was applied to the NAP-25 column and eluted with buffer at the volume specified by the manufacturer to separate and collect the antibody fraction. The separated and collected fractions were then applied to the NAP-25 column again, and this gel filtration purification procedure for elution with buffer was repeated two or three times to obtain the antibody-drug conjugate and remove unbound drug linker, dimethyl sulfoxide, and propylene glycol. If necessary, the concentration of the antibody-drug conjugate solution was adjusted using General Procedures A–C.

[0283] v. General Operation E: Determination of Antibody Concentration of Antibody-Drug Conjugate The concentration of the conjugated drug in the antibody-drug conjugate can be calculated by using the Beer-Lambert law shown below: Equation (I) using the Beer-Lambert law is as follows:

[0284]

number

[0285]

number

number

[0286] The values ​​used in the formulas and the values ​​applied in the examples are explained below. The absorbance A280 used was a measurement of the UV absorbance of an aqueous solution of the antibody-drug conjugate at 280 nm. In terms of molar mass, MW (g mol -1 ) was calculated from the amino acid sequence of the antibody and used as an estimate of the molar mass of the antibody-drug conjugate. The optical path length, l (cm), used in the measurements was 1 cm. The molar absorption coefficient, ε280, of an antibody-drug conjugate can be determined by using the following equation (IV):

[0287]

number

[0288] By using a known calculation method (Protein Science, 1995, vol. 4, 2411-2423), ε Ab,280 can be estimated from the amino acid sequence of the antibody. In the examples, the molar absorption coefficient of the DLL3 antibody (H2-C8-A) used is ε Ab,280 = 220378 (calculated estimate), and the molar absorption coefficient of the DLL3 antibody used (H6-G23-F) is ε Ab,280 = 215353 (calculated estimate), and the molar absorption coefficient of the DLL3 antibody used (H10-O18-A) is ε Ab,280 = 215424 (calculated estimate), and the molar absorption coefficient of the LPS antibody used is ε Ab,280 = 230280 (calculated estimate), and the molar absorption coefficient of the DLL3 antibody used (H2-C8-A-3) is ε Ab,280 = 220420 (calculated estimate), and the molar absorption coefficient of the DLL3 antibody used (H10-O18-A-3) is ε Ab,280 =215380 (calculated estimate). Use ε for DL,280 was calculated from the measured values ​​obtained in each UV measurement. Specifically, the absorbance of a solution containing a conjugate precursor (drug) dissolved at a specific molar concentration was measured, and the Beer-Lambert law (Equation (I)) was applied to the absorbance.

[0289] vi. General Operation F: Determining the Average Number of Drug Molecules Conjugated per Antibody Molecule in Antibody-Drug Conjugates The average number of drug molecules conjugated per antibody molecule in an antibody-drug conjugate can be determined via high performance liquid chromatography (HPLC) using the following method. [F-1. Preparation of Samples for HPLC Analysis (Reduction of Antibody-Drug Conjugates)] A solution of the antibody-drug conjugate (approximately 1 mg / mL, 60 μL) is mixed with an aqueous solution of dithiothreitol (DTT) (100 mM, 15 μL). The mixture is incubated at 37° C. for 30 minutes to prepare a sample in which the disulfide bond between the L chain and H chain of the antibody-drug conjugate has been cleaved. This sample is used for HPLC analysis.

[0290] [F-2.HPLC analysis] The HPLC analysis is carried out under the following conditions: HPLC system: Agilent 1290 HPLC system (Agilent Technologies) Detector: UV absorption spectrometer (measurement wavelength: 280 nm, 329 nm) Column: BEH Phenyl (2.1 x 50 mm, 1.7 μm, Waters Acquity) Column temperature: 75℃ Mobile phase A: 0.1% trifluoroacetic acid (TFA)-15% isopropyl alcohol aqueous solution Mobile phase B: 0.075% TFA-15% isopropyl alcohol acetonitrile solution Gradient Program 1: 14% to 36% (0 to 15 min), 36% to 80% (15 to 17 min), 80% to 14% (17 to 17.1 min), 14% to 14% (17.1 to 23 min) Gradient Program 2: 14% to 50% (0 to 15 min), 50% to 80% (15 to 17 min), 80% to 14% (17 to 17.1 min), 14% to 14% (17.1 to 23 min) Sample injection volume: 5 μL.

[0291] [F-3. Data Analysis] [F-3-1] L chains with conjugated drug molecules (L chains with one conjugated drug molecule: L1) and H chains with conjugated drug molecules (H chains with one conjugated drug molecule: H1, H chains with two conjugated drug molecules: H2, and H chains with three conjugated drug molecules: H3) have hydrophobicity that increases in proportion to the number of conjugated drug molecules and have longer retention times than the L chains (L0) and H chains (H0) of antibodies without any conjugated drug molecules. Therefore, L0, L1, H0, H1, H2, and H3 are eluted in the order shown. In some cases, the order of L1 and H0 is reversed, but H0 without conjugated drug molecules does not absorb at the 329 nm wavelength, which is characteristic of drugs. Therefore, L1 and H0 can be distinguished by checking the absorption at a wavelength of 329 nm. Through comparison of the retention times of L0 and H0, each detected peak can be assigned to L0, L1, H0, H1, H2, or H3.

[0292] [F-3-2] Because each drug linker absorbs UV, the peak area value is corrected according to the number of conjugated drug linker molecules by using the molar absorption coefficients of the light chain, heavy chain, and drug linker as follows:

[0293]

number

[0294] Here, the molar absorption coefficients (280 nm) of the L and H chains of each antibody can be estimated from the amino acid sequences of the antibody L and H chains using a known calculation method (Protein Science, 1995, vol. 4, pp. 2411-2423). In the case of the DLL3 antibody (H2-C8-A), 26,123 was used as the molar absorption coefficient of the L chain estimated from the amino acid sequence, and 84,150 was used as the molar absorption coefficient of the H chain estimated from the amino acid sequence. In the case of the DLL3 antibody (H6-G23-F), 30,105 was used as the molar absorption coefficient of the L chain estimated from the amino acid sequence, and 77,423 was used as the molar absorption coefficient of the H chain estimated from the amino acid sequence. In the case of the DLL3 antibody (H10-O18-A), 26166 was used as the molar absorption coefficient of the light chain predicted from the amino acid sequence, and 81340 was used as the molar absorption coefficient of the heavy chain predicted from the amino acid sequence. In the case of the DLL3 antibody (H2-C8-A-3), 26212 was used as the molar absorption coefficient of the light chain predicted from the amino acid sequence, and 83993 was used as the molar absorption coefficient of the heavy chain predicted from the amino acid sequence. In the case of the DLL3 antibody (H10-O18-A-3), 26212 was used as the molar absorption coefficient of the light chain predicted from the amino acid sequence, and 81478 was used as the molar absorption coefficient of the heavy chain predicted from the amino acid sequence. [F-3-3] The peak area ratio (%) of each chain to the total corrected peak area is calculated by using the following formula:

[0295]

number

[0296] [ka] This is explained below.

[0297] The free drug of the present invention is produced through a process in which the antibody-drug conjugate is transported into tumor cells and then a portion of the linker L in the antibody-drug conjugate is cleaved. The antibody-drug conjugates of the present invention are produced by using production intermediates. The free drug of the antibody-drug conjugate of the present invention may be (a) R 16 and R 17 are combined to form an imine bond (N=C). The intermediate product of the antibody-drug conjugate of the present invention is (b) R 16 is J-La'-Lp'-NH-B'-CH2-O(C=O)- * This corresponds to the case represented by Therefore, l and n in Eq. 1 , E and A, R 9 and R 1 , R 10 and R 2 , R 11 and R 3 , R 12 and R 4 , R 13 and R 5 , R 14 and R 6 , R 15 and R 7 , V and X, W and Y, Group 7 and Group 1, Group 8 and Group 2, Group 9 and Group 3, Group 10 and Group 4, Group 11 and Group 5, and Group 12 and Group 6 are synonyms, respectively. 1 represents an integer of 2 to 8, preferably an integer of 2 to 6, more preferably an integer of 3 to 5. An alkyl chain in which 1 is an integer of 2 to 8, preferably an integer of 2 to 6, more preferably an integer of 3 to 5 may contain a double bond.

[0298] E represents a spiro-bonded 3- to 5-membered saturated hydrocarbon ring or a 3- to 5-membered saturated heterocycle, preferably a 3- to 5-membered saturated hydrocarbon ring (cyclopropane, cyclobutane, or cyclopentane), more preferably cyclopropane or cyclobutane, and most preferably cyclopropane. The spiro-bonded 3- to 5-membered saturated hydrocarbon ring may be substituted with 1 to 4 halogen atoms, preferably 1 or 2 fluorine atoms (e.g., 2,2-difluorocyclopropane). R 9 and R 10 each independently represents a C1-C6 alkoxy group, a C1-C6 alkyl group, a hydrogen atom, a hydroxy group, a thiol group, a C1-C6 alkylthio group, a halogen atom, or -NR'R''; each is preferably a C1-C6 alkoxy group, a C1-C6 alkyl group, or a hydroxy group, more preferably a C1-C3 alkoxy group, and most preferably a methoxy group. R 11 , R 12 , and R 13 is as described in any one of (i) to (iii) below.

[0299] A. Compound Embodiment 1 R 11 and R 12 But R 3 and R 4 When combined with the carbon atom to which it is attached to form a double bond, R 13 represents an aryl or heteroaryl group optionally having one or more substituents selected from Group 7, or a C1-C6 alkyl group optionally having one or more substituents selected from Group 8, preferably an aryl group optionally having one or more substituents selected from Group 7.

[0300] R 13 The "aryl group" in the "aryl group or heteroaryl group optionally having one or more substituents selected from Group 7" is preferably a phenyl group or a naphthyl group, and more preferably a phenyl group. R 13 The "heteroaryl group" in the "aryl group or heteroaryl group optionally having one or more substituents selected from Group 7" is preferably a thienyl group, a pyridyl group, a pyrimidyl group, a quinolyl group, a quinoxalyl group, or a benzothiophenyl group, more preferably a 2-thienyl group, a 3-thienyl group, a 2-pyridyl group, a 3-pyridyl group, or a 4-pyridyl group, and even more preferably a 3-pyridyl group or a 3-thienyl group.

[0301] R 13 Examples of substituents on the aryl or heteroaryl group for the above include, but are not limited to, the following a) to j): a) a C1-C6 alkoxy group optionally substituted with 1 to 3 halogen atoms; b) a C1-C6 alkyl group optionally substituted with 1 to 3 halogen atoms, a hydroxy group, -OCOR', -NR'R'', -C(=NR')-NR''R''', and -NHC(=NR')-NR''R''', c) a halogen atom, d) a C3-C5 cycloalkoxy group, e) a C1-C6 alkylthio group, f)-NR'R'', g)-C(=NR')-NR''R'''', h)-NHC(=NR')-NR''R'''', i) -NHCOR', and j) Hydroxy groups.

