DLL3 antibody-drug conjugate and its use

Abstract

This disclosure provides DLL3 antibody-drug conjugates and their uses. In one embodiment, an antibody-drug conjugate (ADC) of formula (I) or a salt thereof is provided herein, wherein n is an integer from 1 to 8, Ab represents an antibody that binds to DLL3 ("DLL3 antibody"), and the attachment site of the drug conjugate to Ab is via a cysteine ​​or lysine residue of the DLL3 antibody. In certain embodiments, n is 2. In certain embodiments, n is 4. In some embodiments, the ADC is of formula (IA) or a salt thereof.

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims the benefit of U.S. Provisional Patent Application No. 63 / 454,009, filed on March 22, 2023, the disclosure of which is incorporated herein by reference in its entirety.

[0002] Sequence Listing This application includes a computer - readable sequence listing in XML file format submitted together with this application, the entire content of which is incorporated herein by reference. The sequence listing XML file submitted together with this application is entitled "14529 - 133 - 228_SEQ_LISTING.xml", was created on March 20, 2024, and has a size of 49,227 bytes.

[0003] The present disclosure generally relates to antibody - drug conjugates (ADCs) comprising Delta - like canonical Notch ligand 3 (DLL3) antibodies and methods of using them.

Background Art

[0004] Delta-like canonical Notch ligand 3 (DLL3) is a type I transmembrane protein belonging to the DSL family of Notch ligands. DLL3 is normally expressed only in the intracellular membrane, particularly in the Golgi apparatus. Additional Notch family ligands include Delta-like canonical Notch ligand 1 (D1), Delta-like canonical Notch ligand 4 (D4), Jagged canonical Notch ligand 1 (J1), and Jagged canonical Notch ligand 2 (J2). Ligands other than DLL3 can activate Notch signaling. DLL3 functions as an inhibitor of Notch signaling by preventing the binding of Notch to its ligand. DLL3 is highly expressed on the surface of lung tumor cells such as small cell lung cancer (SCLC) and large cell neuroendocrine carcinoma (LCNEC). Normally, DLL3 is expressed only in the intracellular membrane, but it has become a potential therapeutic tumor target for any tumor expressing DLL3, including SCLC and LCNEC. In recent years, the establishment of DLL3 as a unique target in SCLC has accelerated the development of therapeutic agents. However, the success of treatment using antibodies and ADCs targeting DLL3 has not yet been achieved.

[0005] There remains a need in the art for an ADC that can target DLL3 to treat, prevent, or alleviate DLL3-mediated diseases, disorders, or conditions, including lung cancers such as small cell lung cancer (SCLC) or large cell neuroendocrine carcinoma (LCNEC). SUMMARY OF THE INVENTION MEANS FOR SOLVING THE PROBLEM

[0006] In one aspect, an antibody-drug conjugate (ADC) of formula (I),

Chemical formula

[0007] In another embodiment, the ADC of formula (A) is, [ka] Each thick shaded line represents a chain of the DLL3 antibody, the sulfur shown originates from a cysteine ​​residue of the DLL3 antibody, and X represents the following structure or a salt thereof. [ka] In the formula, the dashed line indicates the attachment point of the ADC of formula (A) to the rest of the ADC. An ADC of formula (A) is provided herein. In some embodiments, the cysteine ​​residue is located in the hinge region of the DLL3 antibody. In further embodiments, the cysteine ​​residue forms an interchain disulfide crosslink in the DLL3 antibody that is not directly or indirectly conjugated to X, for example, between two heavy chains, between a heavy chain and a light chain, between two VHH chains, or between two VHH-Fc chains. In further embodiments, the cysteine ​​residue is located at one or more of the EU numbering positions 226 or 229 (also referred to herein as C226 or C229, respectively). In some embodiments, X represents the following structure or a salt thereof. [ka] In other embodiments, X represents the following structure or a salt thereof. [ka]

[0008] In another embodiment, the ADC of formula (B) is, [ka] In the formula, each thick shaded line represents a chain of the DLL3 antibody, the illustrated CH2CH2CH2CH2NH portion originates from a lysine residue in the CH2 domain of the DLL3 antibody, and X represents the following structure: [ka] In the formula, the dashed line indicates the attachment point of the ADC of formula (B) to the rest of the ADC. The ADC of formula (B) is provided herein. In some embodiments, the lysine residue is located at one or more of the EU numbering positions 246, 248, 288, 290, or 317 (also referred herein as K246, K248, K288, K290, or K317, respectively). In some embodiments, X represents the following structure or a salt thereof. [ka] In other embodiments, X represents the following structure or a salt thereof. [ka]

[0009] In another embodiment, the ADC of formula (II) is, [ka] ADCs of formula (II) are provided herein, where n is an integer from 1 to 8 and Ab represents a DLL3 antibody. In some embodiments, the attachment site of the drug conjugate to Ab is via a cysteine ​​or lysine residue of the DLL3 antibody. In certain embodiments, n is 2. In certain embodiments, n is 4. In some embodiments, the ADC is of formula (II-A) or a salt thereof. [ka] In other embodiments, ADC is the one of formula (II-B) or a salt thereof. [ka]

[0010] In another embodiment, the ADC of formula (A) is, [ka] Each thick shaded line represents a chain of the DLL3 antibody, the sulfur atoms shown originate from cysteine ​​residues of the DLL3 antibody, and X represents the following structure: [ka] In the formula, the dashed line indicates the attachment point of the ADC of formula (A) to the rest of the ADC. An ADC of formula (A) is provided herein. In some embodiments, the cysteine ​​residue is located in the hinge region of the DLL3 antibody. In further embodiments, the cysteine ​​residue forms an interchain disulfide crosslink in the DLL3 antibody that is not directly or indirectly conjugated to X, for example, between two heavy chains, between a heavy chain and a light chain, between two VHH chains, or between two VHH-Fc chains. In further embodiments, the cysteine ​​residue is located at one or more of the EU numbering positions 226 or 229 (also referred to herein as C226 or C229, respectively). In some embodiments, X represents the following structure or a salt thereof. [ka] In some embodiments, X represents the following structure or a salt thereof. [ka]

[0011] In another embodiment, the ADC of formula (B) is, [ka] In the formula, each thick shaded line represents a chain of the DLL3 antibody, the illustrated CH2CH2CH2CH2NH portion originates from a lysine residue in the CH2 domain of the DLL3 antibody, and X represents the following structure: [ka] In the formula, the dashed line indicates the attachment point of the ADC of formula (B) to the rest of the ADC. The ADC of formula (B) is provided herein. In some embodiments, the lysine residue is located at one or more of the EU numbering positions 246, 248, 288, 290, or 317 (also referred herein as K246, K248, K288, K290, or K317, respectively). In some embodiments, X represents the following structure or a salt thereof. [ka] In some embodiments, X represents the following structure or a salt thereof. [ka]

[0012] In some embodiments, the DLL3 antibody used herein comprises VHH. In further embodiments, the DLL3 antibody used herein comprises a VHH-Fc fusion, for example, as illustrated in Figure 1A. Additionally or alternatively, the DLL3 antibody comprises CDR1, CDR2, and CDR3 of the heavy chain variable domain (VH), where VH comprises the amino acid sequence described in SEQ ID NOs: 14, 15, or 16, for example, as disclosed in Tables 1 and 2. In further embodiments, the DLL3 antibody comprises the VH described in SEQ ID NOs: 14, 15, or 16. In further embodiments, the DLL3 antibody comprises a homodimer of the VHH-Fc chain described in any one of SEQ ID NOs: 4, 5, or 10.

[0013] In another embodiment, pharmaceutical compositions comprising an ADC described herein and a pharmaceutically acceptable carrier are provided herein. In some embodiments, the pharmaceutical composition has a drug-to-antibody ratio (DAR) of about 1.5 to about 2.5. In further embodiments, the pharmaceutical composition has a DAR of about 1.9 to 2.1. In some embodiments, the pharmaceutical composition has a DAR of about 1 to about 4, for example, about 1 to about 3, about 1 to about 2, about 2 to about 4, or about 3 to about 4. In further embodiments, the pharmaceutical composition has a DAR of about 3.5 to about 4.0, for example, about 3.6, about 3.7, about 3.8, about 3.9, or about 4.0.

[0014] In another embodiment, a method for modulating a DLL3-related immune response in a subject is provided herein, comprising administering an ADC or pharmaceutical composition described herein to the subject such that the immune response is modulated in the subject.

[0015] In another embodiment, a method for treating cancer in a subject is provided herein, comprising administering an ADC or pharmaceutical composition described herein to the subject, wherein the cancer is DLL3-positive or overexpressing. In certain embodiments, the cancer is selected from lung cancer and neuroendocrine cancer. In certain embodiments, the cancer is small cell lung cancer (SCLC), large cell neuroendocrine carcinoma (LCNEC), or colorectal cancer.

[0016] In a further embodiment, a method for producing the ADC described herein is provided herein. [Brief explanation of the drawing]

