Anti-erbb2 antibody-drug conjugate and composition thereof, preparation method therefor, and application thereof

Abstract

The present invention provides an anti-ErbB2 antibody-drug conjugate, a composition comprising the conjugate, a method for preparing the conjugate, and use of the conjugate.

1 . A method for increasing homogeneity of a mixture of different antibody-drug conjugates (ADCs), the method comprising:
 separating the mixture according to a hydrophobic interaction chromatography (HIC) method that utilizes a stationary phase that comprises a phenyl resin to produce an ADC composition that has an increased percentage of an ADC relative to the mixture;   wherein a mobile phase A of the HIC method comprises ammonia sulfate and disodium hydrogen phosphate; and a mobile phase B of the HIC method comprises disodium hydrogen phosphate and isopropanol;   wherein the different antibody-drug conjugates of the mixture comprise the structure of formula I:   
       
         
           
           
               
               
           
         
         wherein A is an anti-ErbB2 antibody or an active fragment or variant thereof; 
         a is an integer selected from the group consisting of 2-10; 
         X is N; 
         Y is CR 1 , and R 1  is H or C 1 -C 10  alkyl; 
         L is a divalent linker; and 
         D is a cytotoxic agent group; 
         wherein all cytotoxic agent groups are conjugated to lysine residues of the same peptide segment of the light chain of the anti-ErbB2 antibody or the active fragment or variant of the ADC composition. 
       
     
     
         2 . The method of  claim 1 , wherein the ratio of ammonia sulfate to disodium hydrogen phosphate in the mobile phase A is 60:1. 
     
     
         3 . The method of  claim 1 , wherein the concentration of ammonia sulfate in the mobile phase A is 25 mM. 
     
     
         4 . The method of  claim 1 , wherein the concentration of isopropanol in the mobile phase B is 10% v/v. 
     
     
         5 . The method of  claim 1 , wherein a gradient elution step of the HIC method comprises:
 applying a gradient from 0-40% of the mobile phase B and 60-100% of the mobile phase A by 20 column volumes (CV); and   applying a gradient from 40-100% of the mobile phase B and 0-60% of the mobile phase A by 30 CV.   
     
     
         6 . The method of  claim 1 , wherein the pH of each of the mobile phase A and the mobile phase B is 7.0. 
     
     
         7 . The method of  claim 1 , wherein a velocity of flow of the mobile phase A and the mobile phase B of the HIC method is 1.0 mL/min. 
     
     
         8 . The method of  claim 1 , wherein the anti-ErbB2 antibody is Trastuzumab. 
     
     
         9 . The method of  claim 8 , wherein a is 2, 3, or 4. 
     
     
         10 . The method of  claim 1 , wherein Lis selected from the group consisting of: 
       
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         wherein each of m and n is an integer individually selected from the group consisting of: 1, 2, 3, 4, 5, and 6. 
       
     
     
         11 . The method of  claim 1 , wherein the cytotoxic agent group is derived from a compound of Formula (D1) or (D2) or a stereoisomer thereof: 
       
         
           
           
               
               
           
         
         wherein: 
         R 2  is selected from the group consisting of: —CH 2 N 3 , —CONHSO 2 (cyclopropyl), thiazol-2-yl, —CH 3 , and —COOH; 
         R 3  is selected from the group consisting of: H and —OH; and 
         R 4  is selected from the group consisting of: H, —NH 2 , Cl, Br, I, —OS(O) 2 R 6 , wherein R 6  is H, C 1 -C 8  alkyl, C 3 -C 8  cycloalkyl, or C 6 -C 14  aryl, and 
         the alkyl, cycloalkyl, and aryl are each optionally substituted with one or more halogen substituents; 
       
       
         
           
           
               
               
           
         
         wherein: 
         R 5  is selected from the group consisting of: —CH(CH 3 )N(CH 3 )C(O)CH 2 CH 2 SH and —CH(CH 3 )N(CH 3 )C(O)CH 2 C(CH 3 ) 2 SH. 
       
     
     
         12 . The method of  claim 1 , wherein the ADC of the ADC composition is selected from the group consisting of: I-1, I-2, and I-3: 
       
         
           
           
               
               
           
         
         wherein A is Trastuzumab. 
       
     
     
         13 . The method of  claim 1 , wherein different values of a in formula I correspond to different ADCs of the mixture. 
     
