Anti-SEZ6 antibody drug conjugates

Abstract

The present disclosure provides SEZ6 antibody drug conjugates (ADCs) comprising a topoisomerase I inhibitor, including compositions and methods of using such ADCs.

Description

[0001] 1. Cross-reference to related applications

[0002] This application claims the benefit of U.S. Provisional Application Serial No. 63 / 373,668, filed on August 26, 2022, which is hereby incorporated by reference in its entirety.

[0003] 2. Sequence Listing

[0004] This application contains a sequence listing, which has been submitted electronically and is hereby incorporated by reference in its entirety. The sequence listing was created on August 24, 2023, is named SeqList_350794-46102 and is 14,966 bytes in size. Technical Field

[0005] The present application relates to an anti-DLL3 antibody drug conjugate (ADC). Background Art

[0006] Seizure-related homologue 6 (SEZ6) is a transmembrane protein found on the cell surface of selected neuronal lineage cells (Shimizu-Nishikawa K et al. Cloning and characterization of seizure-related gene, SEZ6. Biochem Biophys Res Commun. 1995; 216(1): 382-9; Gunnersen JM et al.). SEZ6 is selectively expressed on neuroendocrine tumors including small cell lung cancer (SCLC); Kudoh S et al. Significance of achaete-scute complex homologue 1 (ASCL1) in pulmonary neuroendocrine carcinomas; RNA sequence analyses using small cell lung cancer cells and ASCL1-induced pulmonary neuroendocrine carcinoma cells. Histochem Cell Biol. 2020; 153(6): 443-456). Small cell lung cancer (SCLC) is an aggressive neuroendocrine cancer that accounts for 13%-15% of all lung cancers diagnosed, with approximately 260,000 new cases diagnosed each year worldwide and approximately 33,000 in the United States, resulting in 25,000 deaths. The 5-year survival rate is 2% for extensive disease (approximately 65% ​​of patients) and 15% for limited-stage disease.

[0007] Conventional therapeutic treatments for cancer are often ineffective, and thus there remains a need to develop more targeted and effective therapies for treating cancer.Antibody drug conjugates (ADCs) comprising an antibody targeting DLL3 conjugated to a cytotoxic drug via a chemical linker provide targeted therapies for treating patients with tumors expressing DLL3. Summary of the invention

[0008] Provided herein is an anti-DLL3 antibody drug conjugate of formula (IV):

[0009]

[0010] wherein n is an integer from 1 to 10, and wherein Ab is an anti-DLL3 antibody comprising: a heavy chain variable region comprising CDR-H1, CDR-H2, and CDR-H3; and a light chain variable region comprising CDR-L1, CDR-L2, and CDR-L3, wherein

[0011] CDR-H1 has the amino acid sequence shown in SEQ ID NO: 3,

[0012] CDR-H2 has the amino acid sequence shown in SEQ ID NO:4,

[0013] CDR-H3 has the amino acid sequence shown in SEQ ID NO:5,

[0014] CDR-L1 has the amino acid sequence shown in SEQ ID NO:6,

[0015] CDR-L2 has the amino acid sequence shown in SEQ ID NO:7, and

[0016] CDR-L3 has the amino acid sequence shown in SEQ ID NO:8.

[0017] In some embodiments, the anti-DLL3 antibody of the antibody drug conjugate comprises: (a) a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 1 and (b) a light chain variable region having an amino acid sequence as shown in SEQ ID NO: 2.

[0018] In some embodiments, the anti-DLL3 antibody of the antibody drug conjugate is an IgG1 antibody.

[0019] In some embodiments, the anti-DLL3 antibody of the antibody drug conjugate comprises: (a) a heavy chain having the amino acid sequence shown in SEQ ID NO:9 and (b) a light chain having the amino acid sequence shown in SEQ ID NO:10.

[0020] In some embodiments, the anti-DLL3 antibody drug conjugate has the structure of Formula (IV):

[0021]

[0022] wherein n is an integer from 1 to 10, and wherein Ab is an anti-DLL3 antibody comprising: two heavy chains each having an amino acid sequence as shown in SEQ ID NO: 9 and two light chains each having an amino acid sequence as shown in SEQ ID NO: 10.

[0023] In some embodiments, n of the anti-DLL3 antibody drug conjugate is 6.

[0024] In some embodiments, n of the anti-DLL3 antibody drug conjugate is 2.

[0025] In some embodiments, n of the anti-DLL3 antibody drug conjugate is 4.

[0026] In some embodiments, n of the anti-DLL3 antibody drug conjugate is 8.

[0027] In some embodiments, n of the anti-DLL3 antibody drug conjugate is 10.

[0028] Also provided herein is a composition comprising a therapeutically effective amount of an anti-DLL3 antibody drug conjugate disclosed herein, wherein n of the predominant species of the antibody drug conjugate in the composition is 6.

[0029] Also provided herein is a pharmaceutical composition comprising a therapeutically effective amount of an anti-DLL3 antibody drug conjugate disclosed herein and a pharmaceutically acceptable excipient, wherein the DAR of the composition is 6 or about 6.

[0030] Also provided herein is a method of treating small cell lung cancer, wherein the method comprises administering a therapeutically effective amount of an anti-DLL3 antibody drug conjugate disclosed herein to a patient in need thereof.

[0031] Also provided herein is a method for treating small cell lung cancer, wherein the method administers a therapeutically effective amount of a pharmaceutical composition disclosed herein to a patient in need thereof, the pharmaceutical composition comprising a therapeutically effective amount of an anti-DLL3 antibody-drug conjugate disclosed herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1A-1B Calu-6 (lung adenocarcinoma; Figure 1A ) and A375 (malignant melanoma; Figure 1B ) cell proliferation of cell lines.

[0033] Figure 2A-2H Shown are cell lines positive for SEZ6 mRNA: ( Figure 2A )H526、( Figure 2B )H146、( Figure 2C )H187、( Figure 2D )H1963FP5、( Figure 2E )CORL95, ( Figure 2H )H524; or in two cell lines negative for SEZ6 mRNA: ( Figure 2F )SBC5 and ( Figure 2G ) In vitro activity of SW1271, ADC-1, negative control non-targeted ADC (ADC-2) or TOP1 inhibitor alone (Example 1G) on SCLC cell lines.