[0302] wherein R', R'', and R''' in b) and f) to i) are each independently Each represents a hydrogen atom or a C1-C6 alkyl group, and preferably each independently represents a hydrogen atom or a C1-C3 alkyl group. a) to j) are preferably as follows: a) a C1-C3 alkoxy group optionally substituted with 1 to 3 halogen atoms, more preferably a methoxy group, an ethoxy group, an n-propoxy group, an i-propoxy group, or a trifluoromethoxy group, even more preferably a methoxy group, an ethoxy group, or a trifluoromethoxy group, and most preferably a methoxy group; b) A C1-C3 alkyl group optionally substituted with 1 to 3 halogen atoms, a hydroxy group, -OCOR', -C(=NR')-NR''R''', and -NHC(=NR')-NR''R'''' [wherein R', R'', and R''' are each independently a hydrogen atom or a C1-C3 alkyl group, more preferably a C1-C3 alkyl group optionally substituted with 1 to 3 halogen atoms, a hydroxy group, -OCOR', -C(=NR')-NR''R''', and -NHC( a C1-C3 alkyl group optionally substituted with any one selected from -NR')-NR''R''', where R', R'', and R''' are each independently a hydrogen atom or a methyl group, and more preferably a methyl group, an ethyl group, an n-propyl group, an i-propyl group, a fluoromethyl group, a difluoromethyl group, a trifluoromethyl group, a hydroxymethyl group, -CH2OCOMe, -CH2-NHC(=NH)-NH2, or -CH2-NHC(=NMe)-NH2; c) a halogen atom, preferably a fluorine atom or a chlorine atom; d) a C3-C5 cycloalkoxy group, more preferably a cyclopropoxy group; e) a C1-C3 alkylthio group, more preferably a methylthio group or an ethylthio group; f) -NR'R'' (wherein R' and R'' are each independently a hydrogen atom or a C1-C3 alkyl group, more preferably -NH2, -NHMe, -NMe2, -NHEt, or -NEt2); g) —C(═NR′)—NR″R′″, wherein R′, R″, and R′″ are each independently a hydrogen atom or a C1-C3 alkyl group, more preferably —C(═NH)—NH2 or —C(═NMe)—NH2; h) -NHC(=NR')-NR''R''' [wherein R', R'', and R''' each independently represent a hydrogen atom or a C1-C3 alkyl group, more preferably -NHC(=NH)-NH2 or -NHC(=NMe)-NH2]; i) -NHCOR', where R' is a hydrogen atom or a C1-C3 alkyl group, more preferably -NHCOMe or -NHCOEt; and j) Hydroxy groups.

[0303] R 13 The aryl group (preferably a phenyl group) or heteroaryl group (preferably a pyridyl group) in the formula (I) may have at least one substituent at any position. When multiple substituents are present, the substituents may be the same or different. R 13 When is an aryl group, each substituent is preferably a), b), d), g), h), or j), more preferably a), b), d), or j). R 13 is a phenyl group, R 13 may have a substituent at any position, or may have multiple substituents, preferably one or two substituents at the 3-position and / or 4-position, more preferably one substituent at the 4-position.

[0304] R 5 is a naphthyl group, R 5 may have a substituent at any position, and may have multiple substituents, preferably one substituent at the 6-position. R 13 is a phenyl group, R 13is more preferably a phenyl group, a 4-methoxyphenyl group, a 3-methoxyphenyl group, a 4-ethoxyphenyl group, a 4-(n-propoxy)-phenyl group, a 4-(i-propoxy)-phenyl group, a 4-cyclopropoxy-phenyl group, a 4-trifluoromethylphenyl group, a 4-hydroxymethyl-phenyl group, a 4-acetoxymethyl-phenyl group, or a 4-carbamimidamidomethyl-phenyl group, and even more preferably a phenyl group, a 4-methoxyphenyl group, a 3-methoxyphenyl group, a 4-cyclopropoxy-phenyl group, a 4-hydroxymethyl-phenyl group, a 4-acetoxymethyl-phenyl group, a 4-carbamimidamidomethyl-phenyl group, or a 4-trifluoromethylphenyl group.

[0305] R 13 is a naphthyl group, R 13 is more preferably a naphthyl group or a 6-methoxy-2-naphthyl group, and most preferably a 4-methoxyphenyl group. R 13 When is a heteroaryl group, each substituent is preferably a), b), d), g), h), or j), more preferably a) or b). R 13 When R is a heteroaryl group, 13 R may have at least one substituent at any position. 13 When R is a 3-pyridyl group, the substituents are preferably located at the 6- and / or 5-positions. 13 When R is 2-pyridyl, the substituents are preferably at the 5- and / or 4-positions or the 5- and / or 6-positions. 13 When is 4-pyridyl, the substituents are preferably at the 2- and / or 6-positions. R 13 When R is a heteroaryl group, 13 may have multiple substituents, preferably has one or two substituents, and more preferably has one substituent.

[0306] R 13 is a pyridyl group, R13 is preferably a 6-methoxy-3-pyridyl group or a 6-methyl-3-pyridyl group. R 13 is a 3-thienyl group or a 6-quinoxalyl group, R 13 is preferably unsubstituted. R 13 The "C1-C6 alkyl group" in the "C1-C6 alkyl group optionally having one or more substituents selected from Group 8" is preferably a C1-C3 alkyl group, more preferably a methyl group or an ethyl group. R 13 The substituents in the "C1-C6 alkyl group optionally having one or more substituents selected from Group 8" are each a halogen atom, a hydroxy group, or a C1-C6 alkoxy group (preferably a C1-C3 alkoxy group), preferably a hydroxy group, a methoxy group, or an ethoxy group, more preferably a hydroxy group.

[0307] B. Compound Embodiment 2 R 11 If represents a hydrogen atom, R 12 and R 13 is R 12 and R 13 is combined with the carbon atom to which it is attached to form a 3- to 5-membered saturated hydrocarbon ring or a 3- to 5-membered saturated heterocyclic ring, or CH2=. The 3- to 5-membered saturated hydrocarbon ring may be substituted with 1 to 4 halogen atoms, preferably 1 or 2 fluorine atoms. R 12 and R 13 are preferably combined to form a 3- to 5-membered saturated hydrocarbon ring or CH2=, more preferably to form cyclopropane, cyclobutane, or CH2= (exomethylene group), even more preferably to form cyclopropane. R 12 and R 13are combined to form a 3- to 5-membered saturated hydrocarbon ring or a 3- to 5-membered saturated heterocyclic ring, the 3- to 5-membered saturated hydrocarbon ring or the 3- to 5-membered saturated heterocyclic ring is preferably the same as E. More preferably, E is a 3- to 5-membered saturated hydrocarbon ring, and R 12 and R 13 are combined to form a 3- to 5-membered saturated hydrocarbon ring, and even more preferably, E is a cyclopropane ring and R 12 and R 13 are combined to form a cyclopropane ring.

[0308] C. Compound Embodiment 3 R 11 , R 12 , and R 13 is R 11 and the carbon atom to which R is bonded 12 and R 13 are combined together with the carbon atom to which they are attached to form a benzene ring or a 6-membered heterocyclic ring, optionally bearing one or more substituents selected from Group 9. The benzene ring or heterocycle may have at least one substituent at any position. When multiple substituents are present, the substituents may be the same or different. Each substituent on the benzene ring or heterocycle is a halogen atom, a C1-C6 alkyl group optionally substituted with 1 to 3 halogen atoms, or a C1-C6 alkoxy group, preferably a halogen atom, a C1-C3 alkyl group optionally substituted with 1 to 3 halogen atoms, or a C1-C3 alkoxy group, more preferably a halogen atom, a methyl group, or a methoxy group. The "benzene ring or 6-membered heterocyclic ring optionally having one or more substituents" is preferably an unsubstituted benzene ring. R 11 , R 12 and R 13 Most preferably, satisfies (i) above.

[0309] D. Other Variables and Embodiments R 14 and R 15Each represents a hydrogen atom, or R 14 and R 15 taken together represent an imine bond (C=N). V and W each independently represent an oxygen atom, a nitrogen atom, or a sulfur atom, and are preferably an oxygen atom. R 16 and R 17 teeth, (a)R 16 and R 17 are combined to form an imine bond (N=C); or (b)R 16 is J-La'-Lp'-NH-B'-CH2-O(C=O)- * represents R 17 represents a hydroxy group or a C1-C3 alkoxy group. (b)R 16 is J-La'-Lp'-NH-B'-CH2-O(C=O)- * In the case where: the asterisk in the formula represents the bond to the N10' position of the pyrrolobenzodiazepine ring represented by the formula above.

[0310] B' represents a phenyl group or a heteroaryl group, preferably a 1,4-phenyl group, a 2,5-pyridyl group, a 3,6-pyridyl group, a 2,5-pyrimidyl group, or a 2,5-thienyl group, and more preferably a 1,4-phenyl group. Lp' represents a linker consisting of an amino acid sequence that is cleavable in vivo or in a target cell, for example, by the action of enzymes such as esterases and peptidases. Specific examples of linkers Lp′ include, but are not limited to, -GGVA- (SEQ ID NO: 85), -GG-(D-)VA-, -VA-, -GGFG- (SEQ ID NO: 86), -GGPI- (SEQ ID NO: 87), -GGVCit- (SEQ ID NO: 88), -GGVK- (SEQ ID NO: 89), -GG(D-)PI-, -GGPL- (SEQ ID NO: 90), -EGGVA (SEQ ID NO: 91), -PI-, -GGF-, DGGF- (SEQ ID NO: 92), (D-)D-GGF-, -EGGF- (SEQ ID NO: 93), -SGGF- (SEQ ID NO: 94), -KGGF- (SEQ ID NO: 95), -DGGFG- (SEQ ID NO: 96), -GGFGG- (SEQ ID NO: 97), -DDGGFG- (SEQ ID NO: 98), -KDGGFG- (SEQ ID NO: 99), and -GGFGGGF- (SEQ ID NO: 100).

[0311] Here, "(D-)V" indicates D-valine, "(D)-P" indicates D-proline, and "(D-)D" indicates D-aspartic acid. The linker Lp' is preferably as follows: -GGVA- (SEQ ID NO: 85), -GG-(D-)VA-, -VA-, -GGFG- (SEQ ID NO: 86), -GGPI- (SEQ ID NO: 87), -GGVCit- (SEQ ID NO: 88), -GGVK- (SEQ ID NO: 89), -GG(D-)PI-, or -GGPL- (SEQ ID NO: 90). More preferred examples are -GGVA- (SEQ ID NO: 85), -GGVCit- (SEQ ID NO: 88), and -VA-.

