Antibody-conjugates with improved therapeutic index for targeting her2 tumours and method for improving therapeutic index of antibody-conjugates

Abstract

The present invention concerns novel and improved antibody-conjugates for targeting HER2. The inventors found that when antibody-conjugates were prepared using a specific mode of conjugation, they exhibit an improved therapeutic index. The mode of conjugation comprises a first step (i) of contacting a glycoprotein comprising 1-4 core N-acetylglucosamine moieties with a compound of the formula S(F1)x—P in the presence of a catalyst, wherein S(F1)x is a sugar derivative comprising x functional groups F1 capable of reacting with a functional group Q1, x is 1 or 2 and P is a nucleoside mono- or diphosphate, and wherein the catalyst is capable of transferring the S(F1)x moiety to the core-GlcNAc moiety, to obtain a modified antibody; and a second step (ii) of reacting the modified antibody with a linker-conjugate comprising a functional group Q1 capable of reacting with functional group F1 and a target molecule D connected to Q1 via a linker L2 to obtain the antibody-conjugate wherein linker L comprises S—Z3-L2 and wherein Z3 is a connecting group resulting from the reaction between Q1 and F1. The invention also relates to a use for improving the therapeutic index of an antibody-conjugate and to a method for targeting HER2-expressing cells.

1 . Antibody-conjugate comprising an antibody AB connected to a target molecule D via a linker L, obtainable by:
 (i) contacting a glycoprotein comprising 1-4 core N-acetylglucosamine moieties with a compound of the formula S(F 1 ) x —P in the presence of a catalyst, wherein S(F 1 ) x  is a sugar derivative comprising x functional groups F 1  capable of reacting with a functional group Q 1 , x is 1 or 2 and P is a nucleoside mono- or diphosphate, and wherein the catalyst is capable of transferring the S(F 1 ) x  moiety to the core-GlcNAc moiety, to obtain a modified antibody according to Formula (24):   
       
         
           
           
               
               
           
         
         
           wherein S(F 1 ) x  and x are as defined above; AB represents an antibody; GlcNAc is N-acetylglucosamine; Fuc is fucose; b is 0 or 1; and y is 1, 2, 3 or 4; and 
         
         (ii) reacting the modified antibody with a linker-conjugate comprising a functional group Q 1  capable of reacting with functional group F 1  and a target molecule D connected to Q 1  via a linker L 2  to obtain the antibody-conjugate wherein linker L comprises S—Z 3 -L 2  and wherein Z 3  is a connecting group resulting from the reaction between Q 1  and F 1 , 
         wherein antibody AB is capable of targeting HER2-expressing tumours and target molecule D is selected from the group consisting of taxanes, anthracyclines, camptothecins, epothilones, mytomycins, combretastatins, vinca alkaloids, maytansinoids, calicheamycins and enediynes, duocarmycins, tubulysins, amatoxins, dolastatins and auristatins, pyrrolobenzodiazepine dimers, indolino-benzodiazepine dimers, radioisotopes, therapeutic proteins and peptides (or fragments thereof), kinase inhibitors, MEK inhibitors, KSP inhibitors, and analogues or prodrugs thereof. 
       
     
     
         2 . The antibody-conjugate according to  claim 1 , wherein the antibody is capable of targeting HER2-expressing tumours and is selected from trastuzumab, margetuximab, pertuzumab, HuMax-Her2, ertumaxomab, HB-008, ABP-980, BCD-022, CanMab, Herzuma, HD201, CT-P6, PF-05280014, COVA-208, FS-102, MCLA-128, CKD-10101, HT-19 and functional analogues thereof. 
     
     
         3 . The antibody-conjugate according to  claim 1 , wherein linker L 2  comprises a group according to formula (1) or a salt thereof: 
       
         
           
           
               
               
           
         
         wherein:
 a is 0 or 1; and 
 R 31  is selected from the group consisting of hydrogen, C 1 -C 24  alkyl groups, C 3 -C 24  cycloalkyl groups, C 2 -C 24  (hetero)aryl groups, C 3 -C 24  alkyl(hetero)aryl groups and C 3 -C 24  (hetero)arylalkyl groups, the C 1 -C 24  alkyl groups, C 3 -C 24  cycloalkyl groups, C 2 -C 24  (hetero)aryl groups, C 3 -C 24  alkyl(hetero)aryl groups and C 3 -C 24  (hetero)arylalkyl groups optionally substituted and optionally interrupted by one or more heteroatoms selected from O, S or NR 33  wherein R 33  is independently selected from the group consisting of hydrogen and C 1 -C 4  alkyl groups, or R 31  is an additional target molecule D, wherein the target molecule is optionally connected to N via a spacer moiety. 
 