[0034] Figures 3A-3D It is shown in H1963( Figure 3A ) and H526( Figure 3B ) cell line-derived xenograft (CDX) mouse models and LU95 ( Figure 3C ) and LU149( Figure 3D ) Dose response curve of ADC-1 treatment in a patient-derived xenograft (PDX) mouse model. DETAILED DESCRIPTION

[0035] As used in this disclosure, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. The terms "a" (or "an") and the terms "one or more" and "at least one" are used interchangeably herein unless the context clearly dictates otherwise.

[0036] As used in this disclosure and unless otherwise indicated, the terms "about" and "approximately" generally refer to numerical ranges that are considered by those skilled in the art to be equivalent to the values ​​set forth (i.e., having the same function or result). In many cases, the terms "about" and "approximately" may include numbers rounded to the nearest significant figure. In a specific embodiment, the terms "about" and "approximately" should be interpreted as normal variations that allow for the judgment of those of ordinary skill in the art, such as variations within 20% or 10% or 5%. In a specific embodiment, the terms "about" and "approximately" encompass the exact values ​​set forth. Unless the context clearly indicates otherwise, all numerical values ​​provided herein are modified by the term about.

[0037] Disclosed herein, in certain embodiments, are anti-DLL3 antibody-drug conjugates (ADCs), including anti-DLL3 ADCs comprising a TOP1 inhibitor, and methods of using the ADCs.

[0038] 7.1. Topoisomerase 1 inhibitors (TOP1i)

[0039] Topoisomerase 1 (TOP1) removes supercoils formed during DNA replication. TOP1 inhibitors (TOP1i) can bind and stabilize TOP1-DNA complexes, inducing DNA strand breaks and apoptosis.

[0040] In certain embodiments, provided herein are topoisomerase I inhibitor drugs ("TOP1i drugs") according to structural formula (I), which may be targeted for delivery to cells by conjugation to anti-SEZ6 antibodies.

[0041]

[0042] In certain embodiments, the TOP1i drug is (7S)-14-(3-aminobicyclo[1.1.1]pentan-1-yl)-7-ethyl-7-hydroxy-2H,10H-[1,3]dioxol[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-8,11(7H,13H)-dione.

[0043] In certain embodiments, the TOP1i drugs contemplated herein may be conjugated to antibodies via a linker as shown in structural formula (II):

[0044]

[0045] in Indicates the attachment point of the linker to the TOP1i drug.

[0046] 7.1.1. Linker drug LD1 (Structural formula (III))

[0047] In certain embodiments, the TOP1i linker drug (LD1) is a compound according to formula (III). In certain embodiments, LD1 is (2S)-2-(2-bromoacetylamino)-N-[(2S)-1-({3-[(7S)-7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-2H,10H-[1,3]dioxol[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-14-yl]bicyclo[1.1.1]pentan-1-yl}amino)-1-oxopropane-2-yl]-3-methylbutanamide.

[0048]

[0049] 7.2. Anti-SEZ6 ADC

[0050] In certain embodiments, a TOP1i drug as described herein (i.e., Formula (I)) can be conjugated to an anti-DLL3 antibody to form an anti-DLL3 TOP1i antibody-drug conjugate. The antibody-drug conjugate selectively delivers one or more drug moieties to a target tissue, such as a tumor expressing DLL3. Thus, in certain embodiments, the present disclosure provides an anti-DLL3 TOP1i ADC for therapeutic use in the treatment of small cell lung cancer.

[0051] In certain embodiments, the TOP1i drug is conjugated to the anti-DLL3 antibody via a linker moiety. In certain embodiments, the linker connects the TOP1i drug to the anti-DLL3 antibody by forming a covalent bond with the TOP1i drug at one position and with the antibody at another position. In certain embodiments, the covalent bond is formed by a reaction between a functional group on the linker and a functional group on the TOP1i drug and the anti-DLL3 antibody. In certain embodiments, the anti-DLL3 ADC of the present disclosure is composed of an anti-DLL3 antibody conjugated to a TOP1i linker drug of formula (III).

[0052] In certain embodiments, "Ab" refers to an anti-DLL3 antibody comprising a heavy chain having an amino acid sequence as shown in SEQ ID NO: 9 and a light chain having an amino acid sequence as shown in SEQ ID NO: 10. In certain embodiments, Ab is conjugated to a TOP1i linker drug of formula (III).

[0053] 7.2.1. Anti-SEZ6 Antibody

[0054] In certain embodiments, the Ab is a humanized anti-SEZ6 IgG1 monoclonal antibody.In certain embodiments, the Ab comprises variable regions and CDRs (complementarity determining regions) identified according to rules developed in the art and / or by aligning the sequences with a database of known variable regions.

[0055] 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. For example, CDRs can be identified according to Kabat et al. (1991) Sequences of Proteins of Immunological Interest (5 thEd.), USDept. of Health and Human Services, PHS, NIH, NIH Publication No. 91-3242 (referred to herein as "Kabat"); or identified according to AbM (Oxford Molecular / MSI Pharmacopia) (referred to herein as "AbM"). AbM can be obtained from www.bioinf.org.uk / abs The Abysis database (maintained by AC Martin of the Department of Biochemistry & Molecular Biology University College London).

[0056] In certain embodiments, the Ab comprises a heavy chain having an amino acid sequence as shown in SEQ ID NO: 9 (the variable region is in bold and has an amino acid sequence as shown in SEQ ID NO: 1; the constant region is in italics; the CDRs Underlined and having, in order of appearance, the amino acid sequences shown in SEQ ID NOs: 3, 4 and 5, respectively):

[0057]

[0058] In certain embodiments, the Ab comprises a light chain having an amino acid sequence as shown in SEQ ID NO: 10 (the variable region is in bold and has an amino acid sequence as shown in SEQ ID NO: 2; the constant region is in italics; the CDRs Underlined and having, in order of appearance, the amino acid sequences shown in SEQ ID NOs: 6, 7 and 8, respectively):

[0059]

[0060] In certain embodiments, Ab comprises two heavy chains each having the amino acid sequence set forth in SEQ ID NO:9 and two light chains each having the amino acid sequence set forth in SEQ ID NO:10.