[0312] La' is the following group: -C(=O)-(CH2CH2)n 6 -C(=O)-, -C(=O)-(CH2CH2)n 6 -C(=O)-NH-(CH2CH2)n 7 -C(=O)-, -C(=O)-(CH2CH2)n 6 -C(=O)-NH-(CH2CH2O)n 7 -CH2-C(=O)-, -C(=O)-(CH2CH2)n 6 -NH-C(=O)-(CH2CH2O)n7 -CH2CH2-C(=O)-, -(CH2)n 8 -OC(=O)-, -(CH2)n 12 -C(=O)- and -(CH2CH2)n 13 -C(=O)-NH-(CH2CH2O)n 14 -CH2CH2-C(=O)- wherein n represents any one selected from 6 represents an integer of 1 to 3 (preferably 1 or 2), and n 7 represents an integer of 1 to 5 (preferably an integer of 2 to 4, more preferably an integer of 2 or 4), and n 8 represents an integer of 0 to 2 (preferably 0 or 1), n 12 represents an integer of 2 to 7 (preferably an integer of 2 to 5, more preferably an integer of 2 or 5), n 13 represents an integer of 1 to 3 (preferably 1), and n 14 represents an integer of 6 to 10 (preferably 8).

[0313] La' is preferably selected from the group: -C(=O)-CH2CH2-C(=O)-, -C(=O)-(CH2CH2)2-C(=O)-, -C(=O)-CH2CH2-C(=O)-NH-(CH2CH2)2-C(=O)-, -C(=O)-CH2CH2-C(=O)-NH-(CH2CH2O)2-CH2-C(=O)-, -C(=O)-CH2CH2-NH-C(=O)-(CH2CH2O)4-CH2CH2-C(=O)-, -CH2-OC(=O)-, -OC(=O)-, -(CH2)2-C(=O)-, -(CH2)5-C(=O)-, and -CH2CH2-C(=O)-NH-(CH2CH2O)8-CH2CH2-C(=O)- represents one selected from the above.

[0314] La' is more preferably -C(=O)-CH2CH2-C(=O)-, -C(=O)-(CH2CH2)2-C(=O)-, or -(CH2)5-C(=O)-. J is not limited to a particular structure and may be any cyclic structure, such as an alkyne structure that reacts with an azide group to form a 1,2,3-triazole ring, examples of which include, but are not limited to, compounds represented by the following formula:

[0315] [ka] In the structural formula of J shown above, each asterisk represents a -(C=O) or -(CH2)n at the left end of La'. 8 Represents a bond to Alternatively, J may be a compound that binds to a side chain of an amino acid residue (e.g., cysteine ​​or lysine) or a halogen atom of the antibody Ab, and examples of J include, but are not limited to, a maleimidyl group represented by the following formula:

[0316] [ka] In the maleimidyl group shown above, the asterisk indicates the -(CH2)n group at the left end of La'. 12 or -(CH2CH2)n 13 Represents a bond to

[0317] R 16 is preferably J-La'-Lp'-NH-B'-CH2-O(C=O)- * wherein: B' is a 1,4-phenyl group; Lp' is the following group: -GGVA- (SEQ ID NO: 85), -GG-(D-)VA-, -VA-, -GGFG- (SEQ ID NO: 86), -GGPI- (SEQ ID NO: 87), -GGVCit- (SEQ ID NO: 88), -GGVK- (SEQ ID NO: 89), GG(D-)PI-, and -GGPL- (SEQ ID NO: 90) represents any one selected from; La' is the following group: -C(=O)-CH2CH2-C(=O)-, -C(=O)-(CH2CH2)2-C(=O)-, -C(=O)-CH2CH2-C(=O)-NH-(CH2CH2)2-C(=O)-, -C(=O)-CH2CH2-C(=O)-NH-(CH2CH2O)2-CH2-C(=O)-, -C(=O)-CH2CH2-NH-C(=O)-(CH2CH2O)4-CH2CH2-C(=O)-, -OC(=O)-, -CH2-OC(=O)-, -(CH2)5-C(=O)-, and -CH2CH2-C(=O)-NH-(CH2CH2O)8-CH2CH2-C(=O)- represents any one selected from; J represents one of the following structural formulas:

[0318] [ka] In the formula, in the structural formula of J, Each asterisk represents a bond to La'.

[0319] R 16 is more preferably selected from the group: J 1 —C(═O)—CHCH—C(═O)—GGVA-NH-B′-CH—OC(═O)— (“GGVA” disclosed as SEQ ID NO: 85); J 1 -C(=O)-CH2CH2-C(=O)-GG-(D-)VA-NH-B'-CH2-OC(=O)-, J 1 -C(=O)-CH2CH2-C(=O)-VA-NH-B'-CH2-OC(=O)-, J 1 -C(=O)-(CH2CH2)2-C(=O)-VA-NH-B'-CH2-OC(=O)-, J 1 —C(═O)—CHCH—C(═O)—GGPI-NH-B′-CH—OC(═O)— (“GGPI” disclosed as SEQ ID NO: 87); J1 —C(═O)—CH2CH2—C(═O)-GGFG-NH-B′-CH2-OC(═O)— (“GGFG” disclosed as SEQ ID NO: 86), J 1 —C(═O)—CHCH—C(═O)—GGVCit-NH-B′-CH—OC(═O)— (“GGVCit” disclosed as SEQ ID NO: 88); J 1 —C(═O)—CHCH—C(═O)—GGVK-NH-B′-CH—OC(═O)— (“GGVK” disclosed as SEQ ID NO: 89); J 1 —C(═O)—CHCH—C(═O)—GGPL-NH-B′-CH—OC(═O)— (“GGPL” disclosed as SEQ ID NO: 90); J 1 -C(=O)-CH2CH2-C(=O)-NH-(CH2CH2)2-C(=O)-VA-NH-B'-CH2-OC(=O)-, J 1 -C(=O)-CH2CH2-C(=O)-NH-(CH2CH2O)2-CH2-C(=O)-VA-NH-B'-CH2-OC(=O)-, J 1 -C(=O)-CH2CH2-NH-C(=O)-(CH2CH2O)4-CH2CH2-C(=O)-VA-NH-B'-CH2-OC(=O)-, J 2 —OC(═O)-GGVA-NH-B′-CH—OC(═O)- (“GGVA” disclosed as SEQ ID NO: 85), J 3 —CH—OC(═O)-GGVA-NH-B′-CH—OC(═O)- (“GGVA” disclosed as SEQ ID NO: 85), J 4 —(CH)—C(═O)—GGVA-NH-B′-CH—OC(═O)— (“GGVA” disclosed as SEQ ID NO: 85); J 4 -(CH2)5-C(=O)-VA-NH-B'-CH2-OC(=O)-, and J 4-CH2CH2-C(=O)-NH-(CH2CH2O)8-CH2CH2-C(=O)-VA-NH-B'-CH2-OC(=O)- One of the following is selected from During the ceremony, J. 1 , J 2 , J 3 , and J 4 represents the structural formula represented by:

[0320] [ka] During the ceremony, J. 1 , J 2 , J 3 and J 4 In the structural formula: Each asterisk represents a J 1 , J 2 , J 3 , or J 4 represents a bond to a group adjacent to B' is a 1,4-phenyl group.

[0321] R 16 is most preferably: J 1 —C(═O)—CHCH—C(═O)—GGVA-NH-B′-CH—OC(═O)— (“GGVA” disclosed as SEQ ID NO: 85); J 1 -C(=O)-CH2CH2-C(=O)-VA-NH-B'-CH2-OC(=O)-, J 1 -C(=O)-(CH2CH2)2-C(=O)-VA-NH-B'-CH2-OC(=O)-, J 1 —C(═O)—CHCH—C(═O)—GGVCit-NH-B′-CH—OC(═O)— (“GGVCit” disclosed as SEQ ID NO: 88); J 1 -C(=O)-CH2CH2-C(=O)-NH-(CH2CH2)2-C(=O)-VA-NH-B'-CH2-OC(=O)-, J 1-C(=O)-CH2CH2-C(=O)-NH-(CH2CH2O)2-CH2-C(=O)-VA-NH-B'-CH2-OC(=O)-, J 1 -C(=O)-CH2CH2-NH-C(=O)-(CH2CH2O)4-CH2CH2-C(=O)-VA-NH-B'-CH2-OC(=O)-, J 4 -(CH2)5-C(=O)-VA-NH-B'-CH2-OC(=O)- Either During the ceremony, B' is a 1,4-phenyl group; J 1 and J 4 is represented by the following structural formula for J:

[0322] [ka] During the ceremony, J. 1 and J 4 In the structural formula: Each asterisk represents a J 1 or J 4 represents a bond to a group adjacent to

[0323] R 17 is a hydroxy group or a C1-C3 alkoxy group, preferably a hydroxy group or a methoxy group. R17 may be a bisulfite adduct (OSO3M, where M is a metal cation). R17 is bonded to an asymmetric carbon atom, providing the configuration represented by the following partial structure (VIa) or (VIb). Each wavy line represents a bond to W in the intermediate, and the free drug is represented by the general formula (VI).

[0324] [ka] The free drug is preferably one compound selected from the following group:

[0325] [ka] In some cases, the free drug is released in tumor cells together with a portion of the linker L, but even in such a situation, it is an excellent drug that exhibits excellent antitumor activity. After the free drug is transported to tumor cells, in some cases, it is further oxidized to form R 16 and R 17 However, even in this situation, it exhibits excellent antitumor activity. The product intermediate may also be one compound selected from the following group:

[0326] [ka] The product intermediate may also be one compound selected from the following group:

[0327] [ka]

[0328] VIII. Pharmaceuticals The antibody-drug conjugates of the present invention exhibit cytotoxic activity against cancer cells and may therefore be used as medicines, particularly as therapeutic and / or prophylactic agents for cancer. In some embodiments, the cancer expresses or overexpresses DLL3. Examples of cancers to which the antibody-drug conjugates of the present invention are applicable include small cell lung cancer (SCLC), large cell neuroendocrine carcinoma (LCNEC), neuroendocrine tumors of various tissues including the kidney, genitourinary tract (bladder, prostate, ovary, cervix, and endometrium), gastrointestinal tract (stomach, colon), thyroid (medullary thyroid carcinoma), pancreas, and lung, glioma, or pseudoneuroendocrine tumor (pNET), but are not limited to these, so long as the cancer cells as therapeutic targets express a protein that can be recognized by the antibody in the antibody-drug conjugate.

[0329] The antibody-drug conjugates of the present invention can be preferably administered to mammals, and more preferably to humans. Substances used in pharmaceutical compositions containing the antibody-drug conjugates of the present invention can be suitably selected and applied from formulation adjuvants generally used in the art, taking into consideration the dose or concentration for administration.

[0330] The antibody-drug conjugates of the present invention may be administered as pharmaceutical compositions containing one or more pharmaceutically acceptable ingredients. For example, pharmaceutical compositions typically contain one or more pharmaceutical carriers (e.g., sterile liquids, such as water and oils (petroleum, and animal, vegetable, or synthetic oils, such as peanut oil, soybean oil, mineral oil, and sesame oil)). When the pharmaceutical composition is administered intravenously, water is a more typical carrier. Saline solution, aqueous dextrose, and aqueous glycerol can also be used as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical vehicles are known in the art. If necessary, the composition may also contain minor amounts of humectants, emulsifiers, or pH buffering agents. Examples of suitable pharmaceutical carriers are disclosed in "Remington's Pharmaceutical Sciences" by E.W. Martin. The formulation will depend on the mode of administration.