[0017] [Figure 1A] This shows an antibody containing VHH fused to Fc. The two interchain disulfide bonds may be located between the hinge regions of the two chains. [Figure 1B] As shown in Figure 1A, an exemplary synthetic route for ADCs is provided, in which the antibody may be a conventional antibody containing two heavy chains and two light chains, or a VHH-Fc fusion. [Figure 2] Example 7 provides exemplary RP-HPLC analysis results for linker-drug (2-A), linker-drug (2-B), and linker-drug (2) (a mixture of linker-drug (2-A) and linker-drug (2-B)), which are further described. [Figure 3] Linker-drug (2-B) is shown. [Figure 4A] Exemplary results for mAb300, mAb301, mAb302, and associated ADCs from development assays are shown, as further described in Example 15. mAb302, ADC-013, and ADC-014 are compared using size exclusion chromatography (SEC). [Figure 4B] Exemplary results for mAb300, mAb301, mAb302, and associated ADCs from development assays are shown, as further described in Example 15. mAb300 and mAb302 are compared using hydrophobic interaction chromatography (HIC). [Figure 4C] Exemplary results for mAb300, mAb301, mAb302, and associated ADCs from development assays are shown, as further described in Example 15. mAb300, ADC-009, and ADC-010 are compared using HIC. [Figure 4D]Exemplary results for mAb300, mAb301, mAb302, and associated ADCs from development assays are shown, as further described in Example 15. mAb302, ADC-013, and ADC-014 are compared using HIC. [Figure 5] The following are exemplary results of evaluating the binding properties of mAb300, mAb301, and mAb302 via the Biacore® assay, as further described in Example 16. [Figure 6] The following are exemplary results for evaluating the drug-to-antibody ratio (DAR) of ADC-015 (indicated as mAb301-linker-drug(2) (target DAR=2.0)), ADC-016 (indicated as mAb301-linker-drug(2) (target DAR=4.0)), and ADC-017 (indicated as mAb301-linker-drug(1) (target DAR=4.0)), as further described in Example 17. [Figure 7A]As further described in Example 18, the produced ADC-009-M (mAb300, linker-drug(2), DAR2, Figure 7A), ADC-009-A (mAb300, linker-drug(2-A), DAR2, Figure 7B), ADC-009-B (mAb300, linker-drug(2-B), DAR2, Figure 7C), ADC-015-M (mAb301, linker-drug(2), DAR2, Figure 7D), ADC-015-A (m Ab301, linker-drug (2-A), DAR2, Figure 7E), ADC-015-B (mAb301, linker-drug (2-B), DAR2, Figure 7F), ADC-013-M (mAb302, linker-drug (2), DAR2, Figure 7G), ADC-013-A (mAb302, linker-drug (2-A), DAR2, Figure 7H), ADC-013-B (mAb302, linker-drug (2-B), DAR2, Figure 7I), A DC-018-M (mAb300, linker-drug (2), DAR4, Figure 7J), ADC-018-A (mAb300, linker-drug (2-A), DAR4, Figure 7K), ADC-018-B (mAb300, linker-drug (2-B), DAR4, Figure 7L), ADC-016-M (mAb301, linker-drug (2), DAR4, Figure 7M), ADC-016-A (mAb301, linker-drug (2-A), DA Exemplary SEC-HPLC analysis results are provided for R4 (Figure 7N), ADC-016-B (mAb301, linker-drug (2-B), DAR4, Figure 7O), ADC-020-M (mAb302, linker-drug (2), DAR4, Figure 7P), ADC-020-A (mAb302, linker-drug (2-A), DAR4, Figure 7Q), and ADC-020-B (mAb302, linker-drug (2-B), DAR4, Figure 7R). [Figure 7B]As further described in Example 18, the produced ADC-009-M (mAb300, linker-drug(2), DAR2, Figure 7A), ADC-009-A (mAb300, linker-drug(2-A), DAR2, Figure 7B), ADC-009-B (mAb300, linker-drug(2-B), DAR2, Figure 7C), ADC-015-M (mAb301, linker-drug(2), DAR2, Figure 7D), ADC-015-A (m Ab301, linker-drug (2-A), DAR2, Figure 7E), ADC-015-B (mAb301, linker-drug (2-B), DAR2, Figure 7F), ADC-013-M (mAb302, linker-drug (2), DAR2, Figure 7G), ADC-013-A (mAb302, linker-drug (2-A), DAR2, Figure 7H), ADC-013-B (mAb302, linker-drug (2-B), DAR2, Figure 7I), A DC-018-M (mAb300, linker-drug (2), DAR4, Figure 7J), ADC-018-A (mAb300, linker-drug (2-A), DAR4, Figure 7K), ADC-018-B (mAb300, linker-drug (2-B), DAR4, Figure 7L), ADC-016-M (mAb301, linker-drug (2), DAR4, Figure 7M), ADC-016-A (mAb301, linker-drug (2-A), DA Exemplary SEC-HPLC analysis results are provided for R4 (Figure 7N), ADC-016-B (mAb301, linker-drug (2-B), DAR4, Figure 7O), ADC-020-M (mAb302, linker-drug (2), DAR4, Figure 7P), ADC-020-A (mAb302, linker-drug (2-A), DAR4, Figure 7Q), and ADC-020-B (mAb302, linker-drug (2-B), DAR4, Figure 7R). [Figure 7C]As further described in Example 18, the produced ADC-009-M (mAb300, linker-drug(2), DAR2, Figure 7A), ADC-009-A (mAb300, linker-drug(2-A), DAR2, Figure 7B), ADC-009-B (mAb300, linker-drug(2-B), DAR2, Figure 7C), ADC-015-M (mAb301, linker-drug(2), DAR2, Figure 7D), ADC-015-A (m Ab301, linker-drug (2-A), DAR2, Figure 7E), ADC-015-B (mAb301, linker-drug (2-B), DAR2, Figure 7F), ADC-013-M (mAb302, linker-drug (2), DAR2, Figure 7G), ADC-013-A (mAb302, linker-drug (2-A), DAR2, Figure 7H), ADC-013-B (mAb302, linker-drug (2-B), DAR2, Figure 7I), A DC-018-M (mAb300, linker-drug (2), DAR4, Figure 7J), ADC-018-A (mAb300, linker-drug (2-A), DAR4, Figure 7K), ADC-018-B (mAb300, linker-drug (2-B), DAR4, Figure 7L), ADC-016-M (mAb301, linker-drug (2), DAR4, Figure 7M), ADC-016-A (mAb301, linker-drug (2-A), DA Exemplary SEC-HPLC analysis results are provided for R4 (Figure 7N), ADC-016-B (mAb301, linker-drug (2-B), DAR4, Figure 7O), ADC-020-M (mAb302, linker-drug (2), DAR4, Figure 7P), ADC-020-A (mAb302, linker-drug (2-A), DAR4, Figure 7Q), and ADC-020-B (mAb302, linker-drug (2-B), DAR4, Figure 7R). [Figure 7D]As further described in Example 18, the produced ADC-009-M (mAb300, linker-drug(2), DAR2, Figure 7A), ADC-009-A (mAb300, linker-drug(2-A), DAR2, Figure 7B), ADC-009-B (mAb300, linker-drug(2-B), DAR2, Figure 7C), ADC-015-M (mAb301, linker-drug(2), DAR2, Figure 7D), ADC-015-A (m Ab301, linker-drug (2-A), DAR2, Figure 7E), ADC-015-B (mAb301, linker-drug (2-B), DAR2, Figure 7F), ADC-013-M (mAb302, linker-drug (2), DAR2, Figure 7G), ADC-013-A (mAb302, linker-drug (2-A), DAR2, Figure 7H), ADC-013-B (mAb302, linker-drug (2-B), DAR2, Figure 7I), A DC-018-M (mAb300, linker-drug (2), DAR4, Figure 7J), ADC-018-A (mAb300, linker-drug (2-A), DAR4, Figure 7K), ADC-018-B (mAb300, linker-drug (2-B), DAR4, Figure 7L), ADC-016-M (mAb301, linker-drug (2), DAR4, Figure 7M), ADC-016-A (mAb301, linker-drug (2-A), DA Exemplary SEC-HPLC analysis results are provided for R4 (Figure 7N), ADC-016-B (mAb301, linker-drug (2-B), DAR4, Figure 7O), ADC-020-M (mAb302, linker-drug (2), DAR4, Figure 7P), ADC-020-A (mAb302, linker-drug (2-A), DAR4, Figure 7Q), and ADC-020-B (mAb302, linker-drug (2-B), DAR4, Figure 7R). [Figure 7E]As further described in Example 18, the produced ADC-009-M (mAb300, linker-drug(2), DAR2, Figure 7A), ADC-009-A (mAb300, linker-drug(2-A), DAR2, Figure 7B), ADC-009-B (mAb300, linker-drug(2-B), DAR2, Figure 7C), ADC-015-M (mAb301, linker-drug(2), DAR2, Figure 7D), ADC-015-A (m Ab301, linker-drug (2-A), DAR2, Figure 7E), ADC-015-B (mAb301, linker-drug (2-B), DAR2, Figure 7F), ADC-013-M (mAb302, linker-drug (2), DAR2, Figure 7G), ADC-013-A (mAb302, linker-drug (2-A), DAR2, Figure 7H), ADC-013-B (mAb302, linker-drug (2-B), DAR2, Figure 7I), A DC-018-M (mAb300, linker-drug (2), DAR4, Figure 7J), ADC-018-A (mAb300, linker-drug (2-A), DAR4, Figure 7K), ADC-018-B (mAb300, linker-drug (2-B), DAR4, Figure 7L), ADC-016-M (mAb301, linker-drug (2), DAR4, Figure 7M), ADC-016-A (mAb301, linker-drug (2-A), DA Exemplary SEC-HPLC analysis results are provided for R4 (Figure 7N), ADC-016-B (mAb301, linker-drug (2-B), DAR4, Figure 7O), ADC-020-M (mAb302, linker-drug (2), DAR4, Figure 7P), ADC-020-A (mAb302, linker-drug (2-A), DAR4, Figure 7Q), and ADC-020-B (mAb302, linker-drug (2-B), DAR4, Figure 7R). [Figure 7F]As further described in Example 18, the produced ADC-009-M (mAb300, linker-drug(2), DAR2, Figure 7A), ADC-009-A (mAb300, linker-drug(2-A), DAR2, Figure 7B), ADC-009-B (mAb300, linker-drug(2-B), DAR2, Figure 7C), ADC-015-M (mAb301, linker-drug(2), DAR2, Figure 7D), ADC-015-A (m Ab301, linker-drug (2-A), DAR2, Figure 7E), ADC-015-B (mAb301, linker-drug (2-B), DAR2, Figure 7F), ADC-013-M (mAb302, linker-drug (2), DAR2, Figure 7G), ADC-013-A (mAb302, linker-drug (2-A), DAR2, Figure 7H), ADC-013-B (mAb302, linker-drug (2-B), DAR2, Figure 7I), A DC-018-M (mAb300, linker-drug (2), DAR4, Figure 7J), ADC-018-A (mAb300, linker-drug (2-A), DAR4, Figure 7K), ADC-018-B (mAb300, linker-drug (2-B), DAR4, Figure 7L), ADC-016-M (mAb301, linker-drug (2), DAR4, Figure 7M), ADC-016-A (mAb301, linker-drug (2-A), DA Exemplary SEC-HPLC analysis results are provided for R4 (Figure 7N), ADC-016-B (mAb301, linker-drug (2-B), DAR4, Figure 7O), ADC-020-M (mAb302, linker-drug (2), DAR4, Figure 7P), ADC-020-A (mAb302, linker-drug (2-A), DAR4, Figure 7Q), and ADC-020-B (mAb302, linker-drug (2-B), DAR4, Figure 7R). [Figure 7G]As further described in Example 18, the produced ADC-009-M (mAb300, linker-drug(2), DAR2, Figure 7A), ADC-009-A (mAb300, linker-drug(2-A), DAR2, Figure 7B), ADC-009-B (mAb300, linker-drug(2-B), DAR2, Figure 7C), ADC-015-M (mAb301, linker-drug(2), DAR2, Figure 7D), ADC-015-A (m Ab301, linker-drug (2-A), DAR2, Figure 7E), ADC-015-B (mAb301, linker-drug (2-B), DAR2, Figure 7F), ADC-013-M (mAb302, linker-drug (2), DAR2, Figure 7G), ADC-013-A (mAb302, linker-drug (2-A), DAR2, Figure 7H), ADC-013-B (mAb302, linker-drug (2-B), DAR2, Figure 7I), A DC-018-M (mAb300, linker-drug (2), DAR4, Figure 7J), ADC-018-A (mAb300, linker-drug (2-A), DAR4, Figure 7K), ADC-018-B (mAb300, linker-drug (2-B), DAR4, Figure 7L), ADC-016-M (mAb301, linker-drug (2), DAR4, Figure 7M), ADC-016-A (mAb301, linker-drug (2-A), DA Exemplary SEC-HPLC analysis results are provided for R4 (Figure 7N), ADC-016-B (mAb301, linker-drug (2-B), DAR4, Figure 7O), ADC-020-M (mAb302, linker-drug (2), DAR4, Figure 7P), ADC-020-A (mAb302, linker-drug (2-A), DAR4, Figure 7Q), and ADC-020-B (mAb302, linker-drug (2-B), DAR4, Figure 7R). [Figure 7H]As further described in Example 18, the produced ADC-009-M (mAb300, linker-drug(2), DAR2, Figure 7A), ADC-009-A (mAb300, linker-drug(2-A), DAR2, Figure 7B), ADC-009-B (mAb300, linker-drug(2-B), DAR2, Figure 7C), ADC-015-M (mAb301, linker-drug(2), DAR2, Figure 7D), ADC-015-A (m Ab301, linker-drug (2-A), DAR2, Figure 7E), ADC-015-B (mAb301, linker-drug (2-B), DAR2, Figure 7F), ADC-013-M (mAb302, linker-drug (2), DAR2, Figure 7G), ADC-013-A (mAb302, linker-drug (2-A), DAR2, Figure 7H), ADC-013-B (mAb302, linker-drug (2-B), DAR2, Figure 7I), A DC-018-M (mAb300, linker-drug (2), DAR4, Figure 7J), ADC-018-A (mAb300, linker-drug (2-A), DAR4, Figure 7K), ADC-018-B (mAb300, linker-drug (2-B), DAR4, Figure 7L), ADC-016-M (mAb301, linker-drug (2), DAR4, Figure 7M), ADC-016-A (mAb301, linker-drug (2-A), DA Exemplary SEC-HPLC analysis results are provided for R4 (Figure 7N), ADC-016-B (mAb301, linker-drug (2-B), DAR4, Figure 7O), ADC-020-M (mAb302, linker-drug (2), DAR4, Figure 7P), ADC-020-A (mAb302, linker-drug (2-A), DAR4, Figure 7Q), and ADC-020-B (mAb302, linker-drug (2-B), DAR4, Figure 7R). [Figure 7I]As further described in Example 18, the produced ADC-009-M (mAb300, linker-drug(2), DAR2, Figure 7A), ADC-009-A (mAb300, linker-drug(2-A), DAR2, Figure 7B), ADC-009-B (mAb300, linker-drug(2-B), DAR2, Figure 7C), ADC-015-M (mAb301, linker-drug(2), DAR2, Figure 7D), ADC-015-A (m Ab301, linker-drug (2-A), DAR2, Figure 7E), ADC-015-B (mAb301, linker-drug (2-B), DAR2, Figure 7F), ADC-013-M (mAb302, linker-drug (2), DAR2, Figure 7G), ADC-013-A (mAb302, linker-drug (2-A), DAR2, Figure 7H), ADC-013-B (mAb302, linker-drug (2-B), DAR2, Figure 7I), A DC-018-M (mAb300, linker-drug (2), DAR4, Figure 7J), ADC-018-A (mAb300, linker-drug (2-A), DAR4, Figure 7K), ADC-018-B (mAb300, linker-drug (2-B), DAR4, Figure 7L), ADC-016-M (mAb301, linker-drug (2), DAR4, Figure 7M), ADC-016-A (mAb301, linker-drug (2-A), DA Exemplary SEC-HPLC analysis results are provided for R4 (Figure 7N), ADC-016-B (mAb301, linker-drug (2-B), DAR4, Figure 7O), ADC-020-M (mAb302, linker-drug (2), DAR4, Figure 7P), ADC-020-A (mAb302, linker-drug (2-A), DAR4, Figure 7Q), and ADC-020-B (mAb302, linker-drug (2-B), DAR4, Figure 7R). [Figure 7J]As further described in Example 18, the produced ADC-009-M (mAb300, linker-drug(2), DAR2, Figure 7A), ADC-009-A (mAb300, linker-drug(2-A), DAR2, Figure 7B), ADC-009-B (mAb300, linker-drug(2-B), DAR2, Figure 7C), ADC-015-M (mAb301, linker-drug(2), DAR2, Figure 7D), ADC-015-A (m Ab301, linker-drug (2-A), DAR2, Figure 7E), ADC-015-B (mAb301, linker-drug (2-B), DAR2, Figure 7F), ADC-013-M (mAb302, linker-drug (2), DAR2, Figure 7G), ADC-013-A (mAb302, linker-drug (2-A), DAR2, Figure 7H), ADC-013-B (mAb302, linker-drug (2-B), DAR2, Figure 7I), A DC-018-M (mAb300, linker-drug (2), DAR4, Figure 7J), ADC-018-A (mAb300, linker-drug (2-A), DAR4, Figure 7K), ADC-018-B (mAb300, linker-drug (2-B), DAR4, Figure 7L), ADC-016-M (mAb301, linker-drug (2), DAR4, Figure 7M), ADC-016-A (mAb301, linker-drug (2-A), DA Exemplary SEC-HPLC analysis results are provided for R4 (Figure 7N), ADC-016-B (mAb301, linker-drug (2-B), DAR4, Figure 7O), ADC-020-M (mAb302, linker-drug (2), DAR4, Figure 7P), ADC-020-A (mAb302, linker-drug (2-A), DAR4, Figure 7Q), and ADC-020-B (mAb302, linker-drug (2-B), DAR4, Figure 7R). [Figure 7K]As further described in Example 18, the produced ADC-009-M (mAb300, linker-drug(2), DAR2, Figure 7A), ADC-009-A (mAb300, linker-drug(2-A), DAR2, Figure 7B), ADC-009-B (mAb300, linker-drug(2-B), DAR2, Figure 7C), ADC-015-M (mAb301, linker-drug(2), DAR2, Figure 7D), ADC-015-A (m Ab301, linker-drug (2-A), DAR2, Figure 7E), ADC-015-B (mAb301, linker-drug (2-B), DAR2, Figure 7F), ADC-013-M (mAb302, linker-drug (2), DAR2, Figure 7G), ADC-013-A (mAb302, linker-drug (2-A), DAR2, Figure 7H), ADC-013-B (mAb302, linker-drug (2-B), DAR2, Figure 7I), A DC-018-M (mAb300, linker-drug (2), DAR4, Figure 7J), ADC-018-A (mAb300, linker-drug (2-A), DAR4, Figure 7K), ADC-018-B (mAb300, linker-drug (2-B), DAR4, Figure 7L), ADC-016-M (mAb301, linker-drug (2), DAR4, Figure 7M), ADC-016-A (mAb301, linker-drug (2-A), DA Exemplary SEC-HPLC analysis results are provided for R4 (Figure 7N), ADC-016-B (mAb301, linker-drug (2-B), DAR4, Figure 7O), ADC-020-M (mAb302, linker-drug (2), DAR4, Figure 7P), ADC-020-A (mAb302, linker-drug (2-A), DAR4, Figure 7Q), and ADC-020-B (mAb302, linker-drug (2-B), DAR4, Figure 7R). [Figure 7L]As further described in Example 18, the produced ADC-009-M (mAb300, linker-drug(2), DAR2, Figure 7A), ADC-009-A (mAb300, linker-drug(2-A), DAR2, Figure 7B), ADC-009-B (mAb300, linker-drug(2-B), DAR2, Figure 7C), ADC-015-M (mAb301, linker-drug(2), DAR2, Figure 7D), ADC-015-A (m Ab301, linker-drug (2-A), DAR2, Figure 7E), ADC-015-B (mAb301, linker-drug (2-B), DAR2, Figure 7F), ADC-013-M (mAb302, linker-drug (2), DAR2, Figure 7G), ADC-013-A (mAb302, linker-drug (2-A), DAR2, Figure 7H), ADC-013-B (mAb302, linker-drug (2-B), DAR2, Figure 7I), A DC-018-M (mAb300, linker-drug (2), DAR4, Figure 7J), ADC-018-A (mAb300, linker-drug (2-A), DAR4, Figure 7K), ADC-018-B (mAb300, linker-drug (2-B), DAR4, Figure 7L), ADC-016-M (mAb301, linker-drug (2), DAR4, Figure 7M), ADC-016-A (mAb301, linker-drug (2-A), DA Exemplary SEC-HPLC analysis results are provided for R4 (Figure 7N), ADC-016-B (mAb301, linker-drug (2-B), DAR4, Figure 7O), ADC-020-M (mAb302, linker-drug (2), DAR4, Figure 7P), ADC-020-A (mAb302, linker-drug (2-A), DAR4, Figure 7Q), and ADC-020-B (mAb302, linker-drug (2-B), DAR4, Figure 7R). [Figure 7M]As further described in Example 18, the produced ADC-009-M (mAb300, linker-drug(2), DAR2, Figure 7A), ADC-009-A (mAb300, linker-drug(2-A), DAR2, Figure 7B), ADC-009-B (mAb300, linker-drug(2-B), DAR2, Figure 7C), ADC-015-M (mAb301, linker-drug(2), DAR2, Figure 7D), ADC-015-A (m Ab301, linker-drug (2-A), DAR2, Figure 7E), ADC-015-B (mAb301, linker-drug (2-B), DAR2, Figure 7F), ADC-013-M (mAb302, linker-drug (2), DAR2, Figure 7G), ADC-013-A (mAb302, linker-drug (2-A), DAR2, Figure 7H), ADC-013-B (mAb302, linker-drug (2-B), DAR2, Figure 7I), A DC-018-M (mAb300, linker-drug (2), DAR4, Figure 7J), ADC-018-A (mAb300, linker-drug (2-A), DAR4, Figure 7K), ADC-018-B (mAb300, linker-drug (2-B), DAR4, Figure 7L), ADC-016-M (mAb301, linker-drug (2), DAR4, Figure 7M), ADC-016-A (mAb301, linker-drug (2-A), DA Exemplary SEC-HPLC analysis results are provided for R4 (Figure 7N), ADC-016-B (mAb301, linker-drug (2-B), DAR4, Figure 7O), ADC-020-M (mAb302, linker-drug (2), DAR4, Figure 7P), ADC-020-A (mAb302, linker-drug (2-A), DAR4, Figure 7Q), and ADC-020-B (mAb302, linker-drug (2-B), DAR4, Figure 7R). [Figure 7N]As further described in Example 18, the produced ADC-009-M (mAb300, linker-drug(2), DAR2, Figure 7A), ADC-009-A (mAb300, linker-drug(2-A), DAR2, Figure 7B), ADC-009-B (mAb300, linker-drug(2-B), DAR2, Figure 7C), ADC-015-M (mAb301, linker-drug(2), DAR2, Figure 7D), ADC-015-A (m Ab301, linker-drug (2-A), DAR2, Figure 7E), ADC-015-B (mAb301, linker-drug (2-B), DAR2, Figure 7F), ADC-013-M (mAb302, linker-drug (2), DAR2, Figure 7G), ADC-013-A (mAb302, linker-drug (2-A), DAR2, Figure 7H), ADC-013-B (mAb302, linker-drug (2-B), DAR2, Figure 7I), A DC-018-M (mAb300, linker-drug (2), DAR4, Figure 7J), ADC-018-A (mAb300, linker-drug (2-A), DAR4, Figure 7K), ADC-018-B (mAb300, linker-drug (2-B), DAR4, Figure 7L), ADC-016-M (mAb301, linker-drug (2), DAR4, Figure 7M), ADC-016-A (mAb301, linker-drug (2-A), DA Exemplary SEC-HPLC analysis results are provided for R4 (Figure 7N), ADC-016-B (mAb301, linker-drug (2-B), DAR4, Figure 7O), ADC-020-M (mAb302, linker-drug (2), DAR4, Figure 7P), ADC-020-A (mAb302, linker-drug (2-A), DAR4, Figure 7Q), and ADC-020-B (mAb302, linker-drug (2-B), DAR4, Figure 7R). [Figure 7O]As further described in Example 18, the produced ADC-009-M (mAb300, linker-drug(2), DAR2, Figure 7A), ADC-009-A (mAb300, linker-drug(2-A), DAR2, Figure 7B), ADC-009-B (mAb300, linker-drug(2-B), DAR2, Figure 7C), ADC-015-M (mAb301, linker-drug(2), DAR2, Figure 7D), ADC-015-A (m Ab301, linker-drug (2-A), DAR2, Figure 7E), ADC-015-B (mAb301, linker-drug (2-B), DAR2, Figure 7F), ADC-013-M (mAb302, linker-drug (2), DAR2, Figure 7G), ADC-013-A (mAb302, linker-drug (2-A), DAR2, Figure 7H), ADC-013-B (mAb302, linker-drug (2-B), DAR2, Figure 7I), A DC-018-M (mAb300, linker-drug (2), DAR4, Figure 7J), ADC-018-A (mAb300, linker-drug (2-A), DAR4, Figure 7K), ADC-018-B (mAb300, linker-drug (2-B), DAR4, Figure 7L), ADC-016-M (mAb301, linker-drug (2), DAR4, Figure 7M), ADC-016-A (mAb301, linker-drug (2-A), DA Exemplary SEC-HPLC analysis results are provided for R4 (Figure 7N), ADC-016-B (mAb301, linker-drug (2-B), DAR4, Figure 7O), ADC-020-M (mAb302, linker-drug (2), DAR4, Figure 7P), ADC-020-A (mAb302, linker-drug (2-A), DAR4, Figure 7Q), and ADC-020-B (mAb302, linker-drug (2-B), DAR4, Figure 7R). [Figure 7P]As further described in Example 18, the produced ADC-009-M (mAb300, linker-drug(2), DAR2, Figure 7A), ADC-009-A (mAb300, linker-drug(2-A), DAR2, Figure 7B), ADC-009-B (mAb300, linker-drug(2-B), DAR2, Figure 7C), ADC-015-M (mAb301, linker-drug(2), DAR2, Figure 7D), ADC-015-A (m Ab301, linker-drug (2-A), DAR2, Figure 7E), ADC-015-B (mAb301, linker-drug (2-B), DAR2, Figure 7F), ADC-013-M (mAb302, linker-drug (2), DAR2, Figure 7G), ADC-013-A (mAb302, linker-drug (2-A), DAR2, Figure 7H), ADC-013-B (mAb302, linker-drug (2-B), DAR2, Figure 7I), A DC-018-M (mAb300, linker-drug (2), DAR4, Figure 7J), ADC-018-A (mAb300, linker-drug (2-A), DAR4, Figure 7K), ADC-018-B (mAb300, linker-drug (2-B), DAR4, Figure 7L), ADC-016-M (mAb301, linker-drug (2), DAR4, Figure 7M), ADC-016-A (mAb301, linker-drug (2-A), DA Exemplary SEC-HPLC analysis results are provided for R4 (Figure 7N), ADC-016-B (mAb301, linker-drug (2-B), DAR4, Figure 7O), ADC-020-M (mAb302, linker-drug (2), DAR4, Figure 7P), ADC-020-A (mAb302, linker-drug (2-A), DAR4, Figure 7Q), and ADC-020-B (mAb302, linker-drug (2-B), DAR4, Figure 7R). [Figure 7Q]As further described in Example 18, the produced ADC-009-M (mAb300, linker-drug(2), DAR2, Figure 7A), ADC-009-A (mAb300, linker-drug(2-A), DAR2, Figure 7B), ADC-009-B (mAb300, linker-drug(2-B), DAR2, Figure 7C), ADC-015-M (mAb301, linker-drug(2), DAR2, Figure 7D), ADC-015-A (m Ab301, linker-drug (2-A), DAR2, Figure 7E), ADC-015-B (mAb301, linker-drug (2-B), DAR2, Figure 7F), ADC-013-M (mAb302, linker-drug (2), DAR2, Figure 7G), ADC-013-A (mAb302, linker-drug (2-A), DAR2, Figure 7H), ADC-013-B (mAb302, linker-drug (2-B), DAR2, Figure 7I), A DC-018-M (mAb300, linker-drug (2), DAR4, Figure 7J), ADC-018-A (mAb300, linker-drug (2-A), DAR4, Figure 7K), ADC-018-B (mAb300, linker-drug (2-B), DAR4, Figure 7L), ADC-016-M (mAb301, linker-drug (2), DAR4, Figure 7M), ADC-016-A (mAb301, linker-drug (2-A), DA Exemplary SEC-HPLC analysis results are provided for R4 (Figure 7N), ADC-016-B (mAb301, linker-drug (2-B), DAR4, Figure 7O), ADC-020-M (mAb302, linker-drug (2), DAR4, Figure 7P), ADC-020-A (mAb302, linker-drug (2-A), DAR4, Figure 7Q), and ADC-020-B (mAb302, linker-drug (2-B), DAR4, Figure 7R). [Figure 7R]As further described in Example 18, the produced ADC-009-M (mAb300, linker-drug(2), DAR2, Figure 7A), ADC-009-A (mAb300, linker-drug(2-A), DAR2, Figure 7B), ADC-009-B (mAb300, linker-drug(2-B), DAR2, Figure 7C), ADC-015-M (mAb301, linker-drug(2), DAR2, Figure 7D), ADC-015-A (m Ab301, linker-drug (2-A), DAR2, Figure 7E), ADC-015-B (mAb301, linker-drug (2-B), DAR2, Figure 7F), ADC-013-M (mAb302, linker-drug (2), DAR2, Figure 7G), ADC-013-A (mAb302, linker-drug (2-A), DAR2, Figure 7H), ADC-013-B (mAb302, linker-drug (2-B), DAR2, Figure 7I), A DC-018-M (mAb300, linker-drug (2), DAR4, Figure 7J), ADC-018-A (mAb300, linker-drug (2-A), DAR4, Figure 7K), ADC-018-B (mAb300, linker-drug (2-B), DAR4, Figure 7L), ADC-016-M (mAb301, linker-drug (2), DAR4, Figure 7M), ADC-016-A (mAb301, linker-drug (2-A), DA Exemplary SEC-HPLC analysis results are provided for R4 (Figure 7N), ADC-016-B (mAb301, linker-drug (2-B), DAR4, Figure 7O), ADC-020-M (mAb302, linker-drug (2), DAR4, Figure 7P), ADC-020-A (mAb302, linker-drug (2-A), DAR4, Figure 7Q), and ADC-020-B (mAb302, linker-drug (2-B), DAR4, Figure 7R). [Figure 8A]As further described in Example 18, the produced ADC-009-M (mAb300, linker-drug(2), DAR2, Figure 8A), ADC-009-A (mAb300, linker-drug(2-A), DAR2, Figure 8B), ADC-009-B (mAb300, linker-drug(2-B), DAR2, Figure 8C), ADC-015-M (mAb301, linker-drug(2), DAR2, Figure 8D), ADC-015-A (m Ab301, linker-drug (2-A), DAR2, Figure 8E), ADC-015-B (mAb301, linker-drug (2-B), DAR2, Figure 8F), ADC-013-M (mAb302, linker-drug (2), DAR2, Figure 8G), ADC-013-A (mAb302, linker-drug (2-A), DAR2, Figure 8H), ADC-013-B (mAb302, linker-drug (2-B), DAR2, Figure 8I), A DC-018-M (mAb300, linker-drug (2), DAR4, Figure 8J), ADC-018-A (mAb300, linker-drug (2-A), DAR4, Figure 8K), ADC-018-B (mAb300, linker-drug (2-B), DAR4, Figure 8L), ADC-016-M (mAb301, linker-drug (2), DAR4, Figure 8M), ADC-016-A (mAb301, linker-drug (2-A), DAR4) Exemplary HIC-HPLC analysis results are provided for R4 (Figure 8N), ADC-016-B (mAb301, linker-drug (2-B), DAR4, Figure 8O), ADC-020-M (mAb302, linker-drug (2), DAR4, Figure 8P), ADC-020-A (mAb302, linker-drug (2-A), DAR4, Figure 8Q), and ADC-020-B (mAb302, linker-drug (2-B), DAR4, Figure 8R). [Figure 8B]As further described in Example 18, the produced ADC-009-M (mAb300, linker-drug(2), DAR2, Figure 8A), ADC-009-A (mAb300, linker-drug(2-A), DAR2, Figure 8B), ADC-009-B (mAb300, linker-drug(2-B), DAR2, Figure 8C), ADC-015-M (mAb301, linker-drug(2), DAR2, Figure 8D), ADC-015-A (m Ab301, linker-drug (2-A), DAR2, Figure 8E), ADC-015-B (mAb301, linker-drug (2-B), DAR2, Figure 8F), ADC-013-M (mAb302, linker-drug (2), DAR2, Figure 8G), ADC-013-A (mAb302, linker-drug (2-A), DAR2, Figure 8H), ADC-013-B (mAb302, linker-drug (2-B), DAR2, Figure 8I), A DC-018-M (mAb300, linker-drug (2), DAR4, Figure 8J), ADC-018-A (mAb300, linker-drug (2-A), DAR4, Figure 8K), ADC-018-B (mAb300, linker-drug (2-B), DAR4, Figure 8L), ADC-016-M (mAb301, linker-drug (2), DAR4, Figure 8M), ADC-016-A (mAb301, linker-drug (2-A), DAR4) Exemplary HIC-HPLC analysis results are provided for R4 (Figure 8N), ADC-016-B (mAb301, linker-drug (2-B), DAR4, Figure 8O), ADC-020-M (mAb302, linker-drug (2), DAR4, Figure 8P), ADC-020-A (mAb302, linker-drug (2-A), DAR4, Figure 8Q), and ADC-020-B (mAb302, linker-drug (2-B), DAR4, Figure 8R). [Figure 8C]As further described in Example 18, the produced ADC-009-M (mAb300, linker-drug(2), DAR2, Figure 8A), ADC-009-A (mAb300, linker-drug(2-A), DAR2, Figure 8B), ADC-009-B (mAb300, linker-drug(2-B), DAR2, Figure 8C), ADC-015-M (mAb301, linker-drug(2), DAR2, Figure 8D), ADC-015-A (m Ab301, linker-drug (2-A), DAR2, Figure 8E), ADC-015-B (mAb301, linker-drug (2-B), DAR2, Figure 8F), ADC-013-M (mAb302, linker-drug (2), DAR2, Figure 8G), ADC-013-A (mAb302, linker-drug (2-A), DAR2, Figure 8H), ADC-013-B (mAb302, linker-drug (2-B), DAR2, Figure 8I), A DC-018-M (mAb300, linker-drug (2), DAR4, Figure 8J), ADC-018-A (mAb300, linker-drug (2-A), DAR4, Figure 8K), ADC-018-B (mAb300, linker-drug (2-B), DAR4, Figure 8L), ADC-016-M (mAb301, linker-drug (2), DAR4, Figure 8M), ADC-016-A (mAb301, linker-drug (2-A), DAR4) Exemplary HIC-HPLC analysis results are provided for R4 (Figure 8N), ADC-016-B (mAb301, linker-drug (2-B), DAR4, Figure 8O), ADC-020-M (mAb302, linker-drug (2), DAR4, Figure 8P), ADC-020-A (mAb302, linker-drug (2-A), DAR4, Figure 8Q), and ADC-020-B (mAb302, linker-drug (2-B), DAR4, Figure 8R). [Figure 8D]As further described in Example 18, the produced ADC-009-M (mAb300, linker-drug(2), DAR2, Figure 8A), ADC-009-A (mAb300, linker-drug(2-A), DAR2, Figure 8B), ADC-009-B (mAb300, linker-drug(2-B), DAR2, Figure 8C), ADC-015-M (mAb301, linker-drug(2), DAR2, Figure 8D), ADC-015-A (m Ab301, linker-drug (2-A), DAR2, Figure 8E), ADC-015-B (mAb301, linker-drug (2-B), DAR2, Figure 8F), ADC-013-M (mAb302, linker-drug (2), DAR2, Figure 8G), ADC-013-A (mAb302, linker-drug (2-A), DAR2, Figure 8H), ADC-013-B (mAb302, linker-drug (2-B), DAR2, Figure 8I), A DC-018-M (mAb300, linker-drug (2), DAR4, Figure 8J), ADC-018-A (mAb300, linker-drug (2-A), DAR4, Figure 8K), ADC-018-B (mAb300, linker-drug (2-B), DAR4, Figure 8L), ADC-016-M (mAb301, linker-drug (2), DAR4, Figure 8M), ADC-016-A (mAb301, linker-drug (2-A), DAR4) Exemplary HIC-HPLC analysis results are provided for R4 (Figure 8N), ADC-016-B (mAb301, linker-drug (2-B), DAR4, Figure 8O), ADC-020-M (mAb302, linker-drug (2), DAR4, Figure 8P), ADC-020-A (mAb302, linker-drug (2-A), DAR4, Figure 8Q), and ADC-020-B (mAb302, linker-drug (2-B), DAR4, Figure 8R). [Figure 8E]As further described in Example 18, the produced ADC-009-M (mAb300, linker-drug(2), DAR2, Figure 8A), ADC-009-A (mAb300, linker-drug(2-A), DAR2, Figure 8B), ADC-009-B (mAb300, linker-drug(2-B), DAR2, Figure 8C), ADC-015-M (mAb301, linker-drug(2), DAR2, Figure 8D), ADC-015-A (m Ab301, linker-drug (2-A), DAR2, Figure 8E), ADC-015-B (mAb301, linker-drug (2-B), DAR2, Figure 8F), ADC-013-M (mAb302, linker-drug (2), DAR2, Figure 8G), ADC-013-A (mAb302, linker-drug (2-A), DAR2, Figure 8H), ADC-013-B (mAb302, linker-drug (2-B), DAR2, Figure 8I), A DC-018-M (mAb300, linker-drug (2), DAR4, Figure 8J), ADC-018-A (mAb300, linker-drug (2-A), DAR4, Figure 8K), ADC-018-B (mAb300, linker-drug (2-B), DAR4, Figure 8L), ADC-016-M (mAb301, linker-drug (2), DAR4, Figure 8M), ADC-016-A (mAb301, linker-drug (2-A), DAR4) Exemplary HIC-HPLC analysis results are provided for R4 (Figure 8N), ADC-016-B (mAb301, linker-drug (2-B), DAR4, Figure 8O), ADC-020-M (mAb302, linker-drug (2), DAR4, Figure 8P), ADC-020-A (mAb302, linker-drug (2-A), DAR4, Figure 8Q), and ADC-020-B (mAb302, linker-drug (2-B), DAR4, Figure 8R). [Figure 8F]As further described in Example 18, the produced ADC-009-M (mAb300, linker-drug(2), DAR2, Figure 8A), ADC-009-A (mAb300, linker-drug(2-A), DAR2, Figure 8B), ADC-009-B (mAb300, linker-drug(2-B), DAR2, Figure 8C), ADC-015-M (mAb301, linker-drug(2), DAR2, Figure 8D), ADC-015-A (m Ab301, linker-drug (2-A), DAR2, Figure 8E), ADC-015-B (mAb301, linker-drug (2-B), DAR2, Figure 8F), ADC-013-M (mAb302, linker-drug (2), DAR2, Figure 8G), ADC-013-A (mAb302, linker-drug (2-A), DAR2, Figure 8H), ADC-013-B (mAb302, linker-drug (2-B), DAR2, Figure 8I), A DC-018-M (mAb300, linker-drug (2), DAR4, Figure 8J), ADC-018-A (mAb300, linker-drug (2-A), DAR4, Figure 8K), ADC-018-B (mAb300, linker-drug (2-B), DAR4, Figure 8L), ADC-016-M (mAb301, linker-drug (2), DAR4, Figure 8M), ADC-016-A (mAb301, linker-drug (2-A), DAR4) Exemplary HIC-HPLC analysis results are provided for R4 (Figure 8N), ADC-016-B (mAb301, linker-drug (2-B), DAR4, Figure 8O), ADC-020-M (mAb302, linker-drug (2), DAR4, Figure 8P), ADC-020-A (mAb302, linker-drug (2-A), DAR4, Figure 8Q), and ADC-020-B (mAb302, linker-drug (2-B), DAR4, Figure 8R). [Figure 8G]As further described in Example 18, the produced ADC-009-M (mAb300, linker-drug(2), DAR2, Figure 8A), ADC-009-A (mAb300, linker-drug(2-A), DAR2, Figure 8B), ADC-009-B (mAb300, linker-drug(2-B), DAR2, Figure 8C), ADC-015-M (mAb301, linker-drug(2), DAR2, Figure 8D), ADC-015-A (m Ab301, linker-drug (2-A), DAR2, Figure 8E), ADC-015-B (mAb301, linker-drug (2-B), DAR2, Figure 8F), ADC-013-M (mAb302, linker-drug (2), DAR2, Figure 8G), ADC-013-A (mAb302, linker-drug (2-A), DAR2, Figure 8H), ADC-013-B (mAb302, linker-drug (2-B), DAR2, Figure 8I), A DC-018-M (mAb300, linker-drug (2), DAR4, Figure 8J), ADC-018-A (mAb300, linker-drug (2-A), DAR4, Figure 8K), ADC-018-B (mAb300, linker-drug (2-B), DAR4, Figure 8L), ADC-016-M (mAb301, linker-drug (2), DAR4, Figure 8M), ADC-016-A (mAb301, linker-drug (2-A), DAR4) Exemplary HIC-HPLC analysis results are provided for R4 (Figure 8N), ADC-016-B (mAb301, linker-drug (2-B), DAR4, Figure 8O), ADC-020-M (mAb302, linker-drug (2), DAR4, Figure 8P), ADC-020-A (mAb302, linker-drug (2-A), DAR4, Figure 8Q), and ADC-020-B (mAb302, linker-drug (2-B), DAR4, Figure 8R). [Figure 8H]As further described in Example 18, the produced ADC-009-M (mAb300, linker-drug(2), DAR2, Figure 8A), ADC-009-A (mAb300, linker-drug(2-A), DAR2, Figure 8B), ADC-009-B (mAb300, linker-drug(2-B), DAR2, Figure 8C), ADC-015-M (mAb301, linker-drug(2), DAR2, Figure 8D), ADC-015-A (m Ab301, linker-drug (2-A), DAR2, Figure 8E), ADC-015-B (mAb301, linker-drug (2-B), DAR2, Figure 8F), ADC-013-M (mAb302, linker-drug (2), DAR2, Figure 8G), ADC-013-A (mAb302, linker-drug (2-A), DAR2, Figure 8H), ADC-013-B (mAb302, linker-drug (2-B), DAR2, Figure 8I), A DC-018-M (mAb300, linker-drug (2), DAR4, Figure 8J), ADC-018-A (mAb300, linker-drug (2-A), DAR4, Figure 8K), ADC-018-B (mAb300, linker-drug (2-B), DAR4, Figure 8L), ADC-016-M (mAb301, linker-drug (2), DAR4, Figure 8M), ADC-016-A (mAb301, linker-drug (2-A), DAR4) Exemplary HIC-HPLC analysis results are provided for R4 (Figure 8N), ADC-016-B (mAb301, linker-drug (2-B), DAR4, Figure 8O), ADC-020-M (mAb302, linker-drug (2), DAR4, Figure 8P), ADC-020-A (mAb302, linker-drug (2-A), DAR4, Figure 8Q), and ADC-020-B (mAb302, linker-drug (2-B), DAR4, Figure 8R). [Figure 8I]As further described in Example 18, the produced ADC-009-M (mAb300, linker-drug(2), DAR2, Figure 8A), ADC-009-A (mAb300, linker-drug(2-A), DAR2, Figure 8B), ADC-009-B (mAb300, linker-drug(2-B), DAR2, Figure 8C), ADC-015-M (mAb301, linker-drug(2), DAR2, Figure 8D), ADC-015-A (m Ab301, linker-drug (2-A), DAR2, Figure 8E), ADC-015-B (mAb301, linker-drug (2-B), DAR2, Figure 8F), ADC-013-M (mAb302, linker-drug (2), DAR2, Figure 8G), ADC-013-A (mAb302, linker-drug (2-A), DAR2, Figure 8H), ADC-013-B (mAb302, linker-drug (2-B), DAR2, Figure 8I), A DC-018-M (mAb300, linker-drug (2), DAR4, Figure 8J), ADC-018-A (mAb300, linker-drug (2-A), DAR4, Figure 8K), ADC-018-B (mAb300, linker-drug (2-B), DAR4, Figure 8L), ADC-016-M (mAb301, linker-drug (2), DAR4, Figure 8M), ADC-016-A (mAb301, linker-drug (2-A), DAR4) Exemplary HIC-HPLC analysis results are provided for R4 (Figure 8N), ADC-016-B (mAb301, linker-drug (2-B), DAR4, Figure 8O), ADC-020-M (mAb302, linker-drug (2), DAR4, Figure 8P), ADC-020-A (mAb302, linker-drug (2-A), DAR4, Figure 8Q), and ADC-020-B (mAb302, linker-drug (2-B), DAR4, Figure 8R). [Figure 8J]As further described in Example 18, the produced ADC-009-M (mAb300, linker-drug(2), DAR2, Figure 8A), ADC-009-A (mAb300, linker-drug(2-A), DAR2, Figure 8B), ADC-009-B (mAb300, linker-drug(2-B), DAR2, Figure 8C), ADC-015-M (mAb301, linker-drug(2), DAR2, Figure 8D), ADC-015-A (m Ab301, linker-drug (2-A), DAR2, Figure 8E), ADC-015-B (mAb301, linker-drug (2-B), DAR2, Figure 8F), ADC-013-M (mAb302, linker-drug (2), DAR2, Figure 8G), ADC-013-A (mAb302, linker-drug (2-A), DAR2, Figure 8H), ADC-013-B (mAb302, linker-drug (2-B), DAR2, Figure 8I), A DC-018-M (mAb300, linker-drug (2), DAR4, Figure 8J), ADC-018-A (mAb300, linker-drug (2-A), DAR4, Figure 8K), ADC-018-B (mAb300, linker-drug (2-B), DAR4, Figure 8L), ADC-016-M (mAb301, linker-drug (2), DAR4, Figure 8M), ADC-016-A (mAb301, linker-drug (2-A), DAR4) Exemplary HIC-HPLC analysis results are provided for R4 (Figure 8N), ADC-016-B (mAb301, linker-drug (2-B), DAR4, Figure 8O), ADC-020-M (mAb302, linker-drug (2), DAR4, Figure 8P), ADC-020-A (mAb302, linker-drug (2-A), DAR4, Figure 8Q), and ADC-020-B (mAb302, linker-drug (2-B), DAR4, Figure 8R). [Figure 8K]As further described in Example 18, the produced ADC-009-M (mAb300, linker-drug(2), DAR2, Figure 8A), ADC-009-A (mAb300, linker-drug(2-A), DAR2, Figure 8B), ADC-009-B (mAb300, linker-drug(2-B), DAR2, Figure 8C), ADC-015-M (mAb301, linker-drug(2), DAR2, Figure 8D), ADC-015-A (m Ab301, linker-drug (2-A), DAR2, Figure 8E), ADC-015-B (mAb301, linker-drug (2-B), DAR2, Figure 8F), ADC-013-M (mAb302, linker-drug (2), DAR2, Figure 8G), ADC-013-A (mAb302, linker-drug (2-A), DAR2, Figure 8H), ADC-013-B (mAb302, linker-drug (2-B), DAR2, Figure 8I), A DC-018-M (mAb300, linker-drug (2), DAR4, Figure 8J), ADC-018-A (mAb300, linker-drug (2-A), DAR4, Figure 8K), ADC-018-B (mAb300, linker-drug (2-B), DAR4, Figure 8L), ADC-016-M (mAb301, linker-drug (2), DAR4, Figure 8M), ADC-016-A (mAb301, linker-drug (2-A), DAR4) Exemplary HIC-HPLC analysis results are provided for R4 (Figure 8N), ADC-016-B (mAb301, linker-drug (2-B), DAR4, Figure 8O), ADC-020-M (mAb302, linker-drug (2), DAR4, Figure 8P), ADC-020-A (mAb302, linker-drug (2-A), DAR4, Figure 8Q), and ADC-020-B (mAb302, linker-drug (2-B), DAR4, Figure 8R). [Figure 8L]As further described in Example 18, the produced ADC-009-M (mAb300, linker-drug(2), DAR2, Figure 8A), ADC-009-A (mAb300, linker-drug(2-A), DAR2, Figure 8B), ADC-009-B (mAb300, linker-drug(2-B), DAR2, Figure 8C), ADC-015-M (mAb301, linker-drug(2), DAR2, Figure 8D), ADC-015-A (m Ab301, linker-drug (2-A), DAR2, Figure 8E), ADC-015-B (mAb301, linker-drug (2-B), DAR2, Figure 8F), ADC-013-M (mAb302, linker-drug (2), DAR2, Figure 8G), ADC-013-A (mAb302, linker-drug (2-A), DAR2, Figure 8H), ADC-013-B (mAb302, linker-drug (2-B), DAR2, Figure 8I), A DC-018-M (mAb300, linker-drug (2), DAR4, Figure 8J), ADC-018-A (mAb300, linker-drug (2-A), DAR4, Figure 8K), ADC-018-B (mAb300, linker-drug (2-B), DAR4, Figure 8L), ADC-016-M (mAb301, linker-drug (2), DAR4, Figure 8M), ADC-016-A (mAb301, linker-drug (2-A), DAR4) Exemplary HIC-HPLC analysis results are provided for R4 (Figure 8N), ADC-016-B (mAb301, linker-drug (2-B), DAR4, Figure 8O), ADC-020-M (mAb302, linker-drug (2), DAR4, Figure 8P), ADC-020-A (mAb302, linker-drug (2-A), DAR4, Figure 8Q), and ADC-020-B (mAb302, linker-drug (2-B), DAR4, Figure 8R). [Figure 8M]As further described in Example 18, the produced ADC-009-M (mAb300, linker-drug(2), DAR2, Figure 8A), ADC-009-A (mAb300, linker-drug(2-A), DAR2, Figure 8B), ADC-009-B (mAb300, linker-drug(2-B), DAR2, Figure 8C), ADC-015-M (mAb301, linker-drug(2), DAR2, Figure 8D), ADC-015-A (m Ab301, linker-drug (2-A), DAR2, Figure 8E), ADC-015-B (mAb301, linker-drug (2-B), DAR2, Figure 8F), ADC-013-M (mAb302, linker-drug (2), DAR2, Figure 8G), ADC-013-A (mAb302, linker-drug (2-A), DAR2, Figure 8H), ADC-013-B (mAb302, linker-drug (2-B), DAR2, Figure 8I), A DC-018-M (mAb300, linker-drug (2), DAR4, Figure 8J), ADC-018-A (mAb300, linker-drug (2-A), DAR4, Figure 8K), ADC-018-B (mAb300, linker-drug (2-B), DAR4, Figure 8L), ADC-016-M (mAb301, linker-drug (2), DAR4, Figure 8M), ADC-016-A (mAb301, linker-drug (2-A), DAR4) Exemplary HIC-HPLC analysis results are provided for R4 (Figure 8N), ADC-016-B (mAb301, linker-drug (2-B), DAR4, Figure 8O), ADC-020-M (mAb302, linker-drug (2), DAR4, Figure 8P), ADC-020-A (mAb302, linker-drug (2-A), DAR4, Figure 8Q), and ADC-020-B (mAb302, linker-drug (2-B), DAR4, Figure 8R). [Figure 8N]As further described in Example 18, the produced ADC-009-M (mAb300, linker-drug(2), DAR2, Figure 8A), ADC-009-A (mAb300, linker-drug(2-A), DAR2, Figure 8B), ADC-009-B (mAb300, linker-drug(2-B), DAR2, Figure 8C), ADC-015-M (mAb301, linker-drug(2), DAR2, Figure 8D), ADC-015-A (m Ab301, linker-drug (2-A), DAR2, Figure 8E), ADC-015-B (mAb301, linker-drug (2-B), DAR2, Figure 8F), ADC-013-M (mAb302, linker-drug (2), DAR2, Figure 8G), ADC-013-A (mAb302, linker-drug (2-A), DAR2, Figure 8H), ADC-013-B (mAb302, linker-drug (2-B), DAR2, Figure 8I), A DC-018-M (mAb300, linker-drug (2), DAR4, Figure 8J), ADC-018-A (mAb300, linker-drug (2-A), DAR4, Figure 8K), ADC-018-B (mAb300, linker-drug (2-B), DAR4, Figure 8L), ADC-016-M (mAb301, linker-drug (2), DAR4, Figure 8M), ADC-016-A (mAb301, linker-drug (2-A), DAR4) Exemplary HIC-HPLC analysis results are provided for R4 (Figure 8N), ADC-016-B (mAb301, linker-drug (2-B), DAR4, Figure 8O), ADC-020-M (mAb302, linker-drug (2), DAR4, Figure 8P), ADC-020-A (mAb302, linker-drug (2-A), DAR4, Figure 8Q), and ADC-020-B (mAb302, linker-drug (2-B), DAR4, Figure 8R). [Figure 8O]As further described in Example 18, the produced ADC-009-M (mAb300, linker-drug(2), DAR2, Figure 8A), ADC-009-A (mAb300, linker-drug(2-A), DAR2, Figure 8B), ADC-009-B (mAb300, linker-drug(2-B), DAR2, Figure 8C), ADC-015-M (mAb301, linker-drug(2), DAR2, Figure 8D), ADC-015-A (m Ab301, linker-drug (2-A), DAR2, Figure 8E), ADC-015-B (mAb301, linker-drug (2-B), DAR2, Figure 8F), ADC-013-M (mAb302, linker-drug (2), DAR2, Figure 8G), ADC-013-A (mAb302, linker-drug (2-A), DAR2, Figure 8H), ADC-013-B (mAb302, linker-drug (2-B), DAR2, Figure 8I), A DC-018-M (mAb300, linker-drug (2), DAR4, Figure 8J), ADC-018-A (mAb300, linker-drug (2-A), DAR4, Figure 8K), ADC-018-B (mAb300, linker-drug (2-B), DAR4, Figure 8L), ADC-016-M (mAb301, linker-drug (2), DAR4, Figure 8M), ADC-016-A (mAb301, linker-drug (2-A), DAR4) Exemplary HIC-HPLC analysis results are provided for R4 (Figure 8N), ADC-016-B (mAb301, linker-drug (2-B), DAR4, Figure 8O), ADC-020-M (mAb302, linker-drug (2), DAR4, Figure 8P), ADC-020-A (mAb302, linker-drug (2-A), DAR4, Figure 8Q), and ADC-020-B (mAb302, linker-drug (2-B), DAR4, Figure 8R). [Figure 8P]As further described in Example 18, the produced ADC-009-M (mAb300, linker-drug(2), DAR2, Figure 8A), ADC-009-A (mAb300, linker-drug(2-A), DAR2, Figure 8B), ADC-009-B (mAb300, linker-drug(2-B), DAR2, Figure 8C), ADC-015-M (mAb301, linker-drug(2), DAR2, Figure 8D), ADC-015-A (m Ab301, linker-drug (2-A), DAR2, Figure 8E), ADC-015-B (mAb301, linker-drug (2-B), DAR2, Figure 8F), ADC-013-M (mAb302, linker-drug (2), DAR2, Figure 8G), ADC-013-A (mAb302, linker-drug (2-A), DAR2, Figure 8H), ADC-013-B (mAb302, linker-drug (2-B), DAR2, Figure 8I), A DC-018-M (mAb300, linker-drug (2), DAR4, Figure 8J), ADC-018-A (mAb300, linker-drug (2-A), DAR4, Figure 8K), ADC-018-B (mAb300, linker-drug (2-B), DAR4, Figure 8L), ADC-016-M (mAb301, linker-drug (2), DAR4, Figure 8M), ADC-016-A (mAb301, linker-drug (2-A), DAR4) Exemplary HIC-HPLC analysis results are provided for R4 (Figure 8N), ADC-016-B (mAb301, linker-drug (2-B), DAR4, Figure 8O), ADC-020-M (mAb302, linker-drug (2), DAR4, Figure 8P), ADC-020-A (mAb302, linker-drug (2-A), DAR4, Figure 8Q), and ADC-020-B (mAb302, linker-drug (2-B), DAR4, Figure 8R). [Figure 8Q]As further described in Example 18, the produced ADC-009-M (mAb300, linker-drug(2), DAR2, Figure 8A), ADC-009-A (mAb300, linker-drug(2-A), DAR2, Figure 8B), ADC-009-B (mAb300, linker-drug(2-B), DAR2, Figure 8C), ADC-015-M (mAb301, linker-drug(2), DAR2, Figure 8D), ADC-015-A (m Ab301, linker-drug (2-A), DAR2, Figure 8E), ADC-015-B (mAb301, linker-drug (2-B), DAR2, Figure 8F), ADC-013-M (mAb302, linker-drug (2), DAR2, Figure 8G), ADC-013-A (mAb302, linker-drug (2-A), DAR2, Figure 8H), ADC-013-B (mAb302, linker-drug (2-B), DAR2, Figure 8I), A DC-018-M (mAb300, linker-drug (2), DAR4, Figure 8J), ADC-018-A (mAb300, linker-drug (2-A), DAR4, Figure 8K), ADC-018-B (mAb300, linker-drug (2-B), DAR4, Figure 8L), ADC-016-M (mAb301, linker-drug (2), DAR4, Figure 8M), ADC-016-A (mAb301, linker-drug (2-A), DAR4) Exemplary HIC-HPLC analysis results are provided for R4 (Figure 8N), ADC-016-B (mAb301, linker-drug (2-B), DAR4, Figure 8O), ADC-020-M (mAb302, linker-drug (2), DAR4, Figure 8P), ADC-020-A (mAb302, linker-drug (2-A), DAR4, Figure 8Q), and ADC-020-B (mAb302, linker-drug (2-B), DAR4, Figure 8R). [Figure 8R]As further described in Example 18, the produced ADC-009-M (mAb300, linker-drug(2), DAR2, Figure 8A), ADC-009-A (mAb300, linker-drug(2-A), DAR2, Figure 8B), ADC-009-B (mAb300, linker-drug(2-B), DAR2, Figure 8C), ADC-015-M (mAb301, linker-drug(2), DAR2, Figure 8D), ADC-015-A (m Ab301, linker-drug (2-A), DAR2, Figure 8E), ADC-015-B (mAb301, linker-drug (2-B), DAR2, Figure 8F), ADC-013-M (mAb302, linker-drug (2), DAR2, Figure 8G), ADC-013-A (mAb302, linker-drug (2-A), DAR2, Figure 8H), ADC-013-B (mAb302, linker-drug (2-B), DAR2, Figure 8I), A DC-018-M (mAb300, linker-drug (2), DAR4, Figure 8J), ADC-018-A (mAb300, linker-drug (2-A), DAR4, Figure 8K), ADC-018-B (mAb300, linker-drug (2-B), DAR4, Figure 8L), ADC-016-M (mAb301, linker-drug (2), DAR4, Figure 8M), ADC-016-A (mAb301, linker-drug (2-A), DAR4) Exemplary HIC-HPLC analysis results are provided for R4 (Figure 8N), ADC-016-B (mAb301, linker-drug (2-B), DAR4, Figure 8O), ADC-020-M (mAb302, linker-drug (2), DAR4, Figure 8P), ADC-020-A (mAb302, linker-drug (2-A), DAR4, Figure 8Q), and ADC-020-B (mAb302, linker-drug (2-B), DAR4, Figure 8R). [Figure 9A]This document provides exemplary results from cytotoxic assays investigating the effects of ADC-015, ADC-016, and ADC-017 on hDLL3-overexpressing B16F10 cells, as further described in Example 17. Figure 9A compares various concentrations of MMAE, exatecan, and ADC-017 after 72 hours of incubation. Figure 9B compares various concentrations of MMAE, exatecan, and ADC-017 after 96 hours of incubation. The corresponding IC50 and maximum inhibition percentages are calculated and presented in Figure 9C. Furthermore, Figure 9D compares various concentrations of MMAE, exatecan, ADC-015, ADC-016, ADC-017, IgG1 Fc conjugated to exatecan, and IgG1 Fc conjugated to MMAE after 72 hours of incubation, while Figure 9E presents the corresponding IC50 and maximum inhibition percentages. [Figure 9B] This document provides exemplary results from cytotoxic assays investigating the effects of ADC-015, ADC-016, and ADC-017 on hDLL3-overexpressing B16F10 cells, as further described in Example 17. Figure 9A compares various concentrations of MMAE, exatecan, and ADC-017 after 72 hours of incubation. Figure 9B compares various concentrations of MMAE, exatecan, and ADC-017 after 96 hours of incubation. The corresponding IC50 and maximum inhibition percentages are calculated and presented in Figure 9C. Furthermore, Figure 9D compares various concentrations of MMAE, exatecan, ADC-015, ADC-016, ADC-017, IgG1 Fc conjugated to exatecan, and IgG1 Fc conjugated to MMAE after 72 hours of incubation, while Figure 9E presents the corresponding IC50 and maximum inhibition percentages. [Figure 9C]This document provides exemplary results from cytotoxic assays investigating the effects of ADC-015, ADC-016, and ADC-017 on hDLL3-overexpressing B16F10 cells, as further described in Example 17. Figure 9A compares various concentrations of MMAE, exatecan, and ADC-017 after 72 hours of incubation. Figure 9B compares various concentrations of MMAE, exatecan, and ADC-017 after 96 hours of incubation. The corresponding IC50 and maximum inhibition percentages are calculated and presented in Figure 9C. Furthermore, Figure 9D compares various concentrations of MMAE, exatecan, ADC-015, ADC-016, ADC-017, IgG1 Fc conjugated to exatecan, and IgG1 Fc conjugated to MMAE after 72 hours of incubation, while Figure 9E presents the corresponding IC50 and maximum inhibition percentages. [Figure 9D] This document provides exemplary results from cytotoxic assays investigating the effects of ADC-015, ADC-016, and ADC-017 on hDLL3-overexpressing B16F10 cells, as further described in Example 17. Figure 9A compares various concentrations of MMAE, exatecan, and ADC-017 after 72 hours of incubation. Figure 9B compares various concentrations of MMAE, exatecan, and ADC-017 after 96 hours of incubation. The corresponding IC50 and maximum inhibition percentages are calculated and presented in Figure 9C. Furthermore, Figure 9D compares various concentrations of MMAE, exatecan, ADC-015, ADC-016, ADC-017, IgG1 Fc conjugated to exatecan, and IgG1 Fc conjugated to MMAE after 72 hours of incubation, while Figure 9E presents the corresponding IC50 and maximum inhibition percentages. [Figure 9E]This document provides exemplary results from cytotoxic assays investigating the effects of ADC-015, ADC-016, and ADC-017 on hDLL3-overexpressing B16F10 cells, as further described in Example 17. Figure 9A compares various concentrations of MMAE, exatecan, and ADC-017 after 72 hours of incubation. Figure 9B compares various concentrations of MMAE, exatecan, and ADC-017 after 96 hours of incubation. The corresponding IC50 and maximum inhibition percentages are calculated and presented in Figure 9C. Furthermore, Figure 9D compares various concentrations of MMAE, exatecan, ADC-015, ADC-016, ADC-017, IgG1 Fc conjugated to exatecan, and IgG1 Fc conjugated to MMAE after 72 hours of incubation, while Figure 9E presents the corresponding IC50 and maximum inhibition percentages. [Figure 10A] The following provides exemplary results from in vitro cytotoxic assays investigating the effects of ADC-015-M, ADC-015-A, ADC-015-B, ADC-016-M, ADC-016-A, and ADC-016-B on the DLL3-overexpressing 293F cell line, as further described in Example 20. Figures 10A and 10B plot data from the first experiment, with Figure 10A plotting the concentration of the tested ADC on the x-axis and Figure 10B plotting the calibrated concentration of the linker payload of the tested ADC on the x-axis. Figures 10C and 10D plot data from the second experiment, with Figure 10C plotting the concentration of the tested ADC on the x-axis and Figure 10D plotting the calibrated concentration of the linker payload of the tested ADC on the x-axis. Figure 10E plots data from the third experiment. [Figure 10B]The following provides exemplary results from in vitro cytotoxic assays investigating the effects of ADC-015-M, ADC-015-A, ADC-015-B, ADC-016-M, ADC-016-A, and ADC-016-B on the DLL3-overexpressing 293F cell line, as further described in Example 20. Figures 10A and 10B plot data from the first experiment, with Figure 10A plotting the concentration of the tested ADC on the x-axis and Figure 10B plotting the calibrated concentration of the linker payload of the tested ADC on the x-axis. Figures 10C and 10D plot data from the second experiment, with Figure 10C plotting the concentration of the tested ADC on the x-axis and Figure 10D plotting the calibrated concentration of the linker payload of the tested ADC on the x-axis. Figure 10E plots data from the third experiment. [Figure 10C] The following provides exemplary results from in vitro cytotoxic assays investigating the effects of ADC-015-M, ADC-015-A, ADC-015-B, ADC-016-M, ADC-016-A, and ADC-016-B on the DLL3-overexpressing 293F cell line, as further described in Example 20. Figures 10A and 10B plot data from the first experiment, with Figure 10A plotting the concentration of the tested ADC on the x-axis and Figure 10B plotting the calibrated concentration of the linker payload of the tested ADC on the x-axis. Figures 10C and 10D plot data from the second experiment, with Figure 10C plotting the concentration of the tested ADC on the x-axis and Figure 10D plotting the calibrated concentration of the linker payload of the tested ADC on the x-axis. Figure 10E plots data from the third experiment. [Figure 10D]The following provides exemplary results from in vitro cytotoxic assays investigating the effects of ADC-015-M, ADC-015-A, ADC-015-B, ADC-016-M, ADC-016-A, and ADC-016-B on the DLL3-overexpressing 293F cell line, as further described in Example 20. Figures 10A and 10B plot data from the first experiment, with Figure 10A plotting the concentration of the tested ADC on the x-axis and Figure 10B plotting the calibrated concentration of the linker payload of the tested ADC on the x-axis. Figures 10C and 10D plot data from the second experiment, with Figure 10C plotting the concentration of the tested ADC on the x-axis and Figure 10D plotting the calibrated concentration of the linker payload of the tested ADC on the x-axis. Figure 10E plots data from the third experiment. [Figure 10E] The following provides exemplary results from in vitro cytotoxic assays investigating the effects of ADC-015-M, ADC-015-A, ADC-015-B, ADC-016-M, ADC-016-A, and ADC-016-B on the DLL3-overexpressing 293F cell line, as further described in Example 20. Figures 10A and 10B plot data from the first experiment, with Figure 10A plotting the concentration of the tested ADC on the x-axis and Figure 10B plotting the calibrated concentration of the linker payload of the tested ADC on the x-axis. Figures 10C and 10D plot data from the second experiment, with Figure 10C plotting the concentration of the tested ADC on the x-axis and Figure 10D plotting the calibrated concentration of the linker payload of the tested ADC on the x-axis. Figure 10E plots data from the third experiment. [Figure 11A] The results of a cytotoxic assay investigating the effects of ADC-015, ADC-016, and ADC-017 on H69 cells, as further described in Example 21, are provided as exemplary. Figures 11A, 11C, and 11E compare various concentrations of MMAE, exatecan, ADC-015, ADC-016, and ADC-017 after 120 hours of incubation, while their IC50 and maximum inhibitory percentages are presented in Figures 11B, 11D, and 11F, respectively. [Figure 11B]The results of a cytotoxic assay investigating the effects of ADC-015, ADC-016, and ADC-017 on H69 cells, as further described in Example 21, are provided as exemplary. Figures 11A, 11C, and 11E compare various concentrations of MMAE, exatecan, ADC-015, ADC-016, and ADC-017 after 120 hours of incubation, while their IC50 and maximum inhibitory percentages are presented in Figures 11B, 11D, and 11F, respectively. [Figure 11C] The results of a cytotoxic assay investigating the effects of ADC-015, ADC-016, and ADC-017 on H69 cells, as further described in Example 21, are provided as exemplary. Figures 11A, 11C, and 11E compare various concentrations of MMAE, exatecan, ADC-015, ADC-016, and ADC-017 after 120 hours of incubation, while their IC50 and maximum inhibitory percentages are presented in Figures 11B, 11D, and 11F, respectively. [Figure 11D] The results of a cytotoxic assay investigating the effects of ADC-015, ADC-016, and ADC-017 on H69 cells, as further described in Example 21, are provided as exemplary. Figures 11A, 11C, and 11E compare various concentrations of MMAE, exatecan, ADC-015, ADC-016, and ADC-017 after 120 hours of incubation, while their IC50 and maximum inhibitory percentages are presented in Figures 11B, 11D, and 11F, respectively. [Figure 11E] The results of a cytotoxic assay investigating the effects of ADC-015, ADC-016, and ADC-017 on H69 cells, as further described in Example 21, are provided as exemplary. Figures 11A, 11C, and 11E compare various concentrations of MMAE, exatecan, ADC-015, ADC-016, and ADC-017 after 120 hours of incubation, while their IC50 and maximum inhibitory percentages are presented in Figures 11B, 11D, and 11F, respectively. [Figure 11F]The results of a cytotoxic assay investigating the effects of ADC-015, ADC-016, and ADC-017 on H69 cells, as further described in Example 21, are provided as exemplary. Figures 11A, 11C, and 11E compare various concentrations of MMAE, exatecan, ADC-015, ADC-016, and ADC-017 after 120 hours of incubation, while their IC50 and maximum inhibitory percentages are presented in Figures 11B, 11D, and 11F, respectively. [Figure 12A] The results of a cytotoxic assay investigating the effects of ADC-015, ADC-016, and ADC-017 on CORL279 cells, as further described in Example 21, are