[0061] In certain embodiments, the Ab comprises a CDR-H1 having an amino acid sequence as set forth in SEQ ID NO: 3; a CDR-H2 having an amino acid sequence as set forth in SEQ ID NO: 4; a CDR-H3 having an amino acid sequence as set forth in SEQ ID NO: 5; a CDR-L1 having an amino acid sequence as set forth in SEQ ID NO: 6; a CDR-L2 having an amino acid sequence as set forth in SEQ ID NO 7; and a CDR-L3 having an amino acid sequence as set forth in SEQ ID NO 8. The amino acid sequence of CDR-H1 was identified using the AbM definition, and the remaining CDR amino acid sequences were identified using the Kabat definition.

[0062] In certain embodiments, the Ab comprises a heavy chain variable domain having the amino acid sequence shown in SEQ ID NO:1 and a light chain variable domain having the amino acid sequence shown in SEQ ID NO:2.

[0063] 7.2.2. Number of connected drugs

[0064] In certain embodiments, the ADCs disclosed herein comprise various stoichiometric molar ratios of drug molecules attached to antibody moieties, depending on the configuration of the antibody and at least in part on the method used to achieve conjugation.

[0065] The term "drug load" refers to the number of drug molecules per antibody in an individual ADC molecule. The number of TOPIi drugs linked to an anti-DLL3 ADC can vary and will be limited by the number of available attachment sites on the anti-DLL3 antibody. As contemplated for the anti-DLL3 ADC of the present invention, a linker links a single TOPIi drug to the antibody in the anti-DLL3 ADC. As long as the anti-DLL3 ADC does not exhibit an unacceptable level of aggregation under conditions of use and / or storage, an anti-DLL3 ADC (i.e., Formula (IV)) having n of up to 10 is contemplated. In certain embodiments, n of the anti-DLL3 ADC is in the range of 1-10. In certain embodiments, n of the anti-DLL3 ADC is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In certain embodiments, n is 2, 3, 4, 5, or 6. In certain embodiments, n is 2, 4, 6, 8, or 10. In certain embodiments, n is 2, 4, or 6. In certain embodiments, n is 6. In certain embodiments, the drug loading may include 1 drug molecule, 2 drug molecules, 3 drug molecules, 4 drug molecules, 5 drug molecules, 6 drug molecules, 7 drug molecules, 8 drug molecules, 9 drug molecules, or 10 drug molecules.

[0066] Provided herein is a conjugation method for producing an anti-DLL3 ADC composition, wherein the predominant species of the ADC is n is 6, and the composition has a drug-antibody ratio (DAR) of 6 or about 6. The DAR is the average number of drugs attached to each antibody in the composition. Other conjugation methods are known in the art and can be used to produce ADCs with different numbers of conjugated drugs (n is, for example, 2, 3, 4, 5, 6, or 7) and different DAR compositions (e.g., DAR is 4, 5, 6 or about 4, about 5, or about 6).

[0067] 7.2.3. Example ADC

[0068] In certain embodiments, an anti-DLL3 antibody drug conjugate of formula (IV) is provided:

[0069]

[0070] Wherein the antibody Ab comprises two heavy chains each having an amino acid sequence as shown in SEQ ID NO: 9 and two light chains each having an amino acid sequence as shown in SEQ ID NO: 10. In certain embodiments, the conjugation of the linker-drug to the antibody is via a bond formed with a sulfhydryl group of a cysteine ​​residue of the antibody. In certain embodiments, the value of n is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0071] In certain embodiments, the value of n is 2, 4, 6, 8 or 10. In certain embodiments, n is 6. In certain embodiments, the anti-SEZ6 ADC of formula (IV) comprises an Ab having a heavy chain and a light chain, the heavy chain having the amino acid sequence as shown in SEQ ID NO: 9, the light chain having the amino acid sequence as shown in SEQ ID NO: 10, and the value of n is 2, 4, 6, 8 or 10. In certain embodiments, the anti-SEZ6 ADC of formula (IV) comprises an Ab having a heavy chain and a light chain, the heavy chain having the amino acid sequence as shown in SEQ ID NO: 9, the light chain having the amino acid sequence as shown in SEQ ID NO: 10, and the value of n is 6.

[0072] As used herein, "ADC-1" is an ADC composition comprising an ADC according to structural formula (IV), wherein Ab has two heavy chains each having an amino acid sequence as shown in SEQ ID NO: 9 and two light chains each having an amino acid sequence as shown in SEQ ID NO: 10, and the drug-antibody ratio (DAR) is about 6, wherein the predominant species n is 6. Methods for preparing ADC-1 are described in the following examples. In one embodiment, ADC-1 is a composition comprising a plurality of ADC-1 species having two or more different n values. In some embodiments, the average drug-antibody ratio (DAR) of the ADC-1 composition is about 5.5, about 5.6, about 5.7, about 5.8, about 5.9, about 6.0, about 6.1, about 6.2, about 6.3, about 6.4, or about 6.5. In some embodiments, the composition of ADC-1 has an average of six topoisomerase 1 inhibitor (Top1i) molecules conjugated per anti-DLL3 antibody. In some embodiments, the predominant species of ADC-1 in the composition has n=6. In some embodiments, the average DAR of ADC-1 is about 6.

[0073] 7.3. Usage

[0074] In certain embodiments, methods for treating small cell lung cancer (SCLC) are provided, the methods comprising administering to a subject in need thereof a therapeutically effective amount of an ADC of formula (IV).

[0075] As used herein, the term "subject" refers to a human. The terms "human," "patient," and "subject" are used interchangeably herein.

[0076] As used herein, the terms "treat", "treating" and "treatment" refer to a method of alleviating or eliminating a disease and / or its associated symptoms.

[0077] The phrase "therapeutically effective amount" refers to that amount of an ADC which, when administered for treatment in a particular subject or population of subjects, is sufficient to prevent the development of, or to alleviate to some extent, one or more of the symptoms of, the condition or disorder being treated.

[0078] 8. Examples

[0079] The following examples highlight certain features and characteristics of exemplary embodiments of ADCs, antibodies and TOPIi as described herein and are provided for purposes of illustration.