[0331] The type of cancer to which the anti-DLL3 antibody-drug conjugate of the present invention is applied is not particularly limited, as long as the cancer expresses DLL3 in the cancer cells to be treated. Examples include small cell lung cancer (SCLC), large cell neuroendocrine carcinoma (LCNEC), neuroendocrine tumors of various tissues, including the kidney, genitourinary tract (bladder, prostate, ovary, cervix, and endometrium), gastrointestinal tract (stomach, colon), thyroid (medullary thyroid carcinoma), pancreas, and lung, glioma, or pseudoneuroendocrine tumor (pNET). However, the cancer is not limited, as long as the cancer expresses DLL3. More preferred examples of cancer include small cell lung cancer (SCLC), large cell neuroendocrine carcinoma (LCNEC), and neuroendocrine tumors of various tissues. The anti-DLL3 antibody-drug conjugates of the present invention can be administered preferably to mammals, more preferably to humans. Various delivery systems are known and can be used to administer the antibody-drug conjugate of the present invention. Examples of administration routes include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, and subcutaneous routes. Administration can be by injection or bolus injection, for example. According to a specific preferred embodiment, the above-described ligand-drug conjugate forms are administered by injection. A preferred administration route is parenteral administration.

[0332] According to a representative embodiment, the pharmaceutical composition is formulated according to conventional procedures as a pharmaceutical composition suitable for intravenous administration to humans. Compositions for intravenous administration are typically solutions in sterile and isotonic aqueous buffer. If necessary, the drug may contain a solubilizing agent and a local anesthetic (e.g., lignocaine) to reduce pain at the injection site. Generally, the above ingredients are provided either individually as a dried lyophilized powder or water-free concentrate in individual containers, or as a mixture in unit dosage form, sealed in ampoules or small containers according to the indicated amount of active ingredient. When the pharmaceutical composition is to be administered by injection, it may be administered from an injection bottle containing sterile pharmaceutical-grade water or saline. When the drug is administered by injection, an ampoule of sterile water for injection or saline may be provided so that the aforementioned ingredients can be mixed together prior to administration.

[0333] The pharmaceutical composition of the present invention may be a pharmaceutical composition containing only the antibody-drug conjugate of the present invention, or a pharmaceutical composition containing the antibody-drug conjugate and at least one agent other than the antibody-drug conjugate for treating cancer. The antibody-drug conjugate of the present invention may be administered in combination with another agent for treating cancer, thereby enhancing the anti-cancer effect. The other anti-cancer agent used for such a purpose may be administered to an individual simultaneously with, separately from, or subsequently to the antibody-drug conjugate, and the administration interval between them may also be varied. Examples of such drugs for treating cancer include Abraxane, carboplatin, cisplatin, gemcitabine, irinotecan (CPT-11), paclitaxel, pemetrexed, sorafenib, vinblastine, drugs described in International Publication No. WO2003 / 038043, LH-RH analogs (e.g., leuprorelin, goserelin), estramustine phosphate, estrogen antagonists (e.g., tamoxifen, raloxifene), and aromatase inhibitors (e.g., anastrozole, letrozole, exemestane), but are not limited to these, so long as they are drugs with antitumor activity.

[0334] The pharmaceutical composition can be formulated into a lyophilized or liquid formulation as a formulation having the selected composition and required purity. When formulated as a lyophilized formulation, the pharmaceutical composition may be a formulation containing suitable formulation adjuvants used in the art. In the case of a liquid formulation, the pharmaceutical composition may also be formulated as a liquid formulation containing various formulation adjuvants used in the art.

[0335] The composition and concentration of the pharmaceutical composition can vary depending on the administration method. However, the antibody-drug conjugate contained in the pharmaceutical composition of the present invention can exhibit a pharmaceutical effect even at a low dosage if the antibody-drug conjugate has a higher affinity for the antigen, i.e., a higher affinity (lower Kd value) in terms of the dissociation constant (Kd value) for the antigen. Therefore, when determining the dosage of the antibody-drug conjugate, the dosage can be set taking into account the affinity of the antibody-drug conjugate with the antigen. When the antibody-drug conjugate of the present invention is administered to a human, for example, about 0.001 to 100 mg / kg may be administered in a single dose or in several divided doses at intervals of 1 to 180 days.

[0336] In summary, the disclosed immunoglobulin-related compositions (e.g., antibodies or antigen-binding fragments thereof, such as 2-C8-A, 6-G23-F, 10-O18-A, H2-C8-A, H6-G23-F, or H10-O18-A) and antibody-drug conjugates thereof are useful for treating DLL3-associated cancers. Such treatments can be used in patients identified as having pathologically elevated levels of DLL3 (e.g., those diagnosed by the methods described herein) or patients diagnosed with a disease known to be associated with such pathological levels. In one aspect, the present disclosure provides a method for treating DLL3-associated cancer in a subject in need thereof, comprising administering to the subject an effective amount of an antibody (or antigen-binding fragment thereof) of the present technology. Examples of cancers that can be treated with the antibodies of the present technology include, but are not limited to, small cell lung cancer (SCLC), large cell neuroendocrine carcinoma (LCNEC), neuroendocrine tumors, gliomas, or pseudoneuroendocrine tumors (pNETs) of various tissues, including the kidney, genitourinary tract (bladder, prostate, ovary, cervix, and endometrium), gastrointestinal tract (stomach, colon), thyroid (medullary thyroid carcinoma), pancreas, and lung. The composition of the present technology can optionally be administered to a subject in need thereof as a single bolus. Alternatively, a dosing regimen may include multiple administrations administered at various times after tumor appearance. Administration can be by any suitable route, such as oral, intranasal, parenteral (intravenous, intramuscular, intraperitoneal, or subcutaneous), rectal, intracranial, intratumoral, intrathecal, or topical. Administration includes self-administration and administration by another. It is also to be understood that the various treatment modalities for the described medical conditions are intended to mean "substantial," where substantial includes all treatment, but also includes less than all treatment, that achieves some biologically or medically relevant result. [Example]

[0337] The technology of the present invention is further illustrated by the following examples, which should not be construed as limiting in any way. The following examples demonstrate the preparation, characterization, and use of exemplary anti-DLL3 antibodies and antibody-drug conjugates (ADCs) of the technology of the present invention. However, these examples are not intended to limit the scope of the invention. Furthermore, these examples should not be construed in any way as limiting. In the following examples, unless otherwise specified, individual operations related to genetic engineering were performed according to the methods described in "Molecular Cloning" (Sambrook, J., Fritsch, E. F., and Maniatis, T., published by Cold Spring Harbor Laboratory Press in 1989) or other methods described in laboratory manuals used by those skilled in the art. It should be noted that, when commercially available reagents or kits are used, the examples were performed according to the instructions included with the commercially available products. In describing the present invention, reagents, solvents, and starting materials are readily available from commercially available sources, unless otherwise specified.

[0338] Example 1 Monoclonal antibody generation The extracellular domain (ECD) of DLL3 corresponding to amino acids Ala27-Ala479 with a C-terminal 6xHis tag (SEQ ID NO: 84) (GenBank accession number Q9NY J7-1) generated in HEK293T cells stably expressing full-length DLL3 was used as the immunogen. Ablexis AlivaMAb Kappa Mice (Ablexis, San Diego, CA), which harbor a human immunoglobulin repertoire, were immunized with either soluble DLL3-ECD or stable cells over a 3-week period according to standard immunization techniques. Splenocytes and draining lymph node cells from mice with high DLL3-specific serum titers were harvested and fused with mouse myeloma cells to generate hybridomas using electrofusion. These hybridomas were then screened to identify the presence of antibodies that specifically bound to the soluble DLL3-ECD by ELISA and the presence of full-length DLL3 protein in stably expressing 293 cells by flow cytometry relative to parental 293 cells. Hybridomas were selected for further investigation by flow cytometric ranking for staining intensity in 293DLL3 transfectants along with 4°C / 37°C staining, as described below.

[0339] Example 2 4 / 37 internalization assay using monoclonal antibodies 6-G23-F, 2-C8-A, 7-I1-B, and 10-O18-A Four monoclonal antibodies (6-G23-F, 2-C8-A, 7-I1-B, and 10-O18-A) were compared for their ability to internalize DLL3 by comparing staining at 4°C with staining at 37°C. The reference monoclonal antibody SC16, known to internalize via DLL3 and to have ADC activity and previously reported in the literature, was used as a positive control for internalization. Exponentially growing NCI-H82 cells were harvested with trypsin / EDTA, washed once in RPMI containing 10% fetal calf serum (FCS), and plated at 2 x 10 cells in DMEM supplemented with 10% FCS. 7 The cells were resuspended at 2 x 10 cells / ml. 6100 μg / ml) was added to a U-bottom 96-well plate. Test monoclonal antibodies (6-G23-F, 2-C8-A, 7-I1-B, or 10-O18-A) or reference monoclonal antibodies were added to separate wells to obtain a final concentration of 10 μg / ml in duplicate plates. Both plates were kept at 4°C for 30 minutes, after which both plates were washed twice with cold RPMI supplemented with 10% FCS and resuspended in RPMI supplemented with 10% FCS. One plate was kept at 4°C (control plate), while the other plate was incubated at 37°C in a CO2 incubator (experimental plate). After 4 hours of incubation in a CO2 incubator at 37°C for the experimental plate and 4°C for the control plate, the cells were washed three times with cold wash buffer (PBS containing 0.5% BSA) at 4°C. Samples were then resuspended in cold wash buffer plus R-phycoerythrin-AffiniPure F(ab')2 fragment goat anti-mouse IgG (Jackson 115-116-071) at a final concentration of 7 μg / ml in wash buffer. After a 30-minute incubation at 4°C, cells were washed three times with cold wash buffer, fixed in PBS 0.5% paraformaldehyde, and analyzed by flow cytometry within 48 hours. The mean fluorescence intensity (MFI) ratio, calculated by dividing the MFI obtained from the control plate (incubated with antibody at 4°C) by the corresponding MFI obtained from the experimental plate (incubated with antibody at 37°C), was taken as a relative measure of internalization. Higher values ​​indicated greater internalization. As shown in the table below, all of the monoclonal antibodies (6-G23-F, 2-C8-A, 7-I1-B, and 10-O18-A) were able to internalize DLL3 to which they bound, but not to the same extent as the reference monoclonal antibody.

[0340] [Table 3] These results demonstrate that the immunoglobulin-related compositions of the present technology are internalized via binding to DLL3. Thus, the immunoglobulin-related compositions disclosed herein are useful for delivering therapeutic agents to DLL3-positive cancer cells.