provided as exemplary results. Figures 12A and 12C compare various concentrations of MMAE, exatecan, ADC-015, ADC-016, and ADC-017 after 120 hours of incubation, while their IC50 and maximum inhibitory percentages are presented in Figures 12B and 12D, respectively. [Figure 12B] The results of a cytotoxic assay investigating the effects of ADC-015, ADC-016, and ADC-017 on CORL279 cells, as further described in Example 21, are provided as exemplary results. Figures 12A and 12C compare various concentrations of MMAE, exatecan, ADC-015, ADC-016, and ADC-017 after 120 hours of incubation, while their IC50 and maximum inhibitory percentages are presented in Figures 12B and 12D, respectively. [Figure 12C] The results of a cytotoxic assay investigating the effects of ADC-015, ADC-016, and ADC-017 on CORL279 cells, as further described in Example 21, are provided as exemplary results. Figures 12A and 12C compare various concentrations of MMAE, exatecan, ADC-015, ADC-016, and ADC-017 after 120 hours of incubation, while their IC50 and maximum inhibitory percentages are presented in Figures 12B and 12D, respectively. [Figure 12D]The results of a cytotoxic assay investigating the effects of ADC-015, ADC-016, and ADC-017 on CORL279 cells, as further described in Example 21, are provided as exemplary results. Figures 12A and 12C compare various concentrations of MMAE, exatecan, ADC-015, ADC-016, and ADC-017 after 120 hours of incubation, while their IC50 and maximum inhibitory percentages are presented in Figures 12B and 12D, respectively. [Figure 13] The results of a cytotoxic assay investigating the effects of ADC-015, ADC-016, and ADC-017 on SHP77 cells, as further described in Example 21, are provided. A compares various concentrations of MMAE, exatecan, ADC-015, ADC-016, and ADC-017 after 120 hours of incubation, while the corresponding IC50 and maximum inhibitory percentage are presented in B. [Figure 14] The results of a cytotoxic assay investigating the effects of ADC-015, ADC-016, and ADC-017 on H460 cells, as further described in Example 21, are provided as exemplary results. A compares various concentrations of MMAE, exatecan, ADC-015, ADC-016, and ADC-017 after 120 hours of incubation, while the corresponding IC50 and maximum inhibition percentage are presented in B. [Figure 15A] This document provides exemplary results from cytotoxic assays investigating the effects of ADC-009 and ADC-010 on SHP77 cells (Figure 15A), DMS79 cells (Figure 15B), H69 cells (Figure 15C), and CORL279 cells (Figure 15D), as further described in Example 22. The corresponding IC50 and maximum inhibition percentage were calculated and are presented in Figure 15E. [Figure 15B] This document provides exemplary results from cytotoxic assays investigating the effects of ADC-009 and ADC-010 on SHP77 cells (Figure 15A), DMS79 cells (Figure 15B), H69 cells (Figure 15C), and CORL279 cells (Figure 15D), as further described in Example 22. The corresponding IC50 and maximum inhibition percentage were calculated and are presented in Figure 15E. [Figure 15C] This document provides exemplary results from cytotoxic assays investigating the effects of ADC-009 and ADC-010 on SHP77 cells (Figure 15A), DMS79 cells (Figure 15B), H69 cells (Figure 15C), and CORL279 cells (Figure 15D), as further described in Example 22. The corresponding IC50 and maximum inhibition percentage were calculated and are presented in Figure 15E. [Figure 15D] This document provides exemplary results from cytotoxic assays investigating the effects of ADC-009 and ADC-010 on SHP77 cells (Figure 15A), DMS79 cells (Figure 15B), H69 cells (Figure 15C), and CORL279 cells (Figure 15D), as further described in Example 22. The corresponding IC50 and maximum inhibition percentage were calculated and are presented in Figure 15E. [Figure 15E] This document provides exemplary results from cytotoxic assays investigating the effects of ADC-009 and ADC-010 on SHP77 cells (Figure 15A), DMS79 cells (Figure 15B), H69 cells (Figure 15C), and CORL279 cells (Figure 15D), as further described in Example 22. The corresponding IC50 and maximum inhibition percentage were calculated and are presented in Figure 15E. [Figure 16A] The tumor volume (TV) measurements of mice transplanted with SHP77 small cell lung cancer (SCLC) cells and treated with the indicated compounds, as further described in Example 23, are plotted. Figures 16A, 16C, and 16E plot the data over the observation period, while Figures 16B and 16D compare the data at day 28 (D28), and Figure 16F plots the data at day 24 (D24). [Figure 16B] The tumor volume (TV) measurements of mice transplanted with SHP77 small cell lung cancer (SCLC) cells and treated with the indicated compounds, as further described in Example 23, are plotted. Figures 16A, 16C, and 16E plot the data over the observation period, while Figures 16B and 16D compare the data at day 28 (D28), and Figure 16F plots the data at day 24 (D24). [Figure 16C]The tumor volume (TV) measurements of mice transplanted with SHP77 small cell lung cancer (SCLC) cells and treated with the indicated compounds, as further described in Example 23, are plotted. Figures 16A, 16C, and 16E plot the data over the observation period, while Figures 16B and 16D compare the data at day 28 (D28), and Figure 16F plots the data at day 24 (D24). [Figure 16D] The tumor volume (TV) measurements of mice transplanted with SHP77 small cell lung cancer (SCLC) cells and treated with the indicated compounds, as further described in Example 23, are plotted. Figures 16A, 16C, and 16E plot the data over the observation period, while Figures 16B and 16D compare the data at day 28 (D28), and Figure 16F plots the data at day 24 (D24). [Figure 16E] The tumor volume (TV) measurements of mice transplanted with SHP77 small cell lung cancer (SCLC) cells and treated with the indicated compounds, as further described in Example 23, are plotted. Figures 16A, 16C, and 16E plot the data over the observation period, while Figures 16B and 16D compare the data at day 28 (D28), and Figure 16F plots the data at day 24 (D24). [Figure 16F] The tumor volume (TV) measurements of mice transplanted with SHP77 small cell lung cancer (SCLC) cells and treated with the indicated compounds, as further described in Example 23, are plotted. Figures 16A, 16C, and 16E plot the data over the observation period, while Figures 16B and 16D compare the data at day 28 (D28), and Figure 16F plots the data at day 24 (D24). [Figure 17A]As further described in Example 23, we provide spider plots of mice transplanted with SHP77 SCLC cells and treated with the indicated compounds (Figure 17A, vehicle; Figure 17B, DAR 2 isotype ADC 1 mg / kg; Figure 17C, DAR 4 isotype ADC 1 mg / kg; Figure 17D, ADC-015-B 3 mg / kg; Figure 17E, ADC-015-B 1 mg / kg; Figure 17F, ADC-015-B 0.3 mg / kg × 3; Figure 17G, ADC-015-M 1 mg / kg; Figure 17H, ADC-016-B 3 mg / kg; Figure 17I, ADC-016-B 1 mg / kg; Figure 17J, ADC-016-B 0.3 mg / kg × 3; Figure 17K, ADC-016-M 1 mg / kg). [Figure 17B] As further described in Example 23, we provide spider plots of mice transplanted with SHP77 SCLC cells and treated with the indicated compounds (Figure 17A, vehicle; Figure 17B, DAR 2 isotype ADC 1 mg / kg; Figure 17C, DAR 4 isotype ADC 1 mg / kg; Figure 17D, ADC-015-B 3 mg / kg; Figure 17E, ADC-015-B 1 mg / kg; Figure 17F, ADC-015-B 0.3 mg / kg × 3; Figure 17G, ADC-015-M 1 mg / kg; Figure 17H, ADC-016-B 3 mg / kg; Figure 17I, ADC-016-B 1 mg / kg; Figure 17J, ADC-016-B 0.3 mg / kg × 3; Figure 17K, ADC-016-M 1 mg / kg). [Figure 17C] As further described in Example 23, we provide spider plots of mice transplanted with SHP77 SCLC cells and treated with the indicated compounds (Figure 17A, vehicle; Figure 17B, DAR 2 isotype ADC 1 mg / kg; Figure 17C, DAR 4 isotype ADC 1 mg / kg; Figure 17D, ADC-015-B 3 mg / kg; Figure 17E, ADC-015-B 1 mg / kg; Figure 17F, ADC-015-B 0.3 mg / kg × 3; Figure 17G, ADC-015-M 1 mg / kg; Figure 17H, ADC-016-B 3 mg / kg; Figure 17I, ADC-016-B 1 mg / kg; Figure 17J, ADC-016-B 0.3 mg / kg × 3; Figure 17K, ADC-016-M 1 mg / kg). [Figure 17D] As further described in Example 23, we provide spider plots of mice transplanted with SHP77 SCLC cells and treated with the indicated compounds (Figure 17A, vehicle; Figure 17B, DAR 2 isotype ADC 1 mg / kg; Figure 17C, DAR 4 isotype ADC 1 mg / kg; Figure 17D, ADC-015-B 3 mg / kg; Figure 17E, ADC-015-B 1 mg / kg; Figure 17F, ADC-015-B 0.3 mg / kg × 3; Figure 17G, ADC-015-M 1 mg / kg; Figure 17H, ADC-016-B 3 mg / kg; Figure 17I, ADC-016-B 1 mg / kg; Figure 17J, ADC-016-B 0.3 mg / kg × 3; Figure 17K, ADC-016-M 1 mg / kg). [Figure 17E] As further described in Example 23, we provide spider plots of mice transplanted with SHP77 SCLC cells and treated with the indicated compounds (Figure 17A, vehicle; Figure 17B, DAR 2 isotype ADC 1 mg / kg; Figure 17C, DAR 4 isotype ADC 1 mg / kg; Figure 17D, ADC-015-B 3 mg / kg; Figure 17E, ADC-015-B 1 mg / kg; Figure 17F, ADC-015-B 0.3 mg / kg × 3; Figure 17G, ADC-015-M 1 mg / kg; Figure 17H, ADC-016-B 3 mg / kg; Figure 17I, ADC-016-B 1 mg / kg; Figure 17J, ADC-016-B 0.3 mg / kg × 3; Figure 17K, ADC-016-M 1 mg / kg). [Figure 17F]As further described in Example 23, we provide spider plots of mice transplanted with SHP77 SCLC cells and treated with the indicated compounds (Figure 17A, vehicle; Figure 17B, DAR 2 isotype ADC 1 mg / kg; Figure 17C, DAR 4 isotype ADC 1 mg / kg; Figure 17D, ADC-015-B 3 mg / kg; Figure 17E, ADC-015-B 1 mg / kg; Figure 17F, ADC-015-B 0.3 mg / kg × 3; Figure 17G, ADC-015-M 1 mg / kg; Figure 17H, ADC-016-B 3 mg / kg; Figure 17I, ADC-016-B 1 mg / kg; Figure 17J, ADC-016-B 0.3 mg / kg × 3; Figure 17K, ADC-016-M 1 mg / kg). [Figure 17G] As further described in Example 23, we provide spider plots of mice transplanted with SHP77 SCLC cells and treated with the indicated compounds (Figure 17A, vehicle; Figure 17B, DAR 2 isotype ADC 1 mg / kg; Figure 17C, DAR 4 isotype ADC 1 mg / kg; Figure 17D, ADC-015-B 3 mg / kg; Figure 17E, ADC-015-B 1 mg / kg; Figure 17F, ADC-015-B 0.3 mg / kg × 3; Figure 17G, ADC-015-M 1 mg / kg; Figure 17H, ADC-016-B 3 mg / kg; Figure 17I, ADC-016-B 1 mg / kg; Figure 17J, ADC-016-B 0.3 mg / kg × 3; Figure 17K, ADC-016-M 1 mg / kg). [Figure 17H] As further described in Example 23, we provide spider plots of mice transplanted with SHP77 SCLC cells and treated with the indicated compounds (Figure 17A, vehicle; Figure 17B, DAR 2 isotype ADC 1 mg / kg; Figure 17C, DAR 4 isotype ADC 1 mg / kg; Figure 17D, ADC-015-B 3 mg / kg; Figure 17E, ADC-015-B 1 mg / kg; Figure 17F, ADC-015-B 0.3 mg / kg × 3; Figure 17G, ADC-015-M 1 mg / kg; Figure 17H, ADC-016-B 3 mg / kg; Figure 17I, ADC-016-B 1 mg / kg; Figure 17J, ADC-016-B 0.3 mg / kg × 3; Figure 17K, ADC-016-M 1 mg / kg). [Figure 17I] As further described in Example 23, we provide spider plots of mice transplanted with SHP77 SCLC cells and treated with the indicated compounds (Figure 17A, vehicle; Figure 17B, DAR 2 isotype ADC 1 mg / kg; Figure 17C, DAR 4 isotype ADC 1 mg / kg; Figure 17D, ADC-015-B 3 mg / kg; Figure 17E, ADC-015-B 1 mg / kg; Figure 17F, ADC-015-B 0.3 mg / kg × 3; Figure 17G, ADC-015-M 1 mg / kg; Figure 17H, ADC-016-B 3 mg / kg; Figure 17I, ADC-016-B 1 mg / kg; Figure 17J, ADC-016-B 0.3 mg / kg × 3; Figure 17K, ADC-016-M 1 mg / kg). [Figure 17J] As further described in Example 23, we provide spider plots of mice transplanted with SHP77 SCLC cells and treated with the indicated compounds (Figure 17A, vehicle; Figure 17B, DAR 2 isotype ADC 1 mg / kg; Figure 17C, DAR 4 isotype ADC 1 mg / kg; Figure 17D, ADC-015-B 3 mg / kg; Figure 17E, ADC-015-B 1 mg / kg; Figure 17F, ADC-015-B 0.3 mg / kg × 3; Figure 17G, ADC-015-M 1 mg / kg; Figure 17H, ADC-016-B 3 mg / kg; Figure 17I, ADC-016-B 1 mg / kg; Figure 17J, ADC-016-B 0.3 mg / kg × 3; Figure 17K, ADC-016-M 1 mg / kg). [Figure 17K]As further described in Example 23, we provide spider plots of mice transplanted with SHP77 SCLC cells and treated with the indicated compounds (Figure 17A, vehicle; Figure 17B, DAR 2 isotype ADC 1 mg / kg; Figure 17C, DAR 4 isotype ADC 1 mg / kg; Figure 17D, ADC-015-B 3 mg / kg; Figure 17E, ADC-015-B 1 mg / kg; Figure 17F, ADC-015-B 0.3 mg / kg × 3; Figure 17G, ADC-015-M 1 mg / kg; Figure 17H, ADC-016-B 3 mg / kg; Figure 17I, ADC-016-B 1 mg / kg; Figure 17J, ADC-016-B 0.3 mg / kg × 3; Figure 17K, ADC-016-M 1 mg / kg). [Figure 18A] We provide data obtained from the CXF 742 PDX model, further described in Example 24. Figure 18A provides exemplary IHC stained images of CXF 742 tumor samples. Figure 18B plots the absolute tumor volume of the test groups over the observation period (mean group TV was graphed until two animals were excluded from the group due to the TV endpoint). Figure 18C plots the tumor volume at day 45 (D45) (mixed-effects analysis was performed using Dunnett's post-hoc test compared to the vehicle group). Figures 18D–18G provide spider plots of mice treated with the indicated compounds (Figure 18D, vehicle; Figure 18E, ADC-016-M, 5 mg / kg × 1; Figure 18F, ADC-016-M, 5 mg / kg × 2; Figure 18G, ADC-015-M, 5 mg / kg × 1). Figure 18H plots the relative tumor volume of the test groups over the observation period. Figure 18I plots the body weight of the test group over the observation period. [Figure 18B]We provide data obtained from the CXF 742 PDX model, further described in Example 24. Figure 18A provides exemplary IHC stained images of CXF 742 tumor samples. Figure 18B plots the absolute tumor volume of the test groups over the observation period (mean group TV was graphed until two animals were excluded from the group due to the TV endpoint). Figure 18C plots the tumor volume at day 45 (D45) (mixed-effects analysis was performed using Dunnett's post-hoc test compared to the vehicle group). Figures 18D–18G provide spider plots of mice treated with the indicated compounds (Figure 18D, vehicle; Figure 18E, ADC-016-M, 5 mg / kg × 1; Figure 18F, ADC-016-M, 5 mg / kg × 2; Figure 18G, ADC-015-M, 5 mg / kg × 1). Figure 18H plots the relative tumor volume of the test groups over the observation period. Figure 18I plots the body weight of the test group over the observation period. [Figure 18C] We provide data obtained from the CXF 742 PDX model, further described in Example 24. Figure 18A provides exemplary IHC stained images of CXF 742 tumor samples. Figure 18B plots the absolute tumor volume of the test groups over the observation period (mean group TV was graphed until two animals were excluded from the group due to the TV endpoint). Figure 18C plots the tumor volume at day 45 (D45) (mixed-effects analysis was performed using Dunnett's post-hoc test compared to the vehicle group). Figures 18D–18G provide spider plots of mice treated with the indicated compounds (Figure 18D, vehicle; Figure 18E, ADC-016-M, 5 mg / kg × 1; Figure 18F, ADC-016-M, 5 mg / kg × 2; Figure 18G, ADC-015-M, 5 mg / kg × 1). Figure 18H plots the relative tumor volume of the test groups over the observation period. Figure 18I plots the body weight of the test group over the observation period. [Figure 18D]We provide data obtained from the CXF 742 PDX model, further described in Example 24. Figure 18A provides exemplary IHC stained images of CXF 742 tumor samples. Figure 18B plots the absolute tumor volume of the test groups over the observation period (mean group TV was graphed until two animals were excluded from the group due to the TV endpoint). Figure 18C plots the tumor volume at day 45 (D45) (mixed-effects analysis was performed using Dunnett's post-hoc test compared to the vehicle group). Figures 18D–18G provide spider plots of mice treated with the indicated compounds (Figure 18D, vehicle; Figure 18E, ADC-016-M, 5 mg / kg × 1; Figure 18F, ADC-016-M, 5 mg / kg × 2; Figure 18G, ADC-015-M, 5 mg / kg × 1). Figure 18H plots the relative tumor volume of the test groups over the observation period. Figure 18I plots the body weight of the test group over the observation period. [Figure 18E] We provide data obtained from the CXF 742 PDX model, further described in Example 24. Figure 18A provides exemplary IHC stained images of CXF 742 tumor samples. Figure 18B plots the absolute tumor volume of the test groups over the observation period (mean group TV was graphed until two animals were excluded from the group due to the TV endpoint). Figure 18C plots the tumor volume at day 45 (D45) (mixed-effects analysis was performed using Dunnett's post-hoc test compared to the vehicle group). Figures 18D–18G provide spider plots of mice treated with the indicated compounds (Figure 18D, vehicle; Figure 18E, ADC-016-M, 5 mg / kg × 1; Figure 18F, ADC-016-M, 5 mg / kg × 2; Figure 18G, ADC-015-M, 5 mg / kg × 1). Figure 18H plots the relative tumor volume of the test groups over the observation period. Figure 18I plots the body weight of the test group over the observation period. [Figure 18F]We provide data obtained from the CXF 742 PDX model, further described in Example 24. Figure 18A provides exemplary IHC stained images of CXF 742 tumor samples. Figure 18B plots the absolute tumor volume of the test groups over the observation period (mean group TV was graphed until two animals were excluded from the group due to the TV endpoint). Figure 18C plots the tumor volume at day 45 (D45) (mixed-effects analysis was performed using Dunnett's post-hoc test compared to the vehicle group). Figures 18D–18G provide spider plots of mice treated with the indicated compounds (Figure 18D, vehicle; Figure 18E, ADC-016-M, 5 mg / kg × 1; Figure 18F, ADC-016-M, 5 mg / kg × 2; Figure 18G, ADC-015-M, 5 mg / kg × 1). Figure 18H plots the relative tumor volume of the test groups over the observation period. Figure 18I plots the body weight of the test group over the observation period. [Figure 18G] We provide data obtained from the CXF 742 PDX model, further described in Example 24. Figure 18A provides exemplary IHC stained images of CXF 742 tumor samples. Figure 18B plots the absolute tumor volume of the test groups over the observation period (mean group TV was graphed until two animals were excluded from the group due to the TV endpoint). Figure 18C plots the tumor volume at day 45 (D45) (mixed-effects analysis was performed using Dunnett's post-hoc test compared to the vehicle group). Figures 18D–18G provide spider plots of mice treated with the indicated compounds (Figure 18D, vehicle; Figure 18E, ADC-016-M, 5 mg / kg × 1; Figure 18F, ADC-016-M, 5 mg / kg × 2; Figure 18G, ADC-015-M, 5 mg / kg × 1). Figure 18H plots the relative tumor volume of the test groups over the observation period. Figure 18I plots the body weight of the test group over the observation period. [Figure 18H]We provide data obtained from the CXF 742 PDX model, further described in Example 24. Figure 18A provides exemplary IHC stained images of CXF 742 tumor samples. Figure 18B plots the absolute tumor volume of the test groups over the observation period (mean group TV was graphed until two animals were excluded from the group due to the TV endpoint). Figure 18C plots the tumor volume at day 45 (D45) (mixed-effects analysis was performed using Dunnett's post-hoc test compared to the vehicle group). Figures 18D–18G provide spider plots of mice treated with the indicated compounds (Figure 18D, vehicle; Figure 18E, ADC-016-M, 5 mg / kg × 1; Figure 18F, ADC-016-M, 5 mg / kg × 2; Figure 18G, ADC-015-M, 5 mg / kg × 1). Figure 18H plots the relative tumor volume of the test groups over the observation period. Figure 18I plots the body weight of the test group over the observation period. [Figure 18I] We provide data obtained from the CXF 742 PDX model, further described in Example 24. Figure 18A provides exemplary IHC stained images of CXF 742 tumor samples. Figure 18B plots the absolute tumor volume of the test groups over the observation period (mean group TV was graphed until two animals were excluded from the group due to the TV endpoint). Figure 18C plots the tumor volume at day 45 (D45) (mixed-effects analysis was performed using Dunnett's post-hoc test compared to the vehicle group). Figures 18D–18G provide spider plots of mice treated with the indicated compounds (Figure 18D, vehicle; Figure 18E, ADC-016-M, 5 mg / kg × 1; Figure 18F, ADC-016-M, 5 mg / kg × 2; Figure 18G, ADC-015-M, 5 mg / kg × 1). Figure 18H plots the relative tumor volume of the test groups over the observation period. Figure 18I plots the body weight of the test group over the observation period. [Figure 19A]Data obtained from the LXFS 2156 PDX model are provided, as further described in Example 24. Figure 19A provides exemplary IHC stained images of LXFS 2156 tumor samples. Figure 19B plots the absolute tumor volume of the test groups over the observation period (mean group TV is graphed until two animals are excluded from the group due to the TV endpoint). Figure 19C plots the tumor volume on day 31 (D31) (mixed-effects analysis, Dunnett's post-hoc test compared to the vehicle group). Figures 19D–19G provide spider plots of mice treated with the indicated compounds (Figure 19D, vehicle; Figure 19E, ADC-016-M, 5 mg / kg × 1; Figure 19F, ADC-016-M, 5 mg / kg × 2; Figure 19G, ADC-015-M, 5 mg / kg × 1). Figure 19H plots the relative tumor volume of the test groups over the observation period. Figure 19I plots the body weight of the test group over the observation period. [Figure 19B] Data obtained from the LXFS 2156 PDX model are provided, as further described in Example 24. Figure 19A provides exemplary IHC stained images of LXFS 2156 tumor samples. Figure 19B plots the absolute tumor volume of the test groups over the observation period (mean group TV is graphed until two animals are excluded from the group due to the TV endpoint). Figure 19C plots the tumor volume on day 31 (D31) (mixed-effects analysis, Dunnett's post-hoc test compared to the vehicle group). Figures 19D–19G provide spider plots of mice treated with the indicated compounds (Figure 19D, vehicle; Figure 19E, ADC-016-M, 5 mg / kg × 1; Figure 19F, ADC-016-M, 5 mg / kg × 2; Figure 19G, ADC-015-M, 5 mg / kg × 1). Figure 19H plots the relative tumor volume of the test groups over the observation period. Figure 19I plots the body weight of the test group over the observation period. [Figure 19C]Data obtained from the LXFS 2156 PDX model are provided, as further described in Example 24. Figure 19A provides exemplary IHC stained images of LXFS 2156 tumor samples. Figure 19B plots the absolute tumor volume of the test groups over the observation period (mean group TV is graphed until two animals are excluded from the group due to the TV endpoint). Figure 19C plots the tumor volume on day 31 (D31) (mixed-effects analysis, Dunnett's post-hoc test compared to the vehicle group). Figures 19D–19G provide spider plots of mice treated with the indicated compounds (Figure 19D, vehicle; Figure 19E, ADC-016-M, 5 mg / kg × 1; Figure 19F, ADC-016-M, 5 mg / kg × 2; Figure 19G, ADC-015-M, 5 mg / kg × 1). Figure 19H plots the relative tumor volume of the test groups over the observation period. Figure 19I plots the body weight of the test group over the observation period. [Figure 19D] Data obtained from the LXFS 2156 PDX model are provided, as further described in Example 24. Figure 19A provides exemplary IHC stained images of LXFS 2156 tumor samples. Figure 19B plots the absolute tumor volume of the test groups over the observation period (mean group TV is graphed until two animals are excluded from the group due to the TV endpoint). Figure 19C plots the tumor volume on day 31 (D31) (mixed-effects analysis, Dunnett's post-hoc test compared to the vehicle group). Figures 19D–19G provide spider plots of mice treated with the indicated compounds (Figure 19D, vehicle; Figure 19E, ADC-016-M, 5 mg / kg × 1; Figure 19F, ADC-016-M, 5 mg / kg × 2; Figure 19G, ADC-015-M, 5 mg / kg × 1). Figure 19H plots the relative tumor volume of the test groups over the observation period. Figure 19I plots the body weight of the test group over the observation period. [Figure 19E]Data obtained from the LXFS 2156 PDX model are provided, as further described in Example 24. Figure 19A provides exemplary IHC stained images of LXFS 2156 tumor samples. Figure 19B plots the absolute tumor volume of the test groups over the observation period (mean group TV is graphed until two animals are excluded from the group due to the TV endpoint). Figure 19C plots the tumor volume on day 31 (D31) (mixed-effects analysis, Dunnett's post-hoc test compared to the vehicle group). Figures 19D–19G provide spider plots of mice treated with the indicated compounds (Figure 19D, vehicle; Figure 19E, ADC-016-M, 5 mg / kg × 1; Figure 19F, ADC-016-M, 5 mg / kg × 2; Figure 19G, ADC-015-M, 5 mg / kg × 1). Figure 19H plots the relative tumor volume of the test groups over the observation period. Figure 19I plots the body weight of the test group over the observation period. [Figure 19F] Data obtained from the LXFS 2156 PDX model are provided, as further described in Example 24. Figure 19A provides exemplary IHC stained images of LXFS 2156 tumor samples. Figure 19B plots the absolute tumor volume of the test groups over the observation period (mean group TV is graphed until two animals are excluded from the group due to the TV endpoint). Figure 19C plots the tumor volume on day 31 (D31) (mixed-effects analysis, Dunnett's post-hoc test compared to the vehicle group). Figures 19D–19G provide spider plots of mice treated with the indicated compounds (Figure 19D, vehicle; Figure 19E, ADC-016-M, 5 mg / kg × 1; Figure 19F, ADC-016-M, 5 mg / kg × 2; Figure 19G, ADC-015-M, 5 mg / kg × 1). Figure 19H plots the relative tumor volume of the test groups over the observation period. Figure 19I plots the body weight of the test group over the observation period. [Figure 19G]Data obtained from the LXFS 2156 PDX model are provided, as further described in Example 24. Figure 19A provides exemplary IHC stained images of LXFS 2156 tumor samples. Figure 19B plots the absolute tumor volume of the test groups over the observation period (mean group TV is graphed until two animals are excluded from the group due to the TV endpoint). Figure 19C plots the tumor volume on day 31 (D31) (mixed-effects analysis, Dunnett's post-hoc test compared to the vehicle group). Figures 19D–19G provide spider plots of mice treated with the indicated compounds (Figure 19D, vehicle; Figure 19E, ADC-016-M, 5 mg / kg × 1; Figure 19F, ADC-016-M, 5 mg / kg × 2; Figure 19G, ADC-015-M, 5 mg / kg × 1). Figure 19H plots the relative tumor volume of the test groups over the observation period. Figure 19I plots the body weight of the test group over the observation period. [Figure 19H] Data obtained from the LXFS 2156 PDX model are provided, as further described in Example 24. Figure 19A provides exemplary IHC stained images of LXFS 2156 tumor samples. Figure 19B plots the absolute tumor volume of the test groups over the observation period (mean group TV is graphed until two animals are excluded from the group due to the TV endpoint). Figure 19C plots the tumor volume on day 31 (D31) (mixed-effects analysis, Dunnett's post-hoc test compared to the vehicle group). Figures 19D–19G provide spider plots of mice treated with the indicated compounds (Figure 19D, vehicle; Figure 19E, ADC-016-M, 5 mg / kg × 1; Figure 19F, ADC-016-M, 5 mg / kg × 2; Figure 19G, ADC-015-M, 5 mg / kg × 1). Figure 19H plots the relative tumor volume of the test groups over the observation period. Figure 19I plots the body weight of the test group over the observation period. [Figure 19I]Data obtained from the LXFS 2156 PDX model are provided, as further described in Example 24. Figure 19A provides exemplary IHC stained images of LXFS 2156 tumor samples. Figure 19B plots the absolute tumor volume of the test groups over the observation period (mean group TV is graphed until two animals are excluded from the group due to the TV endpoint). Figure 19C plots the tumor volume on day 31 (D31) (mixed-effects analysis, Dunnett's post-hoc test compared to the vehicle group). Figures 19D–19G provide spider plots of mice treated with the indicated compounds (Figure 19D, vehicle; Figure 19E, ADC-016-M, 5 mg / kg × 1; Figure 19F, ADC-016-M, 5 mg / kg × 2; Figure 19G, ADC-015-M, 5 mg / kg × 1). Figure 19H plots the relative tumor volume of the test groups over the observation period. Figure 19I plots the body weight of the test group over the observation period. [Figure 20A] Data obtained from the LXFS 573 PDX model, further described in Example 24, are provided. Figure 20A provides exemplary IHC stained images of LXFS 573 tumor samples. Figure 20B plots the absolute tumor volume of the test group over the observation period. Figure 20C plots the tumor volume on day 46 (D46) (mixed-effects analysis, Dunnett's post-hoc test compared to the vehicle group). Figures 20D–20G provide spider plots of mice treated with the indicated compounds (Figure 20D, vehicle; Figure 20E, ADC-016-M, 5 mg / kg × 1; Figure 20F, ADC-016-M, 5 mg / kg × 2; Figure 20G, ADC-015-M, 5 mg / kg × 1). Figure 20H plots the relative tumor volume of the test group over the observation period. Figure 20I plots the body weight of the test group over the observation period. [Figure 20B]Data obtained from the LXFS 573 PDX model, further described in Example 24, are provided. Figure 20A provides exemplary IHC stained images of LXFS 573 tumor samples. Figure 20B plots the absolute tumor volume of the test group over the observation period. Figure 20C plots the tumor volume on day 46 (D46) (mixed-effects analysis, Dunnett's post-hoc test compared to the vehicle group). Figures 20D–20G provide spider plots of mice treated with the indicated compounds (Figure 20D, vehicle; Figure 20E, ADC-016-M, 5 mg / kg × 1; Figure 20F, ADC-016-M, 5 mg / kg × 2; Figure 20G, ADC-015-M, 5 mg / kg × 1). Figure 20H plots the relative tumor volume of the test group over the observation period. Figure 20I plots the body weight of the test group over the observation period. [Figure 20C] Data obtained from the LXFS 573 PDX model, further described in Example 24, are provided. Figure 20A provides exemplary IHC stained images of LXFS 573 tumor samples. Figure 20B plots the absolute tumor volume of the test group over the observation period. Figure 20C plots the tumor volume on day 46 (D46) (mixed-effects analysis, Dunnett's post-hoc test compared to the vehicle group). Figures 20D–20G provide spider plots of mice treated with the indicated compounds (Figure 20D, vehicle; Figure 20E, ADC-016-M, 5 mg / kg × 1; Figure 20F, ADC-016-M, 5 mg / kg × 2; Figure 20G, ADC-015-M, 5 mg / kg × 1). Figure 20H plots the relative tumor volume of the test group over the observation period. Figure 20I plots the body weight of the test group over the observation period. [Figure 20D]Data obtained from the LXFS 573 PDX model, further described in Example 24, are provided. Figure 20A provides exemplary IHC stained images of LXFS 573 tumor samples. Figure 20B plots the absolute tumor volume of the test group over the observation period. Figure 20C plots the tumor volume on day 46 (D46) (mixed-effects analysis, Dunnett's post-hoc test compared to the vehicle group). Figures 20D–20G provide spider plots of mice treated with the indicated compounds (Figure 20D, vehicle; Figure 20E, ADC-016-M, 5 mg / kg × 1; Figure 20F, ADC-016-M, 5 mg / kg × 2; Figure 20G, ADC-015-M, 5 mg / kg × 1). Figure 20H plots the relative tumor volume of the test group over the observation period. Figure 20I plots the body weight of the test group over the observation period. [Figure 20E] Data obtained from the LXFS 573 PDX model, further described in Example 24, are provided. Figure 20A provides exemplary IHC stained images of LXFS 573 tumor samples. Figure 20B plots the absolute tumor volume of the test group over the observation period. Figure 20C plots the tumor volume on day 46 (D46) (mixed-effects analysis, Dunnett's post-hoc test compared to the vehicle group). Figures 20D–20G provide spider plots of mice treated with the indicated compounds (Figure 20D, vehicle; Figure 20E, ADC-016-M, 5 mg / kg × 1; Figure 20F, ADC-016-M, 5 mg / kg × 2; Figure 20G, ADC-015-M, 5 mg / kg × 1). Figure 20H plots the relative tumor volume of the test group over the observation period. Figure 20I plots the body weight of the test group over the observation period. [Figure 20F]Data obtained from the LXFS 573 PDX model, further described in Example 24, are provided. Figure 20A provides exemplary IHC stained images of LXFS 573 tumor samples. Figure 20B plots the absolute tumor volume of the test group over the observation period. Figure 20C plots the tumor volume on day 46 (D46) (mixed-effects analysis, Dunnett's post-hoc test compared to the vehicle group). Figures 20D–20G provide spider plots of mice treated with the indicated compounds (Figure 20D, vehicle; Figure 20E, ADC-016-M, 5 mg / kg × 1; Figure 20F, ADC-016-M, 5 mg / kg × 2; Figure 20G, ADC-015-M, 5 mg / kg × 1). Figure 20H plots the relative tumor volume of the test group over the observation period. Figure 20I plots the body weight of the test group over the observation period. [Figure 20G] Data obtained from the LXFS 573 PDX model, further described in Example 24, are provided. Figure 20A provides exemplary IHC stained images of LXFS 573 tumor samples. Figure 20B plots the absolute tumor volume of the test group over the observation period. Figure 20C plots the tumor volume on day 46 (D46) (mixed-effects analysis, Dunnett's post-hoc test compared to the vehicle group). Figures 20D–20G provide spider plots of mice treated with the indicated compounds (Figure 20D, vehicle; Figure 20E, ADC-016-M, 5 mg / kg × 1; Figure 20F, ADC-016-M, 5 mg / kg × 2; Figure 20G, ADC-015-M, 5 mg / kg × 1). Figure 20H plots the relative tumor volume of the test group over the observation period. Figure 20I plots the body weight of the test group over the observation period. [Figure 20H]Data obtained from the LXFS 573 PDX model, further described in Example 24, are provided. Figure 20A provides exemplary IHC stained images of LXFS 573 tumor samples. Figure 20B plots the absolute tumor volume of the test group over the observation period. Figure 20C plots the tumor volume on day 46 (D46) (mixed-effects analysis, Dunnett's post-hoc test compared to the vehicle group). Figures 20D–20G provide spider plots of mice treated with the indicated compounds (Figure 20D, vehicle; Figure 20E, ADC-016-M, 5 mg / kg × 1; Figure 20F, ADC-016-M, 5 mg / kg × 2; Figure 20G, ADC-015-M, 5 mg / kg × 1). Figure 20H plots the relative tumor volume of the test group over the observation period. Figure 20I plots the body weight of the test group over the observation period. [Figure 20I] Data obtained from the LXFS 573 PDX model, further described in Example 24, are provided. Figure 20A provides exemplary IHC stained images of LXFS 573 tumor samples. Figure 20B plots the absolute tumor volume of the test group over the observation period. Figure 20C plots the tumor volume on day 46 (D46) (mixed-effects analysis, Dunnett's post-hoc test compared to the vehicle group). Figures 20D–20G provide spider plots of mice treated with the indicated compounds (Figure 20D, vehicle; Figure 20E, ADC-016-M, 5 mg / kg × 1; Figure 20F, ADC-016-M, 5 mg / kg × 2; Figure 20G, ADC-015-M, 5 mg / kg × 1). Figure 20H plots the relative tumor volume of the test group over the observation period. Figure 20I plots the body weight of the test group over the observation period. [Figure 21A]Data obtained from the CXF 94 PDX model, further described in Example 24, are provided. Figure 21A provides exemplary IHC stained images of CXF 94 tumor samples. Figure 21B plots the absolute tumor volume of the test groups over the observation period (mean group TV is graphed until two animals are excluded from the group due to the TV endpoint). Figure 21C plots the tumor volume on day 31 (D31) (mixed-effects analysis, Dunnett's post-hoc test compared to the vehicle group). Figures 21D–21G provide spider plots of mice treated with the indicated compounds (Figure 21D, vehicle; Figure 21E, ADC-016-M, 5 mg / kg × 1; Figure 21F, ADC-016-M, 5 mg / kg × 2; Figure 21G, ADC-015-M, 5 mg / kg × 1). Figure 21H plots the relative tumor volume of the test groups over the observation period. Figure 21I plots the body weight of the test group over the observation period. [Figure 21B] Data obtained from the CXF 94 PDX model, further described in Example 24, are provided. Figure 21A provides exemplary IHC stained images of CXF 94 tumor samples. Figure 21B plots the absolute tumor volume of the test groups over the observation period (mean group TV is graphed until two animals are excluded from the group due to the TV endpoint). Figure 21C plots the tumor volume on day 31 (D31) (mixed-effects analysis, Dunnett's post-hoc test compared to the vehicle group). Figures 21D–21G provide spider plots of mice treated with the indicated compounds (Figure 21D, vehicle; Figure 21E, ADC-016-M, 5 mg / kg × 1; Figure 21F, ADC-016-M, 5 mg / kg × 2; Figure 21G, ADC-015-M, 5 mg / kg × 1). Figure 21H plots the relative tumor volume of the test groups over the observation period. Figure 21I plots the body weight of the test group over the observation period. [Figure 21C]Data obtained from the CXF 94 PDX model, further described in Example 24, are provided. Figure 21A provides exemplary IHC stained images of CXF 94 tumor samples. Figure 21B plots the absolute tumor volume of the test groups over the observation period (mean group TV is graphed until two animals are excluded from the group due to the TV endpoint). Figure 21C plots the tumor volume on day 31 (D31) (mixed-effects analysis, Dunnett's post-hoc test compared to the vehicle group). Figures 21D–21G provide spider plots of mice treated with the indicated compounds (Figure 21D, vehicle; Figure 21E, ADC-016-M, 5 mg / kg × 1; Figure 21F, ADC-016-M, 5 mg / kg × 2; Figure 21G, ADC-015-M, 5 mg / kg × 1). Figure 21H plots the relative tumor volume of the test groups over the observation period. Figure 21I plots the body weight of the test group over the observation period. [Figure 21D] Data obtained from the CXF 94 PDX model, further described in Example 24, are provided. Figure 21A provides exemplary IHC stained images of CXF 94 tumor samples. Figure 21B plots the absolute tumor volume of the test groups over the observation period (mean group TV is graphed until two animals are excluded from the group due to the TV endpoint). Figure 21C plots the tumor volume on day 31 (D31) (mixed-effects analysis, Dunnett's post-hoc test compared to the vehicle group). Figures 21D–21G provide spider plots of mice treated with the indicated compounds (Figure 21D, vehicle; Figure 21E, ADC-016-M, 5 mg / kg × 1; Figure 21F, ADC-016-M, 5 mg / kg × 2; Figure 21G, ADC-015-M, 5 mg / kg × 1). Figure 21H plots the relative tumor volume of the test groups over the observation period. Figure 21I plots the body weight of the test group over the observation period. [Figure 21E]Data obtained from the CXF 94 PDX model, further described in Example 24, are provided. Figure 21A provides exemplary IHC stained images of CXF 94 tumor samples. Figure 21B plots the absolute tumor volume of the test groups over the observation period (mean group TV is graphed until two animals are excluded from the group due to the TV endpoint). Figure 21C plots the tumor volume on day 31 (D31) (mixed-effects analysis, Dunnett's post-hoc test compared to the vehicle group). Figures 21D–21G provide spider plots of mice treated with the indicated compounds (Figure 21D, vehicle; Figure 21E, ADC-016-M, 5 mg / kg × 1; Figure 21F, ADC-016-M, 5 mg / kg × 2; Figure 21G, ADC-015-M, 5 mg / kg × 1). Figure 21H plots the relative tumor volume of the test groups over the observation period. Figure 21I plots the body weight of the test group over the observation period. [Figure 21F] Data obtained from the CXF 94 PDX model, further described in Example 24, are provided. Figure 21A provides exemplary IHC stained images of CXF 94 tumor samples. Figure 21B plots the absolute tumor volume of the test groups over the observation period (mean group TV is graphed until two animals are excluded from the group due to the TV endpoint). Figure 21C plots the tumor volume on day 31 (D31) (mixed-effects analysis, Dunnett's post-hoc test compared to the vehicle group). Figures 21D–21G provide spider plots of mice treated with the indicated compounds (Figure 21D, vehicle; Figure 21E, ADC-016-M, 5 mg / kg × 1; Figure 21F, ADC-016-M, 5 mg / kg × 2; Figure 21G, ADC-015-M, 5 mg / kg × 1). Figure 21H plots the relative tumor volume of the test groups over the observation period. Figure 21I plots the body weight of the test group over the observation period. [Figure 21G]Data obtained from the CXF 94 PDX model, further described in Example 24, are provided. Figure 21A provides exemplary IHC stained images of CXF 94 tumor samples. Figure 21B plots the absolute tumor volume of the test groups over the observation period (mean group TV is graphed until two animals are excluded from the group due to the TV endpoint). Figure 21C plots the tumor volume on day 31 (D31) (mixed-effects analysis, Dunnett's post-hoc test compared to the vehicle group). Figures 21D–21G provide spider plots of mice treated with the indicated compounds (Figure 21D, vehicle; Figure 21E, ADC-016-M, 5 mg / kg × 1; Figure 21F, ADC-016-M, 5 mg / kg × 2; Figure 21G, ADC-015-M, 5 mg / kg × 1). Figure 21H plots the relative tumor volume of the test groups over the observation period. Figure 21I plots the body weight of the test group over the observation period. [Figure 21H] Data obtained from the CXF 94 PDX model, further described in Example 24, are provided. Figure 21A provides exemplary IHC stained images of CXF 94 tumor samples. Figure 21B plots the absolute tumor volume of the test groups over the observation period (mean group TV is graphed until two animals are excluded from the group due to the TV endpoint). Figure 21C plots the tumor volume on day 31 (D31) (mixed-effects analysis, Dunnett's post-hoc test compared to the vehicle group). Figures 21D–21G provide spider plots of mice treated with the indicated compounds (Figure 21D, vehicle; Figure 21E, ADC-016-M, 5 mg / kg × 1; Figure 21F, ADC-016-M, 5 mg / kg × 2; Figure 21G, ADC-015-M, 5 mg / kg × 1). Figure 21H plots the relative tumor volume of the test groups over the observation period. Figure 21I plots the body weight of the test group over the observation period. [Figure 21I]Data obtained from the CXF 94 PDX model, further described in Example 24, are provided. Figure 21A provides exemplary IHC stained images of CXF 94 tumor samples. Figure 21B plots the absolute tumor volume of the test groups over the observation period (mean group TV is graphed until two animals are excluded from the group due to the TV endpoint). Figure 21C plots the tumor volume on day 31 (D31) (mixed-effects analysis, Dunnett's post-hoc test compared to the vehicle group). Figures 21D–21G provide spider plots of mice treated with the indicated compounds (Figure 21D, vehicle; Figure 21E, ADC-016-M, 5 mg / kg × 1; Figure 21F, ADC-016-M, 5 mg / kg × 2; Figure 21G, ADC-015-M, 5 mg / kg × 1). Figure 21H plots the relative tumor volume of the test groups over the observation period. Figure 21I plots the body weight of the test group over the observation period. [Figure 22A] We provide data obtained from the LXFS 538 PDX model, further described in Example 24. Figure 22A provides exemplary IHC stained images of LXFS 538 tumor samples. Figure 22B plots test versus tumor volume over the observation period (mean group TV is graphed until two animals are excluded from the group due to the TV endpoint). Figure 22C plots tumor volume at day 35 (D35), while Figure 22D plots tumor volume at day 42 (D42) (mixed-effects analysis, Dunnett's post-hoc test compared to the vehicle group). Figures 22E–22H provide spider plots of mice treated with the indicated compounds (Figure 22E, vehicle; Figure 22F, ADC-016-M, 5 mg / kg × 1; Figure 22G, ADC-016-M, 5 mg / kg × 2; Figure 22H, ADC-015-M, 5 mg / kg × 1). Figure 22I plots the relative tumor volume of the test group over the observation period. Figure 22J plots the body weight of the test group over the observation period. [Figure 22B]We provide data obtained from the LXFS 538 PDX model, further described in Example 24. Figure 22A provides exemplary IHC stained images of LXFS 538 tumor samples. Figure 22B plots test versus tumor volume over the observation period (mean group TV is graphed until two animals are excluded from the group due to the TV endpoint). Figure 22C plots tumor volume at day 35 (D35), while Figure 22D plots tumor volume at day 42 (D42) (mixed-effects analysis, Dunnett's post-hoc test compared to the vehicle group). Figures 22E–22H provide spider plots of mice treated with the indicated compounds (Figure 22E, vehicle; Figure 22F, ADC-016-M, 5 mg / kg × 1; Figure 22G, ADC-016-M, 5 mg / kg × 2; Figure 22H, ADC-015-M, 5 mg / kg × 1). Figure 22I plots the relative tumor volume of the test group over the observation period. Figure 22J plots the body weight of the test group over the observation period. [Figure 22C] We provide data obtained from the LXFS 538 PDX model, further described in Example 24. Figure 22A provides exemplary IHC stained images of LXFS 538 tumor samples. Figure 22B plots test versus tumor volume over the observation period (mean group TV is graphed until two animals are excluded from the group due to the TV endpoint). Figure 22C plots tumor volume at day 35 (D35), while Figure 22D plots tumor volume at day 42 (D42) (mixed-effects analysis, Dunnett's post-hoc test compared to the vehicle group). Figures 22E–22H provide spider plots of mice treated with the indicated compounds (Figure 22E, vehicle; Figure 22F, ADC-016-M, 5 mg / kg × 1; Figure 22G, ADC-016-M, 5 mg / kg × 2; Figure 22H, ADC-015-M, 5 mg / kg × 1). Figure 22I plots the relative tumor volume of the test group over the observation period. Figure 22J plots the body weight of the test group over the observation period. [Figure 22D]We provide data obtained from the LXFS 538 PDX model, further described in Example 24. Figure 22A provides exemplary IHC stained images of LXFS 538 tumor samples. Figure 22B plots test versus tumor volume over the observation period (mean group TV is graphed until two animals are excluded from the group due to the TV endpoint). Figure 22C plots tumor volume at day 35 (D35), while Figure 22D plots tumor volume at day 42 (D42) (mixed-effects analysis, Dunnett's post-hoc test compared to the vehicle group). Figures 22E–22H provide spider plots of mice treated with the indicated compounds (Figure 22E, vehicle; Figure 22F, ADC-016-M, 5 mg / kg × 1; Figure 22G, ADC-016-M, 5 mg / kg × 2; Figure 22H, ADC-015-M, 5 mg / kg × 1). Figure 22I plots the relative tumor volume of the test group over the observation period. Figure 22J plots the body weight of the test group over the observation period. [Figure 22E] We provide data obtained from the LXFS 538 PDX model, further described in Example 24. Figure 22A provides exemplary IHC stained images of LXFS 538 tumor samples. Figure 22B plots test versus tumor volume over the observation period (mean group TV is graphed until two animals are excluded from the group due to the TV endpoint). Figure 22C plots tumor volume at day 35 (D35), while Figure 22D plots tumor volume at day 42 (D42) (mixed-effects analysis, Dunnett's post-hoc test compared to the vehicle group). Figures 22E–22H provide spider plots of mice treated with the indicated compounds (Figure 22E, vehicle; Figure 22F, ADC-016-M, 5 mg / kg × 1; Figure 22G, ADC-016-M, 5 mg / kg × 2; Figure 22H, ADC-015-M, 5 mg / kg × 1). Figure 22I plots the relative tumor volume of the test group over the observation period. Figure 22J plots the body weight of the test group over the observation period. [Figure 22F]We provide data obtained from the LXFS 538 PDX model, further described in Example 24. Figure 22A provides exemplary IHC stained images of LXFS 538 tumor samples. Figure 22B plots test versus tumor volume over the observation period (mean group TV is graphed until two animals are excluded from the group due to the TV endpoint). Figure 22C plots tumor volume at day 35 (D35), while Figure 22D plots tumor volume at day 42 (D42) (mixed-effects analysis, Dunnett's post-hoc test compared to the vehicle group). Figures 22E–22H provide spider plots of mice treated with the indicated compounds (Figure 22E, vehicle; Figure 22F, ADC-016-M, 5 mg / kg × 1; Figure 22G, ADC-016-M, 5 mg / kg × 2; Figure 22H, ADC-015-M, 5 mg / kg × 1). Figure 22I plots the relative tumor volume of the test group over the observation period. Figure 22J plots the body weight of the test group over the observation period. [Figure 22G] We provide data obtained from the LXFS 538 PDX model, further described in Example 24. Figure 22A provides exemplary IHC stained images of LXFS 538 tumor samples. Figure 22B plots test versus tumor volume over the observation period (mean group TV is graphed until two animals are excluded from the group due to the TV endpoint). Figure 22C plots tumor volume at day 35 (D35), while Figure 22D plots tumor volume at day 42 (D42) (mixed-effects analysis, Dunnett's post-hoc test compared to the vehicle group). Figures 22E–22H provide spider plots of mice treated with the indicated compounds (Figure 22E, vehicle; Figure 22F, ADC-016-M, 5 mg / kg × 1; Figure 22G, ADC-016-M, 5 mg / kg × 2; Figure 22H, ADC-015-M, 5 mg / kg × 1). Figure 22I plots the relative tumor volume of the test group over the observation period. Figure 22J plots the body weight of the test group over the observation period. [Figure 22H]We provide data obtained from the LXFS 538 PDX model, further described in Example 24. Figure 22A provides exemplary IHC stained images of LXFS 538 tumor samples. Figure 22B plots test versus tumor volume over the observation period (mean group TV is graphed until two animals are excluded from the group due to the TV endpoint). Figure 22C plots tumor volume at day 35 (D35), while Figure 22D plots tumor volume at day 42 (D42) (mixed-effects analysis, Dunnett's post-hoc test compared to the vehicle group). Figures 22E–22H provide spider plots of mice treated with the indicated compounds (Figure 22E, vehicle; Figure 22F, ADC-016-M, 5 mg / kg × 1; Figure 22G, ADC-016-M, 5 mg / kg × 2; Figure 22H, ADC-015-M, 5 mg / kg × 1). Figure 22I plots the relative tumor volume of the test group over the observation period. Figure 22J plots the body weight of the test group over the observation period. [Figure 22I] We provide data obtained from the LXFS 538 PDX model, further described in Example 24. Figure 22A provides exemplary IHC stained images of LXFS 538 tumor samples. Figure 22B plots test versus tumor volume over the observation period (mean group TV is graphed until two animals are excluded from the group due to the TV endpoint). Figure 22C plots tumor volume at day 35 (D35), while Figure 22D plots tumor volume at day 42 (D42) (mixed-effects analysis, Dunnett's post-hoc test compared to the vehicle group). Figures 22E–22H provide spider plots of mice treated with the indicated compounds (Figure 22E, vehicle; Figure 22F, ADC-016-M, 5 mg / kg × 1; Figure 22G, ADC-016-M, 5 mg / kg × 2; Figure 22H, ADC-015-M, 5 mg / kg × 1). Figure 22I plots the relative tumor volume of the test group over the observation period. Figure 22J plots the body weight of the test group over the observation period. [Figure 22J]We provide data obtained from the LXFS 538 PDX model, further described in Example 24. Figure 22A provides exemplary IHC stained images of LXFS 538 tumor samples. Figure 22B plots test versus tumor volume over the observation period (mean group TV is graphed until two animals are excluded from the group due to the TV endpoint). Figure 22C plots tumor volume at day 35 (D35), while Figure 22D plots tumor volume at day 42 (D42) (mixed-effects analysis, Dunnett's post-hoc test compared to the vehicle group). Figures 22E–22H provide spider plots of mice treated with the indicated compounds (Figure 22E, vehicle; Figure 22F, ADC-016-M, 5 mg / kg × 1; Figure 22G, ADC-016-M, 5 mg / kg × 2; Figure 22H, ADC-015-M, 5 mg / kg × 1). Figure 22I plots the relative tumor volume of the test group over the observation period. Figure 22J plots the body weight of the test group over the observation period. [Modes for carrying out the invention]