[0080] Example 1

[0081]

[0082] Example 1

[0083] (2S)-2-(2-Bromoacetylamino)-N-[(2S)-1-({3-[(7S)-7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-2H,10H-[1,3]dioxol[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-14-yl]bicyclo[1.1.1]pentan-1-yl}amino)-1-oxopropan-2-yl]-3-methylbutanamide

[0084]

[0085] Example 1A

[0086] tert-Butyl (3-(methoxy(methyl)carbamoyl)bicyclo[1.1.1]pentan-1-yl)carbamate

[0087] At 10°C, 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxo hexafluorophosphate (8.61 g) was added portionwise to a solution of 3-((tert-butyloxycarbonyl)amino)bicyclo[1.1.1]pentane-1-carboxylic acid (4.9 g), N,O-dimethylhydroxylamine hydrochloride (2.2 g) and N,N-diisopropylethylamine (11.30 mL) in dichloromethane (10 mL). The reaction mixture was stirred at 20°C for 12 hours. Two additional reactions were performed and stirred at 20°C for 12 hours as described above. All three reactions were combined. The reaction was diluted with dichloromethane (200 mL) and added to a 1N aqueous HCl solution (50 mL). The precipitate formed was filtered and the filtrate was separated. The organic layer was washed with saturated aqueous sodium bicarbonate solution (50 mL) and brine (50 mL), dried over anhydrous sodium sulfate, filtered and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel chromatography eluting with 1%-50% ethyl acetate / petroleum ether to give the title compound. 1 H NMR (400MHz, CDCl3) δppm 4.97 (br s, 1H), 3.66 (s, 3H), 3.18 (s, 3H), 2.34 (s, 6H), 1.45 (s, 9H). MS(ESI+)m / z 271.2(M+H) + .

[0088]

[0089] Example 1B

[0090] tert-Butyl (3-(Benzo[d][1,3]dioxole-5-carbonyl)bicyclo[1.1.1]pentan-1-yl)carbamate

[0091] To a solution of 5-bromobenzo[d][1,3]dioxole (8.83 g) in tetrahydrofuran (100 mL) was slowly added n-butyllithium (17.57 mL, 2.5 M in hexane) at -65 ° C under nitrogen. The mixture was stirred at -65 ° C for 30 minutes. A solution of Example 1A (4.75 g) in tetrahydrofuran (40 mL) was slowly added. The mixture was stirred at -65 ° C for 3 hours. Three additional reactions were performed and allowed to stir at -65 ° C for 3 hours. All four reactions were combined. The mixture was quenched with saturated aqueous ammonium chloride solution (500 mL) and extracted with ethyl acetate (3×500 mL). The combined organic layers were washed with brine (500 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel chromatography eluting with 1%-50% ethyl acetate / petroleum ether to give the title compound. 1 HNMR (400MHz, CDCl3) δppm7.63(dd,1H),7.45(d,1H),6.82(d,1H),6.03(s,2H),5.04(br s,1H),2.50(s,6H),1.46(s,9H). MS(ESI+)m / z 354.2(M+Na) + .

[0092]

[0093] Example 1C

[0094] N-(3-(Benzo[d][1,3]dioxole-5-carbonyl)bicyclo[1.1.1]pentan-1-yl)-2,2,2-trifluoroacetamide

[0095] Step 1: To a solution of Example 1B (6.2 g) in dichloromethane (62 mL) was slowly added trifluoroacetic acid (62 mL) at 0°C. The reaction was stirred at 25°C for 4 hours. Two additional reactions were carried out and stirred at 25°C for 4 hours. Each mixture was concentrated under reduced pressure. Each residue was used in the next step without further purification.

[0096] Step 2: N, N-diisopropylethylamine (16.34 mL) and trifluoroacetic anhydride (3.96 mL) were added dropwise to a solution of the above crude product in dichloromethane (62 mL) at 0 ° C. The mixture was stirred at 25 ° C for 2 hours. Two additional reactions were carried out as described and stirred at 25 ° C for 2 hours. All three reactions were combined and poured into water (200 mL) and extracted with dichloromethane (2×200 mL). The organic layer was washed with brine (200 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography eluting with 25% ethyl acetate / petroleum ether to obtain the title compound. 1 H NMR (501MHz, CDCl3) δppm7.61(dd,1H),7.43(d,1H),7.00(s,1H),6.85(d,1H),6.05(s,2H),2.62(s,6H). MS(ESI+)m / z328.2(M+H) + .

[0097]

[0098] Example 1D

[0099] 2,2,2-Trifluoro-N-(3-(6-nitrobenzo[d][1,3]dioxole-5-carbonyl)bicyclo[1.1.1]pentan-1-yl)acetamide

[0100] To a solution of Example 1C (4.3 g) in acetic anhydride (25 mL) was added copper (II) nitrate trihydrate (4.76 g) portionwise at 0°C. The mixture was stirred at 0°C for 3 hours. Three additional reactions were performed and stirred at 0°C for 3 hours as described above. All four reactions were combined. The mixture was poured into water (50 mL) and extracted with ethyl acetate (5×100 mL). The organic layer was dried over anhydrous sodium sulfate, filtered and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography eluting with 75% ethyl acetate / petroleum ether to give the title compound. 1 H NMR (400MHz, CDCl3) δppm 7.61 (s, 1H), 6.77 (br s, 1H), 6.64 (s, 1H), 6.21 (s, 2H), 2.43 (s, 6H). MS(APCI+)m / z 373.1(M+H) + .

[0101]

[0102] Example 1E

[0103] N-(3-(6-aminobenzo[d][1,3]dioxole-5-carbonyl)bicyclo[1.1.1]pentan-1-yl)-2,2,2-trifluoroacetamide

[0104] Under nitrogen, iron (5.4 g) and ammonium chloride (5.17 g) were added to a solution of Example 1D (4 g) in ethanol (40 mL) and water (8 mL). The mixture was stirred at 100 ° C for 3 hours. Three additional reactions were carried out and stirred at 100 ° C for 3 hours as described above. After cooling to ambient temperature, all four reactions were combined. The mixture was poured into water (1 L) and extracted with ethyl acetate (5 × 500 mL). The combined organic phases were dried over anhydrous sodium sulfate, filtered and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography eluting with 10%-75% ethyl acetate / petroleum ether to obtain the title compound. 1 HNMR (400MHz, CDCl3) δppm 7.22 (s, 1H), 6.70 (s, 1H), 6.50 (s, 2H), 6.14 (s, 1H), 5.92 (s, 2H), 2.63 (s, 6H). MS(ESI+)m / z 343.2(M+H) + .