[0341] Example 3 Quenching internalization assay for monoclonal antibodies 6-G23-F, 2-C8-A, 7-I1-B, and 10-O18-A To rank monoclonal antibodies for internalization, we used a quenching internalization assay. This method reflects internalization and entry into the endosomal / lysosomal pathway. Goat anti-mouse IgG1 F(ab) (Jackson Immunoresearch 115-007-185) was double-labeled with Dy light Dy650 NHS ester (Thermofisher 02206) and LICOR IRDye QC1 NHS ester (LICOR 929-7030) (the double-labeled antibody is referred to herein as "F(ab)Dy650-QC1"). The principle of this assay is as follows: Dy light Dy650 fluorescence is quenched by IRDye QC1, so F(ab)Dy650-QC1 does not fluoresce. However, upon internalization, F(ab)Dy650-QC1 is degraded via the endosomal / lysosomal pathway, and the resulting release of IRDye QC1 allows the fluorescence of Dy light Dy650 to be observed. Therefore, the Dy light Dy650 fluorescence signal was taken as a measure of lysosomal-mediated internalization. Briefly, exponentially growing NCI-H82 cells were harvested with trypsin / EDTA, washed once in growth medium RPMI supplemented with 10% FCS, resuspended in growth medium, and plated at 1.25 × 10 cells per well. 680 μl of the monoclonal DLL3 antibody was added to the cells. The monoclonal DLL3 antibody at a concentration of 200 μg / ml was mixed with goat anti-mouse IgG1 Dy650 QC1 at 200 μg / ml for 20 minutes at room temperature, and 20 μl of the mixture was added to the cells. After 30 minutes of incubation at 4°C, the cells were washed twice with growth medium, resuspended in growth medium, and shifted to 37°C in a CO2 incubator for 4 hours to allow for internalization. The cells were then washed twice with ice-cold PBS containing 0.5% BSA and analyzed by flow cytometry to determine the mean fluorescence intensity. The mean fluorescence intensity of the control reference monoclonal was set to 100% internalization. As shown in the table below, all four monoclonal antibodies demonstrated internalization and entry into the endosomal / lysosomal pathway.

[0342] [Table 4]

[0343] These results demonstrate that the immunoglobulin-related compositions of the present technology are internalized through binding to DLL3 and enter the phagosomal / lysosomal compartment of cells. Thus, the immunoglobulin-related compositions disclosed herein are useful for delivering therapeutic agents to DLL3-positive cancer cells.

[0344] Example 4 Fab ZAP assay for anti-DLL3 monoclonal antibodies The Fab ZAP assay was used as an alternative method to measure internalization. The Fab ZAP assay measures the delivery of toxins into cells via the internalization of anti-DLL3 monoclonal antibodies. The Fab ZAP assay uses saporin toxin-conjugated F(ab) anti-mouse heavy and light chains to tag monoclonal antibodies with toxins. A panel of anti-DLL3 monoclonal antibodies was characterized using a kit from Advanced Targeting Systems, following the Fab ZAP assay protocol. Briefly, exponentially growing NCI-H82 cells were harvested with trypsin / EDTA, washed once in RPMI supplemented with 10% FCS, and plated at 5,000 cells / well in 96-well white solid plates in 100 μl of RPMI supplemented with 10% FCS. The following day, 25 μl of purified monoclonal antibody (G23-F, 2-C8-A, 7-I1-B, or 10-O18-A) or a reference monoclonal antibody was added at a starting concentration of 10 μg / ml, and serial...

Claims

1. The formula: 【Chemistry 1】 An antibody-drug conjugate represented by the formula: During the ceremony, m 1 represents an integer of 1 to 2; D has the following formula: 【Chemistry 2】 wherein the asterisk represents a bond to L; L is a linker connecting the N297 glycan of Ab and D; The N297 glycan may optionally be remodeled; Ab represents an anti-DLL3 antibody or a functional fragment of said antibody that specifically binds to DLL-3; antibody-drug conjugate.

2. The anti-DLL3 antibody comprises a heavy chain immunoglobulin variable domain (V H ) and a light chain immunoglobulin variable domain (V L ), (a) V H teeth, (i) SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:5, respectively; (ii) SEQ ID NO:13, SEQ ID NO:14, and SEQ ID NO:15, respectively; (iii) SEQ ID NO:23, SEQ ID NO:24, and SEQ ID NO:25, respectively; and (iv) SEQ ID NO: 33, SEQ ID NO: 34, and SEQ ID NO: 35, respectively. V selected from the group consisting of H - CDR1 sequence, V H - CDR2 sequence, and V H - comprises a CDR3 sequence; and / or (b) V L teeth, (i) SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:10, respectively; (ii) SEQ ID NO:18, SEQ ID NO:19, and SEQ ID NO:20, respectively; (iii) SEQ ID NO:28, SEQ ID NO:29, and SEQ ID NO:30, respectively; and (iv) SEQ ID NO: 38, SEQ ID NO: 39, and SEQ ID NO: 40, respectively. V selected from the group consisting of L - CDR1 sequence, V L - CDR2 sequence, and V L -CDR3 sequence.

3. The anti-DLL3 antibody comprises a heavy chain immunoglobulin variable domain (V H ) and a light chain immunoglobulin variable domain (V L ), (a) V H - CDR1 sequence, V H - CDR2 sequence, and V H - V containing CDR3 sequence H and (b) V L - CDR1 sequence, V L - CDR2 sequence, and V L - V containing CDR3 sequence L The combination of (i) (a) SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:5, and (b) SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:10, respectively; (ii) (a) SEQ ID NO:13, SEQ ID NO:14, and SEQ ID NO:15, and (b) SEQ ID NO:18, SEQ ID NO:19, and SEQ ID NO:20, respectively; (iii) (a) SEQ ID NO:23, SEQ ID NO:24, and SEQ ID NO:25, and (b) SEQ ID NO:28, SEQ ID NO:29, and SEQ ID NO:30, respectively; and (iv) (a) SEQ ID NO:33, SEQ ID NO:34, and SEQ ID NO:35, and (b) SEQ ID NO:38, SEQ ID NO:39, and SEQ ID NO:40, respectively. The antibody-drug conjugate of claim 1 or 2, selected from the group consisting of:

4. The anti-DLL3 antibody has the following characteristics: (a) a light chain immunoglobulin variable domain sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to a light chain immunoglobulin variable domain sequence present in any one of SEQ ID NOs: 7, 17, 27, or 37; and / or (b) a heavy chain immunoglobulin variable domain sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to a heavy chain immunoglobulin variable domain sequence present in any one of SEQ ID NOs: 2, 12, 22, or 32. The antibody drug conjugate of any one of claims 1 to 3, comprising one or more of:

5. The anti-DLL3 antibody has the following characteristics: (a) a light chain immunoglobulin variable domain sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to a light chain immunoglobulin variable domain sequence present in any one of SEQ ID NOs: 17, 27, or 37; and (b) a heavy chain immunoglobulin variable domain sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to a heavy chain immunoglobulin variable domain sequence present in any one of SEQ ID NOs: 12, 22, or 32. The antibody drug conjugate of any one of claims 1 to 4, comprising one or more of:

6. The anti-DLL3 antibody comprises a heavy chain immunoglobulin variable domain (V H ) and a light chain immunoglobulin variable domain (V L (a) V H comprises an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:12, SEQ ID NO:22, and SEQ ID NO:32; and / or (b) V L The antibody drug conjugate of any one of claims 1 to 5, comprising an amino acid sequence selected from the group consisting of SEQ ID NO:7, SEQ ID NO:17, SEQ ID NO:27, and SEQ ID NO:

37.

7. The anti-DLL3 antibody comprises a heavy chain immunoglobulin variable domain (V H ) and a light chain immunoglobulin variable domain (V L (a) V H (b) V comprises an amino acid sequence selected from SEQ ID NO: 12, SEQ ID NO: 22, or SEQ ID NO: 32; L The antibody drug conjugate of any one of claims 1 to 6, comprising an amino acid sequence selected from SEQ ID NO: 17, SEQ ID NO: 27 or SEQ ID NO:

37.

8. V H Amino acid sequence and V L 7. The antibody drug conjugate of claim 6, wherein the amino acid sequences are selected from the group consisting of SEQ ID NO:2 and SEQ ID NO:7 (7-I1-B), respectively; SEQ ID NO:12 and SEQ ID NO:17 (2-C8-A), respectively; SEQ ID NO:22 and SEQ ID NO:27 (10-O18-A), respectively; and SEQ ID NO:32 and SEQ ID NO:37 (6-G23-F), respectively.

9. The antibody-drug conjugate of any one of claims 1 to 8, which undergoes intracellular internalization upon binding of the anti-DLL3 antibody to a DLL3 polypeptide expressed on a cell surface (e.g., a tumor cell surface).

10. 10. The antibody-drug conjugate of any one of claims 1 to 9, wherein the anti-DLL3 antibody comprises an Fc domain of an isotype selected from the group consisting of IgG1 or a variant thereof, IgG2, IgG3, IgG4, IgA1, IgA2, IgM, IgD, and IgE.

11. The antibody-drug conjugate of any one of claims 1 to 10, wherein the anti-DLL3 antibody comprises a heavy chain constant region of SEQ ID NO: 42, 57 or 58.

12. The antibody-drug conjugate of any one of claims 1 to 7, wherein the anti-DLL3 antibody is a monoclonal antibody, a chimeric antibody, a humanized antibody, a human antibody or a bispecific antibody.

13. The antibody, (a) a heavy chain comprising the amino acid sequence of any one of SEQ ID NOs: 59-61, and a light chain comprising the amino acid sequence of SEQ ID NO: 62; (b) a heavy chain comprising the amino acid sequence of any one of SEQ ID NOs: 63-65 and a light chain comprising the amino acid sequence of SEQ ID NO: 66; or (c) a heavy chain comprising any one of the amino acid sequences of SEQ ID NOs: 67 to 69 and a light chain comprising the amino acid sequence of SEQ ID NO: 70 The antibody-drug conjugate according to any one of claims 1 to 12, comprising:

14. The antibody, (a) a heavy chain comprising the amino acid sequence of SEQ ID NO:61 and a light chain comprising the amino acid sequence of SEQ ID NO:62; (b) a heavy chain comprising the amino acid sequence of SEQ ID NO:65 and a light chain comprising the amino acid sequence of SEQ ID NO:66; or (c) a heavy chain comprising the amino acid sequence of SEQ ID NO: 69 and a light chain comprising the amino acid sequence of SEQ ID NO:

70. The antibody-drug conjugate of any one of claims 1 to 13, comprising:

15. 2. The antibody drug conjugate of claim 1, wherein the anti-DLL-3 antibody competes with the antibody of claim 9 or claim V for binding to DLL-3.

16. 16. The antibody-drug conjugate of any one of claims 1 to 15, wherein the anti-DLL3 antibody binds to an epitope on DLL3 that is a conformational epitope or a non-conformational epitope.

