Antibody Drug Conjugates

Abstract

There is disclosed antibody drug conjugates having anthracycline derivative drug moieties that provide improved safety and cell killing efficacy, wherein the anthracycline derivative drug moieties substitute an hydroxymethyl ketone moiety for an hydrazide or hydroxamate moiety. The disclosed cytotoxic agents (i.e., drug moieties) are conjugated to an antibody via either a Cys or a Lys residue. For Lys conjugation, the DAR (drug antibody ratio) of the majority of the ADC is 2 whereas the DAR of the majority of ADC is 4 when conjugation occurs on a Cys residue.

Claims

1. An antibody drug conjugate (ADC) having a structure of Formula I
AbL.sup.1-L.sup.2-D).sub.n   (I) or a pharmaceutically acceptable salt thereof, wherein: Ab is an antibody; L.sup.1 is a connector; L.sup.2 is a linker selected from the group consisting of an amino acid, a peptide, —(CH.sub.2).sub.n—, —(CH.sub.2CH.sub.2O).sub.n—, PAB, Val-Cit-PAB, Val-Ala-PAB, Ala-Ala-Asn-PAB, and combinations thereof; wherein -L.sup.1-L.sup.2 is selected from the group consisting of ##STR00062## D is a drug moiety having a structure of Formula II ##STR00063## wherein Z═O, NH or CH.sub.2; R.sub.1═H, OH, or OMe; and R.sub.2 is a C1-C5 alkyl group. n is an integer from 1-10.

2. The ADC of claim 1, wherein Formula I is a composition selected from the group consisting of ##STR00064## ##STR00065## ##STR00066## ##STR00067##

Description

BRIEF DESCRIPTION OF THE FIGURES

[0016] FIG. 1 shows in vivo efficacy of ADC 20 (anti-Her2 antibody) in an N87 xenograft model.

[0017] FIG. 2 shows in vivo safety of ADC 20 (anti-Her2 antibody) in N87 cells in a xenograft model.

[0018] FIG. 3 shows in vivo efficacy of ADC 35 (anti-Her2 antibody) in an N87 xenograft model.

[0019] FIG. 4 shows in vivo safety of ADC 35 (anti-Her2 antibody) in an N87 xenograft model.

DETAILED DESCRIPTION

[0020] The present disclosure provides examples of the following disclosed antibody conjugates, listed for conjugation to a Lys on an antibody or to a Cys on an antibody.

TABLE-US-00001 TABLE 1 Structures of compounds synthesized (for Lys conjugation) Compound ID Structure 2 [00009] 3 [00010] 4 [00011] 5 [00012] 6 [00013] 7 [00014] 8 [00015]

TABLE-US-00002 TABLE 2 Structures of compounds synthesized (for Cys conjugation) Compound ID Structure 9 [00016] 10 [00017] 11 [00018] 12 [00019] 13 [00020] 14 [00021] 15 [00022] 16 [00023] 17 [00024] 18 [00025]

TABLE-US-00003 TABLE 3 Structures of antibody-drug conjugates synthesized Conjugate ID Structure 19 [00026] 20 [00027] 21 [00028] 22 [00029] 23 [00030] 24 [00031] 25 [00032] 26 [00033] 27 [00034] 28 [00035] 29 [00036] 30 [00037] 31 [00038] 32 [00039] 33 [00040] 34 [00041] 35 [00042] Ab is preferably an IgG class antibody.

Definitions

[0021] As used herein, common organic abbreviations are defined as follows: [0022] Ac Acetyl [0023] aq. Aqueous [0024] BOC or Boc tert-Butoxycarbonyl [0025] BrOP bromo tris(dimethylamino) phosphonium hexafluorophosphate [0026] Bu n-Butyl [0027] ° C. Temperature in degrees Centigrade [0028] DCM methylene chloride [0029] DEPC Diethylcyanophosphonate [0030] DIC diisopropylcarbodiimide [0031] DIEA Diisopropylethylamine [0032] DMA N,N′-Dimethylformamide [0033] DMF N,N′-Dimethylformamide [0034] EDC 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide [0035] Et Ethyl [0036] EtOAc Ethyl acetate [0037] Eq Equivalents [0038] Fmoc 9-Fluorenylmethoxycarbonyl [0039] g Gram(s) [0040] h Hour (hours) [0041] HATU 2-(1H-7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyl uronium hexafluorophosphate [0042] HOBT N-Hydroxybenzotriazole [0043] HOSu N-Hydroxysuccinimide [0044] HPLC High-performance liquid chromatography [0045] LC/MS Liquid chromatography-mass spectrometry [0046] Me Methyl [0047] MeOH Methanol [0048] MeCN Acetonitrile [0049] mL Milliliter(s) [0050] MS mass spectrometry [0051] PAB p-aminobenzyl [0052] RP-HPLC reverse phase HPLC [0053] rt room temperature [0054] t-Bu tert-Butyl [0055] TEA Triethylamine [0056] Tert, t tertiary [0057] TFA Trifluoracetic acid [0058] THF Tetrahydrofuran [0059] TLC Thin-layer chromatography [0060] L Microliter(s)