[0105]

[0106] Example 1F

[0107] (S)-N-(3-(7-ethyl-7-hydroxy-8,11-dioxo-8,10,11,13-tetrahydro-7H-[1,3]dioxol[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-14-yl)bicyclo[1.1.1]pentan-1-yl)-2,2,2-trifluoroacetamide

[0108] To a suspension of Example 1E (3.5 g) and (S)-4-ethyl-4-hydroxy-7,8-dihydro-1H-pyrano[3,4-f]indolizine-3,6,10(4H)-trione (2.69 g) in toluene (140 mL) was added p-toluenesulfonic acid monohydrate (1.945 g). The mixture was stirred at 115 ° C for 12 hours. Three additional reactions were performed and stirred at 115 ° C for 12 hours as described above. After cooling to ambient temperature, all four reactions were combined. The mixture was filtered and the collected solid was ground with acetonitrile (200 mL) to obtain the title compound. 1H NMR (400 MHz, dimethyl sulfoxide-d6) δ ppm 10.28 (s, 1H), 7.64 (s, 1H), 7.52 (s, 1H), 7.24 (s, 1H), 6.47 (s, 1H), 6.30 (dd, 2H), 5.42 (s, 2H), 5.36 (s, 2H), 2.87 (s, 6H), 1.94-1.77 (m, 2H), 0.88 (t, 3H). MS (ESI+) m / z 570.3 (M+H) + .

[0109]

[0110] Example 1G

[0111] (7S)-14-(3-Aminobicyclo[1.1.1]pentan-1-yl)-7-ethyl-7-hydroxy-2H,10H-[1,3]dioxol[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-8,11(7H,13H)-dione

[0112] To a solution of Example 1F (3 g) in methanol (30 mL) was added HCl (60 mL, 4 M in methanol). The mixture was stirred at 65 °C for 4 hours. Four additional reactions were performed and stirred at 65 °C for 4 hours as described above. After cooling to ambient temperature, all five reactions were combined. The mixture was concentrated under reduced pressure, and the residue was triturated with methanol (200 mL) to give the title compound. 1 H NMR (400 MHz, dimethyl sulfoxide-d6) δppm 9.23 (s, 3H), 7.54 (s, 1H), 7.51 (s, 1H), 7.24 (s, 1H), 6.30 (d, 2H), 5.41 (s, 2H), 5.39-5.26 (m, 2H), 2.79 (s, 6H), 1.87 (hept, 2H), 0.88 (t, 3H). MS (ESI+) m / z 474.3 (M+H) + .

[0113]

[0114] Example 1H

[0115] tert-Butyl ((S)-1-(((S)-1-((3-((S)-7-ethyl-7-hydroxy-8,11-dioxo-8,10,11,13-tetrahydro-7H-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-14-yl)bicyclo[1.1.1]pentan-1-yl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamate

[0116] (S)-2-((S)-2-((tert-butyloxycarbonyl)amino)-3-methylbutyrylamino)propanoic acid (2.49 g), 2-hydroxypyridine 1-oxide (1.31 g) and N 1 -((ethylimino)methylene)-N 3 ,N 3 -dimethylpropane-1,3-diamine hydrochloride (2.26 g) in acetonitrile (40 mL) was added 2,6-lutidine (2.74 mL) and the mixture was stirred at ambient temperature for 30 minutes. In a separate flask, Example 1G (4 g) and 2,6-lutidine (2.74 mL) were combined in N,N-dimethylformamide (40 mL) and the above solution was added. The mixture was stirred overnight at ambient temperature. The mixture was concentrated under reduced pressure and the residue was dissolved in dichloromethane (300 mL), washed with saturated aqueous ammonium chloride solution (100 mL), brine (100 mL), dried over anhydrous sodium sulfate, filtered and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography eluting with 0-10% methanol / dichloromethane to give the title compound. 1 HNMR (400MHz, dimethyl sulfoxide-d6) δppm8.65(s,1H),7.89(d,1H),7.61(s,1H),7.49(s,1H),7.22(s,1H),6.77(d,1H),6.46(s,1H),6.29(d,2H),5.41(s,2H),5.32(s,2H),4.29(q,1H),3.87-3.77(m,1H),2.76(s,6H),1.99(q,1H),1.92-1.81(m,2H),1.41(s,9H),1.25(d,3H),0.92-0.80(m,9H). MS(ESI+)m / z 744.4(M+H) + .

[0117]

[0118] Example II

[0119] (S)-2-amino-N-((S)-1-((3-((S)-7-ethyl-7-hydroxy-8,11-dioxo-8,10,11,13-tetrahydro-7H-[1,3]dioxol[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-14-yl)bicyclo[1.1.1]pentan-1-yl)amino)-1-oxopropan-2-yl)-3-methylbutanamide

[0120] Example 1H (5.5 g) was treated with trifluoroacetic acid (30 mL) at ambient temperature for 30 minutes. The mixture was concentrated under reduced pressure and the residue was dissolved in 50% acetonitrile / water (200 mL). The solution was lyophilized to provide the title compound. 1 HNMR (600MHz, dimethyl sulfoxide-d6) δppm8.80(s,1H),8.59(d,1H),8.10(d,3H),7.60(s,1H),7.48(s,1H),7.23(s,1H),6.33-6.26(m,2H),5.41(d,2H),5.35-5.23(m,2H),4.35(p,1H),3.66-3.63(m,1H),2.77(s,6H),2.17-2.06(m,1H),1.91-1.83(m,2H),1.31(d,3H),1.01-0.96(dd,6H),0.89(t,3H). MS(ESI+)m / z 644.4(M+H) + .