17. 17. The antibody-drug conjugate of any one of claims 1 to 16, wherein the anti-DLL3 antibody binds to a mammalian DLL3 polypeptide having an amino acid sequence comprising amino acid residues 27 to 492 of SEQ ID NO:50 or SEQ ID NO:

51.

18. The heavy or light chain of the antibody may be modified by any of the following: N-linked glycosylation, O-linked glycosylation, N-terminal processing, C-terminal processing, deamidation, isomerization of aspartic acid, oxidation of methionine, addition of a methionine residue to the N-terminus, amidation of a proline residue, substitution of two leucine (L) residues at positions 234 and 235 of the heavy chain with alanine (A) (EU numbering) (LALA), a set of amino acid residues, Glu (E) at position 356 and Met (M) at position 358 of the heavy chain ( The antibody-drug conjugate of any one of claims 1 to 17, having one or two or more modifications or sets of amino acid residues selected from the group consisting of: a set of Asp (D) at position 356 and Leucine (L) at position 358 of the heavy chain (EU numbering), or any combination thereof, an N-terminal glutamine or conversion of N-terminal glutamic acid to pyroglutamic acid, and a deletion of one or two amino acids from the carboxyl terminus.

19. The antibody-drug conjugate of claim 18, wherein one or two amino acids are deleted from the carboxyl terminus of the heavy chain.

20. The antibody-drug conjugate of claim 19, wherein one amino acid is deleted from each of both carboxyl termini of the heavy chains.

21. The antibody-drug conjugate of any one of claims 18 to 20, wherein the proline residue at the carboxyl terminus of the heavy chain is further amidated.

22. The antibody-drug conjugate of any one of claims 1 to 21, wherein the anti-DLL3 antibody comprises glycosylation that is regulated to enhance antibody-dependent cellular cytotoxicity.

23. D. 【Chemistry 3】 The antibody-drug conjugate according to any one of claims 1 to 22,

24. D. 【Chemistry 4】 The antibody-drug conjugate according to any one of claims 1 to 22,

25. D. 【Chemistry 5】 The antibody-drug conjugate according to any one of claims 1 to 22,

26. D. 【Chemistry 6】 The antibody-drug conjugate according to any one of claims 1 to 22,

27. The antibody-drug conjugate of any one of claims 23 to 26, wherein -OH is at the 11' position.

28. L is -Lb-La-Lp-NH-B-CH 2 -O(C=O)- * where the asterisk represents the bond to D; B represents a phenyl group or a heteroaryl group; Lp represents a linker consisting of an amino acid sequence cleavable in the target cell; La is selected from the group: -C(=O)-(CH 2 CH 2 )n 2 -C(=O)-、 -C(=O)-(CH 2 CH 2 )n 2 -C(=O)-NH-(CH 2 CH 2 )n 3 -C(=O)-、 -C(=O)-(CH 2 CH 2 )n 2 -C(=O)-NH-(CH 2 CH 2 O)n 3 -CH 2 -C(=O)-、 -C(=O)-(CH 2 CH 2 )n 2 -NH-C(=O)-(CH 2 CH 2 O)n 3 -CH 2 CH 2 -C(=O)-, and -(CH 2 )n 4 -O-C(=O)- represents any one selected from: n 2 represents an integer of 1 to 3; 3 represents an integer from 1 to 5; 4 represents an integer from 0 to 2; The antibody-drug conjugate of any one of claims 1 to 27, wherein Lb represents a spacer linking La to the glycan or remodeled glycan of Ab.

29. The antibody-drug conjugate of claim 28, wherein B is any one selected from a 1,4-phenyl group, a 2,5-pyridyl group, a 3,6-pyridyl group, a 2,5-pyrimidyl group, and a 2,5-thienyl group.

30. The antibody-drug conjugate of claim 29, wherein B is a 1,4-phenyl group.

31. The antibody-drug conjugate according to any one of claims 28 to 30, wherein Lp is an amino acid residue consisting of 2 to 7 amino acids.

32. The antibody-drug conjugate according to any one of claims 28 to 31, wherein Lp is an amino acid residue consisting of an amino acid selected from glycine, valine, alanine, phenylalanine, glutamic acid, isoleucine, proline, citrulline, leucine, serine, lysine, and aspartic acid.

33. The antibody-drug conjugate of any one of claims 28 to 32, wherein Lp is selected from the following group: -GGVA- (SEQ ID NO: 85), -GG-(D-)VA-, -VA-, -GGFG- (SEQ ID NO: 86), -GGPI- (SEQ ID NO: 87), -GGVCit- (SEQ ID NO: 88), -GGVK- (SEQ ID NO: 89), -GG(D-)PI-, and -GGPL- (SEQ ID NO: 90).

34. La is selected from the group: -C(=O)-CH 2 CH 2 -C(=O)-、-C(=O)-(CH 2 CH 2 ) 2 -C(=O)-、 -C(=O)-CH 2 CH 2 -C(=O)-NH-(CH 2 CH 2 ) 2 -C(=O)-、 -C(=O)-CH 2 CH 2 -C(=O)-NH-(CH 2 CH 2 O) 2 -CH 2 -C(=O)-、 -C(=O)-CH 2 CH 2 -NH-C(=O)-(CH 2 CH 2 O) 4 -CH 2 CH 2 -C(=O)-、 -CH 2 —OC(═O)— and —OC(═O)— The antibody-drug conjugate according to any one of claims 28 to 33, selected from the group consisting of

35. Lb is represented by the following formula: 【Chemistry 7】 In each structural formula of Lb shown above, The antibody-drug conjugate of any one of claims 28 to 34, wherein each asterisk represents a bond to La and each wavy line represents a bond to an Ab glycan or a remodeled glycan.

36. L is -Lb-La-Lp-NH-B-CH 2 -O(C=O)- * wherein: B is a 1,4-phenyl group; Lp represents any one selected from the following group: -GGVA- (SEQ ID NO: 85), -GG-(D-)VA-, -VA-, -GGFG- (SEQ ID NO: 86), -GGPI- (SEQ ID NO: 87), -GGVCit- (SEQ ID NO: 88), -GGVK- (SEQ ID NO: 89), and -GGPL- (SEQ ID NO: 90); La is selected from the group: -C(=O)-CH 2 CH 2 -C(=O)-、-C(=O)-(CH 2 CH 2 ) 2 -C(=O)-、 -C(=O)-CH 2 CH 2 -C(=O)-NH-(CH 2 CH 2 ) 2 -C(=O)-、 -C(=O)-CH 2 CH 2 -C(=O)-NH-(CH 2 CH 2 O) 2 -CH 2 -C(=O)-、 -C(=O)-CH 2 CH 2 -NH-C(=O)-(CH 2 CH 2 O) 4 -CH 2 CH 2 -C(=O)-、 -CH 2 —OC(═O)— and —OC(═O)— represents any one selected from: Lb is represented by the following formula: 【Chemistry 8】 wherein in each structural formula of Lb shown above, The antibody-drug conjugate of any one of claims 28 to 35, wherein each asterisk represents a bond to La and each wavy line represents a bond to an Ab glycan or a remodeled glycan.

37. L is selected from the group: -Z 1 -C(=O)-CH 2 CH 2 -C(=O)-GGVA-NH-B-CH 2 -OC(=O)- ("GGVA" disclosed as SEQ ID NO:85), -Z 1 -C(=O)-CH 2 CH 2 -C(=O)-GG-(D-)VA-NH-B-CH 2 -OC(=O)-、 -Z 1 -C(=O)-CH 2 CH 2 -C(=O)-VA-NH-B-CH 2 -OC(=O)-、 -Z 1 -C(=O)-(CH 2 CH 2 ) 2 -C(=O)-VA-NH-B-CH 2 -OC(=O)-、 -Z 1 -C(=O)-CH 2 CH 2 -C(=O)-GGPI-NH-B-CH 2 -OC(=O)- ("GGPI" disclosed as SEQ ID NO:87), -Z 1 -C(=O)-CH 2 CH 2 -C(=O)-GGFG-NH-B-CH 2 -OC(=O)- ("GGFG" disclosed as SEQ ID NO:86), -Z 1 -C(=O)-CH 2 CH 2 -C(=O)-GGVCit-NH-B-CH 2 -OC(=O)- ("GGVCit" disclosed as SEQ ID NO:88), -Z 1 -C(=O)-CH 2 CH 2 -C(=O)-GGVK-NH-B-CH 2 -OC(=O)- ("GGVK" disclosed as SEQ ID NO:89), -Z 1 -C(=O)-CH 2 CH 2 -C(=O)-GGPL-NH-B-CH 2 -OC(=O)- ("GGPL" disclosed as SEQ ID NO:90), -Z 1 -C(=O)-CH 2 CH 2 -C(=O)-NH-(CH 2 CH 2 ) 2 -C(=O)-VA-NH-B-CH 2 -OC(=O)-、 -Z 1 -C(=O)-CH 2 CH 2 -C(=O)-NH-(CH 2 CH 2 O) 2 -CH 2 -C(=O)-VA-NH-B-CH 2 -OC(=O)-、 -Z 1 -C(=O)-CH 2 CH 2 -NH-C(=O)-(CH 2 CH 2 O) 4 -CH 2 CH 2 -C(=O)-VA-NH-B-CH 2 -OC(=O)-、 -Z 2 -OC(=O)-GGVA-NH-B-CH 2 -OC(=O)- ("GGVA" disclosed as SEQ ID NO:85), and -Z 3 -CH 2 -OC(=O)-GGVA-NH-B-CH 2 -OC(=O)- ("GGVA" disclosed as SEQ ID NO:85) is selected from the group consisting of Z 1 has the following structural formula: 【Chemistry 9】 represents Z 2 has the following structural formula: 【Chemistry 10】 represents Z 3 has the following structural formula: 【Chemistry 11】 wherein Z represents 1 , Z 2 , and Z 3 In each structural formula, Each asterisk represents binding to La and each wavy line represents binding of Ab to a glycan or remodeled glycan; The antibody-drug conjugate of any one of claims 28 to 36, wherein B represents a 1,4-phenyl group.

38. L is selected from the group: -Z 1 -C(=O)-CH 2 CH 2 -C(=O)-GGVA-NH-B-CH 2 -OC(=O)- ("GGVA" disclosed as SEQ ID NO:85), -Z 1 -C(=O)-CH 2 CH 2 -C(=O)-VA-NH-B-CH 2 -OC(=O)-、 -Z 1 -C(=O)-(CH 2 CH 2 ) 2 -C(=O)-VA-NH-B-CH 2 -OC(=O)-、 -Z 1 -C(=O)-CH 2 CH 2 -C(=O)-GGVCit-NH-B-CH 2 -OC(=O)- ("GGVCit" disclosed as SEQ ID NO:88), -Z 1 -C(=O)-CH 2 CH 2 -C(=O)-NH-(CH 2 CH 2 ) 2 -C(=O)-VA-NH-B-CH 2 -OC(=O)-、 -Z 1 -C(=O)-CH 2 CH 2 -C(=O)-NH-(CH 2 CH 2 O) 2 -CH 2 -C(=O)-VA-NH-B-CH 2 -OC(=O)-, and -Z 1 -C(=O)-CH 2 CH 2 -NH-C(=O)-(CH 2 CH 2 O) 4 -CH 2 CH 2 -C(=O)-VA-NH-B-CH 2 -OC(=O)- is selected from the group consisting of B is a 1,4-phenyl group; Z 1 has the following structural formula: 【Chemistry 12】 represents Z 1 In the structural formula: Each asterisk represents a 1 and each wavy line represents a bond to a C(=O) adjacent to the glycan of an Ab or a remodeled glycan.