[0018] This disclosure provides antibody-drug conjugates (ADCs) comprising an antibody conjugated to DLL3 and a drug conjugated thereto (directly or indirectly). Such DLL3-ADCs are useful in compositions and methods for treating, preventing, or mitigating DLL3-mediated diseases, disorders, or conditions, including one or more symptoms of a disease, disorder, or condition. Examples of DLL3-mediated diseases, disorders, and conditions include cancers such as lung cancer (e.g., small cell lung cancer (SCLC)), large cell neuroendocrine carcinoma (LCNEC), or colorectal cancer. The DLL3-ADCs described herein comprise a DLL3 antibody conjugated to one or more linker-drug conjugates.

[0019] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art to which the present invention pertains. Methods and materials similar to or equivalent to those described herein may also be used in carrying out or testing the present invention, but preferred methods and materials are described herein. All publications referenced herein are incorporated herein by reference to disclose and describe the methods and / or materials cited in those publications.

[0020] The terms "approximately" and "about" mean a variation of 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less of a given value or range.

[0021] When used herein, comparative terms used herein, such as reduction, decrease, increase, or any grammatical variation thereof, may refer to a particular variation from a reference. In some embodiments, such variation may refer to about 10%, or about 20%, or about 30%, or about 40%, or about 50%, or about 60%, or about 70%, or about 80%, or about 90%, or about 1x, or about 2x, or about 3x, or about 4x, or about 5x, or about 10x, or about 20x, or about 30x, or about 40x, or about 100x or more. In some embodiments, such variation may refer to about 1%, or about 2%, or about 3%, or about 4%, or about 5%, or about 6%, or about 7%, or about 8%, or about 9%, or about 10%, or about 20%, or about 30%, or about 40%, or about 50%, or about 60%, or about 70%, or about 80%, or about 90%, or about 95%, or about 96%, or about 97%, or about 98%, or about 99% of the reference.

[0022] As used in this disclosure and claims, the singular forms "a," "an," and "the" include plural forms unless the context clearly indicates otherwise.

[0023] In some embodiments, the terms "first," "second," "third," "fourth," and similar terms in component names are used to distinguish and identify two or more components that share a particular identity in their names. For example, "first antibody" and "second antibody" are used to distinguish two antibodies.

[0024] Where an embodiment is described herein using the term “including,” it should always be understood that it is also provided in other similar embodiments described in terms of “consisting of” and / or “essentially consisting of.” Similarly, where an embodiment is described herein using the phrase “essentially consisting of,” it should always be understood that other similar embodiments described in terms of “consisting of” are also provided.

[0025] The term "between" or "between A and B" refers to a range that includes both A and B.

[0026] As used herein, the term "and / or" in the phrase "A and / or B" is intended to include both A and B, A or B, A (alone), and B (alone). Similarly, the term "and / or" as used in phrases such as "A, B, and / or C" is intended to include each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone), B (alone), and C (alone).

[0027] The terms "optional" or "optional" mean that the situation described thereafter may or may not occur, and therefore the description includes both the cases in which the situation occurs and the cases in which it does not occur.

[0028] DLL3 antibody For a deeper understanding of this disclosure, the definitions and explanations of relevant terms are provided below.

[0029] The terms “Delta-like canonical Notch ligand 3,” “DLL3,” “SCDO1,” “Drosophila Delta homolog 3,” “Delta-like protein 3,” “Delta3,” “D3,” “Delta (Drosophila)-like 3,” “Delta-like 3 (Drosophila),” “Delta-like 3,” or similar terms refer to any native DLL3 derived from any vertebrate source, including polypeptides ("polypeptide" and "protein" are used interchangeably herein) or mammals, including primates (e.g., humans, cynomolgus macaques (cyno)), dogs, and rodents (e.g., mice and rats), unless otherwise indicated. The term DLL3 encompasses “full-length” DLL3, as well as any form of DLL3 or any fragment thereof resulting from processing in cells. The term DLL3 also encompasses naturally occurring variants of DLL3, such as SNP variants, splice variants, and allele variants. Other related DLL3 polypeptides similarly encompassed by the term DLL3 include fragments, derivatives (e.g., substitution, deletion, cleavage, and insertion variants), fusion polypeptides, and interspecies homologs that retain DLL3 activity. Orthologs to DLL3 polypeptides are also well known in the art. Exemplary DLL3 sequences and additional information can be found in GeneCards:GC19P039498, HGNC:2909, NCBI Entrez Gene:10683, Ensembl:ENSG00000090932, OMIM®:602768, and UniProtKB / Swiss-Prot:Q9NYJ7 (each of which is incorporated herein by reference in its entirety). In further embodiments, an exemplary human DLL3 sequence is provided herein as SEQ ID NO: 11, an exemplary cynomolgus monkey DLL3 sequence is provided herein as SEQ ID NO: 12, and an exemplary mouse DLL3 sequence is provided herein as SEQ ID NO: 13.

[0030] The term "antibody" (e.g., DLL3 antibody) is used in its broadest sense and encompasses all forms of antibodies that exhibit desired biological or binding activity. This term includes, but is not limited to, humanized antibodies, fully human antibodies, chimeric antibodies, and single-domain antibodies (usually sdAbs containing only one chain similar to a heavy chain such as VHH), as well as any fragment of the above, insofar as they exhibit desired antigen-binding activity, including, for example, antibodies containing at least one VHH domain. Conventional antibodies consist of a heavy chain and a light chain. Heavy chains can be classified into μ, δ, γ, α, and ε, which define the antibody isotype as IgM, IgD, IgG, IgA, and IgE, respectively. A heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). A heavy chain may contain one or more constant regions, for example, three constant regions (CH1, CH2, and CH3). A light chain consists of a light chain variable region (VL) and a light chain constant region (CL). The VH and VL regions can be further divided into hypervariable regions (called complementarity-determining regions (CDRs)) interspersed with relatively conserved regions (called framework regions (FRWs)). The VH and VL can contain three CDRs (complementarity-determining regions) and four FRs (framework regions) in the following order: from the N-terminus to the C-terminus, FRW1, CDR1, FRW2, CDR2, FRW3, CDR3, FRW4. Different antibody isotypes may also be used, e.g., IgG (e.g., IgG1, IgG2, IgG3, or IgG4 subtypes), IgA1, IgA2, IgD, IgE, or IgM antibodies.

[0031] A universal numbering system for hypervariable regions, the ImMunoGeneTics (IMGT®) Information System (Lefranc et al., Dev.Comp.Immunol.27(1):55-77(2003)), has been developed and is widely adopted. IMGT® is an integrated information system specifically for human and other vertebrate immunoglobulins (IG), T cell receptors (TR), and major histocompatibility complexes (MHC). In this specification, CDRs are referred to in terms of both amino acid sequence and their position within the light or heavy chain. The “position” of CDRs within the structure of immunoglobulin variable regions is conserved across species and resides within structures called loops; therefore, by using a numbering system that aligns the variable region sequences of structural features, CDRs and framework residues can be easily identified. This information can be used when transplanting and substituting CDR residues from certain immunoglobulins into acceptor frameworks, typically derived from human antibodies. An additional numbering system (AHon) has been developed by Honegger and Pluckthun, J.Mol.Biol.309:657-670 (2001). Correspondence between numbering systems, including, for example, the Kabat numbering and the IMGT® proprietary numbering system, is well known to those skilled in the art (e.g., Kabat (see above), Chothia and Lesk (see above), Martin (see above), Lefranc et al. (see above)), and is also shown below. The various systems known in the art or described herein represent various ways of describing CDRs, and when used to define the same antibody, they are often considered equivalent. The exemplary system shown herein combines Kabat and Chothia. [Table A]

[0032] In this specification, the term “Fc region” is used to define the C-terminal region of an immunoglobulin heavy chain, including, for example, the native sequence Fc region, the recombinant Fc region, and the variant Fc region. The boundary of the Fc region of an immunoglobulin heavy chain may differ, but the human IgG heavy chain Fc region is often defined as extending from the amino acid residue at position Cys226 (according to the EU numbering system) or Pro230 (according to the EU numbering system) to its carboxyl terminus. The C-terminal lysine of the Fc region (residue 447 according to the EU numbering system) can be removed, for example, during antibody production or purification, or by recombinant operation of the nucleic acid encoding the antibody heavy chain.

[0033] A "functional Fc region" possesses "effector function" of a native sequence Fc region. Exemplary "effector functions" include C1q binding, complement-dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), phagocytosis, and downregulation of cell surface receptors (e.g., B cell receptors, BCRs). Such effector functions generally require the Fc region to be combined with a binding region or binding domain (e.g., an antibody variable region or domain containing a VHH domain) and can be evaluated using various assays as disclosed.

[0034] "Natural sequence Fc regions" include amino acid sequences that are identical to the amino acid sequences of Fc regions found in nature and have not undergone any artificial manipulation, modification, and / or alteration (e.g., isolation, purification, selection, inclusion of or combination with other sequences such as variable region sequences). Natural sequence human Fc regions include the Fc regions of natural sequence human IgG1 (non-A and A allotypes), natural sequence human IgG2, natural sequence human IgG3, and natural sequence human IgG4, as well as their natural variants.

[0035] A "variant Fc region" includes an amino acid sequence different from that of the natural sequence Fc region by at least one amino acid modification (e.g., substitution, addition, or deletion), preferably one or more amino acid substitutions. In some embodiments, the variant Fc region has at least one amino acid substitution compared to the natural sequence Fc region or the Fc region of the parent polypeptide, for example, about 1 to about 10 amino acid substitutions, preferably about 1 to about 5 amino acid substitutions, in the natural sequence Fc region or the Fc region of the parent polypeptide. The variant Fc region may have at least about 80% homology, or at least about 90% homology, for example, at least about 95% homology, with the natural sequence Fc region and / or the Fc region of the parent polypeptide. The variant Fc regions described herein may have loss of effector function (e.g., silent Fc).

[0036] The DLL3 antibodies described herein include, but are not limited to, synthetic antibodies, monoclonal antibodies, recombinant antibodies, multispecific antibodies (e.g., including bispecific antibodies), human antibodies, humanized antibodies, chimeric antibodies, intrabodies, single-chain Fv(scFv) (e.g., including monospecific, bispecific, etc.), camelized antibodies, Fab fragments, F(ab') fragments, disulfide-linked Fv(sdFv), anti-idiotype (anti-Id) antibodies, and any of the above epitope-linked fragments.

[0037] The terms “immunoglobulin monovariate domain,” “monovariate domain,” “VHH domain,” “VHH,” or “heavy chain-only antibody monovariate domain” can be used interchangeably herein and refer to a monovariate antigen-binding domain that can bind to an antigen or epitope independently of different monovariate domains. A VHH domain (e.g., a monovariate domain of a heavy chain antibody) represents the smallest known antigen-binding unit produced by an adaptive immune response (Koch-Nolte F. et al., FASEB J. Nov; 21(13): 3490-8. Epub 2007 Jun 15 (2007)). A VHH domain may be a human domain, but also includes single domains derived from other species, such as rodents, nurse sharks, and camelid VHH domains. Camelid VHHs are monovariate domain polypeptides of immunoglobulins derived from species such as camels, llamas, alpacas, dromedaries, and guanacos that naturally produce heavy chain antibodies lacking a light chain. Such VHH domains may be humanized according to standard techniques available in the art and are considered “single-domain antibodies.” As used herein, VHH includes camelid VHH domains and humanized VHH domains.

[0038] The term "humanized antibody" is intended to refer to an antibody in which a CDR sequence derived from the germline of another mammalian species, such as mouse, llama, or alpaca, is grafted onto a human framework sequence. Further modifications of the framework region may be performed within the human framework sequence.

[0039] As used herein, the term "Ka" is intended to refer to the binding rate of a particular antibody-antigen interaction, and as used herein, the term "Kd" is intended to refer to the dissociation rate of a particular antibody-antigen interaction. The Kd value of an antibody can be determined using methods well established in the art. D The term "Kd" refers to the dissociation constant of a specific antibody-antigen interaction, derived from the ratio of Kd to Ka (e.g., Kd / Ka), and expressed as molar concentration (M).D A preferred method for determining this is by using surface plasmon resonance, preferably using a biosensor system such as the Biacore® system.

[0040] As used herein, the terms “specific binding” or “specifically binding” refer to a non-random binding reaction between two molecules, such as between an antibody and an antigen.

[0041] As used herein, the term "high affinity" means 1 × 10⁶ against the target antigen. -7 M or less, more convenient 5×10 -8 M or less, more preferably 1 × 10 -8 M or less, more preferably 5 × 10 -9 M or less, more preferably 1 × 10 -9 K below M D This refers to a DLL3 antibody that possesses [specific characteristic].

[0042] As used herein, the term “epitope” refers to the portion of an antigen to which an immunoglobulin or antibody specifically binds. “Epitope” is also known as an “antigenic determinant.” Epitopes, or antigenic determinants, generally consist of chemically active surface groups of molecules such as amino acids, carbohydrates, or sugar side chains, and generally have a specific three-dimensional structure and specific charge properties. For example, an epitope generally contains at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 consecutive or discontinuous amino acids in a specific stereoconformation that may be “linear” or “conformal.” See, for example, Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, GEMorris, Ed. (1996). In linear epitopes, all interaction sites between a protein and an interacting molecule (e.g., an antibody) are linearly aligned along the primary amino acid sequence of the protein. In three-dimensional epitopes, interaction sites are located across amino acid residues that are geographically separated within the protein. Antibodies can be screened according to their competitiveness in binding to the same epitope by conventional techniques well known to those skilled in the art. For example, experiments on competition or cross-competition can be performed to obtain antibodies that compete or cross-compete with each other for binding to an antigen. A high-throughput method based on their cross-competition for obtaining antibodies that bind to the same epitope is described in international patent application WO03 / 48731.

[0043] As used herein, “isolated antibody” is intended to refer to an antibody that substantially does not contain other antibodies with different antigen specificities (for example, an isolated antibody that specifically binds to the DLL3 protein substantially does not contain antibodies that specifically bind to antigens other than the DLL3 protein). However, an isolated antibody that specifically binds to the human DLL3 protein may cross-react to other antigens, such as DLL3 proteins from other species. Furthermore, an isolated antibody may not substantially contain other cellular material and / or chemical substances.

[0044] Examples of DLL3 antibodies for use in the DLL3-ADCs described herein include, but are not limited to, chimeric antibodies, humanized antibodies, human antibodies, and single-domain antibodies. In some embodiments, the DLL3-ADCs disclosed herein include a DLL3 antibody containing at least one VHH that specifically binds to DLL3. Furthermore, the DLL3 antibody may be a single-domain antibody containing one VHH. For example, a single-domain antibody can selectively bind to a specific antigen (e.g., DLL3). In some embodiments, the DLL3-ADCs disclosed herein include a DLL3 antibody containing a VHH fused to an immunoglobulin Fc region, for example, the Fc region of IgG (e.g., IgG4 or IgG1). In some embodiments, the Fc region is the Fc region of human IgG1. By fusing the VHH to the Fc region, it may be possible to more efficiently mobilize effector function. Also, the fusion of the VHH to the Fc region may help the DLL3 antibody to form dimers, which may also help extend the half-life of the DLL3-ADC in vivo. In some embodiments, the DLL3 antibody for use in the DLL3-ADC disclosed herein comprises two VHH and immunoglobulin Fc regions, each of which is directly or indirectly conjugated to the Fc chain. See, for example, Figure 1A. In some embodiments, the VHH is directly or indirectly conjugated to the N-terminus of the Fc.

[0045] In some embodiments, the DLL3 antibody of the DLL3-ADC disclosed herein is a VHH-Fc fusion protein. As used herein, a VHH-Fc fusion refers to one or more VHHs directly or indirectly conjugated to Fc. In further embodiments, the VHHs are directly or indirectly conjugated to the N-terminus of Fc. In some embodiments, the VHH-Fc fusion comprises two VHHs and an immunoglobulin Fc region, each of which is directly or indirectly conjugated to the Fc chain. In further embodiments, each of which is directly or indirectly conjugated to the N-terminus of the Fc chain. See, for example, Figure 1A. Additionally or alternatively, the VHH-Fc fusion comprises two chains, each of which comprises, from N-terminus to C-terminus, a VHH, a hinge region, CH2, and CH3. In further embodiments, one or more linkers may be present between any two of the VHH, hinge region, CH2, and CH3. In some embodiments, the two strands of the VHH-Fc fusion are identical to each other. In other embodiments, the two strands of the VHH-Fc fusion are different to each other. For example, the first strand of the VHH-Fc fusion contains a first VHH that is different from the VHH of the second strand. Additionally or alternatively, the CH3 domains of the two strands are modified to facilitate the production of the VHH-Fc fusion, for example, by introducing a knob-in-hole mutation. As will be understood by those skilled in the art, in some embodiments, the strands of the VHH-Fc fusion may not contain CH1 between the VHH and the hinge region and are therefore different from the conventionally defined heavy chain of an antibody. Nevertheless, when describing DLL3-ADC or embodiments thereof, and where appropriate, the term “heavy chain” as used herein may be interpreted as referring to the strands of the VHH-Fc fusion protein.

[0046] As is well known in the art, VHH molecules derived from camelid antibodies are among the smallest intact antigen-binding domains known (approximately 15 kDa, i.e., 10 times smaller than conventional IgG), and are therefore well-suited for delivery to dense tissues and access to limited spaces between macromolecules.

[0047] The VHHs disclosed herein can be produced by those skilled in the art according to methods well known in the art or any future methods. For example, VHHs can be obtained, for example, by immunizing camels and thereby obtaining hybridomas, or by cloning a library of VHHs using molecular biology techniques well known in the art and then selecting them using phage display.

[0048] For example, VHH can be obtained by immunizing llamas or alpacas with a desired antigen and subsequently isolating mRNA encoding single-domain heavy chain antibodies. Reverse transcription and polymerase chain reaction produce a gene library of single-domain antibodies containing millions of clones. Screening techniques such as phage display and ribosome display are useful in identifying clones that bind to the antigen. One such technique is phage display, where a library of (e.g., human) antibodies is synthesized on phages, and the library is screened using the antigen of interest or its antibody-binding portion to isolate phages that bind to the antigen and obtain immunoreactive fragments therefrom. Methods for preparing and screening such libraries are well known in the art, and kits for generating phage display libraries are commercially available (e.g., Pharmacia Recombinant Phage Antibody System, catalog no. 27-9400-01, and Stratagene SurfZAP® Phage Display Kit, catalog no. 240612). Other methods and reagents are available for the preparation and screening of antibody display libraries (see, for example, Barbas et al., Proc. Natl. Acad. Sci. USA 88:7978-7982 (1991)).

[0049] If promising clones are identified, their sequences are optimized, for example, through affinity maturation or humanization. Humanization can prevent the human immunological response to the antibody.

[0050] Therefore, VHH can be obtained (1) by isolating the VHH domain from naturally occurring heavy chain antibodies, (2) by expressing nucleotide sequences encoding naturally occurring VHH domains, (3) by "humanizing" naturally occurring VHH domains (as described below) or by expressing nucleic acids encoding such humanized VHH domains, (4) by "camelizing" naturally occurring VH domains from any animal species, especially mammalian species such as humans, or by expressing nucleic acids encoding such camelized VH domains, (5) Ward et al. VHH may be obtained by (6) using synthetic or semi-synthetic techniques for preparing proteins, polypeptides, or other amino acid sequences, (7) using techniques for nucleic acid synthesis to prepare nucleic acids encoding VHH, and subsequently expressing the nucleic acids thus obtained, (8) subjecting the heavy chain antibody or VHH to affinity maturation, mutagenesis (e.g., random mutagenesis or site-directed mutagenesis), and / or any other techniques to enhance the affinity and / or specificity of the VHH, and / or (9) any combination thereof. Suitable methods and techniques for doing the above will be apparent to those skilled in the art based on the disclosure herein, and include, for example, the methods and techniques described in more detail herein.

[0051] Single-domain antibodies are typically produced by PCR cloning a repertoire of variable domains from cDNA of blood, lymph nodes, or spleen obtained from immunized animals into a phage display vector. Antigen-specific single-domain antibodies are generally selected by panning a phage library on immobilized antigens (e.g., antigens coated on the plastic surface of a test tube, biotinylated antigens immobilized on streptavidin beads, or membrane proteins expressed on the cell surface). The affinity of sdAbs can be enhanced by mimicking this strategy in vitro, for example, by site-directed mutagenesis of the CDR region and by performing rounds of further panning of the immobilized antigen under stringency-enhanced conditions (higher temperature, high or low salt concentration, high or low pH, and low antigen concentration) (Wesolowski et al., Single domain antibodies: promising experimental and therapeutic tools in infection and immunity. Med Microbiol Immunol (2009) 198:157-174).

[0052] Methods for preparing VHH that specifically bind to antigens or epitopes are described in the references, for example, R. van der Linden et al., Journal of Immunological Methods, 240(2000)185-195; Li et al., J Biol Chem., 287(2012)13713-13721; Deffer et al., African Journal of Biotechnology Vol.8(12), pp.2645, 17 June, 2009; and WO94 / 04678.

[0053] In some embodiments, VHH may be cleaved at its N-terminus or C-terminus to include only a portion of FRW1 and / or FRW4, or to lack one or both of these framework regions, as long as VHH substantially maintains its antigen-binding and specificity (e.g., substantially maintained, e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%).

[0054] The DLL3 antibodies of DLL3-ADC described herein may be monospecific, bispecific, tripspecific, or higher-order multispecific. Such substances may include antibodies. Multispecific antibodies, such as bispecific antibodies, are monoclonal antibodies that have binding specificity to at least two different targets (e.g., antigens) or two different epitopes on the same target (e.g., a bispecific antibody for DLL3 having a first binding domain for a first epitope of DLL3 and a second binding domain for a second epitope of DLL3). In some embodiments, multispecific (e.g., bispecific) antibodies can be constructed based on the sequence of antibodies described herein. In some embodiments, the multispecific antibodies described herein are bispecific antibodies. In some embodiments, the bispecific antibodies are mouse, chimeric, human antibodies, or humanized antibodies. In some embodiments, one of the binding specificities of the bispecific antibody is for DLL3 and the other is for any other target (e.g., antigen). In some embodiments, a multispecific (e.g., bispecific) antibody may contain two or more target (e.g., antigen) binding domains, where different binding domains are specific to different targets (e.g., a first binding domain that binds to DLL3 and a second binding domain that binds to another target (e.g., antigen), such as an immune checkpoint regulator (e.g., a negative checkpoint regulator)). In some embodiments, a multispecific (e.g., bispecific) antibody molecule may bind to multiple (e.g., two or more) epitopes on the same target (e.g., antigen).In some embodiments, one binding specificity is DLL3, and the other is cytotoxic T-lymphocyte antigen-4 (CTLA-4), CD80, CD86, programmed cell death 1 (PD-1), programmed cell death ligand 1 (PD-L1), programmed cell death ligand 2 (PD-L2), lymphocyte activation gene-3 (LAG-3, also known as CD223), galectin-3, B and T lymphocyte attenuator (BTLA), T-cell membrane protein 3 (TIM3), galectin-9 (GAL9), B7-H1, B7-H3, B7-H4, Ig, and This targets one or more of the following: T-cell immune receptors having an ITIM domain (TIGIT / Vstm3 / WUCAM / VSIG9), V-domain Ig suppressor for T-cell activation (VISTA), glucocorticoid-induced tumor necrosis factor receptor-related (GITR) protein, herpesvirus entry mediator (HVEM), OX40, CD27, CD28, CD137, CGEN-15001T, CGEN-15022, CGEN-15027, CGEN-15049, CGEN-15052, and CGEN-15092.

[0055] Methods for producing multispecific antibodies are well known in the art, for example, by the simultaneous expression of two immunoglobulin heavy / light chain pairs having two heavy chains with different specificities (see, e.g., Milstein and Cuello, 1983, Nature 305:537-40). For further details on the production of multispecific antibodies (e.g., bispecific antibodies), see, for example, Bispecific Antibodies (Kontermann ed., 2011).

[0056] This disclosure provides a humanized antibody that binds to DLL3. Various methods for humanizing non-human antibodies are known in the art. For example, a humanized antibody may have one or more amino acid residues introduced from a non-human source. These non-human amino acid residues are often called “import” residues and usually originate from an “import” variable domain. Humanized antibodies that bind to DLL3 can be produced using techniques known to those skilled in the art (e.g., Zhang et al., Molecular Immunology, 42(12):1445-1451, 2005; Hwang et al., Methods, 36(1):35-42, 2005; Dall'Acqua et al., Methods, 36(1):43-60, 2005; Clark, Immunology Today, 21(8):397-402, 2000; and U.S. Patents No. 6,180,370, No. 6,054,927, No. 5,869,619, No. 5,861,155, No. 5,712,120, and No. 4,816,567).

[0057] As used herein, the term “identity” refers to the relationship between the sequences of two or more polypeptide molecules or two or more nucleic acid molecules as determined by aligning and comparing their sequences. “Identity (%)” means the percentage of identical residues between amino acids or nucleotides within the molecules being compared, calculated based on the size of the smallest of the molecules being compared. For these calculations, alignment gaps (if any) are preferably addressed by a specific mathematical model or computer program (e.g., “algorithm”). Methods that may be used to calculate the identity of aligned nucleic acids or polypeptides include those described in Computational Molecular Biology, (Lesk, AM, ed.), 1988, New York: Oxford University Press; Biocomputing Informatics and Genome Projects, (Smith, DW, ed.), 1993, New York: Academic Press; Computer Analysis of Sequence Data, Part I, (Griffin, AM, and Griffin, HG, eds.), 1994, New Jersey: Humana Press; von Heinje, G., 1987, Sequence Analysis in Molecular Biology, New York: Academic Press; Sequence Analysis Primer, (Gribskov, M. and Devereux, J., eds.), 1991, New York: M. Stockton Press; and Carillo et al, 1988, SIAMJ. Applied Math. 48:1073. Alignment methods such as BLAST and / or Clustal Omega disclosed herein are available to those skilled in the art.

[0058] "Polynucleotide" or "nucleic acid" are used interchangeably herein and refer to polymers of nucleotides of any length, including DNA and RNA. Nucleotides may be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and / or analogs thereof, or any substrate that can be incorporated into the polymer by DNA or RNA polymerase or by synthetic reactions. Polynucleotides may include modified nucleotides such as methylated nucleotides and their analogs. Cells producing the binding molecule antibodies of this disclosure may include parental hybridoma cells, as well as bacterial and eukaryotic host cells into which the nucleic acid encoding the antibody has been introduced. Unless otherwise indicated, the left end of any single-stranded polynucleotide sequence disclosed herein is the 5' end, and the left-side direction of a double-stranded polynucleotide sequence is referred to as the 5' direction. The direction of addition from 5' to 3' of a newly generated RNA transcript is called the transcription direction. The sequence region on the DNA strand that has the same sequence as the RNA transcript and is located 5' to the 5' end of the RNA transcript is called the "upstream sequence," and the sequence region on the DNA strand that has the same sequence as the RNA transcript and is located 3' to the 3' end of the RNA transcript is called the "downstream sequence."