General Procedure—

[0061] Formation of an Activated Ester (e.g. NHS) from an Acid

[0062] An acid was dissolved in DCM and DMF was added to aid dissolution if necessary. N-hydroxysuccinimide (1.5 eq) was added, followed by EDC.HCl (1.5 eq). The reaction mixture was stirred at room temperature for 1 h until most of the acid was consumed. The progress of the reaction was monitored by RP-HPLC. The mixture was then diluted with DCM and washed successively with citric acid (aq. 10%) and brine. The organic layer was dried and concentrated to dryness. The crude product was optionally purified by RP-HPLC or silica gel column chromatography.

Example 1. Preparation of Compound 2

[0063] ##STR00043##

[0064] To compound 41 (72 mg, 0.10 mmol) in 3 mL of DMF was added DIEA (60 μL, 0.34 mmol), and hydroxylamine 58 (45 mg, 0.15 mmol). The mixture was stirred at room termperature for 16 h, then diluted with DCM (30 mL). The mixture was washed with brine. The organic layer was dried and evaporated to dryness. The residue was purified by column (silica gel, DCM:MeOH, 9:1) to give compound 3 (46 mg, 50%). MS m/z 917.4 (M+H).

Example 2. Preparation of Compound 3

[0065] ##STR00044##

[0066] To compound 41 (72 mg, 0.10 mmol) in 3 mL of DMF was added DIEA (60 μL, 0.34 mmol), and amine 42 (42 mg, 0.10 mmol). The mixture was stirred for 16 h, then evaporated and purified by column (silica gel, DCM:MeOH, 9:1) to give compound 3 (70 mg, 68%). MS m/z 1029.4 (M+H).

Example 3. Preparation of Compound 4

[0067] ##STR00045##

[0068] To compound 41 (72 mg, 0.10 mmol) in 3 mL of DMF was added DIEA (60 μL, 0.34 mmol), and hydrazide 59 (43 mg, 0.15 mmol). The mixture was stirred at room temperature for 16 h, then diluted with DCM (30 mL). The mixture was washed with brine. The organic layer was dried and evaporated to dryness. The residue was purified by column (silica gel, DCM:MeOH, 9:1) to give compound 4 (56 mg, 62%). MS m/z 899.4 (M+H).

Example 4. Preparation of Compound 5

[0069] ##STR00046##

[0070] To compound 41 (72 mg, 0.10 mmol) in 3 mL of DMF was added DIEA (60 μL, 0.34 mmol), and hydrazide 60 (50 mg, 0.15 mmol). The mixture was stirred at room temperature for 16 h, then diluted with DCM (30 mL). The mixture was washed with brine. The organic layer was dried and evaporated to dryness. The residue was purified by column (silica gel, DCM:MeOH, 9:1) to give compound 5 (41 mg, 44%). MS m/z 942.5 (M+H).

Example 5. Preparation of Compound 6

[0071] ##STR00047##

[0072] To compound 41 (72 mg, 0.10 mmol) in 3 mL of DMF was added DIEA (60 μL, 0.34 mmol), and hydrazide 61 (87 mg, 0.15 mmol). The mixture was stirred at room temperature for 16 h, then diluted with DCM (50 mL). The mixture was washed with brine. The organic layer was dried and evaporated to dryness. The residue was purified by column (silica gel, DCM:MeOH, 9:1) to give compound 6 (47 mg, 40%). MS m/z 1186.5 (M+H).

Example 6. Preparation of Compound 7

[0073] ##STR00048##

[0074] To compound 41 (72 mg, 0.10 mmol) in 3 mL of DMF was added DIEA (60 μL, 0.34 mmol), and hydrazide 62 (30 mg, 0.15 mmol). The mixture was stirred at room temperature for 16 h, then diluted with DCM (40 mL). The mixture was washed with brine. The organic layer was dried and evaporated to dryness. The residue was purified by column (silica gel, DCM:MeOH, 9:1) to give compound 7 (57 mg, 56%). MS m/z 1015.5 (M+H).