       
     
     
         4 . The antibody-conjugate according to  claim 1 , wherein antibody AB is trastuzumab and target molecule D is selected from the group of maytansinoids, auristatines and pyrrolobenzodiazepine dimer, preferably auristatines selected from the group consisting of MMAD, MMAE, MMAF and functional analogues thereof or maytansinoids selected form the group consisting of DM1, DM3, DM4, Ahx-May and functional analogues thereof. 
     
     
         5 . The antibody-conjugate according to  claim 1 , wherein:
 (I) AB=trastuzumab, wherein S(F 1 ) x  is connected to the core-GlcNAc linked to amino acid N300, S(F 1 ) x =6-azido-6-deoxy-N-acetylgalactosamine, Q 1  is according to formula (9q), L 2 =—CH 2 —O—C(O)—NH—S(O) 2 —NH—(CH 2 —CH 2 —O) 2 —C(O)-Val-Cit-PABC-, D=Ahx-May;   (II) AB=trastuzumab, wherein S(F 1 ) x  is connected to the core-GlcNAc linked to amino acid N300, S(F 1 ) x =6-azido-6-deoxy-N-acetylgalactosamine, Q 1  is according to formula (9q), L 2 =—CH 2 —O—C(O)—NH—(CH 2 ) 3 —C(O)—NH—S(O) 2 —NH—(CH 2 —CH 2 —O) 2 —C(O)-Val-Cit-PABC-, D=Ahx-May;   (III) AB=trastuzumab, wherein S(F 1 ) x  is connected to the core-GlcNAc linked to amino acid N300, S(F 1 ) x =6-azido-6-deoxy-N-acetylgalactosamine, Q 1  is according to formula (9q), L 2 =—CH 2 —O—C(O)—NH—(CH 2 —CH 2 —O) 4 —C(O)-Val-Cit-PABC-, D=Ahx-May;   (IV) AB=trastuzumab, wherein S(F 1 ) x  is connected to the core-GlcNAc linked to amino acid N300, S(F 1 ) x =6-azido-6-deoxy-N-acetylgalactosamine, Q 1  is according to formula (9q), L 2 =—CH 2 —O—C(O)—NH—(CH 2 —CH 2 —O) 4 —C(O)—N(CH 2 —CH 2 —O—C(O)-Val-Cit-PABC-D) 2 , each occurrence of D=Ahx-May;   (V) AB=trastuzumab, wherein S(F 1 ) x  is connected to the core-GlcNAc linked to amino acid N300, S(F 1 ) x =6-azido-6-deoxy-N-acetylgalactosamine, Q 1  is according to formula (9q), L 2 =—CH 2 —O—C(O)—NH—S(O) 2 —NH—(CH 2 —CH 2 —O) 2 —C(O)—NH—S(O) 2 —N(CH 2 —CH 2 —O—C(O)-Val-Cit-PABC-D) 2 , each occurrence of D=Ahx-May;   (VI) AB=trastuzumab, wherein S(F 1 ) x  is connected to the core-GlcNAc linked to amino acid N300, S(F 1 ) x =6-azido-6-deoxy-N-acetylgalactosamine, Q 1  is according to formula (9q), L 2 =—CH 2 —O—C(O)—NH—S(O) 2 —NH—(CH 2 —CH 2 —O) 2 —C(O)-Val-Ala-, D=PBD;   (VII) AB=trastuzumab, wherein S(F 1 ) x  is connected to the core-GlcNAc linked to amino acid N300, S(F 1 ) x =2-azidoacetamido-2-deoxy-galactose, Q 1  is according to formula (9q), L 2 =—CH 2 —O—C(O)—NH—(CH 2 —CH 2 —O) 4 —C(O)—, D=Ahx-May;   (VIII) AB=trastuzumab, wherein S(F 1 ) x  is connected to the core-GlcNAc linked to amino acid N300, S(F 1 ) x =2-azidoacetamido-2-deoxy-galactose, Q 1  is according to formula (9q), L 2 =—CH 2 —O—C(O)—NH—S(O) 2 —NH—(CH 2 —CH 2 —O) 4 —C(O)—, D=Ahx-May;   (IX) AB=trastuzumab, wherein S(F 1 ) x  is connected to the core-GlcNAc linked to amino acid N300, S(F 1 ) x =2-azidodifluoroacetamido-2-deoxy-galactose, Q 1  is according to formula (9q), L 2 =—CH 2 —O—C(O)—NH—S(O) 2 —NH—(CH 2 —CH 2 —O) 4 —C(O)—, D=Ahx-May;   (X) AB=trastuzumab, wherein S(F 1 ) x  is connected to the core-GlcNAc linked to amino acid N300, S(F 1 ) x =2-azidodifluoroacetamido-2-deoxy-galactose, Q 1  is according to formula (9q), L 2 =—CH 2 —O—C(O)—(CH 2 —CH 2 —O) 4 —C(O)—, D=Ahx-May;   (XI) AB=trastuzumab, wherein S(F 1 ) x  is connected to the core-GlcNAc linked to amino acid N300, S(F 1 ) x =6-azido-6-deoxy-N-acetylgalactosamine, Q 1  is according to formula (9n), wherein U=0, L 2 =—C(O)—NH—S(O) 2 —NH—(CH 2 —CH 2 —O) 2 —C(O)—NH—S(O) 2 —N(CH 2 —CH 2 —O—C(O)-Val-Cit-PABC-D) 2 , each occurrence of D=Ahx-May.   (XII) AB=trastuzumab, wherein S(F 1 ) x  is connected to the core-GlcNAc linked to amino acid N300, S(F 1 ) x =6-azido-6-deoxy-N-acetylgalactosamine, Q 1  is according to formula (9zo), L 2 =—C(O)—NH—(CH 2 ) 4 —C(O)—NH—(CH 2 ) 5 —O—C(O)—NH—S(O) 2 —N(CH 2 —CH 2 —O—C(O)-Val-Cit-PABC-D) 2 , each occurrence of D=Ahx-May;   (XIII) AB=trastuzumab, wherein S(F 1 ) x  is connected to the core-GlcNAc linked to amino acid N300, S(F 1 ) x =6-azido-6-deoxy-N-acetylgalactosamine, Q 1  is according to formula (9j), wherein 1=3, L 2 =—O—C(O)—NH—S(O) 2 —NH—(CH 2 —CH 2 —O) 2 —C(O)—NH—S(O) 2 —N(CH 2 —CH 2 —O—C(O)-Val-Cit-PABC-D) 2 , each occurrence of D=Ahx-May;   (XIV) AB=trastuzumab, wherein S(F 1 ) x  is connected to the core-GlcNAc linked to amino acid N300, S(F 1 ) x =6-azido-6-deoxy-N-acetylgalactosamine, Q 1  is according to formula (9q), L 2 =—CH 2 —O—C(O)—NH—S(O) 2 —NH—(CH 2 —CH 2 —O) 2 —C(O)—N(CH 2 —CH 2 —O—C(O)—NH—S(O) 2 —NH—(CH 2 —CH 2 —O) 2 —C(O)-Val-Cit-PABC-D) 2 , each occurrence of D=Ahx-May;   (XV) AB=trastuzumab, wherein S(F 1 ) x  is connected to the core-GlcNAc linked to amino acid N300, S(F 1 ) x =6-azido-6-deoxy-N-acetylgalactosamine, Q 1  is according to formula (9q), L 2 =—CH 2 —O—C(O)—NH—S(O) 2 —NH—(CH 2 —CH 2 —O) 2 —C(O)—N(CH 2 —CH 2 —O—C(O)-Val-Cit-PABC-D) 2 , each occurrence of D=Ahx-May;   (XVI) AB=trastuzumab, wherein S(F 1 ) x  is connected to the core-GlcNAc linked to amino acid N300, S(F 1 ) x =6-azido-6-deoxy-N-acetylgalactosamine, Q 1  is according to formula (9q), L 2 =—CH 2 —O—C(O)—NH—S(O) 2 —NH—(CH 2 —CH 2 —O) 2 —C(O)—N(CH 2 —CH 2 —O—C(O)-Val-Cit-PABC-D) 2 , each occurrence of D=Dox;   (XVII) AB=trastuzumab, wherein S(F 1 ) x  is connected to the core-GlcNAc linked to amino acid N300, S(F 1 ) x =6-azido-6-deoxy-N-acetylgalactosamine, Q 1  is according to formula (9q), L 2 =—CH 2 —O—C(O)—NH—S(O) 2 —NH—(CH 2 —CH 2 —O) 2 —C(O)—N(CH 2 —CH 2 —O—C(O)-Val-Cit-PABC-D) 2 , each occurrence of D=MMAF;   (XVIII) AB=trastuzumab, wherein S(F 1 ) x  is connected to the core-GlcNAc linked to amino acid N300, S(F 1 ) x =6-azido-6-deoxy-N-acetylgalactosamine, Q 1  is according to formula (9q), L 2 =—CH 2 —O—C(O)—NH—S(O) 2 —NH—(CH 2 —CH 2 —O) 2 —C(O)—N(CH 2 —CH 2 —O—C(O)-Val-Cit-PABC-D) 2 , each occurrence of D=MMAE;   (XIX) AB=trastuzumab, wherein S(F 1 ) x  is connected to the core-GlcNAc linked to amino acid N300, S(F 1 ) x =6-azido-6-deoxy-N-acetylgalactosamine, Q 1  is according to formula (9q), L 2 =—CH 2 —O—C(O)—NH—S(O) 2 —NH—(CH 2 —CH 2 —O) 2 —C(O)—NH—S(O) 2 —N(CH 2 —CH 2 —O—C(O)-Val-Cit-PABC-D) 2 , each occurrence of D=MMAE;   (XX) AB=trastuzumab, wherein S(F 1 ) x  is connected to the core-GlcNAc linked to amino acid N300, S(F 1 ) x =6-azido-6-deoxy-N-acetylgalactosamine, Q 1  is according to formula (9q), L 2 =—CH 2 —O—C(O)—NH—S(O) 2 —NH—(CH 2 —CH 2 —O) 2 —C(O)—N(CH 2 —CH 2 —O—C(O)-Val-Cit-PABC-D1)(CH 2 —CH 2 —O—C(O)-Val-Cit-PABC-D2), with D1=MMAE and D2=MMAF;   (XXI) AB=trastuzumab, wherein S(F 1 ) x  is connected to the core-GlcNAc linked to amino acid N300, S(F 1 ) x =6-azido-6-deoxy-N-acetylgalactosamine, Q 1  is according to formula (9q), L 2 =—CH 2 —O—C(O)—NH—S(O) 2 —NH—(CH 2 —CH 2 —O) 2 —C(O)—N(CH 2 —CH 2 —O—C(O)-Val-Cit-PABC-D1)(CH 2 —CH 2 —O—C(O)-Val-Cit-PABC-D2), with D1=MMAE and D2=Dox;   (XXII) AB=trastuzumab, wherein S(F 1 ) x  is connected to the core-GlcNAc linked to amino acid N300, S(F 1 ) x =6-azido-6-deoxy-N-acetylgalactosamine, Q 1  is according to formula (9q), L 2 =—CH 2 —O—C(O)—NH—S(O) 2 —NH—(CH 2 —CH 2 —O) 2 —C(O)—N(CH 2 —CH 2 —O—C(O)-Val-Cit-PABC-D1)(CH 2 —CH 2 —O—C(O)-Val-Cit-PABC-D2), with D1=MMAE and D2=Ahx-May;   (XXIII) AB=trastuzumab, wherein S(F 1 ) x  is connected to the core-GlcNAc linked to amino acid N300, S(F 1 ) x =6-azido-6-deoxy-N-acetylgalactosamine, Q 1  is according to formula (9q), L 2 =—CH 2 —O—C(O)—NH—S(O) 2 —NH—(CH 2 —CH 2 —O) 2 —C(O)-Val-Cit-PABC-D, each occurrence of D=MMAE;   (XXIV) AB=trastuzumab, wherein S(F 1 ) x  is connected to the core-GlcNAc linked to amino acid N300, S(F 1 ) x =6-azido-6-deoxy-N-acetylgalactosamine, Q 1  is according to formula (9q), L 2 =—CH 2 —O—C(O)—NH—(CH 2 —CH 2 —O) 4 —C(O)-Val-Cit-PABC-D, each occurrence of D=MMAE.   
     