[0059] As used herein, the term “vector” refers to a nucleic acid vehicle into which polynucleotides can be inserted. A vector is called an expression vector if it enables the expression of a protein encoded by the polynucleotides inserted therein. A vector may contain genetic material elements that are expressed in a host cell by transformation, transduction, or transfection of the host cell. Vectors are well known to those skilled in the art and include, but are not limited to, plasmids, phages, cosmids; artificial chromosomes such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), or P1-derived artificial chromosomes (PACs); phages such as λ phages or M13 phages; and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papovaviruses (such as SV40). A vector may include, but is not limited to, multiple elements for controlling expression, including a promoter sequence, a transcription start sequence, an enhancer sequence, a selection element, and a reporter gene. Furthermore, the vector may also include an origin of replication.

[0060] As used herein, the term “host cell” refers to a cell into which a vector can be introduced, including but not limited to prokaryotic cells such as E. coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, insect cells such as S2 Drosophila cells or Sf9, and animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells, or human cells.

[0061] As used herein, the terms "transfection" or "transfect" refer to the process by which nucleic acids are introduced into eukaryotic cells, particularly mammalian cells. Transfection protocols and techniques include, but are not limited to, lipid transfection, as well as chemical and physical methods such as electroporation. Many transfection methods are well known in the art and are disclosed herein. See, for example, Graham et al., 1973, Virology 52:456, Sambrook et al., 2001, Molecular Cloning: A Laboratory Manual (see above), Davis et al., 1986, Basic Methods in Molecular Biology, Elsevier, Chu et al, 1981, Gene 13:197.

[0062] The DLL3-ADC disclosed herein includes a DLL3 antibody. DLL3 antibodies include, but are not limited to, chimeric antibodies, humanized antibodies, human antibodies, or single domain antibodies.

[0063] For example, the DLL3 antibodies of the present disclosure that include an antibody comprising at least one VHH domain are characterized by certain functional features or properties of the antibody. In some embodiments, the antibody has any one or more of the following properties. That is, (a) nM-grade EC when measured by ELISA or FACS with human DLL3, cynomolgus monkey DLL3, and mouse DLL3 50 binding, and [[ID=,14]](b) showing dose-dependent internalization ability in human cells engineered to express DLL3, and (c) binding to the human DLL3 extracellular domain (ECD) with a K of 0.1 nM or less when measured by SPR D binding.

[0064] The DLL3-ADCs disclosed herein include a DLL3 antibody that binds to cell surface DLL3 with high affinity. The binding of the antibody, and therefore the DLL3-ADC, of ​​which the antibody is a part, can be evaluated using one or more techniques well established in the art, e.g., ELISA. The binding specificity of DLL3-ADC can also be determined by monitoring the binding of DLL3-ADC to cells expressing the DLL3 protein, e.g., by flow cytometry. For example, the antibody can be tested by a flow cytometry assay (e.g., FACS) in which the DLL3-ADC is reacted with human DLL3-expressing cell lines, such as CHO cells and 293 cells, transfected to express DLL3 on the cell surface. Additionally or alternatively, the binding reaction rate (e.g., K) can be evaluated. D The binding affinity of DLL3-ADCs containing (value) can be tested by the Biacore® binding assay. Other suitable binding assays include, for example, ELISA assays using recombinant DLL3 protein. For example, the DLL3-ADCs disclosed herein can bind to cell surface DLL3 (e.g., human DLL3 ECD) protein at a rate of 1 × 10⁶. -7 M or less, 5×10 -8 M or less, 2×10 -8 M or less, 5×10 -9 M or less, 4×10 -9 M or less, 3×10 -9 M or less, 2×10 -9 M or less, 1×10 -9 M or less, 5×10 -10 M or less, or 1 × 10 -10 K below M D They can be joined together.

[0065] As used herein, the terms “fluorescence-activated cell sorting” or “FACS” refer to a specific type of flow cytometry. This provides a method for separating heterogeneous mixtures of living cells into two or more containers, one cell at a time, based on the specific light scattering and fluorescence properties of each cell (FlowMetric. “Sorting Out Fluorescence Activated Cell Sorting.” Retrieved 2017-11-09.). Instruments for performing FACS are known to those skilled in the art and are generally commercially available. Examples of such instruments include the FACS Star Plus, FACScan, and FACSort instruments sold by Becton Dickinson (Foster City, Calif.), the Epics C sold by Coulter Epics Division (Hialeah, Fla.), and the MoFlo sold by Cytomation (Colorado Springs, Col.).

[0066] As used herein, the terms “SPR” or “Surface Plasmon Resonance” refer to, and include, the optical phenomenon that enables real-time analysis of biospecific interactions by detecting changes in protein concentration within a biosensor matrix, for example, using the BIAcore system (Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, NJ). For further explanation, see Jonsson, U., et al. (1993) Ann. Biol. Clin. 51:19-26, Jonsson, U., et al. (1991) Biotechniques 11:620-627, Johnsson, B., et al. (1995) J. Mol. Recognit. 8:125-131, and Johnsson, B., et al. (1991) Anal. Biochem. 198:268-277.

[0067] When used in this specification, "EC 50The term "median effectiveness concentration" is also referred to as the "median effectiveness concentration" and refers to the concentration of a drug, antibody, or toxin that elicits an intermediate response between baseline and maximum after a specific exposure time. In connection with this disclosure, the EC 50 The unit is expressed as "nM". In some embodiments, the antibodies of this disclosure, when measured by FACS, have an EC of approximately 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM, 0.5 nM, 0.4 nM, 0.3 nM, 0.2 nM, 0.1 nM, 0.09 nM, 0.08 nM, 0.07 nM, 0.06 nM, 0.05 nM, 0.04 nM, 0.03 nM, 0.02 nM, or less than 0.01 nM in cynomolgus monkey or mouse DLL3. 50 They are joined together.

[0068] DLL3 antibody containing VHH CDR In some embodiments, the DLL3-ADCs disclosed herein comprise a DLL3 antibody containing a heavy chain variable domain (VH, such as VHH), where VH comprises CDR1, CDR2, and CDR3, such as VHH or VHH conjugated to Fc. In further embodiments, CDR1 comprises the amino acid sequence described in SEQ ID NO: 1 or 7, CDR2 comprises the amino acid sequence described in SEQ ID NO: 2 or 8, and CDR3 comprises the amino acid sequence described in SEQ ID NO: 3 or 9. In some embodiments, CDR numbering follows a combination of Kabat numbering and AbM numbering. The framework region and the scope of each CDR can be precisely identified using methods known in the art, such as Kabat definitions, Chothia definitions, AbM definitions, Contact definitions, IMGT definitions (all of which are well known in the art), and any combination thereof. For example, Kabat, EA, et al. (1991) Sequences of Proteins of Immunological Interest (Fifth Edition), USDepartment of Health and Human Services, NIH Publication No. 91-3242, Chothia et al., (1989) Nature 342:877, Chothia, C. et al. al. (1987) J. Mol. Biol. 196:901-917, Al-lazikani et al (1997) J. Molec. Antibodies”, chapter 5, 2007. See also hgmp.mrc.ac.uk and bioinf.org.uk / abs.Correspondence or alignment between numbering systems with different definitions can be found, for example, at www.imgt.org / (see also Giudicelli V et al. IMGT (International ImMunoGeneTics Database) Nucleic Acids Res. (1997) 25:206-11, and Lefranc MP et al., IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains. Dev Comp Immunol. (2003) 27:55-77).

[0069] As those skilled in the art will recognize, the precise numbering and arrangement of complementarity-determining regions (CDRs) may vary depending on the numbering system. However, it should be understood that the disclosure of variable heavy chains, variable light chains, and / or VHH sequences includes the disclosure of the relevant (unique) CDRs. Thus, the disclosure of each variable region is the disclosure of each CDR (e.g., CDR1, CDR2, and CDR3). Two antibodies having the same VH, VL, or VHH CDRs mean that their CDRs are the same when determined by the same approach (e.g., the Kabat, AbM, Chothia, Contact, and IMGT numbering approaches well known in the art).

[0070] Variable regions and CDRs in antibody sequences can be identified according to general rules developed in the art (e.g., Kabat, AbM, Chothia, Contact, and IMGT numbering systems) or by aligning the sequence against a database of known variable regions. Methods for identifying these regions are described in Kontermann and Dubel, eds., Antibody Engineering, Springer, New York, NY, 2001 and Dinarello et al., Current Protocols in Immunology, John Wiley and Sons Inc., Hoboken, NJ, 2000. Exemplary databases of antibody sequences are listed on the "Abysis" website (www.bioinf.org.uk / abs) (maintained by AC Martin in the Department of Biochemistry & Molecular Biology, University College London, London, England) and the VBASE2 website (www.vbase2.org) (described in Retter et al., Nucl. Acids Res., 33 (Database issue): D671-D674 (2005)), and can be accessed via them. Preferably, sequences are analyzed using the Abysis database, which integrates sequence data from Kabat, IMGT, and the Protein Data Bank (PDB) with structural data from the PDB. See Dr. Andrew CRMartin's book chapter, Protein Sequence and Structure Analysis of Antibody Variable Domains. See In:Antibody Engineering Lab Manual (Ed.: Duebel, S. and Kontermann, R., Springer-Verlag, Heidelberg, ISBN-13: 978-3540413547, also available on the website bioinforg.uk / abs).The Abysis database website also includes general rules developed to identify CDRs that can be used in accordance with the teachings herein. Unless otherwise indicated, all CDRs mentioned herein, with the exception of the Chothia CDR, are obtained in accordance with the Abysis database website by Kabat (see, for example, Chothia & Lesk (1987) J.Mol.Biol.196:901–917).

[0071] In some embodiments, the DLL3 antibody (VHH or VHH conjugated to Fc, e.g., VHH-Fc fusion) comprises at least one VH containing one, two, and / or three heavy chain CDRs as described in Table 1 or Table 2. [Table 1] [Table 2]

[0072] In some embodiments, the DLL3-ADC disclosed herein comprises a DLL3 antibody containing a heavy chain variable domain (VH), such as VHH or VHH conjugated to Fc, where VH comprises FRW1-CDR1-FRW2-CDR2-FRW3-CDR3-FRW4, where CDR1 comprises the amino acid sequence described in SEQ ID NO: 1 or 7, CDR2 comprises the amino acid sequence described in SEQ ID NO: 2 or 8, and CDR3 comprises the amino acid sequence described in SEQ ID NO: 3 or 9. In some embodiments, the FRW1 and FRW4 at the N-terminus and C-terminus of the VHH contained in the DLL3 antibody may consist of only partial FRW1 and / or FRW4, or the VHH may be cleaved to lack one or both of these framework regions, as long as the VHH substantially maintains antigen-binding and specificity.

[0073] In some embodiments, the DLL3-ADC disclosed herein comprises a DLL3 antibody containing one, two, or all three CDRs of the amino acid sequence described in SEQ ID NO: 14 (e.g., an anti-DLL3 single-domain antibody, VHH, or VHH conjugated to Fc). In some embodiments, the DLL3-ADC comprises a DLL3 antibody containing one, two, or all three CDRs of the amino acid sequence described in SEQ ID NO: 15 (e.g., an anti-DLL3 single-domain antibody, VHH, or VHH conjugated to Fc). In some embodiments, the DLL3-ADC comprises a DLL3 antibody containing one, two, or all three CDRs of the amino acid sequence described in SEQ ID NO: 16 (e.g., an anti-DLL3 single-domain antibody, VHH, or VHH conjugated to Fc). In some embodiments, the DLL3-ADC disclosed herein comprises a DLL3 antibody containing camelid VHH. In some embodiments, the DLL3-ADC disclosed herein comprises a humanized DLL3 antibody. In some embodiments, the DLL3-ADC disclosed herein comprises a DLL3 antibody that includes an acceptor human framework, such as a human immunoglobulin framework or a human consensus framework.

[0074] In some embodiments, the DLL3-ADC disclosed herein comprises a DLL3 antibody containing CDR1 having the amino acid sequence of CDR1 described in SEQ ID NO: 1. In some embodiments, the DLL3-ADC comprises a DLL3 antibody containing CDR2 having the amino acid sequence of CDR2 described in SEQ ID NO: 2. In some embodiments, the DLL3-ADC comprises a DLL3 antibody containing CDR3 having the amino acid sequence of CDR3 described in SEQ ID NO: 3. In some embodiments, the DLL3-ADC comprises DLL3 antibodies containing CDR1 and CDR2 having the amino acid sequences of CDR1 and CDR2 described in SEQ ID NO: 1 and 2, respectively. In some embodiments, the DLL3-ADC comprises DLL3 antibodies containing CDR1 and CDR3 having the amino acid sequences of CDR1 and CDR3 described in SEQ ID NO: 1 and 3, respectively. In some embodiments, the DLL3-ADC comprises DLL3 antibodies containing CDR2 and CDR3 having the amino acid sequences of CDR2 and CDR3 described in SEQ ID NO: 2 and 3, respectively. In some embodiments, DLL3-ADC comprises a DLL3 antibody containing CDR1, CDR2, and CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3 described in SEQ ID NOs: 1, 2, and 3, respectively. In some embodiments, DLL3-ADC comprises a DLL3 antibody (such as an anti-DLL3 single-domain antibody, VHH, or VHH conjugated to Fc) containing one, two, or all three CDRs of the amino acid sequence described in SEQ ID NO: 14. In some embodiments, DLL3-ADC comprises a DLL3 antibody (such as an anti-DLL3 single-domain antibody, VHH, or VHH conjugated to Fc) containing one, two, or all three CDRs of the amino acid sequence described in SEQ ID NO: 15. The CDR sequences can be determined by a well-known numbering system. In some embodiments, the CDRs follow IMGT numbering. In some embodiments, the CDRs follow Kabat numbering. In other embodiments, the CDRs follow Chothia numbering. In other embodiments, the CDRs follow Contact numbering. In some embodiments, the CDR follows AbM numbering. In some embodiments, the DLL3-ADC comprises a DLL3 antibody containing the VHH of a camelid.In some embodiments, DLL3-ADC comprises a humanized DLL3 antibody. In some embodiments, DLL3-ADC comprises a DLL3 antibody comprising an acceptor human framework, such as a human immunoglobulin framework or a human consensus framework.

[0075] In some embodiments, DLL3-ADC includes a DLL3 antibody containing CDR1 having the amino acid sequence of CDR1 described in SEQ ID NO: 7. In some embodiments, DLL3-ADC includes a DLL3 antibody containing CDR2 having the amino acid sequence of CDR2 described in SEQ ID NO: 8. In some embodiments, DLL3-ADC includes a DLL3 antibody containing CDR3 having the amino acid sequence of CDR3 described in SEQ ID NO: 9. In some embodiments, DLL3-ADC includes DLL3 antibodies containing CDR1 and CDR2 having the amino acid sequences of CDR1 and CDR2 described in SEQ ID NO: 7 and 8, respectively. In some embodiments, DLL3-ADC includes DLL3 antibodies containing CDR1 and CDR3 having the amino acid sequences of CDR1 and CDR3 described in SEQ ID NO: 7 and 9, respectively. In some embodiments, DLL3-ADC includes DLL3 antibodies containing CDR2 and CDR3 having the amino acid sequences of CDR2 and CDR3 described in SEQ ID NO: 8 and 9, respectively. In some embodiments, DLL3-ADC comprises a DLL3 antibody containing CDR1, CDR2, and CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3 described in SEQ ID NOs. 7, 8, and 9, respectively. In some embodiments, DLL3-ADC comprises a DLL3 antibody (such as an anti-DLL3 single-domain antibody, VHH, or VHH conjugated to Fc) containing one, two, or all three CDRs of the amino acid sequence described in SEQ ID NO. 16. The CDR sequences can be determined by a well-known numbering system. In some embodiments, the CDRs follow IMGT numbering. In some embodiments, the CDRs follow Kabat numbering. In other embodiments, the CDRs follow Chothia numbering. In other embodiments, the CDRs follow Contact numbering. In some embodiments, the CDRs follow AbM numbering. In some embodiments, DLL3-ADC comprises a DLL3 antibody containing camelid VHH. In some embodiments, DLL3-ADC comprises a humanized DLL3 antibody. In some embodiments, the DLL3-ADC comprises a DLL3 antibody that includes an acceptor human framework, such as a human immunoglobulin framework or a human consensus framework.

[0076] In some embodiments, a DLL3-ADC is provided herein that comprises a DLL3 antibody having the following structure: FRW1-CDR1-FRW2-CDR2-FRW3-CDR3-FRW4, where (i) CDR1 comprises the amino acid sequence described in any one of SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 20, SEQ ID NO: 23, SEQ ID NO: 24, or SEQ ID NO: 27; (ii) CDR2 comprises the amino acid sequence described in any one of SEQ ID NO: 2, SEQ ID NO: 21, SEQ ID NO: 25, SEQ ID NO: 28, SEQ ID NO: 56, or SEQ ID NO: 30; and / or (iii) CDR3 comprises the amino acid sequence described in any one of SEQ ID NO: 3, SEQ ID NO: 22, SEQ ID NO: 26, or SEQ ID NO: 29. In some embodiments, the DLL3-ADC disclosed herein comprises a DLL3 antibody containing camelid VHH. In some embodiments, the DLL3-ADC disclosed herein comprises a humanized DLL3 antibody. In some embodiments, the DLL3-ADC disclosed herein comprises a DLL3 antibody that includes an acceptor human framework, such as a human immunoglobulin framework or a human consensus framework.

[0077] In some embodiments, CDR1 of the DLL3 antibody of DLL3-ADC includes the exemplary amino acid sequence described in SEQ ID NO: 1, CDR2 includes the exemplary amino acid sequence described in SEQ ID NO: 2, and CDR3 includes the exemplary amino acid sequence described in SEQ ID NO: 3. In some embodiments, CDR1 of the DLL3 antibody of DLL3-ADC includes the amino acid sequence described in SEQ ID NO: 20, according to IMGT numbering, CDR2 includes the amino acid sequence described in SEQ ID NO: 21, according to IMGT numbering, and CDR3 includes the amino acid sequence described in SEQ ID NO: 22, according to IMGT numbering. In some embodiments, CDR1 of the DLL3 antibody of DLL3-ADC includes the amino acid sequence described in SEQ ID NO: 23, according to Kabat numbering, CDR2 includes the amino acid sequence described in SEQ ID NO: 2, according to Kabat numbering, and CDR3 includes the amino acid sequence described in SEQ ID NO: 3, according to Kabat numbering. In some embodiments, CDR1 of the DLL3 antibody of DLL3-ADC includes the amino acid sequence described in SEQ ID NO: 24, according to Chothia numbering; CDR2 includes the amino acid sequence described in SEQ ID NO: 25, according to Chothia numbering; and CDR3 includes the amino acid sequence described in SEQ ID NO: 26, according to Chothia numbering. In some embodiments, CDR1 of the DLL3 antibody of DLL3-ADC includes the amino acid sequence described in SEQ ID NO: 27, according to Contact numbering; CDR2 includes the amino acid sequence described in SEQ ID NO: 28 or 56, according to Contact numbering; and CDR3 includes the amino acid sequence described in SEQ ID NO: 29, according to Contact numbering. In some embodiments, CDR1 of the DLL3 antibody of DLL3-ADC includes the amino acid sequence described in SEQ ID NO: 1, according to AbM numbering; CDR2 includes the amino acid sequence described in SEQ ID NO: 30, according to AbM numbering; and CDR3 includes the amino acid sequence described in SEQ ID NO: 3, according to AbM numbering. In some embodiments, the DLL3-ADC disclosed herein comprises a DLL3 antibody containing a camelid VHH. In some embodiments, the DLL3-ADC comprises a humanized DLL3 antibody.In some embodiments, the DLL3-ADC disclosed herein comprises a DLL3 antibody that includes an acceptor human framework, such as a human immunoglobulin framework or a human consensus framework.

[0078] In some embodiments, CDR1 of the DLL3 antibody of DLL3-ADC includes the exemplary amino acid sequence described in SEQ ID NO: 7, CDR2 includes the exemplary amino acid sequence described in SEQ ID NO: 8, and CDR3 includes the exemplary amino acid sequence described in SEQ ID NO: 9. In some embodiments, CDR1 of the DLL3 antibody of DLL3-ADC includes the amino acid sequence described in SEQ ID NO: 42, according to IMGT numbering, CDR2 includes the amino acid sequence described in SEQ ID NO: 43, according to IMGT numbering, and CDR3 includes the amino acid sequence described in SEQ ID NO: 44, according to IMGT numbering. In some embodiments, CDR1 of the DLL3 antibody of DLL3-ADC includes the amino acid sequence described in SEQ ID NO: 45, according to Kabat numbering, CDR2 includes the amino acid sequence described in SEQ ID NO: 8, according to Kabat numbering, and CDR3 includes the amino acid sequence described in SEQ ID NO: 9, according to Kabat numbering. In some embodiments, CDR1 of the DLL3 antibody in DLL3-ADC includes the amino acid sequence described in SEQ ID NO: 46, according to Chothia numbering; CDR2 includes the amino acid sequence described in SEQ ID NO: 47, according to Chothia numbering; and CDR3 includes the amino acid sequence described in SEQ ID NO: 48, according to Chothia numbering. In some embodiments, CDR1 of the DLL3 antibody in DLL3-ADC includes the amino acid sequence described in SEQ ID NO: 49, according to Contact numbering; CDR2 includes the amino acid sequence described in SEQ ID NO: 50, according to Contact numbering; and CDR3 includes the amino acid sequence described in SEQ ID NO: 51, according to Contact numbering. In some embodiments, CDR1 of the DLL3 antibody in DLL3-ADC includes the amino acid sequence described in SEQ ID NO: 7, according to AbM numbering; CDR2 includes the amino acid sequence described in SEQ ID NO: 52, according to AbM numbering; and CDR3 includes the amino acid sequence described in SEQ ID NO: 9, according to AbM numbering. In some embodiments, the DLL3-ADC disclosed herein comprises a DLL3 antibody containing a camelid VHH. In some embodiments, the DLL3-ADC disclosed herein comprises a humanized DLL3 antibody.In some embodiments, the DLL3-ADC disclosed herein comprises a DLL3 antibody that includes an acceptor human framework, such as a human immunoglobulin framework or a human consensus framework.

[0079] In some embodiments, DLL3-ADC includes DLL3 comprising a single-domain antibody comprising one or more frameworks derived from a VHH domain containing the sequence described in SEQ ID NO: 14. In some embodiments, DLL3-ADC includes DLL3 antibody comprising a single-domain antibody comprising one or more frameworks derived from a VHH domain containing the sequence described in SEQ ID NO: 15. In some embodiments, DLL3-ADC includes DLL3 antibody comprising a single-domain antibody comprising one or more frameworks derived from a VHH domain containing the sequence described in SEQ ID NO: 16.

[0080] In some embodiments, the DLL3-ADC provided herein comprises a single-domain antibody which is a humanized single-domain antibody. The framework region described herein is determined based on the boundaries of a CDR numbering system. In other words, if the CDR is determined by, for example, IMGT, Kabat, Chothia, Contact, or AbM, the framework region is an amino acid residue that surrounds the CDR within the variable region from N-terminus to C-terminus in the format FRW1-CDR1-FRW2-CDR2-FRW3-CDR3-FRW4. For example, FRW1 is defined as the N-terminal amino acid residue of a CDR1 amino acid residue as defined by, for example, the IMGT numbering system, Kabat numbering system, Chothia numbering system, Contact numbering system, or AbM numbering system; FRW2 is defined as the amino acid residue between the CDR1 amino acid residue and the CDR2 amino acid residue and base as defined by, for example, the IMGT numbering system, Kabat numbering system, Chothia numbering system, Contact numbering system, or AbM numbering system; FRW3 is defined as the amino acid residue between the CDR2 amino acid residue and the CDR3 amino acid residue as defined by, for example, the IMGT numbering system, Kabat numbering system, Chothia numbering system, Contact numbering system, or AbM numbering system; and FRW4 is defined as the C-terminal amino acid residue of the CDR3 amino acid residue as defined by, for example, the IMGT numbering system, Kabat numbering system, Chothia numbering system, Contact numbering system, or AbM numbering system.

[0081] In some embodiments, the disclosure provides a DLL3-ADC comprising an isolated DLL3 antibody comprising a VHH domain having the amino acid sequence described in SEQ ID NO: 14. In some embodiments, the disclosure provides a DLL3-ADC comprising a polypeptide comprising the amino acid sequence described in SEQ ID NO: 14. In some embodiments, the disclosure provides a DLL3-ADC comprising an isolated DLL3 antibody comprising a VHH domain having the amino acid sequence described in SEQ ID NO: 15. In some embodiments, the disclosure provides a DLL3-ADC comprising a polypeptide comprising the amino acid sequence described in SEQ ID NO: 15. In some embodiments, the disclosure provides a DLL3-ADC comprising an isolated DLL3 antibody comprising a VHH domain having the amino acid sequence described in SEQ ID NO: 16. In some embodiments, the disclosure provides a DLL3-ADC comprising a polypeptide comprising the amino acid sequence described in SEQ ID NO: 16.

[0082] Anti-DLL3 antibody containing the VHH sequence In some embodiments, the DLL3-ADC disclosed herein comprises a DLL3 antibody containing a heavy chain variable domain (VH) (e.g., VHH), (a) The amino acid sequence described in any one of Sequence IDs 14-16, (b) an amino acid sequence that is at least 85%, at least 90%, or at least 95% identical to any one of sequence numbers 14-16, or (c) an amino acid sequence having one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acid additions, deletions, and / or substitutions compared to any one of sequence numbers 14-16.

[0083] The percentage of identity between two amino acid sequences can be determined using the algorithm of E. Meyers and W. Miller (Comput.Appl.Biosci.,4:11-17(1988)), which is incorporated into the ALIGN program (version 2.0), using the PAM120 weighted residue table, gap length penalty = 12, and gap penalty = 4. Furthermore, the percentage of identity between two amino acid sequences can be determined using the algorithm of Needleman and Wunsch (J.Mol.Biol.48:444-453(1970)), which is incorporated into the GAP program of the GCG software package (available at www.gcg.com), using either the Blossum62 matrix or the PAM250 matrix, and gap weights of 16, 14, 12, 10, 8, 6, or 4, and length weights of 1, 2, 3, 4, 5, or 6.

[0084] Additionally or alternatively, the protein (antibody) sequences of this disclosure can be used as further "query sequences" to perform searches against public databases to identify, for example, relevant sequences. Such searches may be performed using the XBLAST program (version 2.0) of Altschul et al. (1990) J.Mol.Biol.215:403-10. A BLAST protein search can be performed using the XBLAST program, score=50, and word length=3 to obtain amino acid sequences homologous to the antibody molecules of this disclosure. To obtain alignments including gaps for comparison purposes, Gapped BLAST can be used as described in Altschul et al., (1997) Nucleic Acids Res.25(17):3389-3402. When using the BLAST and Gapped BLAST programs, the default parameters of each program (e.g., XBLAST and NBLAST) may be used. See www.ncbi.nlm.nih.gov.

[0085] In some embodiments, the amino acid sequence of VH (such as VHH) may be at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of sequence numbers 14-16.

[0086] In some further embodiments, the DLL3 antibody may contain conserved amino acid substitutions or modifications in the variable regions of the heavy and / or light chain, such as VH or VHH. It is understood in the art that certain conserved sequence modifications can be made without removing antigen binding. For example, see Brummell et al. (1993) Biochem 32:1180-8, de Wildt et al. (1997) Prot.Eng.10:835-41, Komissarov et al. (1997) J.Biol.Chem.272:26864-26870, Hall et al. (1992) J.Immunol.149:1605-12, Kelley and O'Connell (1993) Biochem.32:6862-35, Adib-Conquy et al. (1998) Int.Immunol.10:341-6, and Beers et al. (2000) Clin.Can.Res.6:2835-43.

[0087] As stated above, as used herein, the term “conservative substitution” refers to an amino acid substitution that does not adversely affect or alter the essential properties of a protein / polypeptide, including its amino acid sequence. For example, conservative substitutions can be introduced by standard techniques well known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions include substitutions in which an amino acid residue is replaced by another amino acid residue having a similar side chain to the corresponding amino acid residue, for example, a physically or functionally similar residue (e.g., having similar size, shape, charge, and chemical properties including the ability to form covalent or hydrogen bonds). Families of amino acid residues with similar side chains are defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, and histidine), amino acids with acidic side chains (e.g., aspartic acid and glutamic acid), amino acids with non-charged side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, and tryptophan), amino acids with nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, and methionine), amino acids with β-branched side chains (e.g., threonine, valine, and isoleucine), and amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, and histidine). Therefore, the corresponding amino acid residue is preferably substituted with another amino acid residue from the same side chain family. Methods for identifying conserved amino acid substitutions are well known in the art (see, for example, Brummell et al., Biochem. 32:1180-1187 (1993), Kobayashi et al., Protein Eng. 12(10):879-884 (1999), and Burks et al., Proc. Natl. Acad. Sci. USA 94:412-417 (1997) (incorporated herein by reference)).

[0088] In some embodiments, DLL3-ADC comprises a DLL3 antibody containing at least one VHH, wherein the VHH comprises the amino acid sequence described in any one of SEQ ID NOs: 14-16.

[0089] In some embodiments, DLL3-ADC is a DLL3 antibody comprising VHH (hereinafter referred to as VHH-Fc fusion) fused to the Fc region of human IgG1 or IgG4, wherein VHH comprises the amino acid sequence described in any one of SEQ ID NOs: 14-16. In some embodiments, DLL3-ADC comprises a DLL3 antibody comprising VHH and the Fc region of human IgG1. In some further embodiments, DLL3-ADC comprises a DLL3 antibody that is a humanized antibody comprising VHH and the Fc region of human IgG1. In some embodiments, the antibody or its antigen-binding portion comprises a VHH domain comprising the amino acid sequence described in any one of SEQ ID NOs: 14-16, and an Fc region comprising the amino acid sequence described in SEQ ID NO: 17. In some embodiments, DLL3-ADC comprises a DLL3 antibody comprising a polypeptide having the amino acid sequence described in any one of SEQ ID NOs: 4, 5, or 10. In further embodiments, DLL3-ADC comprises a DLL3 antibody comprising a homodimer of a polypeptide having the amino acid sequence described in any one of SEQ ID NOs: 4, 5, or 10.

[0090] In some embodiments, at least one addition, deletion, and / or substitution of amino acids in the VHH region is not present in any of the CDR sequences but is present in the framework (FRW) sequence. For example, the antibody or antigen-binding moiety described above may include one or more amino acid substitutions in the framework sequence of the VHH region, e.g., FRW1, FRW2, FRW3, and / or FRW4.

[0091] In some embodiments, the antibody or its antigen-binding portion provided herein includes any suitable framework region (FRW) sequence, as long as the antigen-binding domain specifically binds to DLL3.

[0092] As described above, an antibody or its antigen-binding moiety may contain one or more amino acid modifications in the variable region and / or constant region, including cases where the modification is a conservative substitution. It is understood in the art that certain conservative sequence modifications can be made without removing antigen binding. For example, see Brummell et al. (1993) Biochem 32:1180-8, de Wildt et al. (1997) Prot.Eng.10:835-41, Komissarov et al. (1997) J.Biol.Chem.272:26864-26870, Hall et al. (1992) J.Immunol.149:1605-12, Kelley and O'Connell (1993) Biochem.32:6862-35, Adib-Conquy et al. (1998) Int.Immunol.10:341-6, and Beers et al. (2000) Clin.Can.Res.6:2835-43.

[0093] The antigen-binding domain of the DLL3 antibody in DLL3-ADC is not limited to the VHH form and can take various other forms, such as Fab, Fab', F(ab')2, Fv fragment, or single-chain antibody molecule (scFv), although these are not limited to these. In some embodiments, the antigen-binding domain is an Fv fragment having a VH region and a VL region in separate chains held together by a tight non-covalent interaction.

[0094] Fc region containing IgG constant domain The DLL3-ADCs disclosed herein comprise a DLL3 antibody further comprising an Fc region containing one or more human IgG constant domains. The human IgG constant domain may be a human IgG1, IgG2, IgG3, or IgG4 constant domain, preferably a human IgG1 constant domain. An example of an amino acid sequence of an Fc region containing a human IgG1 constant domain is described in SEQ ID NO: 17. In some embodiments, the DLL3-ADC comprises a DLL3 antibody comprising the amino acid sequence described in any one of SEQ ID NOs: 4, 5, and 10. In some embodiments, the Fc region is a human IgG1 Fc region such as a wild-type Fc region or an Fc variant containing one or more amino acid modifications (e.g., Leu234Ala / Leu235Ala or LALA) that alter antibody-dependent cytotoxicity (ADCC) or other effector function.

[0095] In some embodiments, Fc modifications include LALA mutations with EU numbering, e.g., L234A and L235A mutations, as described in Kabat et al. The Kabat numbering system is generally used to refer to residues in the variable domain (approximately residues 1-107 of the light chain and residues 1-113 of the heavy chain) (e.g., Kabat et al., Sequences of Immunological Interest. 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). The "EU numbering system" or "EU index" is generally used to refer to residues within the constant region of the immunoglobulin heavy chain (e.g., the EU index reported by Kabat et al. (cited above)). See www.imgt.org / IMGTScientificChart / Numbering / Hu_IGHGnber.html. "Kabat-like EU numbering" or "Kabat-like EU index" refers to the residue numbering of human IgG1 EU antibodies. Unless otherwise specified herein, the designation of residue numbers within the constant domain of an antibody refers to the numbering of residues according to the EU numbering system.

[0096] Modification of antibodies is also included within the scope of this disclosure. Covalent modifications include reacting target amino acid residues of an antibody with an organic derivatizer that can react with selected side chains or N-terminal or C-terminal residues of the antibody, such as those disclosed herein. Other modifications include deamidation of glutaminyl and asparaginyl residues to their corresponding glutamyl and aspartyl residues, respectively; hydroxylation of proline and lysine; phosphorylation of hydroxyl groups of ceryl or threonyl residues; methylation of α-amino groups of lysine, arginine, and histidine side chains (e.g., Creighton, Proteins: Structure and Molecular Properties 79-86 (1983)); glycosylation (such as N-linked glycosylation); isomerization and oxidation of Asp; acetylation of N-terminal amines; and amidation of any C-terminal carboxyl group.

[0097] Nucleic acid molecule encoding the DLL3 antibody The nucleic acid molecules encoding the DLL3 antibodies disclosed herein can be readily selected by those skilled in the art. In one embodiment, the DLL3 antibody for DLL3-ADC provided herein may be produced using a nucleic acid molecule containing a nucleic acid sequence encoding the amino acid sequence described in SEQ ID NO: 14. In one embodiment, the DLL3 antibody for DLL3-ADC provided herein may be produced using a nucleic acid molecule containing a nucleic acid sequence encoding the amino acid sequence described in SEQ ID NO: 15. In one embodiment, the DLL3 antibody for DLL3-ADC provided herein may be produced using a nucleic acid molecule containing a nucleic acid sequence encoding the amino acid sequence described in SEQ ID NO: 16.

[0098] In some embodiments, the degree of identity (%) stems from the degeneracy of the genetic code, and the encoded protein sequence remains unchanged.

[0099] host cell The host cells disclosed herein may be any cells suitable for expressing the antibodies disclosed herein, such as yeast, bacteria, plants, and mammalian cells. Examples of mammalian host cells for expressing the antibodies disclosed herein include Chinese hamster ovary cells (CHO cells) (e.g., including dhfr CHO cells described in Urlaub and Chasin, (1980) Proc. Natl. Acad. ScL USA 77:4216-4220, used with the DHFR selection marker described in RJ Kaufman and PA Sharp (1982) J. Mol. Biol. 159:601-621), 293F cells, NSO myeloma cells, COS cells, and SP2 cells. Specifically, another expression system for use with NSO myeloma cells is the GS gene expression system disclosed in WO87 / 04462, WO89 / 01036, and EP338,841.Additionally, the study included SV4010-transformed monkey kidney CV1 cell line (COS-7, ATCC CRL 1651), human fetal kidney cell line (293 cells, or 293 cells subcloned in suspension culture for proliferation, Graham et al., J. Gen Virol. 36:59 (1977)), baby hamster kidney cells (BHK, ATCC CCL), Chinese hamster ovary cells / -DHFR (CHO, Urlaub et al., 1980, Proc. Natl. Acad. Sci. USA 77:4216), mouse Sertoli cells (TM4, Mather, 1980, Biol. Reprod. 23:243-251), monkey kidney cells (CV1 ATCC CCL 70), African green monkey kidney cells (VERO-76, ATCC CRL-1587), and human cervical cancer cells (HELA, ATCC CCL). 2) Mouse myeloma cells such as canine kidney cells (MDCK, ATCC CCL 34), buffalo rat liver cells (BRL 3A, ATCC CRL 1442), human lung cells (W138, ATCC CCL 75), human liver cells (Hep G2, HB 8065), mouse mammary cancer cells (MMT 060562, ATCC CCL 51), TRI cells (Mather et al., 1982, Annals NYAcad.Sci.383:44-68), MRC-5 cells, FS4 cells, NSO (e.g., RCB0213, 1992, Bio / Technology 10:169) and SP2 / 0 cells (e.g., SP2 / 0-Ag14 cells, ATCC CRL 1581), YB2 / 0 cells (e.g., YB2 / 3HL.P2.G11.16Ag.20 cells, ATCC Examples include rat myeloma cells such as CRL 1662, PER.C6 cells, and human hepatocellular carcinoma cells (Hep G2). CHO cells are one of the cell lines available for use herein, with CHO-K1, DUK-B11, CHO-DP12, CHO-DG44 (Somatic Cell and Molecular Genetics 12:555(1986)), and Lec13 being exemplary host cell lines. In the case of CHO-K1, DUK-B11, DG44, or CHO-DP12 host cells, they can be modified to lack the ability to fucosylate the proteins they express.In some embodiments, the host cells herein are selected from CHO, CHO-S, HEK, HEK293, HEK-293F, Expi293F, PER.C6, or NSO cells or lymphocytes.

[0100] Suitable prokaryotes for this purpose include eubacteria such as Gram-negative or Gram-positive organisms, such as Enterobacteriaceae such as Escherichia, such as E. coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella such as Salmonella typhimurium, Serratia such as Serratia marcescens, and Shigella, as well as Bacilli such as B. subtilis and B. licheniformis, P. aeruginosa, and Streptomyces.

[0101] Besides prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeast are also suitable cloning or expression hosts for antibody-coding vectors. Budding yeast, or common baker's yeast, is the most commonly used among lower eukaryotic host microorganisms. However, Schizosaccharomyces pombe, Kluyveromyces host, for example, K. lactis, K. fragilis (ATCC 12,424), K. bulgaricus (ATCC 16,045), K. wickerhamii (ATCC 24,178), K. waltii (ATCC 56,500), K. drosophilarum (ATCC 36,906), K. thermotlerans, and K. marxianus, as well as yarrowia (EP5 402,226), Pichia pastoris (EP183,070), Candida, Trichoderma reesia (EP244,234), Neurospora crassa, Schwanniomyces, for example, Schwanniomyces Numerous other genera, species, and strains of filamentous fungi, such as Neurospora, Penicillium, Tolypocladium, and Aspergillus, as well as hosts such as A. nidulans and A. niger, are commonly available and useful herein.

[0102] When a recombinant expression vector encoding an antibody is introduced into mammalian host cells, the antibody is produced by culturing the host cells for a sufficient time for the antibody to be expressed within the host cells, or for a sufficient time for the antibody to be secreted into the culture medium in which the host cells are grown. The antibody can be recovered from the culture medium using standard protein purification methods.

[0103] Linker - Drug Conjugate DLL3-ADCs contain a DLL3 antibody conjugated to a drug via a linker. The linker and the drug may collectively be referred to as "linker-drug" in this specification.