Example 7. Preparation of Compound 8

[0075] ##STR00049##

[0076] To compound 41 (72 mg, 0.10 mmol) in 3 mL of DMF was added DIEA (75 μL), and amine. TFA 63 (86 mg, 0.12 mmol). The mixture was stirred at room temperature for 3 h, then diluted with DCM (40 mL). The mixture was washed with brine. The organic layer was dried and evaporated to dryness. The residue was purified by column (silica gel, DCM:MeOH, 9:1) to give compound 8 (63 mg, 52%). MS m/z 1214.5 (M+H).

Example 8. Preparation of Compound 9

[0077] ##STR00050##

[0078] To compound 44 (3.3 mg, 7.7 umol) in 2 mL of DMF was added DIEA (2.6 μL, 15 umol), PyBrOP (2.3 mg, 5 μmol), and amine 43 (2.5 mg, 3 μmol). The mixture was stirred for 10 min, then purified by column (silicagel, DCM:MeOH, 95:5) to give compound 9 (2.0 mg, 54%). MS m/z 1228.3 (M+H).

Example 9. Preparation of Compound 10

[0079] ##STR00051##

[0080] To compound 64 (10 mg, 23 umol) in 2 mL of DMF was added DIEA (8 μL, 50 umol), PyBrOP (7 mg, 15 μmol), and amine 43 (8 mg, 10 μmol). The mixture was stirred for 10 min, then purified by column (silicagel, DCM:MeOH, 90:10) to give compound 10 (5.0 mg, 42%). MS m/z 1202.3 (M+H).

Example 10. Preparation of Compound 11

[0081] ##STR00052## ##STR00053##

Preparation of Compound 47

[0082] To compound 45 (17.7 mg, 28 μmol) in 2 mL of DMF was added DIEA (5 μL, 30 μmol), HATU (11 mg, 29 μmol), and amine 46 (48 mg, 28 μmol). The mixture was stirred for 30 min, then 100 μL of pipridine added. After 15 min, the mixture was evaporated and purified by HPLC to give compound 47 (18 mg, 30%). MS m/z 1974.7 (M+H).

Preparation of Compound 11

[0083] To compound 48 (13.6 mg, 40 μmol) in 2 mL of DCM was added DIC (2.5 mg, 20 μmol), and amine 47 (18 mg, 9 μmol). The mixture was stirred for 30 min, then purified by HPLC to give compound 11 (9 mg, 43%). MS m/z 2296.8 (M+H).

Example 11. Preparation of Compound 12

[0084] ##STR00054##

[0085] To compound 45 (45 mg, 72 μmol) in 2 mL of DMF was added DIEA (13 μL, 80 μmol), HATU (28 mg, 74 μmol), and amine 49 (36 mg, 72 μmol). The mixture was stirred for 30 min, then 100 μL of pipridine added. After 15 min, the mixture was evaporated and purified by HPLC to give compound 50 (16 mg, 25%). MS m/z 889.4 (M+H).

[0086] To compound 48 (13.6 mg, 40 μmol) in 2 mL of DCM was added DIC (2.5 mg, 20 μmol), and amine 50 (16 mg, 18 μmol). The mixture was stirred for 30 min, then purified by HPLC to give compound 12 (7 mg, 32%). MS m/z 1212.3 (M+H).

Example 12. Preparation of Compound 13

[0087] ##STR00055##

[0088] To compound 45 (45 mg, 72 μmol) in 2 mL of DMF was added DIEA (13 μL, 80 μmol), HATU (28 mg, 74 μmol), and amine 51 (49 mg, 72 μmol). The mixture was stirred for 30 min, then 100 μL of pipridine added. After 15 min, the mixture was evaporated and purified by HPLC to give compound 52 (27 mg, 35%). MS m/z 1074.4 (M+H).

[0089] To compound 53 (15 mg, 40 μmol) in 2 mL of DCM was added DIC (2.5 mg, 20 μmol), and amine 52 (21 mg, 20 μmol). The mixture was stirred for 30 min, then purified by HPLC to give compound 13 (13 mg, 47%). MS m/z 1416.3 (M+H).