     
         14 . A method for preparing the ADC composition of  claim 1 , wherein the method comprises:
 preparing a compound of Formula (I-A):   
       
         
           
           
               
               
           
         
         wherein X is N; 
         Y is CR 1 , and R 1  is H or C 1 -C 10  alkyl; 
         L is a divalent linker; and 
         D is a cytotoxic agent group; 
         obtaining a compound of Formula I-A-G by activating the compound of Formula I-A: 
       
       
         
           
           
               
               
           
         
         wherein G is selected from the group consisting of: —F, —Cl, —Br, —I, —N 3 , —OR, —SR, —ONRR′, RC(O)O—, —OP(O)RR′, RSO 2 —O— and 
       
       
         
           
           
               
               
           
         
       
       wherein each of R and R′ at each occurrence is independently C 1 -C 10  alkyl, C 6 -C 14  aryl, heterocyclyl having 5 to 10 ring atoms, or phenoxy, and each of the alkyl, aryl, heterocyclyl and phenoxy is unsubstituted or independently substituted with one or more substituents selected from the group consisting of: halogen, hydroxy, C 1 -C 4  alkyl, C 1 -C 4  alkoxy, C 3 -C 8  cycloalkyl, heterocyclyl having 5 to 8 ring atoms, C 6 -C 10  aryl, and heteroaryl having 5 to 10 ring atoms;
 obtaining the mixture of  claim 1  having different drug-to-antibody ratio (DAR) values by conjugating the compound of Formula (I-A-G) with the anti-ErbB2 antibody or the active fragment or variant thereof of  claim 1 ; and 
 increasing homogeneity of the mixture by the method of  claim 1 . 
 
     
     
         15 . The method of  claim 14 , wherein G is selected from the group consisting of: —ONRR′ and —OP(O)RR′; wherein each of R and R′ at each occurrence is phenoxy. 
     
     
         16 . The method of  claim 14 , wherein the compound of Formula (I-A-G) is a compound of Formula (I-B) formed by reacting the compound of Formula (I-A) with pentafluorophenol: 
       
         
           
           
               
               
           
         
         wherein D, L, X and Y are as defined in  claim 1  for Formula (I), and the reaction is performed using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, N-hydroxysuccinimide, and/or dichloromethane. 
       
     
     
         17 . The ADC composition produced by the method of  claim 14 . 
     
     
         18 . The ADC composition of  claim 17 , wherein the anti-ErbB2 antibody is Trastuzumab. 
     
     
         19 . The ADC composition of  claim 17 , wherein a is 2, 3, or 4. 
     
     
         20 . The ADC composition of  claim 17 , wherein the percentage of ADC in the ADC composition having a DAR of 2 is greater than the percentage of ADC in the mixture having a DAR of 2. 
     
     
         21 . The ADC composition of  claim 17 , wherein L is selected from the group consisting of: 
       
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         wherein each of m and n is an integer individually selected from the group consisting of: 1, 2, 3, 4, 5, and 6. 
       
     
     
         22 . The ADC composition of  claim 17 , wherein the cytotoxic agent group is derived from a compound of Formula (D1), (D2), or a stereoisomer thereof: 
       
         
           
           
               
               
           
         
         wherein: 
         R 2  is selected from the group consisting of: —CH 2 N 3 , —CONHSO 2 (cyclopropyl), thiazol-2-yl, —CH 3 , and —COOH; 
         R 3  is selected from the group consisting of: H and —OH; and 
         R 4  is selected from the group consisting of: H, —NH 2 , Cl, Br, I, —OS(O) 2 R 6 , wherein R 6  is H, C 1 -C 8  alkyl, C 3 -C 8  cycloalkyl, or C 6 -C 14  aryl, and 
         the alkyl, cycloalkyl, and aryl are each optionally substituted with one or more halogen substituents; 
       
       
         
           
           
               
               
           
         
         wherein: 
         R 5  is selected from the group consisting of: —CH(CH 3 )N(CH 3 )C(O)CH 2 CH 2 SH and —CH(CH 3 )N(CH 3 )C(O)CH 2 C(CH 3 ) 2 SH. 
       
     
     
         23 . The ADC composition of  claim 12 . 
     
     
         24 . A pharmaceutical composition comprising the ADC composition of  claim 23  and a pharmaceutically acceptable carrier. 
     
     
         25 . A method for the prophylaxis or treatment of cancer, comprising administering to a patient in need thereof the pharmaceutical composition of  claim 24 . 
     
     
         26 . The method of  claim 25 , wherein the cancer is selected from the group consisting of: breast cancer, gastric cancer, ovarian cancer, non-small cell lung cancer, liver cancer, endometrial cancer, salivary gland cancer, kidney cancer, colon cancer, thyroid cancer, pancreas cancer, and bladder cancer. 
     
     
         27 . The method of  claim 25 , wherein the cancer is breast cancer.