[0104] In one embodiment, the linker structure is [ka] or a salt thereof, where the wavy line indicates the attachment point to the drug. In further embodiments, the linker structure is the (R)-epimer or a salt thereof of the above linker structure. Thus, the linker structure is [ka] or a salt thereof. In other embodiments, the linker structure is the (S)-epimer or a salt thereof of the above linker structure. Thus, the linker structure is [ka] or its salt.

[0105] In one embodiment, the drug is auristatin, for example, MMAE. The structure of MMAE is provided below. [ka] MMAE is also known as (2S)-N-[(2S)-1-[[(3R,4S,5S)-1-[(2S)-2-[(1R,2R)-3-[[(1S,2R)-1-hydroxy-1-phenylpropane-2-yl]amino]-1-methoxy-2-methyl-3-oxopropyl]pyrrolidine-1-yl]-3-methoxy-5-methyl-1-oxoheptan-4-yl]-methylamino]-3-methyl-1-oxobutan-2-yl]-3-methyl-2-(methylamino)butanamide.

[0106] In one embodiment, the drug is a topoisomerase inhibitor, such as camptothecin or a derivative thereof, such as exatecan. The structure of exatecan is provided below. [ka] This is also known as (1S,9S)-1-amino-9-ethyl-5-fluoro-9-hydroxy-4-methyl-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4':6,7]indolidino[1,2-b]quinoline-10,13-dione.

[0107] In one embodiment, the linker-drug is linker-drug (1), [ka] or a salt thereof, where "Ac" is an acetyl group. Linker-drug (1) (or a salt thereof) comprises a linker conjugated to monomethyl auristatin E (MMAE). In further embodiments, the linker-drug is the (R)-epimer of linker-drug (1) or a salt thereof. Thus, the linker-drug is [ka] (This is also referred to herein as linker-drug(1) form A or linker-drug(1-A)), or a salt thereof. In other embodiments, the linker-drug is the (S)-epimer of linker-drug(1) or a salt thereof. Thus, the linker-drug is, [ka] (This is also referred to herein as Linker-drug(1) form B or Linker-drug(1-B)), or a salt thereof.

[0108] In one embodiment, the linker-drug is linker-drug (2), [ka] or a salt thereof, where "Ac" is an acetyl group. Linker-drug (2) (or a salt thereof) comprises a linker conjugated to exatecan. In further embodiments, the linker-drug is the (R)-epimer of linker-drug (2) or a salt thereof. Thus, the linker-drug is [ka] (This is also referred to herein as linker-drug(2) form A or linker-drug(2-A)), or a salt thereof. In other embodiments, the linker-drug is the (S)-epimer of linker-drug(2) or a salt thereof. Thus, the linker-drug is, [ka] (This is also referred to herein as Linker-drug(2) form B or Linker-drug(2-B)), or a salt thereof.

[0109] Other suitable linker-drugs may be found in International PCT Patent Applications PCT / JP2021 / 162299, PCT / JP2022 / 036852 (International Patent Application Publication WO2023 / 054714, published on April 6, 2023), and PCT / JP2022 / 036835 (International Patent Application Publication WO2023 / 054706, published on April 6, 2023) (each of these is incorporated herein by reference in its entirety).

[0110] As used herein, the term “salt” includes, for example, salts of inorganic acids, salts of organic acids, salts of inorganic bases, salts of organic bases, and salts of amino acids. Examples of salts of inorganic acids include salts of hydrogen chloride, hydrogen bromide, phosphoric acid, sulfuric acid, and nitric acid. Examples of salts of organic acids include salts of formic acid, acetic acid, trifluoroacetic acid, lactic acid, tartaric acid, fumaric acid, oxalic acid, maleic acid, citric acid, succinic acid, malic acid, benzenesulfonic acid, and p-toluenesulfonic acid. Examples of salts of inorganic bases include salts of alkali metals (e.g., sodium, potassium), alkaline earth metals (e.g., calcium, magnesium), other metals such as zinc and aluminum, and ammonium. Examples of salts of organic bases include salts of trimethylamine, triethylamine, propylenediamine, ethylenediamine, pyridine, ethanolamine, monoalkylethanolamine, dialkylethanolamine, diethanolamine, and triethanolamine. Examples of amino acid salts include salts of basic amino acids (e.g., arginine, histidine, lysine, ornithine) and acidic amino acids (e.g., aspartic acid, glutamic acid). The salts are preferably salts of inorganic acids (e.g., hydrogen chloride) or organic acids (e.g., trifluoroacetic acid).

[0111] Antibody-linker-drug conjugation In some embodiments, the antibody may be modified before conjugation to a linker-drug. Modification of the antibody can produce a modified antibody containing one or more reactive groups suitable for conjugation to a linker-drug.

[0112] In some embodiments, the antibody may be modified at one or more amino acid residues to provide one or more reactive groups suitable for linker-drug conjugation. For example, amino acid residues and their side chains in the antibody may be chemically modified as described below and conjugated to a linker-drug. In certain embodiments, the attachment site of the linker-drug (e.g., drug conjugate portion) to Ab is via a cysteine ​​or lysine residue of the DLL3 antibody, as described below.

[0113] Chemical conjugation at lysine residues In some embodiments, the antibody can be conjugated to a linker-drug using the chemical conjugation with an affinity peptide (CCAP) method disclosed in US Patent Application Publication No. 2021 / 0139549A1 (incorporated herein by reference in its entirety) (Ajinomoto Co. Inc.). Specifically, the lysine residues of the DLL3 antibody disclosed herein are contacted with an affinity peptide conjugated to a thiophenol activating moiety, as shown in Schemes 1 and 2 below, to react the thiophenol with the lysine residues on the antibody and conjugate the affinity peptide to the lysine via a peptide bond, thereby chemically modifying it.

Chemical formula

[0114] Subsequently, the bond between the carboxyl (C=O) and sulfur can be cleaved to obtain a modified lysine residue having a terminal sulfhydryl group, as shown in Scheme 2 below.

Chemical formula

[0115] The modification occurs at lysine (e.g., K246 or K248 according to EU numbering) on each chain of the antibody, thereby providing two conjugation sites, as shown in Scheme 3 below.

Chemical formula

[0116] In some embodiments, the modified lysine residue is located at one or more of the following EU numbering positions: 246, 248, 288, 290, or 317 (also referred to herein as K246, K248, K288, K290, or K317, respectively). In further embodiments, the lysine residue is EU numbering K246 and / or K248. In further embodiments, the lysine residue is EU numbering K248.

[0117] As used herein, K246, K248, K288, K290, or K317 refer to amino acid residues (lysine, Lys, K) of immunoglobulins identified by EU numbering. As will be understood by those skilled in the art, such numbering therefore represents amino acid residues of polypeptides aligned to those identified by immunoglobulins, such as those shown at www.imgt.org / IMGTScientificChart / Numbering / Hu_IGHGnber.html. For example, in some embodiments, K246 refers to the 150th amino acid residue of SEQ ID NO: 10, which is lysine; K248 refers to the 152nd amino acid residue of SEQ ID NO: 10, which is also lysine; K288 refers to the 192nd amino acid residue of SEQ ID NO: 10; K290 refers to the 194th amino acid residue of SEQ ID NO: 10; and K317 refers to the 221st amino acid residue of SEQ ID NO: 10. In another embodiment, K246 refers to the 148th amino acid residue of SEQ ID NO: 4 or 5, which is lysine; K248 refers to the 150th amino acid residue of SEQ ID NO: 4 or 5, which is similarly lysine; K288 refers to the 190th amino acid residue of SEQ ID NO: 4 or 5; K290 refers to the 192nd amino acid residue of SEQ ID NO: 4 or 5; and K317 refers to the 219th amino acid residue of SEQ ID NO: 4 or 5.

[0118] Accordingly, DLL3 antibodies are provided herein, having one or more side-chain lysyl (-(CH2)4NH2) of its lysine residues modified to -(CH2)4NHCO(CH2)2SH, for example, as illustrated in Scheme 3. In some embodiments, the DLL3 antibody comprises a VHH comprising CDR1, CDR2, and CDR3 as disclosed herein. In further embodiments, the DLL3 antibody comprises a VHH as disclosed herein. In further embodiments, the DLL3 antibody comprises a VHH-Fc fusion as disclosed herein, comprising, for example, the amino acid sequence described in any one of SEQ ID NOs: 4, 5, or 10. Additionally or alternatively, the modified lysine residue is selected from one or more of the following: K246, K248, K288, K290, or K317. In further embodiments, the modified lysine residue is K246 and / or K248. In further embodiments, the modified lysine residue is K248. These modified DLL3 antibodies of the present invention are useful, for example, as synthetic intermediates in the production of DLL3 ADCs disclosed herein.

[0119] According to Scheme 4, the terminal sulfhydryl group can be reacted with the maleimide moiety on the linker drug, such as the maleimide moiety on linker drug (1) (or its salt), linker drug (1-A) (or its salt), linker drug (1-B) (or its salt), linker drug (2) (or its salt), linker drug (2-A) (or its salt), or linker drug (2-B) (or its salt), to conjugate the linker drug to them. [ka]

[0120] In one embodiment, the present disclosure is a method for preparing DLL3-ADC of formula (I) or a salt thereof as disclosed herein, a) A step of providing a solution containing DLL3 antibody, b) The step of contacting the solution of a) with the affinity peptide conjugated to the thiophenol activation site, The step of contacting the solution of c)b) with a solution containing the linker drug (1) or a salt thereof, [ka] The present invention provides a method comprising the steps for producing an ADC. In some embodiments, linker-drug(1) is linker-drug(1-A). In some embodiments, linker-drug(1) is linker-drug(1-B). In some embodiments, linker-drug(1) is a mixture of linker-drug(1-A) and linker-drug(1-B).

[0121] In another embodiment, the present disclosure is a method for preparing DLL3-ADC of formula (II) or a salt thereof as disclosed herein, a) A step of providing a solution containing DLL3 antibody, b) The step of contacting the solution of a) with the affinity peptide conjugated to the thiophenol activation site, The step of contacting the solution of c)b) with a solution containing the linker-drug (2) or a salt thereof, [ka] The present invention provides a method comprising the steps for producing an ADC. In some embodiments, linker-drug(2) is linker-drug(2-A). In some embodiments, linker-drug(2) is linker-drug(2-B). In some embodiments, linker-drug(2) is a mixture of linker-drug(2-A) and linker-drug(2-B).

[0122] In one embodiment, the affinity peptide is the peptide of SEQ ID NO: 6. In one embodiment, the affinity peptide conjugated to the thiophenol activation moiety is [ka] In the formula, Ac is the acetyl moiety, and the line connecting the sulfur of the cysteine ​​residue represents a disulfide crosslink. Other suitable affinity peptides and thiophenol activating moieties can be found, for example, in U.S. Patent Application Publication No. 2021 / 0139549A1.

[0123] Stochastic conjugation of reduced interchain disulfide bonds In some embodiments, the DLL3 antibody is conjugated to a linker-drug via stochastic conjugation to reduced interchain disulfide bonds in the antibody. For example, the IgG1 antibody consists of two heavy chains containing four polypeptide chains, VH, CH1, and Fc (e.g., hinge, CH2, and CH3) domains, and two light chains containing VL and CL domains linked by interchain cysteine ​​disulfide (-SS-) bonds (e.g., interchain disulfide bonds between two heavy chains and interchain disulfide bonds between two hinge heavy chains). In certain embodiments, when these disulfide bonds are broken under reducing conditions, eight reactive cysteine ​​sulfhydryl moieties are produced. In certain embodiments, each of the eight reactive cystine sulfhydryl moieties is a linker-drug attachment site such that up to eight (n=8) linker-drugs can attach to the reducing antibody. In a VHH-Fc fusion antibody, the antibody may comprise two chains, each containing a VHH domain fused to Fc (e.g., hinge, CH2, and CH3), and the two chains are linked via a disulfide bond between the two hinge chains (see, for example, Figure 1A). In certain embodiments, when these disulfide bonds are broken under reducing conditions, four reactive cysteine ​​sulfhydryl moieties are produced. In certain embodiments, each of the four reactive cysteine ​​sulfhydryl moieties is a linker-drug attachment site, such that up to four (n=4) linker-drugs can attach to the reducing antibody.

[0124] In some embodiments, the interchain disulfide bond is between two cysteine residues, is broken under reducing conditions, and results in two reactive cysteine sulfhydryl moieties. In further embodiments, the interchain disulfide bridge in the DLL3 antibody is between the heavy chain and the light chain. Additionally or alternatively, the interchain disulfide bridge in the DLL3 antibody is between two heavy chains. In some embodiments, the interchain disulfide bridge in the DLL3 antibody is between two VHH chains. In some embodiments, the interchain disulfide bridge in the DLL3 antibody is between two VHH-Fc chains. In some embodiments, the cysteine residue is in the hinge region of the DLL3 antibody. In some embodiments, the cysteine residue is at any one or more of positions 226 or 229 according to EU numbering (also referred to herein as C226 or C229, respectively).

[0125] As used herein, C226 and C229 refer to the amino acid residues (cysteine, Cys, C) of an immunoglobulin identified according to EU numbering. As will be understood by those skilled in the art, such numbering thus represents the amino acid residues of a polypeptide aligned to those identified in an immunoglobulin, such as that shown at www.imgt.org / IMGTScientificChart / Numbering / Hu_IGHGnber.html. For example, in some embodiments, C226 refers to the 130th amino acid residue of SEQ ID NO: 10, which is cysteine, and C229 refers to the 133rd amino acid residue of SEQ ID NO: 10, which is also cysteine. In another embodiment, C226 refers to the 128th amino acid residue of SEQ ID NO: 4 or 5, which is cysteine, and C229 refers to the 131st amino acid residue of SEQ ID NO: 4 or 5, which is also cysteine.

[0126] In certain embodiments, the DLL3 antibody disclosed herein comprises two interchain disulfide bonds in the hinge region, which may be reduced to break the bonds and reveal a reactive sulfhydryl moiety that can be conjugated with a maleimide moiety on a linker-drug, such as a maleimide moiety on linker-drug (1) (or a salt thereof) or linker-drug (2) (or a salt thereof).

[0127] In one embodiment, the present disclosure relates to a method for producing DLL3-ADC of formula (I) or a salt thereof, a) A step of providing a solution containing DLL3 antibody, b) The step of bringing the solution of a) into contact with a reducing agent, The step of contacting the solution of c)b) with a solution containing the linker drug (1) or a salt thereof, [ka] The present invention provides a method comprising the steps for producing an ADC. In some embodiments, linker-drug(1) is linker-drug(1-A). In some embodiments, linker-drug(1) is linker-drug(1-B). In some embodiments, linker-drug(1) is a mixture of linker-drug(1-A) and linker-drug(1-B).

[0128] In one embodiment, the present disclosure relates to a method for preparing DLL3-ADC of formula (II) or a salt thereof, a) A step of providing a solution containing DLL3 antibody, b) The step of bringing the solution of a) into contact with a reducing agent, The step of contacting the solution of c)b) with a solution containing the linker-drug (2) or a salt thereof, [ka] The present invention provides a method comprising the steps for producing an ADC. In some embodiments, linker-drug(2) is linker-drug(2-A). In some embodiments, linker-drug(2) is linker-drug(2-B). In some embodiments, linker-drug(2) is a mixture of linker-drug(2-A) and linker-drug(2-B).

[0129] In one embodiment, the reducing agent is tris(2-carboxyethyl)phosphine (TCEP).

[0130] DLL3-ADCS DLL3-ADC can be produced by conjugating any of the DLL3 antibodies described herein with any of the linker-drugs or salts thereof described herein.

[0131] In one embodiment, the present disclosure relates to the ADC of formula (I), [ka] or a salt thereof n is an integer between 1 and 8. Ab provides an ADC of formula (I) or a salt thereof, representing an antibody that binds to DLL3 ("DLL3 antibody"). In some embodiments, the ADC is the (R)-epimer of formula (I) or a salt thereof. Thus, the ADC is of the following formula (IA): [ka] or a salt thereof. In some embodiments, ADC is the (S)-epimer of formula (I) or a salt thereof. Thus, ADC is the one of the following formula (IB), [ka] or its salt.

[0132] In one embodiment, DLL3-ADC of formula (I), (IA), (IB), or any salt thereof comprises (1) CDR1, CDR2, and CDR3 of VHH, wherein the VHH comprises the amino acid sequence described in SEQ ID NO: 14, 15, or 16, or (2) at least one VHH, wherein the at least one VHH comprises CDR1, CDR2, and CDR3, and the DLL3 antibody comprises (i) CDR1 comprising the amino acid sequence described in SEQ ID NO: 1 or 7, (ii) CDR2 comprising the amino acid sequence described in SEQ ID NO: 2 or 8, and (iii) CDR3 comprising the amino acid sequence described in SEQ ID NO: 3 or 9.

[0133] In one embodiment, DLL3-ADC of formula (I), (IA), (IB), or any salt thereof comprises a DLL3 antibody comprising (a) CDR1 as described in SEQ ID NO: 1, CDR2 as described in SEQ ID NO: 2, and CDR3 as described in SEQ ID NO: 3, or (b) CDR1 as described in SEQ ID NO: 7, CDR2 as described in SEQ ID NO: 8, and CDR3 as described in SEQ ID NO: 9. In one embodiment, DLL3-ADC of formula (I), (IA), (IB), or any salt thereof comprises a DLL3 antibody comprising VHH containing the amino acid sequence described in any one of SEQ ID NOs: 14 to 16. In one embodiment, DLL3-ADC of formula (I), (IA), (IB), or any salt thereof comprises a DLL3 antibody further comprising a human IgG constant domain. In one embodiment, human IgG is human IgG1. In one embodiment, DLL3-ADC of formula (I), (IA), (IB), or any salt thereof comprises a DLL3 antibody further comprising an Fc fragment. In one embodiment, the Fc fragment comprises a homodimer of the amino acid sequence described in SEQ ID NO: 17. In one embodiment, DLL3-ADC of formula (I), (IA), (IB), or a salt of any of them comprises a DLL3 antibody containing at least one VHH domain. In one embodiment, DLL3-ADC of formula (I), (IA), (IB), or a salt of any of them comprises a DLL3 antibody containing two VHH domains (each of which is conjugated to the Fc fragment chain). In one embodiment, DLL3-ADC of formula (I), (IA), (IB), or a salt of any of them comprises a homodimer of the amino acid sequence described in any one of SEQ ID NOs: 4, 5, or 10.

[0134] In one embodiment, n is 1 in formula (I), (IA), or (IB) (or a salt thereof). In one embodiment, n is 2. In one embodiment, n is 3. In one embodiment, n is 4. In one embodiment, n is 5. In one embodiment, n is 6. In one embodiment, n is 7. In one embodiment, n is 8.

[0135] In one embodiment, the present disclosure relates to the ADC of formula (II), [ka] or a salt thereof n is an integer between 1 and 8. Ab provides an ADC of formula (II) or a salt thereof, representing an antibody that binds to DLL3 ("DLL3 antibody"). In some embodiments, the ADC is the (R)-epimer of formula (II) or a salt thereof. Thus, the ADC is the one of formula (II-A), [ka] or a salt thereof. In some embodiments, ADC is the (S)-epimer of formula (II) or a salt thereof. Thus, ADC is the one of formula (II-B), [ka] or its salt.

[0136] In one embodiment, DLL3-ADC of formula (II), (II-A), (II-B), or any salt thereof comprises (1) CDR1, CDR2, and CDR3 of VHH, wherein the VHH comprises the amino acid sequence described in SEQ ID NO: 14, 15, or 16, or (2) at least one VHH, wherein the at least one VHH comprises CDR1, CDR2, and CDR3, and the DLL3 antibody comprises (i) CDR1 comprising the amino acid sequence described in SEQ ID NO: 1 or 7, (ii) CDR2 comprising the amino acid sequence described in SEQ ID NO: 2 or 8, and (iii) CDR3 comprising the amino acid sequence described in SEQ ID NO: 3 or 9.

[0137] In one embodiment, a DLL3-ADC of formula (II), (II-A), (II-B), or any salt thereof comprises a DLL3 antibody comprising (a) CDR1 as described in SEQ ID NO: 1, CDR2 as described in SEQ ID NO: 2, and CDR3 as described in SEQ ID NO: 3, or (b) CDR1 as described in SEQ ID NO: 7, CDR2 as described in SEQ ID NO: 8, and CDR3 as described in SEQ ID NO: 9. In one embodiment, a DLL3-ADC of formula (II), (II-A), (II-B), or any salt thereof comprises a DLL3 antibody comprising VHH as described in any one of SEQ ID NOs: 14 to 16. In one embodiment, a DLL3-ADC of formula (II), (II-A), (II-B), or any salt thereof comprises a DLL3 antibody further comprising a human IgG constant domain. In one embodiment, human IgG is human IgG1. In one embodiment, a DLL3-ADC of formula (II), (II-A), (II-B), or any salt thereof comprises a DLL3 antibody further comprising an Fc fragment. In one embodiment, the Fc fragment comprises a homodimer of the amino acid sequence described in SEQ ID NO: 17. In one embodiment, DLL3-ADC of formula (II), (II-A), (II-B), or a salt thereof comprises a DLL3 antibody containing at least one VHH domain. In one embodiment, DLL3-ADC of formula (II), (II-A), (II-B), or a salt thereof comprises a DLL3 antibody containing two VHH domains (each of which is conjugated to the Fc fragment chain). In one embodiment, DLL3-ADC of formula (II), (II-A), (II-B), or a salt thereof comprises a homodimer of the amino acid sequence described in any one of SEQ ID NOs: 4, 5, or 10.

[0138] In one embodiment, n is 1 in formula (II), (II-A), or (II-B) (or a salt thereof). In one embodiment, n is 2. In one embodiment, n is 3. In one embodiment, n is 4. In one embodiment, n is 5. In one embodiment, n is 6. In one embodiment, n is 7. In one embodiment, n is 8.

[0139] In one embodiment, the DLL3-ADC is defined by the following formula (A): [ka] Each thick shaded line represents a chain of antibody that binds to DLL3 ("DLL3 antibody"), the sulfur shown originates from a cysteine ​​residue of the DLL3 antibody, and X represents the following structure or a salt thereof. [ka] In the formula, the dashed line represents the attachment point of the ADC of formula (A) to the rest of the antibody. In some embodiments, the cysteine ​​residue is located in the hinge region of the DLL3 antibody. In further embodiments, the cysteine ​​residue forms an interchain disulfide crosslink in the DLL3 antibody that is not directly or indirectly conjugated to X, for example, between two heavy chains, between a heavy chain and a light chain, between two VHH chains, or between two VHH-Fc chains. In further embodiments, the cysteine ​​residue is located at one or more of the EU numbering positions 226 or 229 (also referred to herein as C226 or C229, respectively). In some embodiments, X is the (R)-epimer or a salt thereof of the above structure. Thus, X is [ka] or a salt thereof, where the dashed line represents the attachment point of ADC to the remainder of equation (A). In some embodiments, X is the (S)-epimer or a salt thereof of the above structure. Thus, X is [ka] or its salt, where the dashed line indicates the attachment point of ADC to the remainder of equation (A).

[0140] In one embodiment, the DLL3-ADC is defined by the following formula (B): [ka] In the formula, each thick shaded line represents a chain of antibody that binds to DLL3 ("DLL3 antibody"), the illustrated CH2CH2CH2CH2NH portion originates from a lysine residue in the CH2 domain of the DLL3 antibody, and X represents the following structure or a salt thereof. [ka] In the formula, the dashed line indicates the attachment point of the ADC of formula (B) to the rest of the molecule. In some embodiments, the lysine residue is located at one or more of the EU numbering positions 246, 248, 288, 290, or 317 (also referred to herein as K246, K248, K288, K290, or K317, respectively). In some embodiments, X is the (R)-epimer or a salt thereof of the above structure. Thus, X is [ka] or a salt thereof, where the dashed line represents the attachment point of ADC to the remainder of equation (B). In some embodiments, X is the (S)-epimer or a salt thereof of the above structure. Thus, X is [ka] or its salt, where the dashed line indicates the attachment point of ADC to the remainder of equation (B).

[0141] In one embodiment, DLL3-ADC is the one shown in formula (A) above, or a salt thereof, where each thick shaded line represents a chain of antibody bound to DLL3 ("DLL3 antibody"), the sulfur shown is derived from a cysteine ​​residue of the DLL3 antibody, and X represents the following structure or a salt thereof. [ka] In the formula, the dashed line represents the attachment point to each maleimide moiety illustrated in formula (A). In some embodiments, the cysteine ​​residue is located in the hinge region of the DLL3 antibody. In further embodiments, the cysteine ​​residue forms an interchain disulfide crosslink in the DLL3 antibody that is not directly or indirectly conjugated to X, for example, between two heavy chains, between a heavy chain and a light chain, between two VHH chains, or between two VHH-Fc chains. In further embodiments, the cysteine ​​residue is located at one or more of the EU numbering positions 226 or 229 (also referred to herein as C226 or C229, respectively). In some embodiments, X is the (R)-epimer or a salt thereof of the above structure. Thus, X is [ka] or a salt thereof, where the dashed line represents the attachment point of ADC to the remainder of equation (A). In some embodiments, X is the (S)-epimer or a salt thereof of the above structure. Thus, X is [ka] or its salt, where the dashed line indicates the attachment point of ADC to the remainder of equation (A).

[0142] In one embodiment, the DLL3-ADC is represented by formula (B) shown above, where each thick shaded line represents a chain of antibody bound to DLL3 ("DLL3 antibody"), the illustrated CH2CH2CH2CH2NH portion is derived from a lysine residue in the CH2 domain of the DLL3 antibody, and X represents the following structure or a salt thereof. [ka] In the formula, the dashed lines represent attachment points to each maleimide moiety illustrated in formula (B). In some embodiments, the lysine residue is located at one or more of the EU numbering positions 246, 248, 288, 290, or 317 (also referred to herein as K246, K248, K288, K290, or K317, respectively). In some embodiments, X is the (R)-epimer or a salt thereof of the above structure. Thus, X is [ka] or a salt thereof, where the dashed line represents the attachment point of ADC to the remainder of equation (B). In some embodiments, X is the (S)-epimer or a salt thereof of the above structure. Thus, X is [ka] or its salt, where the dashed line indicates the attachment point of ADC to the remainder of equation (B).

[0143] In some embodiments, the DLL3 antibody of formula (A) or formula (B) comprises the amino acid sequence described in SEQ ID NO: 4, where the lysine residue is K248 of the DLL3 antibody, for example, the 150th amino acid residue of SEQ ID NO: 4. In some embodiments, the DLL3 antibody of formula (A) or formula (B), or a salt of either thereof, comprises the amino acid sequence described in SEQ ID NO: 5, where the lysine residue is K248 of the DLL3 antibody, for example, the 150th amino acid residue of SEQ ID NO: 5. In some embodiments, the DLL3 antibody of formula (A) or formula (B), or a salt of either thereof, comprises the amino acid sequence described in SEQ ID NO: 10, where the lysine residue is K248 of the DLL3 antibody, for example, the 152nd amino acid residue of SEQ ID NO: 10.

[0144] Additionally or alternatively, the DLL3 antibody of formula (A) or formula (B) comprises the amino acid sequence described in SEQ ID NO: 4, where the lysine residue is K246 of the DLL3 antibody, for example, the 148th amino acid residue of SEQ ID NO: 4. In some embodiments, the DLL3 antibody of formula (A) or formula (B), or a salt of either thereof, comprises the amino acid sequence described in SEQ ID NO: 5, where the lysine residue is K246 of the DLL3 antibody, for example, the 148th amino acid residue of SEQ ID NO: 5. In some embodiments, the DLL3 antibody of formula (A) or formula (B), or a salt of either thereof, comprises the amino acid sequence described in SEQ ID NO: 10, where the lysine residue is K246 of the DLL3 antibody, for example, the 150th amino acid residue of SEQ ID NO: 10.

[0145] In one embodiment, the DLL3-ADC is defined by the following formula (C): [ka] or a salt thereof, where each thick shaded line represents a chain of antibody bound to DLL3 ("DLL3 antibody"), and the illustrated CH2CH2CH2CH2NH portion is derived from a lysine residue of the CH2 domain of the DLL3 antibody. In some embodiments, DLL3-ADC is the (R)-epimer of formula (C) or a salt thereof. [ka] In some embodiments, DLL3-ADC is the (S)-epimer of formula (C), for example, formula (CB), or a salt thereof. [ka]

[0146] In one embodiment, the DLL3-ADC is defined by the following formula (D): [ka] or a salt thereof, where each thick shaded line represents a chain of antibody bound to DLL3 ("DLL3 antibody"), and the illustrated CH2CH2CH2CH2NH portion is derived from a lysine residue in the CH2 domain of the DLL3 antibody. In some embodiments, DLL3-ADC is the (R)-epimer of formula (D), e.g., formula (DA), or a salt thereof. [ka] In some embodiments, DLL3-ADC is the (S)-epimer of formula (D), for example, formula (DB), or a salt thereof. [ka]

[0147] In some embodiments, the DLL3 antibody of formula (C) or formula (D), or a salt thereof, comprises the amino acid sequence described in SEQ ID NO: 4, where the lysine residue is K248 of the DLL3 antibody, for example, the 150th amino acid residue of SEQ ID NO: 4. In some embodiments, the DLL3 antibody of formula (C) or formula (D), or a salt thereof, comprises the amino acid sequence described in SEQ ID NO: 5, where the lysine residue is K248 of the DLL3 antibody, for example, the 150th amino acid residue of SEQ ID NO: 5. In some embodiments, the DLL3 antibody of formula (C) or formula (D), or a salt thereof, comprises the amino acid sequence described in SEQ ID NO: 10, where the lysine residue is K152 of the DLL3 antibody, for example, the 152nd amino acid residue of SEQ ID NO: 10.

[0148] Additionally or alternatively, the DLL3 antibodies of formula (C), (CA), (CB), (D), (DA), (DB), or any salt thereof, contain the amino acid sequence described in SEQ ID NO: 4, where the lysine residue is K246 of the DLL3 antibody, for example, the 148th amino acid residue of SEQ ID NO: 4. In some embodiments, the DLL3 antibodies of formula (C), (CA), (CB), (D), (DA), (DB), or any salt thereof, contain the amino acid sequence described in SEQ ID NO: 5, where the lysine residue is K246 of the DLL3 antibody, for example, the 148th amino acid residue of SEQ ID NO: 5. In some embodiments, the DLL3 antibodies of formula (C), (CA), (CB), (D), (DA), (DB), or any salt thereof, contain the amino acid sequence described in SEQ ID NO: 10, where the lysine residue is K246 of the DLL3 antibody, for example, the 150th amino acid residue of SEQ ID NO: 10.

[0149] In some embodiments, DLL3-ADC is represented by formula (I), (IA), (IB), (II), (II-A), (II-B), or a salt thereof, where Ab is a DLL3 antibody comprising a VHH containing one or more CDRs having the same or substantially the same amino acid sequence as one or more of SEQ ID NOs. 1-3 and 7-9.

[0150] In some embodiments, DLL3-ADC is represented by formula (I), (IA), (IB), (II), (II-A), (II-B), or a salt thereof, where Ab is a DLL3 antibody containing a VHH having the same or substantially the same amino acid sequence as one or more of SEQ ID NOs. 14-16.

[0151] In one embodiment, a DLL3-ADC of formula (I), (IA), (IB), or any salt thereof comprises an antibody containing a VHH having the same or substantially the same amino acid sequence as SEQ ID NO: 14, where n is 2. In one embodiment, a DLL3-ADC of formula (I), (IA), (IB), or any salt thereof comprises an antibody containing a VHH having the same or substantially the same amino acid sequence as SEQ ID NO: 14, where n is 4. In one embodiment, a DLL3-ADC of formula (I), (IA), (IB), or any salt thereof comprises linker-drug (1), linker-drug (1-A), linker-drug (1-B), or any salt thereof, which are conjugated to a DLL3 antibody of formula (I), (IA), (IB), or any salt thereof via the K248 residue of each chain of the DLL3 antibody, where the DLL3 antibody contains a VHH having the same or substantially the same amino acid sequence as SEQ ID NO: 14. Therefore, n is 2. In one embodiment, a DLL3-ADC of formula (I), (IA), (IB), or any salt thereof comprises a linker-drug (1), a linker-drug (1-A), a linker-drug (1-B), or any salt thereof, which are conjugated to a DLL3 antibody of formula (I), (IA), (IB), or any salt thereof via one or more reduced disulfide crosslinks on each chain of the DLL3 antibody, the antibody comprising a VHH having the same or substantially the same amino acid sequence as SEQ ID NO: 14. In some embodiments, the one or more disulfide crosslinks before reduction are selected from those formed between C226 and C229 of two antibody chains. Thus, n is 2 or 4. In some embodiments, both C226 and C229 of both antibody chains are modified to be directly or indirectly conjugated to the linker-drugs disclosed herein, and thus n is 4.

[0152] In one embodiment, a DLL3-ADC of formula (I), (IA), (IB), or any salt thereof comprises an antibody containing a VHH having the same or substantially the same amino acid sequence as SEQ ID NO: 15, where n is 2. In one embodiment, a DLL3-ADC of formula (I), (IA), (IB), or any salt thereof comprises an antibody containing a VHH having the same or substantially the same amino acid sequence as SEQ ID NO: 15, where n is 4. In one embodiment, a DLL3-ADC of formula (I), (IA), (IB), or any salt thereof comprises linker-drug (1), linker-drug (1-A), linker-drug (1-B), or any salt thereof, which are conjugated to a DLL3 antibody of formula (I), (IA), (IB), or any salt thereof via the K248 residue of each chain of the DLL3 antibody, where the DLL3 antibody contains a VHH having the same or substantially the same amino acid sequence as SEQ ID NO: 15. Therefore, n is 2. In one embodiment, a DLL3-ADC of formula (I), (IA), (IB), or any salt thereof comprises a linker-drug (1), a linker-drug (1-A), a linker-drug (1-B), or any salt thereof, which are conjugated to a DLL3 antibody of formula (I), (IA), (IB), or any salt thereof via one or more reduced disulfide crosslinks on each chain of the DLL3 antibody, the antibody comprising a VHH having the same or substantially the same amino acid sequence as SEQ ID NO: 15. In some embodiments, the one or more disulfide crosslinks before reduction are selected from those formed between C226 and C229 of two antibody chains. Thus, n is 2 or 4. In some embodiments, both C226 and C229 of both antibody chains are modified to be directly or indirectly conjugated to the linker-drugs disclosed herein, and thus n is 4.

[0153] In one embodiment, a DLL3-ADC of formula (I), (IA), (IB), or any salt thereof comprises an antibody containing a VHH having the same or substantially the same amino acid sequence as SEQ ID NO: 16, where n is 2. In one embodiment, a DLL3-ADC of formula (I), (IA), (IB), or any salt thereof comprises an antibody containing a VHH having the same or substantially the same amino acid sequence as SEQ ID NO: 16, where n is 4. In one embodiment, a DLL3-ADC of formula (I), (IA), (IB), or any salt thereof comprises linker-drug (1), linker-drug (1-A), linker-drug (1-B), or any salt thereof, which are conjugated to a DLL3 antibody of formula (I), (IA), (IB), or any salt thereof via the K248 residue of each chain of the DLL3 antibody, where the DLL3 antibody contains a VHH having the same or substantially the same amino acid sequence as SEQ ID NO: 16. Therefore, n is 2. In one embodiment, a DLL3-ADC of formula (I), (IA), (IB), or any salt thereof comprises a linker-drug (1), a linker-drug (1-A), a linker-drug (1-B), or any salt thereof, which are conjugated to a DLL3 antibody of formula (I), (IA), (IB), or any salt thereof via one or more reduced disulfide crosslinks on each chain of the DLL3 antibody, the antibody comprising a VHH having the same or substantially the same amino acid sequence as SEQ ID NO: 16. In some embodiments, the one or more disulfide crosslinks before reduction are selected from those formed between C226 and C229 of two antibody chains. Thus, n is 2 or 4. In some embodiments, both C226 and C229 of both antibody chains are modified to be directly or indirectly conjugated to the linker-drugs disclosed herein, and thus n is 4.

[0154] In one embodiment, DLL3-ADC of formula (II), (II-A), (II-B), or a salt thereof comprises an antibody containing VHH having the same or substantially the same amino acid sequence as SEQ ID NO: 14, where n is 2. In another embodiment, DLL3-ADC of formula (II), (II-A), (II-B), or a salt thereof comprises an antibody containing VHH having the same or substantially the same amino acid sequence as SEQ ID NO: 14, where n is 4. In one embodiment, DLL3-ADC of formula (II), (II-A), (II-B), or any salt thereof comprises linker-drug (2), linker-drug (2-A), linker-drug (2-B), or any salt thereof, which are conjugated to DLL3 antibody of formula (II), (II-A), (II-B), or any salt thereof via the K248 residue of each chain of DLL3 antibody, and the DLL3 antibody contains VHH having the same or substantially the same amino acid sequence as SEQ ID NO: 14. Therefore, n is 2. In one embodiment, DLL3-ADC of formula (II), (II-A), (II-B), or a salt thereof comprises linker-drug (2), linker-drug (2-A), linker-drug (2-B), or a salt thereof, which are conjugated to DLL3 antibody of formula (II), (II-A), (II-B), or a salt thereof via one or more reduced disulfide crosslinks on each chain of the DLL3 antibody, and the antibody comprises VHH having the same or substantially the same amino acid sequence as SEQ ID NO: 14. In some embodiments, the one or more disulfide crosslinks before reduction are selected from those formed between C226 and C229 of the two antibody chains. Thus, n is 2 or 4. In some embodiments, both C226 and C229 of both antibody chains are modified to be directly or indirectly conjugated to the linker-drugs disclosed herein, and thus n is 4.

[0155] In one embodiment, DLL3-ADC of formula (II), (II-A), (II-B), or a salt thereof comprises an antibody containing VHH having the same or substantially the same amino acid sequence as SEQ ID NO: 15, where n is 2. In another embodiment, DLL3-ADC of formula (II), (II-A), (II-B), or a salt thereof comprises an antibody containing VHH having the same or substantially the same amino acid sequence as SEQ ID NO: 15, where n is 4. In one embodiment, DLL3-ADC of formula (II), (II-A), (II-B), or any salt thereof comprises linker-drug (2), linker-drug (2-A), linker-drug (2-B), or any salt thereof, which are conjugated to DLL3 antibody of formula (II), (II-A), (II-B), or any salt thereof via the K248 residue of each chain of DLL3 antibody, and the DLL3 antibody contains VHH having the same or substantially the same amino acid sequence as SEQ ID NO: 15. Therefore, n is 2. In one embodiment, a DLL3-ADC of formula (II), (II-A), (II-B), or any salt thereof comprises a linker-drug (2), a linker-drug (2-A), a linker-drug (2-B), or any salt thereof, which are conjugated to a DLL3 antibody of formula (II), (II-A), (II-B), or any salt thereof via one or more reduced disulfide crosslinks on each chain of the DLL3 antibody, wherein the antibody contains a VHH having the same or substantially the same amino acid sequence as SEQ ID NO: 15. In some embodiments, the one or more disulfide crosslinks before reduction are selected from those formed between C226 and C229 of two antibody chains. Thus, n is 2 or 4. In some embodiments, both C226 and C229 of both antibody chains are modified to be directly or indirectly conjugated to the linker-drugs disclosed herein, and thus n is 4.