Example 13. Preparation of Compound 14

[0090] ##STR00056##

[0091] To a solution of compound 50 (18 mg, 0.02 mmol) in DCM (2 mL) was added compound 65 (15 mg), followed by DIEA (5 μL). The mixture was stirred at room temperature for 10 min. The reaction was then diluted with DCM (30 mL) and washed with aq. saturated NaHCO.sub.3. The organic layer was concentrated and residue was purified by RP-HPLC to give compound 14 as a red solid after lyophilization (7 mg, 29%). MS m/z 1231.3 (M+H).

Example 14. Preparation of Compound 15

[0092] ##STR00057##

[0093] To compound 55 (9 mg, 20 μmol) in 2 mL of DCM was added PyBrOP (9 mg, 20 μmol), DIEA (8 μL, 80 μmol), and amine 54 (15 mg, 20 μmol). The mixture was stirred for 30 min, then evaporated and purified by HPLC to give compound 15 (9 mg, 37%). MS m/z 1253.2 (M+H).

Example 15. Preparation of Compound 16

[0094] ##STR00058##

[0095] To compound 55 (9 mg, 20 μmol) in 2 mL of DCM was added PyBrOP (9 mg, 20 μmol), DIEA (8 μL, 80 μmol), and amine 56 (15 mg, 20 μmol). The mixture was stirred for 30 min, then evaporated and purified by HPLC to give compound 16 (8 mg, 33%). MS m/z 1196.2 (M+H).

Example 16. Preparation of Compound 17

[0096] ##STR00059##

[0097] To compound 57 (12 mg, 20 μmol) in 2 mL of DCM was added PyBrOP (9 mg, 20 μmol), DIEA (8 μL, 80 μmol), and amine 54 (15 mg, 20 μmol). The mixture was stirred for 30 min, then evaporated and purified by HPLC to give compound 17 (13 mg, 47%). MS m/z 1419.3 (M+H).

Example 17. Preparation of Compound 18

[0098] ##STR00060##

[0099] To a solution of compound 45 (63 mg, 0.1 mmol) in DMF (3 mL) was added compound 66 (75 mg, 0.1 mmol), followed by DIEA (70 μL) and HATU (40 mg). The mixture was stirred at room temperature for 5 min, then diluted with DCM (50 mL). The mixture was washed with aq. saturated NaHCO.sub.3 and brine. The organic layer was dried and concentrated. The crude product was purified by column chromatography (silica gel, MeOH/DCM: 1/19, /v/v) to give compound 67 as a red solid (81 mg, 61%)

[0100] Compound 67 (66 mg, 0.05 mmol) was dissolved in DMF (2 mL). Pipridine (100 μL) was added. The mixture was stirred at room temperature for 30 min and then concentrated to dryness under reduced pressure. The residue was redissolved in DCM (3 mL). Anhydride 65 (42 mg) was added, followed by DIEA (18 μL). After 30 min, the reaction was concentrated and the crude product was purified by RP-HPLC to give compound 18 as a red solid (52 mg, 72%). MS m/z 1444.5 (M+H).

Example 18

[0101] This example provides the results of EC50 assays of the designated drug conjugated antibodies measured in vitro in specified cells. ADC 70 was synthesized from an unmodified PNU-159682 (WO 2010/009124 A2) conjugated to an anti-Her 2 antibody as a comparison. Most of ADCs disclosed here showed much improved safety characteristics (ADC 21-29, 31, and 35) and some ADCs showed improved cell king efficacy (ADC 26, 30, 31, and 34).

TABLE-US-00004 MDA- MDA- MDA- SBKR3 HCC1954 MCF7 MDAMB BT474 MB-453 MB-175 MB-361 Conjugate (Her2+++) (Her2+++) (Her2+) 468 (−) (Her2+++) (Her2++) (Her2+) (Her2+++) ID EC50 [nM] 20 0.030 0.400 11.660 5.648 21 0.072 29.130 0.885 0.740 22 0.074 13.570 0.778 9.566 0.697 26 0.022 14.370 0.374 0.403 0.124 28 0.031 0.541 ~80 14.910 ~30 11.820 29 0.049 0.343 ~100 31.480 ~50 ~20 30 0.023 0.066 18.740 3.393 0.143 0.169 0.264 0.063 31 0.028 0.128 12.560 0.283 0.270 0.087 32 0.034 0.775 0.108 0.210 0.057 0.093 34 0.011 0.144 1.619 0.923 0.167 0.655 35 0.062 0.111 59.180 0.296 0.194 0.137 0.057 70 0.033 10 5

##STR00061##

Example 19

[0102] This example shows in vivo efficacy of ADC 20 (an anti-Her2 antibody conjugate) in a Subcutaneous N87 Xenograft Model. FIG. 1 shows a single dose of conjugate 20 administered to BALB/c nude mice by intravenous administration. There were 8 mice in each group and total 3 groups of mice were studied: 1 group of mice was injected with T-DM1 (Trastuzumab-DM1 conjugate); one group of mice was injected with ADC 20; and one vehicle control. All the drugs were administered in the same manner (single dose). A single dose of ADC-20 iv. at 1 mg/kg outperformed T-DM1 at 2 mg/kg and completely inhibited tumor growth up to 58 days.