[0121]

[0122] Example 1J

[0123] (2S)-2-(2-Bromoacetylamino)-N-[(2S)-1-({3-[(7S)-7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-2H,10H-[1,3]dioxol[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-14-yl]bicyclo[1.1.1]pentan-1-yl}amino)-1-oxopropan-2-yl]-3-methylbutanamide

[0124] To a solution of 2-bromoacetic acid (1.435 g) in N,N-dimethylformamide (26 mL) was added ethyl 2-ethoxyquinoline-1(2H)-carboxylate (2.55 g). The mixture was stirred at ambient temperature for 10 minutes. In a separate flask, Example II (4.5 g) and 2,6-lutidine (3.61 mL) were combined in N,N-dimethylformamide (26 mL) and the above solution was added. The mixture was stirred at ambient temperature for 30 minutes. The mixture was acidified with trifluoroacetic acid (4 mL) and Purify by reverse phase HPLC on a Teledyne Isco system using a Luna column (250 x 50 mm, 10 mm) eluting with 5% to 75% acetonitrile / water containing 0.1% trifluoroacetic acid over 30 minutes to give the title compound after lyophilization. 1 H NMR (600 MHz, dimethyl sulfoxide-d6) δppm8.57 (s, 1H), 8.32 (d, 1H), 8.16 (d, 1H), 7.67 (s, 1H), 7.52 (s, 1H), 7.24 (s, 1H), 6.30 (dd, 2H), 5.42 (s, 2H), 5.38 (d, 2H), 4.28-4.19 (m, 2H), 4.03-3.91 (m, 2H), 2.76 (s, 6H), 2.02 (h, 1H), 1.86 (ddp, 2H), 1.25 (d, 3H), 0.93-0.84 (m, 9H). MS (ESI+) m / z 764.46 (M+H) + .

[0125] Example 2: Cell proliferation

[0126] The effect of TOP1i drugs on cell viability was evaluated by monitoring the presence of ATP in metabolically active cells. The results are shown in Figure 1A , Figure 1B and Table 1. Using CellTiter- Luminescent quantification of proliferation of Calu-6 cells (lung adenocarcinoma) and A375 cells (malignant melanoma). Figure 1A , Figure 1B As shown in Table 1, the compound of formula (I) (Example 1G) showed efficacy in reducing proliferation of Calu-6 cells (0.57 nM, Figure 1A ) and A375 (0.50 nM, Figure 1B ) activity. This subnanomolar potency was improved over that of the TOP1i drug SN-38 in the same assay, demonstrating that EC 50 6.58nM (Calu-6) and 2.30nM (A375).

[0127]

[0128]

[0129] SN-38

[0130] Example 3: Preparation and purification of antibodies

[0131] Antibody Ab was expressed in Chinese hamster ovary (CHO) cells as an IgG1 antibody, and its heavy and light chain sequences were SEQ ID NOs: 9 and 10, respectively. Ab was isolated and purified prior to conjugation.

[0132] Example 4: Conjugation Procedure (ADC-1)

[0133] Ab was conjugated to a linker drug as described below (Example 1J) to form antibody drug conjugate ADC-1.

[0134] The Ab antibody from Example 3 was fully reduced with an excess of reducing agent and then conjugated with an excess of drug linker. This produced a final drug product comprising an ADC containing primarily six drug molecules, i.e., an ADC of formula (IV) with n being 6 and a DAR of 6 or about 6. The DAR of ADC-1 was determined by LC-MS. LC-MS analysis was performed using an Agilent 1100 HPLC system connected to an Agilent LC / MSD TOF 6220 ESI mass spectrometer. ADC-1 was treated with 5 mM (final concentration) Bond- TCEP solution (Thermo Scientific, Rockford, IL) was reduced, loaded onto a Protein Microtrap (Michrom Bioresorces, Auburn, CA) desalting cartridge column, and eluted with a gradient of 10% B to 75% B in 0.2 minutes at ambient temperature. Mobile phase A was H2O with 0.1% formic acid (FA), mobile phase B was acetonitrile with 0.1% FA, and the flow rate was 0.2 mL / min. Electrospray ionization time-of-flight mass spectra of co-eluted light and heavy chains were acquired using Agilent MassHunterTM acquisition software. The extracted intensity and m / z spectra were deconvoluted using the maximum entropy feature of MassHunter software to determine the mass of each reduced antibody fragment. The DAR was calculated from the deconvoluted spectrum by adding the intensity of the bare and modified peaks of the light and heavy chains, normalizing by multiplying the intensity by the number of attached drugs. The added normalized intensities were divided by the sum of the intensities and the sum of the two light chains and the two heavy chains gave the final average DAR value for intact ADC-1.

[0135] The Ab antibody solution at 4°C was treated with PBSE (125 mM potassium phosphate, 150 mM NaCl, 6.3 mM EDTA, pH 7.2) to a final concentration of 0.1 mM. Tris(2-carboxyethyl)phosphine (TCEP, 10 mM, 10 molar equivalents, Bond Breaker TM ,Thermo Scientific TM ) was added to the Ab antibody solution (approximately 10-20 mg / mL in 0.5X PBSE) with gentle mixing on a stir plate (225 rpm) for 5 minutes. After incubation at 4°C for 20 hours, the solution was allowed to reach 22°C.

[0136] use 3 0.57m 2 The Ab antibody was then ultrafiltered (UFDF) into a PBSE buffer using a Millipore 400 µl diafiltration kit. The Ab antibody was ultrafiltered (UF) until the Ab antibody reached a concentration of 40 mg / mL. The PBSE was diafiltered (DF) with 10 diafiltration volumes (DV).

[0137] A solution of 6.6 molar equivalents of 10 mM linker drug (Example 1J) in N,N-dimethylacetamide was gently mixed on a stirring plate and incubated at 23° C. for 16 hours. After incubation, 8.0 equivalents of 100 mM N-acetyl-L-cysteine ​​(NAC, Sigma Aldrich) prepared in sterile purified water was added to the solution, gently mixed, and then incubated at 23° C. for 2 hours.

[0138] use 30.57m 2 The antibody drug conjugate (ADC-1) buffer was exchanged into a mixture of DMSO (dimethyl sulfoxide) and 15 mM histidine buffer (pH 6) using UFDF in a cartridge (Millipore), and then exchanged into 15 mM histidine buffer. Ultrafiltration and diafiltration were performed. The ADC-1 solution was collected and then further diluted with buffer. The final ADC-1 solution was sterile filtered with a 0.22 μm filter and stored at -80°C.

[0139] Example 5A: Control ADC (ADC-2)

[0140] As used herein, ADC-2 is an ADC having antibody Ab2 conjugated to LD1 (Formula (III), Example 1J). Ab2 is MSL109, which is a monoclonal antibody that binds to CMV glycoprotein H. MSL109 is used as a non-targeting control antibody / negative control antibody.