39. The antibody-drug conjugate of any one of claims 1 to 38, wherein the antibody is an IgG.

40. The antibody-drug conjugate of claim 39, wherein the antibody is IgG1 or a variant thereof, IgG2, or IgG4.

41. The antibody-drug conjugate of any one of claims 1 to 40, wherein the antibody binds to, is taken up by, and is internalized in tumor cells.

42. The antibody-drug conjugate of claim 41, wherein the antibody further has an anti-tumor effect.

43. The antibody-drug conjugate of any one of claims 1 to 42, wherein the N297 glycan is a remodeled glycan.

44. The N297 glycan is N297-(Fuc)MSG1, N297-(Fuc)MSG2, or a mixture thereof, or N297-(Fuc)SG, where N297-(Fuc)MSG1, N297-(Fuc)MSG2, and N297-(Fuc)SG have the following formula: 【Chemistry 13】 (In the formula, The wavy line represents antibody binding to Asn297; L(PEG) is -(CH 2 CH 2 -O)n 5 -CH 2 CH 2 represents -NH-, where the amino group at the right end is linked to a carboxylic acid via an amide bond at the 2-position of the sialic acid at the non-reducing end of the 1-3 branch of β-Man in the N297 glycan; The asterisk represents the bond to the linker L; n 5 represents an integer from 2 to 10) 【Chemistry 14】 (In the formula, The wavy line represents antibody binding to Asn297; L(PEG) is -(CH 2 CH 2 -O)n 5 -CH 2 CH 2 represents -NH-, where the amino group at the right end is linked to a carboxylic acid via an amide bond at the 2-position of the sialic acid at the non-reducing end of the 1-6 branched chain of β-Man in the N297 glycan; The asterisk represents the bond to the linker L; n 5 represents an integer from 2 to 10), and 【Chemistry 15】 (In the formula, The wavy line represents antibody binding to Asn297; L(PEG) is -(CH 2 CH 2 -O)n 5 -CH 2 CH 2 represents -NH-, where the amino group at the right end is linked to a carboxylic acid via an amide bond at the 2-position of the sialic acid at the non-reducing end of each of the 1-3 and 1-6 branches of β-Man in the N297 glycan; The asterisk represents the bond to the linker L; n 5 represents an integer from 2 to 10) The antibody-drug conjugate of claim 43, having a structure represented by:

45. n 5 The antibody-drug conjugate of claim 44, wherein is an integer from 2 to 5.

46. m 2 represents an integer of 1 or 2; L is one of the following groups: -Z 1 -C(=O)-CH 2 CH 2 -C(=O)-GGVA-NH-B-CH 2 -OC(=O)- ("GGVA" disclosed as SEQ ID NO:85), -Z 1 -C(=O)-CH 2 CH 2 -C(=O)-VA-NH-B-CH 2 -OC(=O)-、 -Z 1 -C(=O)-(CH 2 CH 2 ) 2 -C(=O)-VA-NH-B-CH 2 -OC(=O)-、 -Z 1 -C(=O)-CH 2 CH 2 -C(=O)-GGVCit-NH-B-CH 2 -OC(=O)- ("GGVCit" disclosed as SEQ ID NO:88), -Z 1 -C(=O)-CH 2 CH 2 -C(=O)-NH-(CH 2 CH 2 ) 2 -C(=O)-VA-NH-B-CH 2 -OC(=O)-、 -Z 1 -C(=O)-CH 2 CH 2 -C(=O)-NH-(CH 2 CH 2 O) 2 -CH 2 -C(=O)-VA-NH-B-CH 2 -OC(=O)-, and -Z 1 -C(=O)-CH 2 CH 2 -NH-C(=O)-(CH 2 CH 2 O) 4 -CH 2 CH 2 -C(=O)-VA-NH-B-CH 2 -OC(=O)- is selected from the group consisting of B is a 1,4-phenyl group; Z 1 has the following structural formula: 【Chemistry 16】 represents Z 1 In the structural formula: Each asterisk represents a 1 each wavy line represents a bond to the C(=O) adjacent to the N297 glycan of an Ab; Ab represents IgG antibody; The N297 glycan of the Ab represents any one of N297-(Fuc)MSG1, N297-(Fuc)MSG2, and a mixture thereof, and N297-(Fuc)SG, where N297-(Fuc)MSG1, N297-(Fuc)MSG2, and N297-(Fuc)SG have the following formula: 【Chemistry 17】 (In the formula, Each wavy line represents antibody binding to Asn297, L(PEG) in the N297 glycan is -NH-CH 2 CH 2 -(O-CH 2 CH 2 ) n 5 - * wherein: n 5 represents an integer from 2 to 5, the amino group at the left end is linked to a carboxylic acid via an amide bond at the 2-position of the sialic acid at the non-reducing end of each or any one of the 1-3 and 1-6 branches of β-Man in the N297 glycan, and each asterisk represents a Z 1 represents a bond to a nitrogen atom at the 1- or 3-position of the triazole ring The antibody-drug conjugate of any one of claims 1 to 45, having a structure represented by:

47. An antibody-drug conjugate selected from the group consisting of: 【Chemistry 18-1】 【Chemistry 18-2】 [wherein, in each structural formula shown above, m 2 represents an integer of 1 or 2; Ab represents an anti-DLL3 IgG antibody or a functional fragment of said antibody; The N297 glycan of the Ab represents any one of N297-(Fuc)MSG1, N297-(Fuc)MSG2, and a mixture thereof, and N297-(Fuc)SG, where N297-(Fuc)MSG1, N297-(Fuc)MSG2, and N297-(Fuc)SG have the following formula: 【Chemistry 19】 (In the formula, Each wavy line represents antibody binding to Asn297, L(PEG) in the N297 glycan is -NH-CH 2 CH 2 -(O-CH 2 CH 2 ) 3 - * wherein: The amino group at the left end is linked to a carboxylic acid via an amide bond at position 2 of a sialic acid at the non-reducing end of each or either one of the 1-3 and 1-6 branches of β-Man in the N297 glycan, and each asterisk represents a bond to a nitrogen atom at position 1 or 3 of the triazole ring in the corresponding structural formula).

48. 48. The antibody drug conjugate of claim 47, wherein the antibody comprises a heavy chain constant region of human IgG1 or a variant thereof, human IgG2, or human IgG4.

49. The antibody-drug conjugate of claim 47 or 48, wherein the antibody comprises a heavy chain constant region of SEQ ID NO: 42, 57 or 58.

50. The anti-DLL3 antibody comprises a heavy chain immunoglobulin variable domain (V H ) and a light chain immunoglobulin variable domain (V L ), (a) V H teeth, (i) SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:5, respectively; (ii) SEQ ID NO:13, SEQ ID NO:14, and SEQ ID NO:15, respectively; (iii) SEQ ID NO:23, SEQ ID NO:24, and SEQ ID NO:25, respectively; and (iv) SEQ ID NO: 33, SEQ ID NO: 34, and SEQ ID NO: 35, respectively. V selected from the group consisting of H - CDR1 sequence, V H - CDR2 sequence, and V H - comprises a CDR3 sequence; and / or (b) V L teeth, (i) SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:10, respectively; (ii) SEQ ID NO:18, SEQ ID NO:19, and SEQ ID NO:20, respectively; (iii) SEQ ID NO:28, SEQ ID NO:29, and SEQ ID NO:30, respectively; and (iv) SEQ ID NO: 38, SEQ ID NO: 39, and SEQ ID NO: 40, respectively. V selected from the group consisting of L - CDR1 sequence, V L - CDR2 sequence, and V L - The antibody drug conjugate of any one of claims 46 to 49, comprising a CDR3 sequence.

51. The anti-DLL3 antibody comprises a heavy chain immunoglobulin variable domain (V H ) and a light chain immunoglobulin variable domain (V L (a) V H - CDR1 sequence, V H - CDR2 sequence, and V H - V containing CDR3 sequence H and (b) V L - CDR1 sequence, V L - CDR2 sequence, and V L - V containing CDR3 sequence L The combination of (i) (a) SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:5, and (b) SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:10, respectively; (ii) (a) SEQ ID NO:13, SEQ ID NO:14, and SEQ ID NO:15, and (b) SEQ ID NO:18, SEQ ID NO:19, and SEQ ID NO:20, respectively; (iii) (a) SEQ ID NO:23, SEQ ID NO:24, and SEQ ID NO:25, and (b) SEQ ID NO:28, SEQ ID NO:29, and SEQ ID NO:30, respectively; and (iv) (a) SEQ ID NO:33, SEQ ID NO:34, and SEQ ID NO:35, and (b) SEQ ID NO:38, SEQ ID NO:39, and SEQ ID NO:40, respectively.

51. The antibody drug conjugate of claim 50, selected from the group consisting of:

52. The antibody is selected from the following combinations (1) to (4): (1) a light chain comprising a variable domain sequence of SEQ ID NO: 7 and a heavy chain comprising a variable domain sequence of SEQ ID NO: 2; (2) a light chain comprising a variable domain sequence of SEQ ID NO: 17 and a heavy chain comprising a variable domain sequence of SEQ ID NO: 12; (3) a light chain comprising a variable domain sequence of SEQ ID NO: 27 and a heavy chain comprising a variable domain sequence of SEQ ID NO: 22; and (4) a light chain comprising a variable domain sequence of SEQ ID NO: 37 and a heavy chain comprising a variable domain sequence of SEQ ID NO: 32 The antibody-drug conjugate according to any one of claims 47 to 51, which is an antibody comprising a light chain and a heavy chain in any one combination selected from the group consisting of:

53. The antibody-drug conjugate of claim 52, wherein the antibody comprises a light chain comprising the variable domain sequence of SEQ ID NO: 17 and a heavy chain comprising the variable domain sequence of SEQ ID NO:

12.

54. The antibody-drug conjugate of claim 52, wherein the antibody comprises a light chain comprising the variable domain sequence of SEQ ID NO:27 and a heavy chain comprising the variable domain sequence of SEQ ID NO:

22.

55. The antibody-drug conjugate of claim 52, wherein the antibody comprises a light chain comprising the variable domain sequence of SEQ ID NO:37 and a heavy chain comprising the variable domain sequence of SEQ ID NO:

32.

56. The antibody-drug conjugate of claim 52, wherein the antibody comprises a light chain comprising the variable domain sequence of SEQ ID NO:7 and a heavy chain comprising the variable domain sequence of SEQ ID NO:

2.