     
         6 . A method comprising using a mode of conjugation for increasing the therapeutic index of a bioconjugate, wherein the mode of conjugation is being used to connect a biomolecule B with a target molecule D via a linker L, wherein the mode of conjugation comprises:
 (i) contacting a glycoprotein comprising 1-4 core N-acetylglucosamine moieties with a compound of the formula S(F 1 ) x —P in the presence of a catalyst, wherein S(F 1 ) x  is a sugar derivative comprising x functional groups F 1  capable of reacting with a functional group Q 1 , x is 1 or 2 and P is a nucleoside mono- or diphosphate, and wherein the catalyst is capable of transferring the S(F 1 ) x  moiety to the core-GlcNAc moiety, to obtain a modified glycoprotein according to Formula (24):   
       
         
           
           
               
               
           
         
         
           wherein S(F 1 ) x  and x are as defined above; AB represents an antibody; GlcNAc is N-acetylglucosamine; Fuc is fucose; b is 0 or 1; and y is 1, 2, 3 or 4; and 
         
         (ii) reacting the modified glycoprotein with a linker-conjugate comprising a functional group Q 1  capable of reacting with functional group F 1  and a target molecule D connected to Q 1  via a linker L 2  to obtain the antibody-conjugate wherein linker L comprises S—Z 3 -L 2  and wherein Z 3  is a connecting group resulting from the reaction between Q 1  and F 1 . 
       
     
     
         7 . The method according to  claim 6 , wherein the antibody AB is capable of targeting tumours that express an antigen selected from 5T4 (TPBG), αv-integrin/ITGAV, BCMA, C4.4a, CA-IX, CD19, CD19b, CD22, CD25, CD30, CD33, CD37, CD40, CD56, CD70, CD74, CD79b, c-KIT (CD117), CD138/SDC1, CEACAM5 (CD66e), Cripto, CS1, DLL3, EFNA4, EGFR, EGFRvIII, Endothelin B Receptor (ETBR), ENPP3 (AGS-16), EpCAM, EphA2, FGFR2, FGFR3, FOLR1 (folate receptor a), gpNMB, guanyl cyclase C (GCC), HER2, Erb-B2, Lamp-1, Lewis Y antigen, LIV-1 (SLC39A6, ZIP6), Mesothelin (MSLN), MUC1 (CA6, huDS6), MUC16/EA-125, NaPi2b, Nectin-4, Notch3, P-cadherin, PSMA/FOLH1, PTK7, SLITRK6 (SLC44A4), STEAP1, TF (CD142), Trop-1, Trop-2/EGP-1, Trop-3, Trop-4, preferably HER2-expressing tumours. 
     
     
         8 . The method according to  claim 6 , wherein increasing the therapeutic index of an antibody-conjugate is selected from:
 (a) increasing the therapeutic efficacy of the antibody-conjugate; and/or   (b) increasing the tolerability of the antibody-conjugate.   
     
     
         9 . The method according to  claim 6 , wherein the reaction of step (ii) is a(n) (cyclo)alkyne-azide conjugation reaction to from a connecting moiety Z 3  that is represented by (10e), (10i), (10g), (10j) or (10k), preferably by (10g), as represented by: 
       
         
           
           
               
               
           
         
         wherein cycle A is a 7-10-membered (hetero)cyclic moiety. 
       
     
     
         10 . The method according to  claim 6 , wherein one of F 1  is an azide moiety, Q 1  is an (cyclo)alkyne moiety, and Z 3  is a triazole moiety. 
     
     
         11 . The method according to  claim 6 , wherein x is 1 or 2, preferably wherein x is 1. 
     
     
         12 . The method according to  claim 6 , wherein S(F 1 ) x  is 6-azido-6-deoxy-N-acetylgalactosamine. 
     