[0141] Example 5B: Conjugation Procedure (ADC-2)

[0142] Ethylenediaminetetraacetic acid disodium salt aqueous solution (EDTA, 0.5 M, 974 μL, Sigma Aldrich) was added to 243.3 mL of purified Ab2 solution (12.3 mg / mL in 20 mM Tris buffer, pH 7.4). Tris(2-carboxyethyl)phosphine (TCEP, 500 mM, 6.0 molar equivalents, 242.4 μL, Bond Breaker TM , Thermo Scientific TM ) was added to the Ab2 solution with 2 mM EDTA and kept at 4° C. for 20 hours with gentle stirring. Tris buffer (1.0 M, pH 8.0) at 4° C. was added to the solution (24.5 mL, 10% v / v). Linker-drug LD1 (Example 1J, (2S)-2-(2-bromoacetamido)-N-[(2S)-1-({3-[(7S)-7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-2H,10H-[1,3]dioxol[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-14-yl]bicyclo[1.1.1]pentan-1-yl}amino)-1-oxopropan-2-yl]-3-methylbutanamide) was added to the reduced antibody at pH 8.0 (10 equivalents, 22.5 mL of a 10 mM solution of LD1 (Example 1J) in N,N-dimethylacetamide, 90% purity). The conjugate was gently stirred and allowed to stand in an ambient temperature water bath for 1.5 hours. 4 equivalents of N-acetyl-L-cysteine ​​in water (NAC, Sigma Aldrich, 100 mM solution in 1X DPBS, 0.8 mL) were added to quench the conjugation. The mixture was gently stirred and allowed to stand at ambient temperature for 1 hour. Tris buffer (15%, Sigma, 1.0 M, pH 7.4, 44 mL) was added to adjust the resulting ADC-2 solution to pH 7.5. The ADC-2 solution was stored at 4°C overnight. The ADC-2 solution was concentrated and buffer exchanged via ultrafiltration / diafiltration ( 3 0.22m 2 The final ADC-2 solution was filtered through a 0.22 μm filter and the resulting ADC-2 sample was stored at 4° C. The final volume of ADC-2 was 138 mL.

[0143] Example 6: In vitro SEZ6 expression and cell viability assay

[0144] SCLC cell lines NCI-H526 ("H526"), NCI-H524 ("H524"), NCI-H146 ("H146"), NCI-H1963 ("H1963"), and SW-1271 were obtained from the American Type Culture Collection (ATCC). COR-L95 cells were obtained from Millipore Sigma (#96020733). SBC-5 cells were obtained from the Japan Research Bioresource Cell Bank (JCRB).

[0145] SCLC cell lines (shown in column 1 of Table 2) were seeded in a white 384-well plate ( 3765). The cell plates were incubated overnight at 37°C in 5% CO2. Dose responses using stock concentrations of ADC or solutions containing Example 1G and DMSO (dimethyl sulfoxide) were applied to the cells on day 0 using a Tecan D300e digital dispenser and incubated for an additional 10 days (ADC) or 3 days (Example 1G) at 37°C in 5% CO2. Samples were run in duplicate. TM The luminescent cell viability assay (Promega, #G7570) was used to assess cell viability according to the manufacturer's instructions, except that the plates were shaken for 30 minutes before reading. Luminescence was read on a BioTek Synergy Neo2 and data were analyzed using GraphPad Prism software.

[0146] result :

[0147] like Figure 2A-2H As shown in Table 2, ADC-1 targeting SEZ6 has strong activity, showing sub-nM to low nM growth inhibition IC against five SEZ6 mRNA-positive SCLC cell lines (NCI-H524, NCI-H526, COR-L96, NCI-H146, NCI-H1963FP5). 50 In contrast, ADC-1 was inactive in two SCLC cell lines (SW-1271 and SBC-5) that were negative for SEZ6 mRNA. In addition, the non-targeted ADC-2 was inactive in all cell lines, confirming the SEZ6-dependent cell killing specificity of ADC-1. All cell lines were sensitive to Example 1G, as shown by sub-nM to low nM IC 50 These data demonstrate the SEZ6-specific activity of ADC-1 in SCLC cell lines expressing SEZ6.

[0148] Table 2. In vitro SEZ6 expression and cell viability

[0149]

[0150] nd = not determined due to lack of activity

[0151] a Fragments per kilobase of transcript per million mapped reads (FPKM) of SEZ6 as determined by the Cancer Cell Line Encyclopedia (CCLE), a publicly available database.

[0152] b IC in nanomolar (nM) 50 Values ​​represent 50% growth inhibition at 10 days compared to vehicle control (0%) and staurosporine (100%) treatment, with 95% confidence intervals for replicate titrations indicated in parentheses.

[0153] c IC in nanomolar (nM) 50 Values ​​represent 50% cell growth inhibition at 3 days compared to vehicle control (0%) and staurosporine (100%) treatment, with 95% confidence intervals for replicate titrations indicated in parentheses.

[0154] Example 7: ADC-1 inhibits the growth of cancer xenografts in vivo

[0155] Figure 3A and Figure 3B : Mouse CDX Model

[0156] Adult female SCID-bg (FoxChase SCID BeigeMouse) or SCID CB.17 (Fox Chase SCID Mouse) mice (approximately 22 g) were obtained from Charles River (Wilmington, MA). Mice were housed in solid bottom Plexiglas cages equipped with constant air circulation in groups of ten in an institution approved by the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC). Animals were tested during the light phase of a 12-hour light: dark cycle.

[0157] NCI-H69 ("H69"), NCI-H1963 ("H1963"), and NCI-H526 ("H526") cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA). Cells were maintained in 10% RPMI-1640 medium. Cell culture medium was obtained from Invitrogen (Carlsbad, CA) and supplemented with 10% fetal bovine serum (Hyclone, Logan, UT).