57. The antibody, (a) a heavy chain comprising the amino acid sequence of any one of SEQ ID NOs: 59-61, and a light chain comprising the amino acid sequence of SEQ ID NO: 62; (b) a heavy chain comprising the amino acid sequence of any one of SEQ ID NOs: 63-65 and a light chain comprising the amino acid sequence of SEQ ID NO: 66; or (c) a heavy chain comprising any one of the amino acid sequences of SEQ ID NOs: 67 to 69 and a light chain comprising the amino acid sequence of SEQ ID NO: 70 The antibody-drug conjugate of any one of claims 47 to 56, comprising:

58. The antibody, (a) a heavy chain comprising the amino acid sequence of SEQ ID NO:61 and a light chain comprising the amino acid sequence of SEQ ID NO:62; (b) a heavy chain comprising the amino acid sequence of SEQ ID NO:65 and a light chain comprising the amino acid sequence of SEQ ID NO:66; or (c) a heavy chain comprising the amino acid sequence of SEQ ID NO: 69 and a light chain comprising the amino acid sequence of SEQ ID NO:

70. The antibody-drug conjugate of any one of claims 47 to 57, comprising:

59. The heavy or light chain may have N-linked glycosylation, O-linked glycosylation, N-terminal processing, C-terminal processing, deamidation, isomerization of aspartic acid, oxidation of methionine, addition of a methionine residue to the N-terminus, amidation of a proline residue, substitution of two leucine (L) residues at positions 234 and 235 of the heavy chain with alanine (A) (according to the EU index) (LALA), a set of amino acid residues, Glu (E) at position 356 and Met (M) at position 358 of the heavy chain (according to the EU index) (LALA). The antibody-drug conjugate of any one of claims 47 to 58, having one or two or more modifications or sets of amino acid residues selected from the group consisting of: a set of Asp (D) at position 356 and Leucine (L) at position 358 of the heavy chain (according to the EU index), or any combination thereof, an N-terminal glutamine or conversion of the N-terminal glutamic acid to pyroglutamic acid, and a deletion of one or two amino acids from the carboxyl terminus.

60. An antibody-drug conjugate represented by the formula: 【Chemistry 20】 [In the formula, m 2 represents an integer of 1 or 2; Ab represents an anti-DLL3 IgG antibody comprising a heavy chain of the amino acid sequence of SEQ ID NO:61 and a light chain of the amino acid sequence of SEQ ID NO:62; The N297 glycan of the Ab represents any one of N297-(Fuc)MSG1, N297-(Fuc)MSG2, and a mixture thereof, and N297-(Fuc)SG, where N297-(Fuc)MSG1, N297-(Fuc)MSG2, and N297-(Fuc)SG have the following formula: 【Chemistry 21】 (In the formula, Each wavy line represents antibody binding to Asn297, L(PEG) in the N297 glycan is -NH-CH 2 CH 2 -(O-CH 2 CH 2 ) 3 - * wherein: The amino group at the left end is linked to a carboxylic acid via an amide bond at position 2 of the sialic acid at the non-reducing end of each or either one of the 1-3 and 1-6 branches of β-Man in the N297 glycan, and each asterisk represents a bond to a nitrogen atom at position 1 or 3 of the triazole ring in the corresponding structural formula. [0043] The compound has a structure represented by the formula:

61. An antibody-drug conjugate represented by the formula: 【Chemical 22】 [In the formula, m 2 represents an integer of 1 or 2; Ab represents an anti-DLL3 IgG antibody comprising a heavy chain of the amino acid sequence of SEQ ID NO:65 and a light chain of the amino acid sequence of SEQ ID NO:66; The N297 glycan of the Ab represents any one of N297-(Fuc)MSG1, N297-(Fuc)MSG2, and a mixture thereof, and N297-(Fuc)SG, where N297-(Fuc)MSG1, N297-(Fuc)MSG2, and N297-(Fuc)SG have the following formula: 【Chemistry 23】 (In the formula, Each wavy line represents antibody binding to Asn297, L(PEG) in the N297 glycan is -NH-CH 2 CH 2 -(O-CH 2 CH 2 ) 3 - * wherein: The amino group at the left end is linked to a carboxylic acid via an amide bond at position 2 of the sialic acid at the non-reducing end of each or either one of the 1-3 and 1-6 branches of β-Man in the N297 glycan, and each asterisk represents a bond to a nitrogen atom at position 1 or 3 of the triazole ring in the corresponding structural formula. [0043] The compound has a structure represented by the formula:

62. An antibody-drug conjugate represented by the formula: 【Chemistry 24】 [In the formula, m 2 represents an integer of 1 or 2; Ab represents an anti-DLL3 IgG antibody comprising a heavy chain of the amino acid sequence of SEQ ID NO:69 and a light chain of the amino acid sequence of SEQ ID NO:70; The N297 glycan of the Ab represents any one of N297-(Fuc)MSG1, N297-(Fuc)MSG2, and a mixture thereof, and N297-(Fuc)SG, where N297-(Fuc)MSG1, N297-(Fuc)MSG2, and N297-(Fuc)SG have the following formula: 【Chemistry 25】 (In the formula, Each wavy line represents antibody binding to Asn297, L(PEG) in the N297 glycan is -NH-CH 2 CH 2 -(O-CH 2 CH 2 ) 3 - * wherein: The amino group at the left end is linked to a carboxylic acid via an amide bond at position 2 of a sialic acid at the non-reducing end of each or either one of the 1-3 and 1-6 branches of β-Man in the N297 glycan, and each asterisk represents a bond to a nitrogen atom at position 1 or 3 of the triazole ring in the corresponding structural formula).

63. The N297 glycan of Ab represents N297-(Fuc)MSG1, wherein N297-(Fuc)MSG1 has the following formula: and having a structure represented by The antibody-drug conjugate of any one of claims 60 to 62, wherein the left-most amino group of L(PEG) in the N297 glycan is bound to a carboxylic acid via an amide bond at the 2-position of sialic acid at the non-reducing end of the 1-3 branched chain of β-Man in the N297 glycan.

64. An antibody-drug conjugate described in any one of claims 60 to 63, wherein the average number of drug molecules conjugated per antibody molecule in the antibody-drug conjugate is 1 to 3.

65. An antibody-drug conjugate described in any one of claims 1 to 59, wherein the average number of drug molecules conjugated per antibody molecule in the antibody-drug conjugate is 1 to 3 or 3 to 5.

66. A pharmaceutical composition comprising the antibody-drug conjugate according to any one of claims 1 to 65, a salt thereof, or a hydrate of the conjugate or salt.

67. 67. The pharmaceutical composition of claim 66, which is an antitumor drug.

68. 68. The pharmaceutical composition of claim 66 or 67, for use in treating a tumor, wherein the tumor is a DLL3-expressing tumor.

69. 69. The pharmaceutical composition of claim 67 or 68, wherein the tumor is small cell lung cancer (SCLC); large cell neuroendocrine carcinoma (LCNEC); neuroendocrine tumors of various tissues including kidney, genitourinary tract (bladder, prostate, ovary, cervix, and endometrium), gastrointestinal tract (stomach, colon), thyroid (medullary thyroid carcinoma), pancreas, and lung; glioma; or pseudoneuroendocrine tumor (pNET).

70. 1. A method for producing a glycan remodeled antibody, comprising: i) culturing a host cell containing nucleic acid encoding an anti-DLL3 antibody and harvesting the targeted antibody from the resulting culture; ii) treating the antibody obtained in step i) with a hydrolase to produce a (Fucα1,6)GlcNAc-antibody; and iii)-1. A step of reacting a glycan donor molecule with a (Fucα1,6)GlcNAc-antibody in the presence of a transglycosidase, in which the glycan donor molecule is obtained by introducing a PEG linker having an azide group into the carbonyl group of the carboxylic acid at the 2-position of the sialic acid in MSG(9) or SG(10) and oxazolinating the reducing end; or iii)-2 A step of reacting a (Fucα1,6)GlcNAc-antibody with a glycan donor molecule in the presence of a transglycosidase, in which the glycan donor molecule is obtained by introducing a PEG linker having an azide group into the carbonyl group of the carboxylic acid at the 2-position of the sialic acid in (MSG-)Asn or (SG-)Asn, whose α-amino group may be protected, and the carbonyl group of the carboxylic acid in Asn, causing the action of a hydrolase, and then oxazolinating the reducing end, or iii)-3 A step of reacting the (Fucα1,6)GlcNAc-antibody with a glycan donor molecule in the presence of two types of glycosyltransferases, the glycan donor molecule being (SG-)Asn or (MSG-)Asn having an azide group introduced into its sialic acid. The method includes:

71. 71. The method of claim 70, further comprising purifying the (Fucα1,6)GlcNAc-antibody by purifying the reaction solution in step ii) on a hydroxyapatite column.

72. A method for producing an antibody-drug conjugate formulation according to any one of claims 1 to 65, comprising the steps of: i) producing a glycan-remodeled antibody by using the method of claim 70 or 71; and ii) reacting the drug linker with DBCO and the azide groups in the glycans of the glycan-remodeled antibody produced in step i). The method includes:

73. 72. A glycan-remodeled antibody obtained by using the method of claim 70 or 71.

74. An antibody-drug conjugate obtained by using the method of claim 72.

75. 66. Use of the antibody-drug conjugate according to any one of claims 1 to 65, a salt of the antibody-drug conjugate, or a hydrate of the antibody-drug conjugate or salt in the manufacture of a medicament for treating a tumor.

76. 76. The use of claim 75, wherein the tumor is a DLL3-expressing tumor.

77. 77. The use of claim 75 or 76, wherein the tumor is small cell lung cancer (SCLC); large cell neuroendocrine carcinoma (LCNEC); neuroendocrine tumors of various tissues including kidney, genitourinary tract (bladder, prostate, ovary, cervix, and endometrium), gastrointestinal tract (stomach, colon), thyroid (medullary thyroid carcinoma), pancreas, and lung; glioma; or pseudoneuroendocrine tumor (pNET).

78. A pharmaceutical composition comprising at least one component selected from an antibody-drug conjugate described in any one of claims 1 to 65, a salt thereof, and a hydrate of the conjugate or salt, for use in a method for treating a tumor, said method comprising administering to an individual said pharmaceutical composition and at least one anti-tumor drug simultaneously, separately or sequentially.

79. The pharmaceutical composition of claim 78, wherein the tumor is a tumor that expresses DLL3.

80. The pharmaceutical composition of claim 78 or 79, wherein the tumor is small cell lung cancer (SCLC); large cell neuroendocrine carcinoma (LCNEC); neuroendocrine tumors of various tissues including the kidney, genitourinary tract (bladder, prostate, ovary, cervix, and endometrium), gastrointestinal tract (stomach, colon), thyroid (medullary thyroid carcinoma), pancreas, and lung; glioma; or pseudoneuroendocrine tumor (pNET).

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