[0156] In one embodiment, DLL3-ADC of formula (II), (II-A), (II-B), or a salt thereof comprises an antibody containing VHH having the same or substantially the same amino acid sequence as SEQ ID NO: 16, where n is 2. In another embodiment, DLL3-ADC of formula (II), (II-A), (II-B), or a salt thereof comprises an antibody containing VHH having the same or substantially the same amino acid sequence as SEQ ID NO: 16, where n is 4. In one embodiment, DLL3-ADC of formula (II), (II-A), (II-B), or any salt thereof comprises linker-drug (2), linker-drug (2-A), linker-drug (2-B), or any salt thereof, which are conjugated to DLL3 antibody of formula (II), (II-A), (II-B), or any salt thereof via the K248 residue of each chain of DLL3 antibody, and the DLL3 antibody contains VHH having the same or substantially the same amino acid sequence as SEQ ID NO: 16. Therefore, n is 2. In one embodiment, a DLL3-ADC of formula (II), (II-A), (II-B), or any salt thereof comprises a linker-drug (2), a linker-drug (2-A), a linker-drug (2-B), or any salt thereof, which are conjugated to a DLL3 antibody of formula (II), (II-A), (II-B), or any salt thereof via one or more reduced disulfide crosslinks on each chain of the DLL3 antibody, and the antibody contains a VHH having the same or substantially the same amino acid sequence as SEQ ID NO: 16. In some embodiments, the one or more disulfide crosslinks before reduction are selected from those formed between C226 and C229 of the two antibody chains. Thus, n is 2 or 4. In some embodiments, both C226 and C229 of both antibody chains are modified to be directly or indirectly conjugated to the linker-drugs disclosed herein, and thus n is 4.

[0157] In some embodiments, DLL3-ADC is represented by formula (I), (IA), (IB), (II), (II-A), (II-B), or a salt thereof, where Ab is a DLL3 antibody having the same or substantially the same amino acid sequence as one of SEQ ID NOs: 4, 5, and 10. In some embodiments, the cysteine ​​or lysine residue of the DLL3 antibody is modified as disclosed herein and conjugated to a linker-drug as disclosed herein.

[0158] In one embodiment, DLL3-ADC comprises a DLL3 antibody and two or more linker-drugs (1) conjugated thereto, or salts thereof. In one embodiment, DLL3-ADC comprises a DLL3 antibody and two or more linker-drugs (1-A) conjugated thereto, or salts thereof. In one embodiment, DLL3-ADC comprises a DLL3 antibody and two or more linker-drugs (1-B) conjugated thereto, or salts thereof.

[0159] In one embodiment, a DLL3-ADC of formula (I), (IA), (IB), or any salt thereof comprises a DLL3 antibody having the same or substantially the same amino acid sequence as SEQ ID NO: 4, where n is 2. In one embodiment, a DLL3-ADC of formula (I), (IA), (IB), or any salt thereof comprises a DLL3 antibody having the same or substantially the same amino acid sequence as SEQ ID NO: 4, where n is 4. In one embodiment, a DLL3-ADC of formula (I), (IA), (IB), or any salt thereof comprises linker-drug (1), linker-drug (1-A), linker-drug (1-B), or any salt thereof, which are conjugated to a DLL3 antibody of formula (I), (IA), (IB), or any salt thereof via the K248 residue of each chain of the DLL3 antibody, and the antibody has the same or substantially the same amino acid sequence as SEQ ID NO: 4. Therefore, n is 2. In one embodiment, a DLL3-ADC of formula (I), (IA), (IB), or any salt thereof comprises linker-drug (1), linker-drug (1-A), linker-drug (1-B), or any salt thereof, which are conjugated to a DLL3 antibody of formula (I), (IA), (IB), or any salt thereof via one or more reduced disulfide crosslinks on each chain of the DLL3 antibody, the antibody having the same or substantially the same amino acid sequence as SEQ ID NO: 4. In some embodiments, the one or more disulfide crosslinks before reduction are selected from those formed between C226 and C229 of the two antibody chains. Thus, n is 2 or 4. In some embodiments, both C226 and C229 of both antibody chains are modified to be directly or indirectly conjugated to the linker-drugs disclosed herein, and thus n is 4.

[0160] In one embodiment, a DLL3-ADC of formula (I), (IA), (IB), or any salt thereof comprises a DLL3 antibody having the same or substantially the same amino acid sequence as SEQ ID NO: 5, where n is 2. In one embodiment, a DLL3-ADC of formula (I), (IA), (IB), or any salt thereof comprises a DLL3 antibody having the same or substantially the same amino acid sequence as SEQ ID NO: 5, where n is 4. In one embodiment, a DLL3-ADC of formula (I), (IA), (IB), or any salt thereof comprises linker-drug (1), linker-drug (1-A), linker-drug (1-B), or any salt thereof, which are conjugated to a DLL3 antibody of formula (I), (IA), (IB), or any salt thereof via the K248 residue of each chain of the DLL3 antibody, and the antibody has the same or substantially the same amino acid sequence as SEQ ID NO: 5. Therefore, n is 2. In one embodiment, a DLL3-ADC of formula (I), (IA), (IB), or any salt thereof comprises linker-drug (1), linker-drug (1-A), linker-drug (1-B), or any salt thereof, which are conjugated to a DLL3 antibody of formula (I), (IA), (IB), or any salt thereof via one or more reduced disulfide crosslinks on each chain of the DLL3 antibody, the antibody having the same or substantially the same amino acid sequence as SEQ ID NO: 5. In some embodiments, the one or more disulfide crosslinks before reduction are selected from those formed between C226 and C229 of the two antibody chains. Thus, n is 2 or 4. In some embodiments, both C226 and C229 of both antibody chains are modified to be directly or indirectly conjugated to the linker-drugs disclosed herein, and thus n is 4.

[0161] In one embodiment, a DLL3-ADC of formula (I), (IA), (IB), or any salt thereof comprises a DLL3 antibody having the same or substantially the same amino acid sequence as SEQ ID NO: 10, where n is 2. In one embodiment, a DLL3-ADC of formula (I), (IA), (IB), or any salt thereof comprises a DLL3 antibody having the same or substantially the same amino acid sequence as SEQ ID NO: 10, where n is 4. In one embodiment, a DLL3-ADC of formula (I), (IA), (IB), or any salt thereof comprises linker-drug (1), linker-drug (1-A), linker-drug (1-B), or any salt thereof, which are conjugated to a DLL3 antibody of formula (I), (IA), (IB), or any salt thereof via the K248 residue of each chain of the DLL3 antibody, and the antibody has the same or substantially the same amino acid sequence as SEQ ID NO: 10. Therefore, n is 2. In one embodiment, a DLL3-ADC of formula (I), (IA), (IB), or any salt thereof comprises linker-drug (1), linker-drug (1-A), linker-drug (1-B), or any salt thereof, which are conjugated to a DLL3 antibody of formula (I), (IA), (IB), or any salt thereof via one or more reduced disulfide crosslinks on each chain of the DLL3 antibody, the antibody having the same or substantially the same amino acid sequence as SEQ ID NO: 10. In some embodiments, the one or more disulfide crosslinks before reduction are selected from those formed between C226 and C229 of two antibody chains. Thus, n is 2 or 4. In some embodiments, both C226 and C229 of both antibody chains are modified to be directly or indirectly conjugated to the linker-drugs disclosed herein, and thus n is 4.

[0162] In one embodiment, the DLL3-ADC comprises a DLL3 antibody and two or more linker-drugs (2) conjugated thereto, or salts thereof.

[0163] In one embodiment, DLL3-ADC of formula (II), (II-A), (II-B), or a salt thereof comprises a DLL3 antibody having the same or substantially the same amino acid sequence as SEQ ID NO: 4, where n is 2. In one embodiment, DLL3-ADC of formula (II), (II-A), (II-B), or a salt thereof comprises a DLL3 antibody having the same or substantially the same amino acid sequence as SEQ ID NO: 4, where n is 4. In one embodiment, DLL3-ADC of formula (II), (II-A), (II-B), or a salt thereof comprises linker-drug (2), linker-drug (2-A), linker-drug (2-B), or a salt thereof, which are conjugated to DLL3 antibodies of formula (II), (II-A), (II-B), or a salt thereof via the K248 residue of each chain of the DLL3 antibody, and the antibody has the same or substantially the same amino acid sequence as SEQ ID NO: 4. Therefore, n is 2. In one embodiment, DLL3-ADC of formula (II), (II-A), (II-B), or a salt of any of them comprises linker-drug (2), linker-drug (2-A), linker-drug (2-B), or a salt of any of them, which are conjugated to DLL3 antibody of formula (II), (II-A), (II-B), or a salt of any of them via one or more reduced disulfide crosslinks on each chain of the DLL3 antibody, and the antibody contains the same or substantially the same amino acid sequence as SEQ ID NO: 4. In some embodiments, the one or more disulfide crosslinks before reduction are selected from those formed between C226 and C229 of the two antibody chains. Therefore, n is 2 or 4. In some embodiments, both C226 and C229 of both antibody chains are modified to be directly or indirectly conjugated to the linker-drugs disclosed herein, and therefore n is 4.

[0164] In one embodiment, DLL3-ADC of formula (II), (II-A), (II-B), or a salt thereof comprises a DLL3 antibody having the same or substantially the same amino acid sequence as SEQ ID NO: 5, where n is 2. In one embodiment, DLL3-ADC of formula (II), (II-A), (II-B), or a salt thereof comprises a DLL3 antibody having the same or substantially the same amino acid sequence as SEQ ID NO: 5, where n is 4. In one embodiment, DLL3-ADC of formula (II), (II-A), (II-B), or a salt thereof comprises linker-drug (2), linker-drug (2-A), linker-drug (2-B), or a salt thereof, which are conjugated to DLL3 antibodies of formula (II), (II-A), (II-B), or a salt thereof via the K248 residue of each chain of the DLL3 antibody, and the antibody has the same or substantially the same amino acid sequence as SEQ ID NO: 5. Therefore, n is 2. In one embodiment, DLL3-ADC of formula (II), (II-A), (II-B), or a salt of any of them comprises linker-drug (2), linker-drug (2-A), linker-drug (2-B), or a salt of any of them, which are conjugated to DLL3 antibody of formula (II), (II-A), (II-B), or a salt of any of them via one or more reduced disulfide crosslinks on each chain of the DLL3 antibody, and the antibody contains the same or substantially the same amino acid sequence as SEQ ID NO: 5. In some embodiments, the one or more disulfide crosslinks before reduction are selected from those formed between C226 and C229 of the two antibody chains. Therefore, n is 2 or 4. In some embodiments, both C226 and C229 of both antibody chains are modified to be directly or indirectly conjugated to the linker-drugs disclosed herein, and therefore n is 4.

[0165] In one embodiment, DLL3-ADC of formula (II), (II-A), (II-B), or a salt thereof comprises a DLL3 antibody having the same or substantially the same amino acid sequence as SEQ ID NO: 10, where n is 2. In one embodiment, DLL3-ADC of formula (II), (II-A), (II-B), or a salt thereof comprises a DLL3 antibody having the same or substantially the same amino acid sequence as SEQ ID NO: 10, where n is 4. In one embodiment, DLL3-ADC of formula (II), (II-A), (II-B), or a salt thereof comprises linker-drug (2), linker-drug (2-A), linker-drug (2-B), or a salt thereof, which are conjugated to DLL3 antibody of formula (II), (II-A), (II-B), or a salt thereof via the K248 residue of each chain of the DLL3 antibody, and the antibody has the same or substantially the same amino acid sequence as SEQ ID NO: 10. Therefore, n is 2. In one embodiment, DLL3-ADC of formula (II), (II-A), (II-B), or a salt of any of them comprises linker-drug (2), linker-drug (2-A), linker-drug (2-B), or a salt of any of them, which are conjugated to DLL3 antibody of formula (II), (II-A), (II-B), or a salt of any of them via one or more reduced disulfide crosslinks on each chain of the DLL3 antibody, and the antibody contains the same or substantially the same amino acid sequence as SEQ ID NO: 10. In some embodiments, the one or more disulfide crosslinks before reduction are selected from those formed between C226 and C229 of the two antibody chains. Therefore, n is 2 or 4. In some embodiments, both C226 and C229 of both antibody chains are modified to be directly or indirectly conjugated to the linker-drugs disclosed herein, and therefore n is 4.

[0166] The DLL3-ADC of the present invention can possess excellent properties such as a long retention time in the body, a high monomer ratio (low aggregation rate), high release of functional substances in human cells, high stability (e.g., in mouse plasma), and desired cytotoxicity against target cells (such as DLL3-expressing or overexpressing cancer cells).

[0167] composition In another embodiment, the Disclosure provides compositions comprising DLL3-ADCs disclosed herein. A composition may comprise a plurality of DLL3-ADCs disclosed herein, each DLL3-ADC in the composition independently comprising formula (I), (IA), (IB), (II), (II-A), (II-B), or a salt thereof, where n is independently 1, 2, 3, or 4. In other words, each antibody molecule in the composition may be conjugated to 1, 2, 3, or 4 linker-drugs. Thus, compositions may be characterized by a “drug-to-antibody” ratio (DAR) ranging from about 1 to about 4. Methods for determining the DAR are well known to those skilled in the art and include methods using reversed-phase chromatography or HPLC-MS.

[0168] For example, in any embodiment, a composition comprising multiple DLL3-ADCs may exhibit DARs of about 1 to about 4, about 1 to about 3, about 1 to about 2, about 2 to about 4, about 2 to about 3, about 3 to about 4, about 1.5 to about 2, about 1.8 to about 2, about 1.9 to about 2, about 3.5 to about 4, about 3.6 to about 4, about 3.7 to about 4, about 3.8 to about 4, about 3.9 to about 4, about 3.5 to about 3.9, about 3.6 to about 3.9, about 3.7 to about 3.9, about 3.8 to about 3.9, about 3.5 to about 3.8, about 3.6 to about 3.8, about 3.7 to about 3.8, about 3.5 to about 3.7, about 3.6 to about 3.7, or about 3.5 to about 3.6. In one embodiment, the DAR is about 1 to about 2. In one embodiment, DAR is about 1.1, about 1.2, about 1.3, about 1.4, about 1.5, about 1.6, about 1.7, about 1.8, about 1.9, or about 2. In one embodiment, DAR is about 2 to about 4. In one embodiment, DAR is about 2, about 2.1, about 2.2, about 2.4, about 2.4, about 2.5, about 2.6, about 2.7, about 2.8, about 2.9, about 3, about 3.1, about 3.2, about 3.3, about 3.4, about 3.5, about 3.6, about 3.7, about 3.8, about 3.9, or about 4.

[0169] In some embodiments, the DLL3-ADC comprises a DLL3 antibody disclosed herein, conjugated to a linker-drug disclosed herein via a lysine residue of the antibody disclosed herein, such as K246, K248, K288, K290, or K317. Thus, compositions comprising DLL3-ADC are provided herein. In some embodiments, the compositions comprising DLL3-ADC exhibit a DAR of about 1.5 to about 2.5. In some embodiments, the compositions exhibit a DAR of 1.5 or greater, 1.6 or greater, 1.7 or greater, 1.8 or greater, or 1.9 or greater. Additionally or alternatively, the compositions exhibit a DAR of 2.5 or less, 2.4 or less, 2.3 or less, 2.2 or less, or 2.1 or less. In some embodiments, the compositions exhibit a DAR of 1.6 to 2.4, 1.7 to 2.3, 1.8 to 2.2, or 1.9 to 2.1. In some embodiments, compositions containing DLL3-ADC exhibit a DAR of about 1.9 to about 2.1.

[0170] In one embodiment, a composition comprising DLL3-ADC disclosed herein may exhibit a DAR of about 2, and the linker-drug is conjugated to the lysine of the DLL3 antibody of DLL3-ADC (e.g., K246, K248, K288, K290, or K317 in the CH2 domain) via site-specific conjugation as described herein. In one embodiment, a composition comprising DLL3-ADC disclosed herein may exhibit a DAR of about 4, and the linker-drug is conjugated to, for example, a reduced cysteine ​​portion of an interchain disulfide crosslink in the hinge region of the DLL3 antibody of DLL3-ADC.

[0171] Administration, formulation, and dosage In another aspect, the Disclosure provides a pharmaceutical composition comprising a DLL3-ADC disclosed herein and a pharmaceutically acceptable carrier. In another aspect, the Disclosure provides a pharmaceutical composition ("DLL3-ADC Pharmaceutical Composition") comprising a composition of a DLL3-ADC disclosed herein and a pharmaceutically acceptable carrier. The DLL3-ADC Pharmaceutical Composition may comprise a plurality of DLL3-ADCs disclosed herein, each DLL3-ADC in the Pharmaceutical Composition independently comprises formula (I), (IA), (IB), (II), (II-A), (II-B), or a salt thereof, where n is independently 1, 2, 3, or 4. In other words, each antibody molecule in the DLL3-ADC Pharmaceutical Composition may be conjugated to 1, 2, 3, or 4 linker-drugs. Thus, the DLL3-ADC Pharmaceutical Composition may be characterized by a DAR in the range of about 1 to about 4.

[0172] In one embodiment, the DLL3-ADC pharmaceutical composition may exhibit a DAR of about 2, and the linker-drug is conjugated to the lysine of the DLL3 antibody of the DLL3-ADC (e.g., K246, K248, K288, K290, or K317 of the CH2 domain) via site-directed conjugation as described herein. In one embodiment, the DLL3-ADC pharmaceutical composition may exhibit a DAR of about 4, and the linker-drug is conjugated to, for example, the reduced cysteine ​​portion of an interchain disulfide crosslink within the hinge region of the DLL3 antibody.

[0173] In further embodiments, the DLL3-ADC pharmaceutical composition comprises 20 mM histidine. Additionally or alternatively, the DLL3-ADC pharmaceutical composition comprises 5% trehalose. Additionally or alternatively, the DLL3-ADC pharmaceutical composition has a pH of about 5 to about 6, for example, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, or 6.0. In further embodiments, the DLL3-ADC pharmaceutical composition comprises 20 mM histidine and 5% trehalose, and has a pH of about 5 to about 6, for example, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, or 6.0. In further embodiments, the DLL3-ADC pharmaceutical composition comprises 20 mM histidine and 5% trehalose, and has a pH of 6.0.

[0174] The pharmaceutical compositions of this disclosure, though not limited to those described herein, can be administered to subjects requiring administration by various routes, including oral, intravenous, intra-arterial, subcutaneous, parenteral, intranasal, intramuscular, intracranial, intracardiac, ventricular, intratracheal, buccal, rectal, intraperitoneal, intradermal, topical, transdermal, and subarachnoid, or otherwise implanted or inhaled. The compositions can be formulated as solid, semi-solid, liquid, or gaseous preparations, including tablets, capsules, powders, granules, ointments, solutions, suppositories, enemas, injections, inhalants, and aerosols. The appropriate formulation and route of administration can be selected according to the intended use and treatment regimen.

[0175] Formulations suitable for enteral administration include hard or soft gelatin capsules, pills, coated tablets, elixirs, suspensions, syrups, or inhalants, and controlled-release forms thereof.

[0176] Suitable formulations for parenteral administration (e.g., by injection) include aqueous or non-aqueous isotonic pyrogen-free sterile solutions (e.g., solutions, suspensions) in which the active ingredient is provided in a dissolved, suspended, or otherwise (e.g., within liposomes or other microparticles). Such liquids may further contain other pharmaceutically acceptable components such as antioxidants, buffers, preservatives, stabilizers, bacteriostatic agents, suspending agents, thickeners, and solutes that make the formulation isotonic with the blood (or other relevant body fluids) of the recipient to whom the formulation is intended. Examples of excipients include, for example, water, alcohol, polyols, glycerol, and vegetable oils. Examples of isotonic carriers suitable for use in such formulations include sodium chloride injection, Ringer's solution, or Ringer's lactate injection. Similarly, specific administration regimens, including dosage, timing, and repetition, are determined in accordance with the specific individual and their medical history, as well as empirical considerations such as pharmacokinetics (e.g., half-life, clearance rate, etc.).

[0177] The frequency of administration may be determined and adjusted during the treatment period based on the reduction of the number of proliferating or oncogenic cells, the maintenance of such reduction in tumor cells, the reduction of tumor cell proliferation, or the delay of metastasis development. In some embodiments, the dose may be adjusted or reduced to manage potential side effects and / or toxicity. Alternatively, a sustained-release formulation of the therapeutic composition of the subject may be appropriate.

[0178] Those skilled in the art will recognize that the appropriate dose may vary from patient to patient. Determining the optimal dosage generally involves balancing the level of therapeutic benefit against the risks or adverse side effects. In particular, the dosage level selected will depend on a variety of factors, including, but are not limited to, the activity of the particular compound, the route of administration, the time of administration, the rate of excretion of the compound, the duration of treatment, other drugs, compounds and / or substances used concomitantly, the patient's species, sex, age, weight, condition, overall health, and prior medical history. The amount of compound and the route of administration are ultimately left to the discretion of the physician, veterinarian, or clinician, but generally the dosage is selected to obtain a local concentration at the site of action that produces the desired effect without causing significant harm or adverse side effects.

[0179] In general, the DLL3-ADCs of this disclosure can be administered in a variety of ranges. These ranges include approximately 5 μg / kg body weight to approximately 100 mg / kg body weight per dose, approximately 50 μg / kg body weight to approximately 5 mg / kg body weight per dose, approximately 100 μg / kg body weight to approximately 10 mg / kg body weight per dose, and any value within the above ranges. Other ranges include approximately 100 μg / kg body weight to approximately 20 mg / kg body weight per dose, and approximately 0.5 mg / kg body weight to approximately 20 mg / kg body weight per dose. In some embodiments, the doses are at least approximately 100 μg / kg body weight, at least approximately 250 μg / kg body weight, at least approximately 750 μg / kg body weight, at least approximately 3 mg / kg body weight, at least approximately 5 mg / kg body weight, and at least approximately 10 mg / kg body weight.

[0180] In any case, the antibody or antigen-binding portion of the present disclosure is preferably administered as needed to multiple subjects (or subjects) that require administration. The frequency of administration can be determined by a person skilled in the art, such as a physician, based on considerations such as the condition being treated, the age of the subject being treated, the severity of the condition being treated, and the general health status of the subject being treated.

[0181] In some embodiments, a treatment course involving the DLL3-ADC of this disclosure includes multiple administrations of a selected drug over several weeks or months. For example, the DLL3-ADC of this disclosure may be administered once daily, every two days, every four days, weekly, every ten days, every two weeks, every three weeks, monthly, every six weeks, every two months, every ten weeks, or every three months. It will be recognized that the dosage may be modified or the intervals adjusted based on the patient's response and clinical practice.

[0182] Dosages and regimens may also be empirically determined for disclosed therapeutic compositions in individuals that have received one or more doses. For example, an increasing dose of a therapeutic composition prepared as described herein may be administered to an individual. In some embodiments, the dose may be gradually increased, decreased, or attenuated based on empirically determined or observed side effects or toxicity, respectively. To evaluate the effectiveness of the selected composition, markers of specific diseases, disorders, or conditions may be tracked, as described above. In the case of cancer, this includes direct measurement of tumor size by palpation or visual observation, indirect measurement of tumor size by X-ray or other imaging techniques, improvement assessed by direct tumor biopsy and microscopic examination of tumor specimens, measurement of indirect tumor markers (e.g., PSA for prostate cancer) or oncogenic antigens, reduction of pain or paralysis; improvement of speech, vision, breathing, or other tumor-related impairments, increased appetite, or improvement in quality of life as measured by accepted tests or extension of survival time. It will be apparent to those skilled in the art that the dose will vary depending on the individual, the type of tumor condition, the stage of the tumor condition, whether the tumor condition has begun to metastasize to other parts of the individual, and past and present treatments.

[0183] Formulations suitable for parenteral administration (e.g., intravenous injection) may contain the DLL3-ADC disclosed herein at concentrations ranging from about 10 μg / mL to about 100 mg / mL. In some embodiments, the concentration of DLL3-ADC (e.g., antibody or its antigen-binding moiety) may include 20 μg / mL, 40 μg / mL, 60 μg / mL, 80 μg / mL, 100 μg / mL, 200 μg / mL, 300 μg / mL, 400 μg / mL, 500 μg / mL, 600 μg / mL, 700 μg / mL, 800 μg / mL, 900 μg / mL, or 1 mg / mL. In some embodiments, the concentration of DLL3-ADC includes 2 mg / mL, 3 mg / mL, 4 mg / mL, 5 mg / mL, 6 mg / mL, 8 mg / mL, 10 mg / mL, 12 mg / mL, 14 mg / mL, 16 mg / mL, 18 mg / mL, 20 mg / mL, 25 mg / mL, 30 mg / mL, 35 mg / mL, 40 mg / mL, 45 mg / mL, 50 mg / mL, 60 mg / mL, 70 mg / mL, 80 mg / mL, 90 mg / mL, or 100 mg / mL.

[0184] When used herein, the term "pharmaceutically acceptable" means that the vehicle, diluent, excipient, and / or salt thereof are chemically and / or physically compatible with the other components of the formulation and physiologically compatible with the recipient.

[0185] As used herein, the term “pharmaceutically acceptable carrier and / or excipient” means a carrier, stabilizer, and / or excipient that is pharmacologically and / or physiologically compatible with the subject and activator, as is well known in the art (see, for example, Remington's Pharmaceutical Sciences, Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), but is not limited to, pH modifiers, surfactants, adjuvants, or ionic strength enhancers. For example, pH modifiers include, but are not limited to, phosphate buffers; surfactants include, but are not limited to, cationic, anionic, or nonionic surfactants, such as Tween®-80; and ionic strength enhancers include, but are not limited to, sodium chloride. The carrier, excipient, or stabilizer is nontoxic to cells or mammals to which it is exposed at the doses and concentrations used. In most cases, the carrier is a pH-buffered aqueous solution. Examples of carriers include buffers such as phosphates, citrates, and other organic acids; antioxidants, including ascorbic acid; low molecular weight (e.g., less than about 10 amino acid residues) polypeptides; proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates such as glucose, mannose, or dextrin; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and / or nonionic surfactants such as TWEEN®, polyethylene glycol (PEG), and PLURONICS®. The term “carrier” can also refer to diluents, adjuvants (e.g., Freund’s adjuvants (complete or incomplete)), excipients, or vehicles to which the therapeutic agent is administered together. Such carriers may be sterilizing solutions such as water and oil, including those derived from petroleum, animals, plants, or synthetic sources such as peanut oil, soybean oil, mineral oil, and sesame oil.Water is an exemplary carrier when a composition (e.g., a pharmaceutical composition) is administered intravenously. Physiological saline and aqueous dextrose and glycerol solutions can also be used as liquid carriers, particularly in injectable solutions. Suitable excipients (e.g., pharmaceutical excipients) include starch, glucose, lactose, sucrose, gelatin, malt, rice, wheat flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk powder, glycerol, propylene, glycol, water, and ethanol. The composition may also contain trace amounts of wetting agents or emulsifiers, or pH buffers, as needed. The composition can be in the form of liquids, suspensions, emulsions, tablets, pills, capsules, powders, sustained-release formulations, etc. Oral compositions containing formulations may contain standard carriers such as pharmaceutical-grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, and magnesium carbonate. Examples of suitable carriers are described in Remington's Pharmaceutical Sciences (1990), Mack Publishing Co., Easton, PA. Compositions containing pharmaceutical compounds may include, together with a suitable amount of carrier, a prophylactic or therapeutically effective amount of a D3 conjugate (e.g., an anti-D3 antibody), for example, in an isolated or purified form, to provide a form for appropriate administration to a subject (e.g., a patient). The formulation should be suitable for the method of administration.

[0186] As used herein, the term “adjuvant” refers to a non-specific immunostimulant that, when administered to an organism together with an antigen or prior to it, can enhance the immune response to an antigen or alter the type of immune response within an organism. Examples of adjuvants, but not limited to these, include aluminum adjuvants (e.g., aluminum hydroxide), Freund’s adjuvants (e.g., complete and incomplete Freund’s adjuvants), Corynebacterium parvum, lipopolysaccharides, and cytokines. Freund’s adjuvants are the most commonly used adjuvants in animal studies. Aluminum hydroxide adjuvants are more commonly used in clinical trials.

[0187] Treatment method Methods for treating, preventing, or mitigating DLL3-mediated diseases, disorders, or conditions, including one or more symptoms of a DLL3-mediated disease, disorder, or condition, using the DLL3-ADCs described herein. In some embodiments, this disclosure provides methods for treating, preventing, or mitigating DLL3-positive cancer or DLL3-overexpressing cancer using the DLL3-ADCs described herein. In some embodiments, the cancer is, for example, lung cancer, including small cell lung cancer (SCLC), large cell neuroendocrine carcinoma (LCNEC), or colorectal cancer. Methods for killing tumor cells using the DLL3-ADCs described herein are also provided herein. In some embodiments, the method for killing tumor cells comprises contacting tumor cells with DLL3-ADCs of formula (I), (IA), (IB), (II), (II-A), (II-B), or salts thereof. In some embodiments, the contact is in vivo or in vitro.

[0188] The term "subject" includes all human or non-human animals, preferably humans.

[0189] The terms “DLL3-related condition” or “DLL3-related condition,” as used herein, refer to any condition caused, exacerbated by, or otherwise associated with an increase or decrease (generally an increase) in the expression or activity of DLL3 (e.g., human DLL3).

[0190] As used herein, the term “cancer” refers to the growth, proliferation, or proliferation of any tumor or malignant cell, whether primary or metastatic, including solid tumors and non-solid tumors such as leukemia.

[0191] As used herein in the context of treating a condition, the terms “treat,” “to treat,” or “to be treated” generally include, in relation to treatment or therapy, any desired therapeutic effect, whether in humans or animals, achieved, such as inhibition of the progression of a condition, including reduction of the rate of progression, cessation of the rate of progression, regression of the condition, improvement of the condition, and cure of the condition. Treatment as a preventive measure (e.g., preventive measures, prophylaxis) is also included. In the case of cancer, “to treat” may mean to weaken or slow the growth, proliferation, or metastasis of a tumor or malignant cells, or a combination thereof. With respect to tumors, “treatment” includes the removal of all or part of a tumor, inhibition or delay of tumor growth and metastasis, prevention or delay of tumor development, or any combination thereof.

[0192] As used herein, the term “therapeutic dose” refers to the amount of an active compound, or a substance, composition, or dosage form containing an active compound, that is effective in producing some desired therapeutic effect, given a reasonable benefit / risk ratio, when administered according to a desired therapeutic regimen. For example, the “therapeutic dose” of DLL3-ADC refers to the amount or concentration effective in treating a human DLL3-related disease or condition.

[0193] Treatment of diseases including cancer In one embodiment, the Disclosure provides a method for treating a disorder or disease in a mammal, comprising administering a therapeutically effective amount of DLL3-ADC disclosed herein to a subject in need of treatment (e.g., a human) in the form of a DLL3-ADC pharmaceutical composition or the like. In one embodiment, the Disclosure provides DLL3-ADC disclosed herein for use in the treatment of a disease or disorder. In another embodiment, the Use of DLL3-ADC disclosed herein for the manufacture of a drug for treating a disease or disorder is provided herein. The disorder or disease may be cancer.

[0194] Various cancers related to DLL3, whether malignant or benign, primary or secondary, can be treated or prevented in the manner provided by this disclosure. These cancers, however, may include, but are not limited to, lung cancer (including various subtypes, e.g., small cell lung cancer and non-small cell lung cancer), adrenal cancer, liver cancer, kidney cancer, bladder cancer, breast cancer, gastric cancer, ovarian cancer, cervical cancer, uterine cancer, esophageal cancer, colorectal cancer, prostate cancer, pancreatic cancer, thyroid cancer, carcinomas, sarcomas, glioblastomas, and various head and neck tumors. Examples of cancers include, for example, small cell lung cancer, large cell neuroendocrine carcinoma, glioblastoma, Ewing's sarcoma, and cancers with a neuroendocrine phenotype.

[0195] The DLL3-ADCs disclosed herein, as well as compositions and pharmaceutical compositions containing them, may be used to treat bronchogenic lung cancer, non-small cell lung cancer, squamous cell carcinoma, small cell carcinoma, large cell carcinoma, and adenocarcinoma, such as lung adenocarcinoma. Lung cancer may be refractory, recurrent, or resistant to platinum-based drugs (e.g., carboplatin, cisplatin, oxaliplatin, topotecan) and / or taxanes (e.g., docetaxel, paclitaxel, larotaxel, or cabazitaxel).

[0196] Cancers treated with DLL3-ADC disclosed herein may include large cell neuroendocrine carcinoma (LCNEC), medullary thyroid carcinoma, glioblastoma, neuroendocrine prostate cancer (NEPC), high-grade gastrointestinal pancreatic cancer (GEP), and malignant melanoma. DLL3-ADC disclosed herein, as well as compositions and pharmaceutical compositions containing the same, may be used to treat neuroendocrine tumors (both NETs and pNETs) occurring in the kidneys, urogenital tract (bladder, prostate, ovaries, cervix, and endometrium), gastrointestinal tract (colon, stomach), thyroid (medullary thyroid carcinoma), and lungs (small cell lung cancer and large cell neuroendocrine carcinoma).

[0197] As described above, DLL3-ADC, as well as compositions and pharmaceutical compositions containing it, are particularly effective in treating lung cancer, including the following subtypes: small cell lung cancer and non-small cell lung cancer (e.g., squamous cell non-small cell lung cancer or squamous cell small cell lung cancer), and large cell neuroendocrine carcinoma.

[0198] Stimulation of the immune response This disclosure also provides a method for enhancing (e.g., stimulating) an immune response in a subject, which includes administering the subject DLL3-ADC of this disclosure to such an extent that the immune response in the subject is enhanced. In one embodiment, this disclosure provides DLL3-ADC for use in enhancing (e.g., stimulating) an immune response in a subject. In another embodiment, the use of DLL3-ADC for the manufacture of a drug for enhancing (e.g., stimulating) an immune response in a subject is provided herein. In some embodiments, the subject is a mammal. In some embodiments, the subject is a human.

[0199] The term "enhance the immune response," or its grammatical variations, means stimulating, inducing, increasing, improving, or enhancing any response of the mammalian immune system. The immune response may be a cellular response (e.g., cell-mediated, such as cytotoxic T lymphocyte-mediated) or a humoral response (e.g., antibody-mediated), and may be a primary or secondary immune response. An example of immune response enhancement is CD4 +Enhanced immune responses include increased helper T cell activity and the generation of cytolytic T cells. Enhanced immune responses can be evaluated using several in vitro or in vivo measurements known to those skilled in the art, including, but not limited to, cytotoxic T lymphocyte assays, cytokine release (e.g., IL-2 production or IFN-γ production), tumor regression, survival of tumor-carrying animals, antibody production, immune cell proliferation, expression of cell surface markers, and cytotoxic activity. For example, the methods of this disclosure are useful for enhancing the immune response in mammals compared to the immune response in untreated mammals or animals not treated with the methods disclosed herein.

[0200] DLL3-ADC may be used as monotherapy alone, or in combination with chemotherapy, radiotherapy, targeted therapy, or cellular immunotherapy.

[0201] Combination with chemotherapy DLL3-ADC can be used in combination with chemotherapy, for example, anticancer agents, cytotoxic agents, or chemotherapeutic agents.

[0202] The terms “anticancer agent” or “antiproliferative agent” mean any agent that can be used to treat cell proliferation disorders such as cancer, and include, but are not limited to, cytotoxic agents, cell proliferation inhibitors, anti-angiogenic agents, weight-reducing agents, chemotherapeutic agents, radiotherapy and radiotherapy agents, targeted anticancer agents, BRMs, therapeutic antibodies, cancer vaccines, cytokines, hormone therapy, radiotherapy and anti-metastatic agents and immunotherapeutic agents. As described above, in some embodiments, such anticancer agents may include conjugates that can be conjugated with DLL3-ADCs disclosed herein before administration. For example, in some embodiments, a selected anticancer agent is conjugated to an unpaired cysteine ​​of an engineered antibody to provide an engineered conjugate (e.g., an antibody-drug conjugate) as described herein. Thus, such an engineered conjugate is expressly intended to be within the scope of this disclosure. In some embodiments, the disclosed anticancer agents are administered in combination with DLL3-ADCs containing different therapeutic agents as described above.

[0203] As used herein, the term “cytotoxic agent” refers to a substance that is toxic to cells and impairs or inhibits cellular function, and / or causes cell destruction. In some embodiments, the substance is a naturally occurring molecule of biological origin. Examples of cytotoxic agents, but not limited to, include bacterial small molecule toxins or enzymatically active toxins (e.g., diphtheria toxin, Pseudomonas aeruginosa endotoxins and exotoxins, Staphylococcus aureus enterotoxin A), fungi (e.g., α-sarcin, restrictosin), plants (e.g., abrin, lysine, modexin, biscumin, pokeweed antiviral protein, saporin, geronin, momorizin, tricosanthin, barley toxin, Aleurites fordii protein, dianthin protein, Phytolacca mericana protein (PAPI, PAPII, and PAP-S), Momordica charantia inhibitor, curcin, crotin, and Saponaria. Examples include officinalis inhibitors, geronin, mitegerin, restrictosin, phenomycin, neomycin, and trichothecene) or animals (e.g., cytotoxic RNases such as extracellular pancreatic RNases, their fragments and / or variants, and DNase I).

[0204] For the purposes of this disclosure, “chemotherapeutic agents” include compounds (e.g., cytotoxic agents or cell proliferation inhibitors) that nonspecifically reduce or inhibit the growth, proliferation, and / or survival of cancer cells. Such chemicals often target intracellular processes necessary for cell proliferation or division and are therefore generally particularly effective against cancer cells that grow and divide rapidly. For example, vincristine depolymerizes microtubules and inhibits cells from entering mitosis. Generally, chemotherapeutic agents may include any chemicals that inhibit or are designed to inhibit cancer cells or cells that are likely to become cancerous or give rise to tumorigenic offspring (e.g., TICs). Such agents are often administered in combination with regimens such as CHOP or FOLFIRI, and are often most effective in this context.

[0205] Examples of anticancer agents that may be used in combination with the DLL3-ADC of this disclosure (either as a component of a site-specific conjugate or in an unconjugated state) include, but are not limited to, alkylating agents, alkyl sulfonates, aziridines, ethyleneimine and methylamelamine, acetogenins, camptothecin, bryostatin, calistatin, CC-1065, cryptophycin, dorastatin, duocalmycin, eleutherobin, pancratistatin, sarcodictyin, spongystatin, nitrogen mustard, antibiotics, engine antibiotics, dynemycin, bisphosphonates, esperamicin, and chromoprotein engine antibiotics. Chromophore, acrasinomycin, actinomycin, autramycin, azaserin, bleomycin, kactinomycin, carabicin, carminomycin, cardinophilin, chromomycin, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, ADRIAMYCIN®, doxorubicin, epirubicin, esorubicin, idarubicin, marcelomycin, mitomycin, mycophenolic acid, nogaramycin, olibomycin, peplomycin, potfiromycin, puromycin, queramycin, rhodorubicin, streptonigrin, streptozocin, tubercidine, ubenimex, dinostatin, zorubicin;Antimetabolites, erlotinib, vemurafenib, crizotinib, sorafenib, ibrutinib, enzalutamide, folate analogs, purine analogs, androgens, anti-adrenal drugs, folate supplements, such as frolinic acid. Acid), acegraton, aldofamide glycoside, aminolevulinic acid, enyluracil, amsacrin, bestrabusil, bisanthren, edatraxate, defofamine, demecolsin, diazicone, elfornithine, eriptinium acetate, epotilon, etoglucide, gallium nitrate, hydroxyurea, lentinan, lonidainine, mytansinoid, mitogluzone, mitoxantrone, mopidanmol, nitraerine, pentostatin, fenamet, pirarubicin, losoxantrone, podophyllic acid, 2-ethylhydrazide, procarbazine, PSK (registered trademark) polysaccharide complex (JHS Natural Products, Eugene, OR), Lazoxane; Rhizoxin; Schizophyllan; Spirogermanium; Tenuazonic Acid; Triadicone; 2,2',2”-Trichlorotriethylamine; Trichothecene (especially T-2 toxin, verracurin A, loridine A, and anguidin); Urethane; Vindesine; Dacarbazine; Mannomustine; Mitobronitol; Mitractol; Pipobroman; Gacytosine; Arabinoside ("Ara-C"); Cyclophosphamide; Thiotepa; Taxoid, Chloranbucil; GEMZAR (Registered Trademark), Gemcitabine; 6-Thiogunine; Mercaptopurine; Methotrexate; Platinum Analogue, Vinblastine; Platinum; Etoposide (VP -16); Ifosfamide; Mitoxantrone; Vincristine; NAVELBINE® Vinorelbine; Novantrone; Teniposide; Edatrexate; Daunomycin; Aminopterin; Xeloda; Ibandronate; Irinotecan (Camptosar, CPT-11), topoisomerase inhibitor RFS2000; Difluoromethylornithine; Retinoids; Capecitabine; Combretastatin; Leucovorin;Examples include oxaliplatin; inhibitors of PKC-alpha, Raf, H-Ras, EGFR, and VEGF-A that reduce cell proliferation, as well as pharmaceutically acceptable salts, acids, or derivatives of any of the above. Also included in this definition are anti-estrogens and selective estrogen receptor modulators, aromatase inhibitors that inhibit the enzyme aromatase (which regulate estrogen production in the adrenal gland), and anti-androgens; as well as troxacitabine (1,3-dioxolane nucleoside cytosine analog); antisense oligonucleotides, ribozymes, e.g., VEGF expression inhibitors and HER2 expression inhibitors; vaccines, PROLEUKIN® rIL-2; LURTOTECAN® topoisomerase 1 inhibitor; ABARELIX® rmRH; vinorelbine and esperamicin, as well as pharmaceutically acceptable salts, acids, or derivatives of any of the above, and other antihormone agents that modulate or inhibit the hormonal effects on tumors.