Example 20

[0103] This example shows in vivo safety of ADC 20 (an anti-Her2 antibody conjugate) in a Subcutaneous N87 Xenograft Model. FIG. 2 shows a single dose of conjugate 20 administered to BALB/c nude mice by intravenous administration. There were 8 mice in each group and total 3 groups of mice were studied: 1 group of mice was injected with T-DM1 (Trastuzumab-DM1 conjugate); 1 group of mice was injected with ADC 20; and one vehicle control. All the drugs were administered in the same manner (single dose). A single dose of ADC-20 iv. at 1 mg/kg did not retard body weight gain and was comparable to that of T-DM1

Example 21

[0104] This example shows in vivo efficacy of ADC 35 (an anti-Her2 antibody conjugate) in a Subcutaneous N87 Xenograft Model. FIG. 3 shows a single dose of conjugate 30 administered to BALB/c nude mice by intravenous administration. There were 8 mice in each group and total 3 groups of mice were studied: 1 group of mice was injected with T-DM1 (Trastuzumab-DM1 conjugate); 1 group of mice was injected with ADC 20; and one vehicle control. All the drugs were administered in the same manner (single dose). A single dose of ADC-35 iv. at 1 mg/kg outperformed T-DM1 at 2 mg/kg and completely inhibited tumor growth up to 58 days.

Example 22

[0105] This example shows in vivo safety of ADC 35 (an anti-Her2 antibody conjugate) in a Subcutaneous N87 Xenograft Model. FIG. 4 shows a single dose of conjugate 30 administered to BALB/c nude mice by intravenous administration. There were 8 mice in each group and total 3 groups of mice were studied: 1 group of mice was injected with T-DM1 (Trastuzumab-DM1 conjugate); 1 group of mice was injected with ADC 20; and one vehicle control. All the drugs were administered in the same manner (single dose). A single dose of ADC-35 iv. at 1 mg/kg did not retard body weight gain and was comparable to that of T-DM1.

Example 23

[0106] This example shows a general conjugation procedure for synthesizing antibody drug conjugates 19, 20, 21, 22, 23, 24 and 25 (Table 3 above). To a solution of 0.5-50 mg/mL of antibody in buffer at pH 6.0-9.0 with 0-30% organic solvent, was added 0.1-10 eq of activated drug linker conjugate (2, or 3, or 4, or 5, or 6, or 7, or 8) in a manner of portion wise or continuous flow. The reaction was performed at 0-40° C. for 0.5-50 hours with gentle stirring or shaking, monitored by HIC-HPLC. The resultant crude ADC product underwent necessary down-stream steps of desalt, buffet changes/formulation, and optionally, purification, using the state-of-art procedures. The ADC product was characterized by HIC-HPLC, SEC, RP-HPLC, and optionally LC-MS.

Example 24

[0107] This example shows the general conjugation procedure for synthesizing antibody drug conjugates 26, 27, 28, 29, 30, 31, 32, 33, 34 and 35 (Table 3 above). To a solution of antibody, 0.5-50 mgs/mL, in a certain buffet at pH 5.0-9.0, such as PBS, was added 0.5-100 eq of reducing agent such as TCEP and DTT. The reduction was performed at 0-40° C. for 0.5-40 hours with gentle stirring or shaking, and then the reducing agent was removed by column or ultrafiltration. To the reduced antibody, 0.5-50 mg/mL, in a certain buffet at pH 5.0-9.0, such as PBS, with 0-30% of organic co-solvent such as DMA, was added 0.5-10 eq of the drug-linker reactant (selected from compound 9). The reaction was conducted at 0-40° C. for 0.5-40 hours with gentle stirring or shaking, monitored by HIC-HPLC. The resultant crude ADC product underwent necessary down-stream steps of desalt, buffet changes/formulation, and optionally, purification, using the state-of-art procedures. The final ADC product was characterized by HIC-HPLC, SEC, RP-HPLC, and optionally LC-MS.