[0158] Five million cells were inoculated subcutaneously into the right flank of mice on day 0. The injection volume was 0.1 mL, consisting of a 1:1 mixture of culture medium and matrigel (BD, Franklin Lakes, NJ). The size of the tumor was matched to approximately 200 mm 3 . Treatment was initiated 24 hours after tumor size matching. There were four treatment groups: (1) vehicle; (2) ADC-1; (3) ADC-2; and (4) topotecan (ADC-1, ADC-2, and topotecan formulated in vehicle). Each was administered once daily. Tumor volume was calculated two to three times per week. Tumor length (L) and width (W) measurements were obtained via electronic calipers and volume was calculated according to the following formula: V = L x W 2 / 2.

[0159] Figure 3C-Figure 3D :Mouse PDX Model

[0160] SCLC patient-derived xenograft (PDX) models were established from stage IV tumors as previously described (Anderson et al. 2015 PLoS One; PMID: 25955027) and passaged in mice to generate sufficient cell numbers to inject single cell suspensions subcutaneously into the right lower mammary fat pad of NOD / SCID immunocompromised mice for efficacy studies. Once tumor cells were injected, tumor volume was collected using caliper measurements of length and width, and tumor volume was calculated using the formula for ellipsoids (volume = length × width × width × 0.5, where width is shorter than length; units: mm 3 ). When the tumor reaches 90 to 300 mm 3 After the range of , mice were randomly divided into treatment groups of five to ten mice per group, each group having approximately the same average tumor volume. Mice in the randomized treatment groups received the following: vehicle as a single intraperitoneal (ip) dose; topotecan standard of care (Hospira; 0.5 mg / kg once a day, a total of 5 doses ip administration; formulated with 0.9% saline); or ADC (ADC-1 or ADC-2; formulated in vehicle), each administered as a single ip dose. Efficacy was measured by monitoring tumor volume over time, and the data were plotted in GraphPad Prism.

[0161] The magnitude parameter of the therapeutic response (TGI max ) and persistence parameters (TGD) are used to refer to the efficacy of the drug. TGI max The maximum tumor growth inhibition during the experiment. v / C v ) to calculate tumor growth inhibition (TGI), where T v and C v are the mean tumor volumes of the treated and control groups, respectively. TGD or tumor growth delay is the time at which the treated tumor reaches 1 cm relative to the control group. 3 The extension time required to change the volume. TGD is calculated by 100*(T t / C t -1) calculation, where T t and C t The treatment group and the control group reached 1cm 3 The median time.

[0162] result :

[0163] When administered as a single agent in H1963 tumor-bearing mice, ADC-1 significantly inhibited tumor growth with (TGI max =94%, p=0.002) and induced a durable response with TGD>472%. In the H526 cell line, ADC-1 inhibitor significantly inhibited tumor growth with (TGI max =97.4%, p=<0.001) and durable response, TGD was 336.7% ( Figure 3A and Figure 3B ).

[0164] ADC-1 treatment produced durable responses (no detectable disease) in (1) cell line xenograft (CDX; H1963) and (2) patient-derived xenograft (PDX; LU95 and LU149) models. Strong efficacy (>30 days without measurable disease) was observed in the rapidly growing and aggressive H526CDX model, where vehicle-treated tumors grew to >2000 mm in 17 days. 3 Efficacy responses were SEZ6 target specific, as responses were rare with the nontargeted ADC-2 (modest to no tumor growth delay). ADC-1 was more effective than second-line standard of care topotecan for SCLC, which is administered as a single cycle at a dose and schedule close to human clinical exposure ( Figure 3A-3D ).

[0165]

[0166]

Claims

1. An anti-DLL3 antibody drug conjugate of formula (IV): wherein n is an integer from 1 to 10, and wherein Ab is an anti-DLL3 antibody comprising: a heavy chain variable region comprising CDR-H1, CDR-H2, and CDR-H3; and a light chain variable region comprising CDR-L1, CDR-L2 and CDR-L3, wherein CDR-H1 has the amino acid sequence shown in SEQ ID NO: 3, CDR-H2 has the amino acid sequence shown in SEQ ID NO:4, CDR-H3 has the amino acid sequence shown in SEQ ID NO:5, CDR-L1 has the amino acid sequence shown in SEQ ID NO:6, CDR-L2 has the amino acid sequence shown in SEQ ID NO:7, and CDR-L3 has the amino acid sequence shown in SEQ ID NO:

8.

2. The anti-DLL3 antibody-drug conjugate according to claim 1, wherein the antibody is an IgG1 antibody.

3. The anti-DLL3 antibody drug conjugate according to claim 1, wherein the antibody comprises (a) a heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 1, and (b) a light chain variable region having the amino acid sequence shown in SEQ ID NO:

2.

4. The anti-DLL3 antibody-drug conjugate according to claim 1, wherein the antibody comprises (a) a heavy chain having the amino acids shown in SEQ ID NO: 9, and (b) a light chain having the amino acid sequence shown in SEQ ID NO:

10.

5. An anti-DLL3 antibody drug conjugate of formula (IV): wherein n is an integer from 1 to 10, and wherein Ab is an anti-DLL3 antibody comprising: two heavy chains each having an amino acid sequence as shown in SEQ ID NO: 9 and two light chains each having an amino acid sequence as shown in SEQ ID NO:

10. 6 . The anti-DLL3 antibody drug conjugate according to any one of claims 1 to 5 , wherein n is 6.

7. The anti-DLL3 antibody drug conjugate according to any one of claims 1 to 5, wherein n is 2.

8. The anti-DLL3 antibody drug conjugate according to any one of claims 1 to 5, wherein n is 4.

9. The anti-DLL3 antibody drug conjugate according to any one of claims 1 to 5, wherein n is 8. 10 . The anti-DLL3 antibody drug conjugate according to claim 1 , wherein n is 10.

11. A composition comprising a therapeutically effective amount of the antibody drug conjugate according to any one of claims 1 to 5, wherein n of the predominant species of the antibody drug conjugate in the composition is 6.

12. A pharmaceutical composition comprising a therapeutically effective amount of the antibody drug conjugate according to any one of claims 1 to 5 and a pharmaceutically acceptable excipient, wherein the DAR of the composition is 6 or about 6.

13. A method for treating small cell lung cancer, the method comprising administering a therapeutically effective amount of the antibody drug conjugate according to any one of claims 1 to 5 to a patient in need thereof.

14. A method for treating small cell lung cancer, comprising administering a therapeutically effective amount of the pharmaceutical composition according to claim 12 to a patient in need thereof.

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