[0206] Combined use with radiation therapy This disclosure also provides the use of DLL3-ADC in combination with radiotherapy (e.g., any mechanism for inducing localized DNA damage within tumor cells, such as gamma irradiation, X-rays, ultraviolet irradiation, microwaves, or electron emission). Combination therapies using directed delivery of radioisotopes to tumor cells are also envisioned, and the disclosed DLL3-ADC may be used in combination with targeted anticancer agents or other targeting means. Generally, radiotherapy is administered in pulses over a period of about 1 to 2 weeks. Radiotherapy may be administered to subjects with head and neck cancer over a period of about 6 to 7 weeks. Depending on the circumstances, radiotherapy may be administered as a single dose or as multiple sequential doses.

[0207] Modifications that do not substantially affect the activity of the various embodiments described herein are also provided within the scope of the subject matter definitions described herein. Accordingly, the following examples are intended to illustrate the invention and are not intended to limit the disclosure.

[0208] Sequence List Overview This application is accompanied by a sequence listing containing numerous amino acid sequences. Table 3 below provides an overview of the included sequences, where underlined sequences represent CDRs, and bold underlined lysine (K) residues are modification sites for linker-drug conjugation via site-directed modification / conjugation. [Table 3-1] [Table 3-2] [Table 3-3] [Table 3-4] [Examples]

[0209] Example 1. Preparation of DLL3 antibody antigen production DNA sequences encoding the extracellular domain (ECD) of cynomolgus monkey DLL3 (Uniprot number A0A2K5WSR4) and mouse DLL3 (Uniprot number O88516) were synthesized at Sangon Biotech (Shanghai, China) and subcloned into modified pcDNA3.3 expression vectors having an MBP tag at the N-terminus and an AVI-His tag or human Fc tag at the C-terminus. Human DLL3 (Uniprot number Q9NYJ7) was purchased from AcroBiosystems (catalog DL3-H52H4).

[0210] The DNA sequence encoding a cleavage isoform of human DLL3 (disclosed in WO2017 / 021349, Bispecific Antibody Constructs Binding D3 and CD3) was synthesized at Sangon Biotech (Shanghai, China) and then subcloned into a modified pcDNA3.3 expression vector containing MBP and AVI-His tags at the C-terminus.

[0211] The purified expression vector was transfected into Expi293 cells (Invitrogen-A14527). The cells were cultured for 5 days, and the supernatant was collected for protein purification using a Ni-NTA column (GE Healthcare, catalog 175248) or a Protein A column (GE Healthcare, catalog 175438). The resulting mouse DLL3, cynomolgus monkey DLL3, and cleaved human DLL3 were analyzed by SDS-PAGE and SEC, and then stored at -80°C.

[0212] Human DLL3 (ACRO DL3-H52H4) was named WT115-hPro1.ECD.His. The resulting mouse DLL3 was named WT115-MBP-mPro1.ECD.hFc. The cleavage isoforms of human DLL3 are WT115-hPro1.V1.ECD.MBP.AVI.His (DSL domain + EGF1~6 domains + proximal membrane), WT115-hPro1.V2.ECD.MBP.AVI.His (EGF1~6 domains + proximal membrane), WT115-hPro1.V3.ECD.MBP.AVI.His (EGF2~6 domains + proximal membrane), and WT115-hPro1. These were named V4.ECD.MBP.AVI.His (EGF3-6 domains + proximal membrane), WT115-hPro1.V5.ECD.MBP.AVI.His (EGF4-6 domains + proximal membrane), WT115-hPro1.V6.ECD.MBP.AVI.His (EGF5-6 domains + proximal membrane), and WT115-hPro1.V7.ECD.MBP.AVI.His (EGF6 domain + proximal membrane).

[0213] Construction of an expression vector for BMK antibodies Two DLL3 antibodies were used as benchmark antibodies, referred to herein as WT115-BMK1 and WT115-BMK2. The DNA sequences encoding the variable regions of WT115-BMK1 (sequences 212 and 213 of US2019 / 0046656 (which is incorporated herein by reference in its entirety)) and WT115-BMK2 (sequences 37 and 38 of WO2017 / 021349 (which is incorporated herein by reference in its entirety)) were synthesized at Sangon Biotech (Shanghai, China) and then subcloned into a modified pcDNA3.3 expression vector encoding the Fc region of human IgG1.

[0214] Plasmids containing the VH and VL genes were co-transfected into Expi293 cells. The cells were cultured for 5 days, and the supernatant was collected for protein purification using a Protein A column (GE Healthcare, 175438). The resulting antibodies were analyzed by SDS-PAGE and SEC, and then stored at -80°C.

[0215] Establishment of stable cell lines / cell pools Using lipofectamine 2000, 293F cells were transfected with an expression vector containing the gene encoding full-length human DLL3 (UniProt, Q9NYJ7-1). Flpin293 cells were transfected with an expression vector containing the gene encoding full-length cynomolgus monkey DLL3. Cells were cultured in a medium containing appropriate selection markers. A stable cell line with high human DLL3 expression (WT115-293F.hPro1.2E5) was selected after limiting dilution, and a stable cell pool with high cynomolgus monkey DLL3 expression (WT115.Flpin293.cPro1.pool) was selected with appropriate selection antibiotics.

[0216] Antibody biotinylation To perform NHS-PEO4 biotinylation, 1–10 mg / mL of antibody (IgG) was incubated with a 20-fold molar excess of NHS-PEO4 biotin reagent in a metal bath at 25°C for 75 minutes, or on ice for 2 hours. Next, excess biotin was removed using a desalting spin column, and the purified protein sample was recovered from the flow-through solution. The level of biotin incorporation into the protein was measured by a HABA assay. The biotinylated sample was diluted 10-fold with HABA / avidin solution, and the absorbance of the mixed solution at A500 was measured. The number of moles of biotin per mole of protein was calculated based on the A500 value.

[0217] Example 2. Production of DLL3 antibody containing VHH Generation of anti-DLL3 VHH The DLL3 antibody VHH was generated using immunization and phage display techniques in camelid animals. Briefly, alpacas (Vicugna pacos) were subcutaneously immunized with hFc-tagged human DLL3 ECD protein (ACRO, DL3-H5255). Following immunization, peripheral blood was collected to construct a phage library displaying VHH fragments. After biopanning using the corresponding target ECD protein, positive VHH clones that bind to DLL3 were selected.

[0218] VHH sequencing Positive E. coli clones selected by target-specific binding ELISA and FACS using E. coli supernatant were sent to Biosune (Shanghai, China) for nucleotide sequencing of the VHH gene. The sequencing results were analyzed using the CLC Main Workbench (Qiagen, Hilden, Germany). The sequences of the three unique positive VHH clones were VHH300, VHH301, and VHH302, as shown in Tables 4 and 5. [Table 4] [Table 5]

[0219] Generation of human Fc fusion antibodies containing VHH Three unique positive VHH clones were converted into VHH-Fc(hIgG1) fusion antibodies. Briefly, the VHH gene was PCR amplified from a pET-bac vector using VHH-specific cloning primers containing appropriate restriction sites, and cloned by fusion to a modified human hIgG1 expression pcDNA3.3 vector to produce corresponding clones of VHH-Fc(hIgG1) chimeric antibodies. The vectors were transiently transfected into 293F or Expi293 cells to express the antibodies. Cell culture supernatants containing the antibodies were collected and purified using protein A chromatography. The generated antibodies were named mAb300, mAb301, and mAb302, respectively. The obtained antibodies were analyzed by SDS-PAGE and HPLC-SEC, and then stored at -80°C.

[0220] Humanization Humanization of VHH was performed using a "best-fit" approach. Briefly, a blast search was performed on the amino acid sequence of the VHH framework region in the human germline V gene database, and the human CDR sequence of the top hit was replaced with the VHH CDR sequence using Kabat's CDR definition to generate the humanized VHH sequence. Next, key residues within the framework that play an important role in antibody affinity or developmentability were reversed to parental residues, either individually or in combination. Each variant was codon-optimized for expression in mammals and synthesized using GENEWIZ (SuZhou, China). The designed VHH variants and parental VHH proteins were cloned into a human IgG1 expression vector to create the human IgG1 construct. Antibodies were produced in HEK293 cells and purified using protein A chromatography. Variants with the desired affinity were ultimately selected as humanized leads.

[0221] Example 3: Characterization of DLL3-conjugated antibody Human DLL3 binding by FACS: WT115-293F.hPro1.2E5 cells (1 × 10⁻¹⁰) 5 Cells (per well) were incubated with antibodies of various concentrations (5-fold serial dilutions from 200 nM to 0.0128 nM) at 4°C for 1 hour. After washing with 1× PBS / 1% BSA, R-PE labeled goat anti-human IgG (1:150, Jackson ImmunoResearch, 109-115-098) was added as a secondary antibody, and the cells were incubated with the antibody at 4°C in the dark for 1 hour. Anti-human DLL3 antibodies WT115-BMK1 and WT115-BMK2 were used as positive controls. Human IgG1 isotype antibodies were used as negative controls. The cells were washed and resuspended in 4% paraformaldehyde. Cellular filtration efficiency (MFI) was measured by flow cytometry and analyzed by FlowJo.

[0222] FACS-mediated binding of DLL3 to cynomolgus monkeys: WT115-Flpin293.cPro1. Pooled cells (1×10⁶) 5 Cells (per well) were incubated with antibodies of various concentrations (4-fold serial dilutions from 10 nM to 0.00061 nM) at 4°C for 1 hour. After washing with 1× PBS / 1% BSA, the secondary antibody, Alexa Fluor 647-labeled goat anti-human IgG (1:150, Jackson ImmunoResearch, 109-605-098), was added, and the cells were incubated with the antibody at 4°C in the dark for 1 hour. Anti-human DLL3 antibodies WT115-BMK1 and WT115-BMK2 were used as positive controls. Human IgG1 isotype antibodies were used as negative controls. Cells were washed with 1× PBS / 1% BSA, resuspended in 4% paraformaldehyde, and incubated with the antibody at 4°C in the dark for 0.5 hours. The buffer was then changed to 1× PBS / 1% BSA, and the cells were filtered. Cellular Filtration Intake (MFI) was measured by flow cytometry and analyzed with FlowJo.

[0223] Mouse DLL3 conjugation by ELISA: Plates were pre-coated overnight at 4°C with WT115-MBP-mPro1.ECD.hFc at 1 μg / mL per well. Antigen was diluted from stock solution with coating buffer. The following day, plates were washed once with 1×PBST and blocked with 200 μL of 1×PBS / 2%BSA. Antibodies were serially diluted with blocking buffer (5-fold serial dilutions from 20 nM to 0.000256 nM). After 1 hour of blocking, plates were washed three times with 1×PBST, then the antibodies were added to the plates and incubated at ambient temperature for 1 hour. Anti-human DLL3 antibodies WT115-BMK1-biotin and WT115-BMK2-biotin were used as positive controls. WT114-BMK1-biotin antibody was used as a negative control. Antibody binding to immobilized mouse DLL3 was detected using HRP-labeled secondary antibody (Invitrogen, SNN1004) diluted to a concentration of 1:30000 in 1×PBS / 2%BSA. After incubation, the plate was washed six times with 1×PBST. 100 μL of TMB substrate was dispensed to induce color development, and the reaction was stopped by adding 100 μL of 2M HCl. Absorbance was read at 450 nm and 540 nm using a microplate spectrophotometer. All samples were tested in double strips.

[0224] Internalization: WT115-293F.hPro1.2E5 cells (4 × 10 4Cells (per well) were seeded into 96-well plates, and after centrifugation, the culture medium was removed from the plates. Dilutions of 1× final maximum concentration primary antibody (5-fold serial dilutions from 40 nM to 0.00256 nM, or from 200 nM to 0.0128 nM) and pHrodo (amine-reactive, Thermo Fisher, P36011)-labeled secondary antibody (Affinipure F(ab')2 fragment goat anti-human IgG, Jackson ImmunoResearch, 109-006-098, ratio = 1:1) were prepared and added to the plates along with the cell culture medium. The plates were incubated at 37°C for 5 hours. Anti-human DLL3 antibodies WT115-BMK1 and WT115-BMK2 were used as positive controls. Human IgG1 isotype antibodies were used as negative controls. After incubation, cells were stained with reagents (nucleus - Hoechst 33342, 1000 ng / ml; cytoplasm - calcein AM, diluted 1:2000 in DPBS), and plates were incubated at 37°C for 15 minutes. Finally, cells were imaged using Operatta CLS, and antibody endocytosis was analyzed using the parameter "number of spots per cell".

[0225] The data for mAb301 and the two WT BMK antibodies are summarized in Table 6. [Table 6]

[0226] Kinetic Binding Affinity of DLL3 Antibodies: The binding affinity of DLL3 antibodies to human DLL3 ECD protein was detected by an SPR assay using Biacore T200. Each antibody tested was captured on an anti-human IgGFc antibody-immobilized CM5 sensor chip (GE). Different concentrations of WT115-hPro1.ECD.His were injected onto the sensor chip at a flow rate of 30 μL / min over a 180-second binding phase followed by a 3600-second dissociation phase. The chip was regenerated with 10 mM glycine (pH 1.5) after each binding cycle.

[0227] As shown in Table 7, experimental data for human DLL3 were fitted using a steady-state affinity model. Experimental data for WT-115-BMK1 against human DLL3 were fitted using a heteroligand model. Other experimental data were fitted using a 1:1 model with Langmuir analysis. The sensorgrams of the blank surface and buffer channels were subtracted from the test sensorgrams. The molar concentration of the analyte was calculated using a molecular weight of 34 kDa. The affinity of the test antibody against human DLL3 is shown in Table 7. [Table 7]

[0228] Stability of human serum Human serum was freshly isolated from healthy donors by centrifugation. The sample was diluted with serum, and the serum volume accounted for more than 90% of the total volume. Five aliquots of the sample were incubated at 37°C. The samples were then collected on days 0, 1, 4, 7, and 14, and rapidly frozen together until analysis.

[0229] The stability of the samples was tested by binding to human DLL3 using ELISA. Briefly, plates were pre-coated overnight at 4°C with 100 μL / well of 1 μg / mLWT115-hPro1.ECD.his (in-house). The following day, plates were washed once with 1×PBST (PBS containing 0.05% tween®-20) and blocked with 200 μL of 1×PBS / 2% BSA per well. During blocking, the test antibody was added to the plates at various concentrations (4-fold serial dilutions from 3 nM to 0.00018 nM). These plates were incubated at room temperature for 1 hour. The binding of each antibody to immobilized human DLL3 was detected by goat anti-human IgG-Fc fragment cross-absorption antibody HRP (Bethyl, A80-304P) and mouse IgG-Fc fragment cross-adsorption antibody HRP (Bethyl, A90-231P), diluted 1:5000 in 1×PBS / 2% BSA. After incubation, plates were washed six times with 1×PBST. 100 μL of TMB substrate was dispensed to induce color development, and the reaction was stopped by adding 100 μL of 2M HCl. Absorbance was read at 450 nM and 540 nM using a microplate spectrophotometer. Human IgG1 isotype antibodies were used as negative controls. All samples were tested in double denominations. After incubation in human serum at 37°C for up to 14 days, the binding profile of mAb301 to human DLL3 protein remained unchanged (data not shown).

[0230] Example 4. Synthesis of linker-drug (1) The linker-drug (1) was prepared according to the following procedure: Synthesis of intermediate (2) [ka] Ac-Glu(OtBu)-Val-Cit-OH (19.9 mg, 39.7 μmol) was dissolved in N,N-dimethylformamide (400 μL), and 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (18.1 mg, 47.6 μmol) and 2,4,6-trimethylpyridine (6.27 μL, 47.6 μmol) were added, and the mixture was stirred at room temperature for 10 minutes. Next, methyl 4-aminomandelate (8.63 mg, 47.6 μmol) was added, and the mixture was stirred at room temperature for 21.5 hours, after which it was purified by reverse-phase fractional chromatography. The fraction containing the product was collected, concentrated under vacuum to remove acetonitrile, and lyophilized to obtain alcohol (2) (28.5 mg, quantified). 1 H NMR(400MHz,DMSO-d6)δ9.95(s,1H),8.07(d,J=7.4Hz,1H),7.99(d,J=8.0Hz,1H),7.66(d,J=8.4Hz,1H ),7.50(d,J=8.4Hz,2H),7.25(d,J=8.4Hz,2H),5.92(brs,1H),5.36(brs,2H),5.01(s,1H),4.34-4.29( m,1H),4.26-4.20(m,1H),4.14-4.10(m,1H),3.53(s,3H),3.00-2.83(m,2H),2.18-2.13(m,2H),1.94-1 .89(m,2H),1.84-1.23(m,17H),0.79(d,J=6.8Hz,3H),0.75(d,J=6.8Hz,3H).MS(ESI)m / z:665.30[M+H] + [ka] Ac-Glu(t-Bu)-Glu(t-Bu)-Val-Cit-OH (50.0 mg, 72.8 μmol) was dissolved in N,N-dimethylformamide (800 μL), and 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (33.2 mg, 87.4 μmol) and 2,4,6-trimethylpyridine (11.5 μL, 87.4 μmol) were added, and the mixture was stirred at room temperature for 10 minutes. Next, methyl 4-aminomandelate (15.8 mg, 87.4 μmol) was added, and the mixture was stirred at room temperature for 16 hours, and then purified by reverse-phase fractional chromatography. The product-containing fraction was collected, concentrated under vacuum to remove acetonitrile, and lyophilized to obtain alcohol (12) (54.0 mg, 63.5 μmol).

[0231] 1 H NMR(400MHz,DMSO-d6)δ10.00(s,1H),8.26-7.88(m,3H),7.68-7.60(m,1H),7.57(d,J =8.4Hz,2H),7.32(d,J=8.4Hz,2H),6.00-5.97(m,1H),5.43(brs,2H),5.08(s,1H),4. 40-4.37(m,1H),4.32-4.19(m,3H),3.60(s,3H),3.09-2.90(m,2H),2.25-2.18(m,4H) ,2.03-1.53(m,10H),1.46-1.36(m,20H),0.86(d,J=6.8Hz,3H),0.82(d,J=6.8Hz,3H).

[0232] MS(ESI)m / z:850.40[M+H] +

[0233] Synthesis of intermediate (43) [ka] Intermediate (2) (140 mg, 0.165 mmol) was dissolved in N,N-dimethylformamide (4 mL), and the mixture was stirred for 5 minutes while cooling on ice. Then, bis(4-nitrophenyl) carbonate (108 mg, 0.355 mmol) and N,N-diisopropylethylamine (100 μL, 0.574 mmol) were added, and the mixture was stirred at room temperature under a nitrogen atmosphere for 18 hours. The organic solvent was removed via an evaporator, and then a 1:1 solution of acetonitrile and water was added, and the mixture was purified by reverse-phase fractional chromatography. The product-containing fraction was collected, concentrated under vacuum to remove acetonitrile, and lyophilized to obtain intermediate (43) (130 mg, 0.128 mmol). MS(ESI) m / z: 1015.6[M+H] +

[0234] Synthesis of intermediate (44) [ka] Intermediate (43) (85 mg, 0.084 mmol) was dissolved in N,N-dimethylformamide (2 mL), and 1-hydroxybenzotriazole (20 mg, 0.13 mmol) and commercially available monomethyl auristatin E (MMAE, 63 mg, 0.088 mmol) were added at room temperature. Next, diisopropylethylamine (75 μL, 0.43 mmol) was added, and the mixture was stirred at room temperature under a nitrogen atmosphere for 23 hours. The organic solvent was removed via evaporator, and then a 1:1 solution of acetonitrile and water was added, and the mixture was purified by reverse-phase fractional chromatography. The product-containing fraction was collected, concentrated under vacuum to remove acetonitrile, and lyophilized to obtain intermediate (44) (94 mg, 0.059 mmol). MS (ESI) m / z: 1593.6 [M+H] +

[0235] Synthesis of intermediate (45) [ka] Intermediate (44) (94 mg, 0.059 mmol) was dissolved in tetrahydrofuran (5 mL) and water (2 mL). While cooling the mixture on ice, lithium hydroxide (1.0 M, 0.6 mL, 0.6 mmol) was added, and the mixture was stirred for 1 hour. Hydrochloric acid was added to the reaction solution to adjust the pH to 5, and then a 1:1 solution of acetonitrile and water was added. The mixture was purified by reverse-phase fractional chromatography. The product-containing fraction was collected, concentrated under vacuum to remove acetonitrile, and freeze-dried to obtain intermediate (45) (77 mg, 0.049 mmol). MS(ESI) m / z: 1579.7[M+H] +

[0236] Synthesis of intermediate (46) [ka] Intermediate (45) (77 mg, 0.049 mmol) was dissolved in N,N-dimethylformamide (3 mL), then cooled on ice, and N,N-diisopropylethylamine (50 μL, 0.29 mmol) and 1H-benzotriazole-1-yloxytripyrrolidinophosphonium hexafluorophosphate (87 mg, 0.17 mmol) were added. Next, N-(5-aminopentyl)maleimide hydrochloride (35 mg, 0.16 mmol) was added, and the mixture was allowed to return to room temperature and stirred for 20 hours. After the reaction was complete, purification was carried out by reverse-phase fractional chromatography. The product-containing fraction was collected, concentrated under vacuum to remove acetonitrile, and then lyophilized to obtain intermediate (46) (65 mg, 0.037 mmol). MS(ESI) m / z: 1744.7[M+H] +

[0237] Conversion from intermediate (46) to linker-drug (1) [ka] Acetonitrile (2 mL) and an 85% by weight aqueous phosphoric acid solution (1.00 mL, 14.6 mmol) were sequentially added to compound (46) (65 mg, 0.037 mmol), and the mixture was stirred at room temperature for 6 hours. Once the reaction was complete, water (2 mL) was added, and the reaction solution was purified by reverse-phase fractional chromatography. The product-containing fraction was collected, concentrated under vacuum to remove acetonitrile, and lyophilized to obtain linker-drug (1) (49 mg, 0.030 mmol). MS(ESI) m / z: 1631.6[M+H] +

[0238] Example 5. Synthesis of linker-drug (2) Synthesis of intermediate (43) [ka] Intermediate (2) (140 mg, 0.165 mmol), prepared as described in Example 1, was dissolved in N,N-dimethylformamide (4 mL). The mixture was stirred for 5 minutes while cooling on ice. Then, bis(4-nitrophenyl) carbonate (108 mg, 0.355 mmol) and N,N-diisopropylethylamine (100 μL, 0.574 mmol) were added, and the mixture was stirred at room temperature under a nitrogen atmosphere for 18 hours. The organic solvent was removed via an evaporator, and then a 1:1 solution of acetonitrile and water was added. The mixture was purified by reverse-phase fractional chromatography. The product-containing fraction was collected, concentrated under vacuum to remove acetonitrile, and lyophilized to obtain compound 43 (130 mg, 0.128 mmol). MS(ESI) m / z: 1015.6[M+H] +

[0239] Synthesis of intermediate (48) [ka] Intermediate (43) (67 mg, 0.066 mmol) was dissolved in N,N-dimethylformamide (2 mL), and 1-hydroxybenzotriazole (17 mg, 0.11 mmol) and commercially available exatecan mesylate (CAS: 169869-90-3, 35 mg, 0.066 mmol) were added at room temperature. Next, diisopropylethylamine (50 μL, 0.29 mmol) was added, and the mixture was stirred at room temperature under a nitrogen atmosphere for 4 hours. The organic solvent was removed via evaporator, and then a 1:1 solution of acetonitrile and water was added, and the mixture was purified by reverse-phase fractional chromatography. The product-containing fraction was collected, concentrated under vacuum to remove acetonitrile, and lyophilized to obtain compound 48 (57 mg, 0.043 mmol). MS (ESI) m / z: 1311.7 [M+H] +

[0240] Synthesis of intermediate (49) [ka] Intermediate (48) (57 mg, 0.043 mmol) was dissolved in tetrahydrofuran (3 mL) and water (1.5 mL). While cooling the mixture on ice, lithium hydroxide (1.0 M, 0.5 mL, 0.5 mmol) was added, and the mixture was stirred for 1 hour. Hydrochloric acid was added to the reaction solution to adjust the pH to 5, and then a 1:1 solution of acetonitrile and water was ...

Claims

1. Antibody-drug conjugate (ADC) of formula (I), 【Chemistry 1】 or a salt thereof, in the formula, n is an integer from 1 to 8, An antibody-drug conjugate (ADC) or salt thereof of formula (I), wherein Ab represents an antibody that binds to DLL3 ("DLL3 antibody").

2. The aforementioned ADC is of the form of formula (I-A), 【Chemistry 2】 ADC according to claim 1, or a salt thereof.

3. The aforementioned ADC is of the form of formula (I-B), 【Transformation 3】 ADC according to claim 1, or a salt thereof.

4. An antibody-drug conjugate (ADC) of formula (A), 【Chemistry 4】 In the formula, each thick shaded line represents a chain of antibody that binds to DLL3 ("DLL3 antibody"), the sulfur shown originates from a cysteine ​​residue (optionally C226 and / or C229) of the DLL3 antibody, and X represents the following structure or a salt thereof. 【Transformation 5】 In the formula, the wavy line represents the attachment point of the ADC of formula (A) to the rest of the antibody-drug conjugate (ADC) of formula (A).

5. An antibody-drug conjugate (ADC) of formula (B), 【Transformation 6】 In the formula, each thick shaded line represents a chain of antibody that binds to DLL3 ("DLL3 antibody"), and the CH shown in the diagram ２ CH ２ CH ２ CH ２ The NH portion is derived from a lysine residue of the DLL3 antibody (optionally, one or more of K246, K248, K288, K290, or K317 according to EU numbering), and X represents the following structure or a salt thereof. 【Transformation 7】 In the formula, the wavy line represents the attachment point of the ADC of formula (B) to the rest of the antibody-drug conjugate (ADC) of formula (B).

6. The ADC according to claim 5, wherein the DLL3 antibody comprises the amino acid sequence described in SEQ ID NO: 4, and the lysine residue is the 150th amino acid residue of SEQ ID NO: 4 (for example, K248 according to EU numbering).

7. The ADC according to claim 5, wherein the DLL3 antibody comprises the amino acid sequence described in SEQ ID NO: 5, and the lysine residue is the 150th amino acid residue of SEQ ID NO: 5 (for example, K248 according to EU numbering).

8. The ADC according to claim 5, wherein the DLL3 antibody comprises the amino acid sequence described in SEQ ID NO: 10, and the lysine residue is the 152nd amino acid residue of SEQ ID NO: 10 (for example, K248 according to EU numbering).

9. The above X represents the following structure or a salt thereof, 【Transformation 8】 The ADC according to any one of claims 4 to 8, wherein the dashed line in the formula represents the point of attachment of the ADC of formula (A) or the remainder of the ADC of formula (B).

10. The above X represents the following structure or a salt thereof, 【Chemistry 9】 The ADC according to any one of claims 4 to 8, wherein the dashed line in the formula represents the point of attachment of the ADC of formula (A) or the remainder of the ADC of formula (B).

11. The ADC according to any one of claims 1 to 10, wherein the DLL3 antibody is (1) CDR1, CDR2, and CDR3 of heavy chain-only antibody variable domains (VHH), wherein the VHH comprises the amino acid sequence described in SEQ ID NO: 14, 15, or 16, or (2) at least one VH, wherein the VH comprises the at least one VH comprising CDR1, CDR2, and CDR3, (i) the CDR1 comprises the amino acid sequence described in SEQ ID NO: 1 or 7, (ii) the CDR2 comprises the amino acid sequence described in SEQ ID NO: 2 or 8, and (iii) the CDR3 comprises the amino acid sequence described in SEQ ID NO: 3 or 9.

12. The aforementioned DLL3 antibody, (a) CDR1 described in Sequence ID No. 1, CDR2 described in Sequence ID No. 2, and CDR3 described in Sequence ID No. 3, or (b) The ADC according to any one of claims 1 to 11, comprising CDR1 as described in Sequence ID No. 7, CDR2 as described in Sequence ID No. 8, and CDR3 as described in Sequence ID No.

9.

13. The ADC according to any one of claims 1 to 12, wherein the DLL3 antibody comprises a VHH having the amino acid sequence described in any one of SEQ ID NOs: 14 to 16.

14. The ADC according to any one of claims 1 to 13, wherein the DLL3 antibody further comprises a human IgG constant domain.

15. The ADC according to any one of claims 1 to 14, wherein the DLL3 antibody further comprises an Fc fragment.

16. The ADC according to claim 14, wherein the human IgG is human IgG1.

17. The ADC according to claim 15, wherein the Fc fragment comprises a homodimer of the amino acid sequence described in SEQ ID NO:

17.

18. The ADC according to any one of claims 1 to 17, wherein the DLL3 antibody comprises at least one VHH single domain.

19. The ADC according to any one of claims 1 to 18, wherein the DLL3 antibody comprises two VHH single domains, each of which is conjugated to a chain of Fc fragments.

20. The ADC according to any one of claims 1 to 19, wherein the DLL3 antibody comprises a homodimer of the amino acid sequence described in any one of SEQ ID NOs: 4, 5, or 10.

21. The ADC according to any one of claims 1 to 3 and 11 to 20, wherein n is 1.

22. The ADC according to any one of claims 1 to 20, wherein n is 2.

23. The ADC according to any one of claims 1 to 3 and 11 to 20, wherein n is 3.

24. The ADC according to any one of claims 1 to 4 and 9 to 20, wherein n is 4.

25. Antibody-drug conjugate (ADC) of formula (II), 【Chemistry 10】 or a salt thereof, in the formula, n is an integer from 1 to 8, An antibody-drug conjugate (ADC) or salt thereof of formula (II), wherein Ab represents an antibody that binds to DLL3 ("DLL3 antibody").

26. The aforementioned ADC is given by equation (II-A), 【Chemistry 11】 The ADC according to claim 25, or a salt thereof.

27. The aforementioned ADC is given by equation (II-B), 【Chemistry 12】 The ADC according to claim 25, or a salt thereof.

28. An antibody-drug conjugate (ADC) of formula (A), 【Chemistry 13】 In the formula, each thick shaded line represents a chain of antibody that binds to DLL3 ("DLL3 antibody"), the sulfur shown originates from a cysteine ​​residue (optionally C226 and / or C229) of the DLL3 antibody, and X represents the following structure or a salt thereof. 【Chemistry 14】 In the formula, the wavy line represents the attachment point of the ADC of formula (A) to the rest of the antibody-drug conjugate (ADC) of formula (A).

29. An antibody-drug conjugate (ADC) of formula (B), 【Chemistry 15】 In the formula, each thick shaded line represents a chain of antibody that binds to DLL3 ("DLL3 antibody"), and the CH shown in the diagram ２ CH ２ CH ２ CH ２ The NH portion is derived from a lysine residue of the CH2 domain of the DLL3 antibody (optionally, one or more of K246, K248, K288, K290, or K317 according to EU numbering), and X represents the following structure or a salt thereof. 【Chemistry 16】 In the formula, the wavy line represents the attachment point of the ADC of formula (B) to the rest of the antibody-drug conjugate (ADC) of formula (B).

30. The ADC according to claim 29, wherein the DLL3 antibody comprises the amino acid sequence described in SEQ ID NO: 4, and the lysine residue is the 150th amino acid residue of SEQ ID NO: 4 (for example, K248 according to EU numbering).

31. The ADC according to claim 29, wherein the DLL3 antibody comprises the amino acid sequence described in SEQ ID NO: 5, and the lysine residue is the 150th amino acid residue of SEQ ID NO: 5 (for example, K248 according to EU numbering).

32. The ADC according to claim 29, wherein the DLL3 antibody comprises the amino acid sequence described in SEQ ID NO: 10, and the lysine residue is the 152nd amino acid residue of SEQ ID NO: 10 (for example, K248 according to EU numbering).

33. The above X represents the following structure or a salt thereof, 【Chemistry 17】 The ADC according to any one of claims 28 to 32, wherein the dashed line in the formula represents the point of attachment of the ADC of formula (A) or the remainder of the ADC of formula (B).

34. The above X represents the following structure or a salt thereof, [Chemistry 18] The ADC according to any one of claims 28 to 32, wherein the dashed line in the formula represents the point of attachment of the ADC of formula (A) or the remainder of the ADC of formula (B).

35. The ADC according to any one of claims 25 to 34, wherein the DLL3 antibody is (1) CDR1, CDR2, and CDR3 of heavy chain-only antibody variable domains (VHH), wherein the VHH comprises the amino acid sequence described in SEQ ID NO: 14, 15, or 16, or (2) at least one VH, wherein the VH comprises the at least one VH, wherein the VH comprises the amino acid sequence described in SEQ ID NO: 1, CDR2, and CDR3, and (i) the CDR1 comprises the amino acid sequence described in SEQ ID NO: 1 or 7, (ii) the CDR2 comprises the amino acid sequence described in SEQ ID NO: 2 or 8, and (iii) the CDR3 comprises the amino acid sequence described in SEQ ID NO: 3 or 9.

36. The aforementioned DLL3 antibody, (a) CDR1 described in Sequence ID No. 1, CDR2 described in Sequence ID No. 2, and CDR3 described in Sequence ID No. 3, or (b) The ADC according to any one of claims 25 to 35, comprising CDR1 as described in Sequence ID No. 7, CDR2 as described in Sequence ID No. 8, and CDR3 as described in Sequence ID No.

9.

37. The ADC according to any one of claims 25 to 36, wherein the DLL3 antibody comprises a VH having the amino acid sequence described in any one of SEQ ID NOs. 14 to 16.

38. The ADC according to any one of claims 25 to 37, wherein the DLL3 antibody further comprises a human IgG constant domain.

39. The ADC according to any one of claims 25 to 38, wherein the DLL3 antibody further comprises an Fc fragment.

40. The ADC according to claim 38, wherein the human IgG is human IgG1.

41. The ADC according to claim 39, wherein the Fc fragment comprises a homodimer of the amino acid sequence described in SEQ ID NO:

17.

42. The ADC according to any one of claims 25 to 41, wherein the DLL3 antibody comprises at least one VHH single domain.

43. The ADC according to any one of claims 25 to 42, wherein the DLL3 antibody comprises two VHH single domains, each of which is conjugated to a chain of Fc fragments.

44. The ADC according to any one of claims 25 to 43, wherein the DLL3 antibody comprises a homodimer of the amino acid sequence described in any one of SEQ ID NOs: 4, 5, or 10.

45. The ADC according to any one of claims 25 to 27 and 35 to 44, wherein n is 1.

46. The ADC according to any one of claims 25 to 44, wherein n is 2.

47. The ADC according to any one of claims 25 to 27 and 35 to 44, wherein n is 3.

48. The ADC according to any one of claims 25 to 28 and 35 to 44, wherein n is 4.

49. A pharmaceutical composition comprising one or more ADCs according to any one of claims 1 to 48 and a pharmaceutically acceptable carrier.

50. The pharmaceutical composition according to claim 49, wherein the composition is characterized by a drug-to-antibody ratio ("DAR") of about 1 to about 8.

51. The pharmaceutical composition according to claim 50, wherein the DAR is about 1 to about 2.

52. The pharmaceutical composition according to claim 50 or 51, wherein the DAR is about 1.1, about 1.2, about 1.3, about 1.4, about 1.5, about 1.6, about 1.7, about 1.8, about 1.9, or about 2.

53. The pharmaceutical composition according to claim 50, wherein the DAR is approximately 2 to approximately 4.

54. The pharmaceutical composition according to claim 50 or 53, wherein the DAR is approximately 2, approximately 2.1, approximately 2.2, approximately 2.4, approximately 2.4, approximately 2.5, approximately 2.6, approximately 2.7, approximately 2.8, approximately 2.9, approximately 3, approximately 3.1, approximately 3.2, approximately 3.3, approximately 3.4, approximately 3.5, approximately 3.6, approximately 3.7, approximately 3.8, approximately 3.9, or approximately 4.

55. A method for modulating a DLL3-related immune response in a subject, comprising administering to the subject an ADC according to any one of claims 1 to 48 or a pharmaceutical composition according to any one of claims 49 to 54, such that the immune response is modulated in the subject.

56. A method for treating cancer in a subject, comprising administering to the subject an ADC according to any one of claims 1 to 48 or a pharmaceutical composition according to any one of claims 49 to 54, wherein the cancer is DLL3-positive or DLL3-overexpressing.

57. The method according to claim 56, wherein the cancer is selected from lung cancer, neuroendocrine cancer, and colorectal cancer.

58. The method according to claim 56 or 57, wherein the cancer is SCLC or LCNEC.

59. Use of the ADC according to any one of claims 1 to 48 or the pharmaceutical composition according to any one of claims 49 to 54 in the manufacture of a drug for diagnosing or treating the aforementioned DLL3-positive cancer.

60. An ADC according to any one of claims 1 to 48 or a pharmaceutical composition according to any one of claims 49 to 54, for use in treating the aforementioned DLL3-positive cancer.

61. A method for producing an ADC according to any one of claims 1 to 24, a) A step of providing a solution containing the DLL3 antibody, b) A step of bringing the solution from a) into contact with a reducing agent, c) A step of bringing the solution from b) into contact with a solution containing the linker-drug (1), 【Chemistry 19】 The method comprising the step of manufacturing the ADC.

62. A method for producing an ADC according to any one of claims 25 to 48, a) A step of providing a solution containing the DLL3 antibody, b) A step of bringing the solution from a) into contact with a reducing agent, c) A step of bringing the solution from b) into contact with a solution containing the linker-drug (2), 【Chemistry 20】 The method comprising the step of manufacturing the ADC.

63. The method according to claim 61 or 62, wherein the reducing agent is tris(2-carboxyethyl)phosphine (TCEP).

64. A method for producing an ADC according to any one of claims 1 to 24, a) A step of providing a solution containing the DLL3 antibody, b) A step of contacting the solution from a) with the affinity peptide conjugated to the thiophenol activating portion, c) A step of bringing the solution from b) into contact with a solution containing the linker-drug (1), 【Chemistry 21】 The method comprising the step of manufacturing the ADC.

65. A method for producing an ADC according to any one of claims 25 to 48, a) A step of providing a solution containing the DLL3 antibody, b) A step of contacting the solution from a) with the affinity peptide conjugated to the thiophenol activating portion, c) A step of bringing the solution from b) into contact with a solution containing the linker-drug (2), 【Chemistry 22】 The method comprising the step of manufacturing the ADC.

66. The method according to claim 64 or 65, wherein the affinity peptide is the peptide of Sequence ID No.

6.

67. The affinity peptide conjugated to the thiophenol activation portion 【Chemistry 23】 The method according to any one of claims 64 to 66.

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