     
         13 . The method according to  claim 6 , wherein the antibody-conjugate is represented by Formula (40) or (40b): 
       
         
           
           
               
               
           
         
         wherein:
 R 31  is independently selected from the group consisting of hydrogen, halogen, —OR 35 , —NO 2 , —CN, —S(O) 2 R 35 , C 1 -C 24  alkyl groups, C 6 -C 24  (hetero)aryl groups, C 7 -C 24  alkyl(hetero)aryl groups and C 7 -C 24  (hetero)arylalkyl groups and wherein the alkyl groups, (hetero)aryl groups, alkyl(hetero)aryl groups and (hetero)arylalkyl groups are optionally substituted, wherein two substituents R 31  may be linked together to form an annelated cycloalkyl or an annelated (hetero)arene substituent, and wherein R 35  is independently selected from the group consisting of hydrogen, halogen, C 1 -C 24  alkyl groups, C 6 -C 24  (hetero)aryl groups, C 7 -C 24  alkyl(hetero)aryl groups and C 7 -C 24  (hetero)arylalkyl groups; 
 X is C(R 31 ) 2 , O, S or NR 32 , wherein R 32  is R 31  or L 3 (D) r , wherein L 3  is a linker, and D is as defined in  claim 1 ; 
 r is 1-20; 
 q is 0 or 1, with the proviso that if q is 0 then X is N-L 2 (D) r ; 
 aa is 0, 1, 2, 3, 4, 5, 6, 7 or 8; 
 aa′ is 0, 1, 2, 3, 4, 5, 6, 7 or 8; and 
 aa+aa′<10. 
 b is 0 or 1; 
 pp is 0 or 1; 
 M is —N(H)C(O)CH 2 —, —N(H)C(O)CF 2 —, —CH 2 —, —CF 2 — or a 1,4-phenylene containing 0-4 fluorine substituents, preferably 2 fluorine substituents which are preferably positioned on C2 and C6 or on C3 and C5 of the phenylene; 
 y is 1-4; 
 Fuc is fucose 
 
         or wherein the antibody-conjugate the antibody-conjugate is represented by Formula (40d): 
       
       
         
           
           
               
               
           
         
         wherein AB, L 2 , D, S, b, pp, x, y and M are as defined above and wherein 1 is 0-10. 
       
     
     
         14 . The use according to  claim 6 , wherein D is an active substance, preferably an anti-cancer agent. 
     
     
         15 . A method for targeting HER2-expressing cells, comprising administering to a subject in need thereof an antibody-conjugate, comprising an antibody AB connected to a target molecule D via a linker L, wherein the antibody-conjugate is obtainable by:
 (i) contacting a glycoprotein comprising 1-4 core N-acetylglucosamine moieties with a compound of the formula S(F 1 ) x —P in the presence of a catalyst, wherein S(F 1 ) x  is a sugar derivative comprising x functional groups F 1  capable of reacting with a functional group Q 1 , x is 1 or 2 and P is a nucleoside mono- or diphosphate, and wherein the catalyst is capable of transferring the S(F 1 ) x  moiety to the core-GlcNAc moiety, to obtain a modified antibody according to Formula (24):   
       
         
           
           
               
               
           
         
         
           wherein S(F 1 ) x  and x are as defined above; AB represents an antibody; GlcNAc is N-acetylglucosamine; Fuc is fucose; b is 0 or 1; and y is 1, 2, 3 or 4; and 
         
         (ii) reacting the modified antibody with a linker-conjugate comprising a functional group Q 1  capable of reacting with functional group F 1  and a target molecule D connected to Q 1  via a linker L 2  to obtain the antibody-conjugate wherein linker L comprises S—Z 3 -L 2  and wherein Z 3  is a connecting group resulting from the reaction between Q 1  and F 1 , 
         wherein antibody AB is capable of targeting HER2-expressing tumours. 
       
     
     
         16 . The method according to  claim 15 , wherein the targeting HER2-expressing cells includes one or more of treating, imaging, diagnosing, preventing the proliferation of, containing and reducing HER2-expressing cells, in particular HER2-expressing tumours. 
     
     
         17 . The method according to  claim 15 , wherein the subject suffers from a disorder selected from breast cancer, bladder cancer, pancreatic cancer, gastric cancer. 
     
     
         18 . The method according to  claim 15 , wherein target molecule D is an anti-cancer agent, preferably a cytotoxin. 
     
     
         19 . The method according to  claim 15 , wherein the bioconjugate comprises two distinct target molecules D, preferably two distinct anti-cancer agents, most preferably two distinct cytotoxins.