Modified antibody, antibody-conjugate and process for the preparation thereof

1. An antibody-conjugate, obtainable by a process comprising the steps of:

(i) reacting an antibody comprising a core N-acetylglucosamine (GlcNAc) substituent with a compound of the formula S(A) x -P in the presence of a suitable catalyst,

wherein:

the core N-acetylglucosamine substituent is present at an N-glycosylation site of the antibody and is optionally fucosylated,

S(A) x -P is a substrate for the catalyst,

wherein:

 S is selected from the group consisting of galactose, mannose, N-acetylglucosamine, glucose, N-acetylgalactosamine, glucuronic acid, fucose, and N-acetylneuraminic acid, comprising x functional groups A,

 A is an azido group,

 x is 1 or 2, and

 P is selected from the group consisting of uridine diphosphate (UDP), guanosine diphosphate (GDP), and cytidine diphosphate (CDP),

to obtain a modified antibody, defined as an antibody comprising a GlcNAc-S(A) x substituent bonded to the antibody via C1 of the N-acetylglucosamine of the GlcNAc-S(A) x substituent, according to Formula (4):

wherein:

AB represents an antibody;

GlcNAc is N-acetylglucosamine;

Fuc is fucose;

b is 0 or 1; and

y is 1 to 4; and

(ii) reacting the modified antibody with a modified active substance, wherein the modified active substance has the Formula (15), (16), (17), or (19):

wherein:

F 3 is selected from the group consisting of —[C(R 5 ) 2 C(R 5 ) 2 O] q —, —X—, —NR 5 —, —C(X)NR 5 —, —C(R 5 ) 2 X—, —C(X)—, —C(X)X—, —S(O)—, —S(O) 2 —, —S(O) 2 O—, —S(O)NR 5 —, —S(O) 2 NR 5 —, —OS(O)—, —OS(O) 2 —, —OS(O)O—, —OS(O) 2 O—, —P(O)(R 5 )(O)—, —P(O)(OR 5 )—, —OP(O)(OR 5 )—, —Si(R 5 ) 2 —, —XC(X)—, —XC(X)X—, —XC(X)N(R 5 )—, —N(R 5 )C(X)—, —N(R 5 )C(X)X—, —N(R 5 )C(X)N(R 5 )—, and combinations of two or more thereof;

L is selected from the group consisting of selected from the group consisting of linear or branched C 1 -C 200 alkylene groups, C 2 -C 200 alkenylene groups, C 2 -C 200 alkynylene groups, C 3 -C 200 cycloalkylene groups, C 5 -C 200 cycloalkenylene groups, C 8 -C 200 cycloalkynylene groups, C 7 -C 200 alkylarylene groups, C 7 -C 200 arylalkylene groups, C 8 -C 200 arylalkenylene groups, and C 9 -C 200 arylalkynylene groups, wherein the groups are optionally substituted with an oxo group and/or interrupted by one or more heteroatoms selected from N and O:

F 4 is selected from the group consisting of —[C(R 5 ) 2 C(R 5 ) 2 O] q —, —X—, —NR 5 —, —C(X)NR 5 —, —C(R 5 ) 2 X—, —C(X)—, —C(X)X—, —S(O)—, —S(O) 2 —, —S(O) 2 O—, —S(O)NR 5 —, —S(O) 2 NR 5 —, —OS(O)—, —OS(O) 2 —, —OS(O)O—, —OS(O) 2 O—, —P(O)(R)(O)—, —P(O)(OR 5 )—, —OP(O)(OR 5 )—, —Si(R 5 ) 2 —, —XC(X)—, —XC(X)X—, —XC(X)N(R 5 )—, —N(R 5 )C(X)—, —N(R)C(X)X—, —N(R 5 )C(X)N(R 5 )—, cleavable linker, and combinations of two or more thereof;

wherein:

 g is in the range of 1 to 200;

 X is independently oxygen or sulphur; and

 R 5 is independently selected from the group consisting of hydrogen, 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;

provided that together F 3 -L-F 4 comprise at least one —[C(R 5 ) 2 C(R 5 ) 2 O] q — group;

R 1a , R 1 , and R 3 are each hydrogen;

R 4 is selected from the group consisting of hydrogen, —F 3 -L-(F 4 -D) r , —(CH 2 ) n —F 3 -L-(F 4 -D) r , and halogen;

D is an active substance, wherein an active substance is a substance that is biologically and/or pharmaceutically active;

a is 0, 1, or 2;

a′ is 0, 1, or 2;

r is an integer from 1 to 8; and

n is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

2. The antibody-conjugate according to claim 1 , wherein the antibody is a whole antibody, and wherein x is 1 and y is 2.

3. The antibody-conjugate according to claim 1 , wherein the modified active substance has the Formula (17):

4. The antibody-conjugate according to claim 1 , wherein the modified active substance has the Formula (19):

5. The antibody-conjugate according to claim 1 , wherein the modified active substance has the Formula (15):

wherein: r is an integer from 1 to 4.

6. The antibody-conjugate according to claim 1 , wherein the modified active substance has the Formula (16):

wherein: r is an integer from 1 to 4.

7. The antibody-conjugate according to claim 1 , wherein F 4 comprise at least one vc-PABC group.

8. The antibody-conjugate according to claim 1 , wherein n is 1, 2, 3, 4, or 5.

9. The antibody-conjugate according to claim 3 , wherein r is an integer from 1 to 4.

10. The antibody-conjugate according to claim 4 , wherein r is an integer from 1 to 4.

11. The antibody-conjugate according to claim 5 , wherein r is an integer from 1 to 4.

12. The antibody-conjugate according to claim 6 , wherein r is an integer from 1 to 4.

13. An antibody-conjugate according to the Formula (15b), (15c), (16b), (16c), (21), (22), or (22b):

wherein:

b is 0 or 1;

p is 0 or 1;

Q is —N(H)C(O)CH 2 — or —CH 2 —;

x is 1 or 2;

y is 1-4;

AB is an antibody;

S is selected from the group consisting of galactose, mannose, N-acetylglucosamine, glucose, N-acetylgalactosamine, glucuronic acid, fucose, and N-acetylneuraminic acid;

GlcNAc is N-acetylglucosamine and is present at an N-glycosylation site of the antibody;

Fuc is fucose;

F 3 is selected from the group consisting of —[C(R 5 ) 2 C(R 5 ) 2 O] q —, —X—, —NR 5 —, —C(X)NR 5 —, —C(R 5 ) 2 X—, —C(X)—, —C(X)X—, —S(O)—, —S(O) 2 —, —S(O) 2 O—, —S(O)NR 5 —, —S(O) 2 NR 5 —, —OS(O)—, —OS(O) 2 —, —OS(O)O—, —OS(O) 2 O—, —P(O)(R 5 )(O)—, —P(O)(OR 5 )—, —OP(O)(OR 5 )—, —Si(R 5 ) 2 —, —XC(X)—, —XC(X)X—, —XC(X)N(R 5 )—, —N(R 5 )C(X)—, —N(R 5 )C(X)X—, —N(R 5 )C(X)N(R 5 )—, and combinations of two or more thereof;

L is selected from the group consisting of selected from the group consisting of linear or branched C 1 -C 200 alkylene groups, C 2 -C 200 alkenylene groups, C 2 -C 200 alkynylene groups, C 3 -C 200 cycloalkylene groups, C 5 -C 200 cycloalkenylene groups, C 8 -C 200 cycloalkynylene groups, C 7 -C 200 alkylarylene groups, C 7 -C 200 arylalkylene groups, C 8 -C 200 arylalkenylene groups, and C 9 -C 200 arylalkynylene groups, wherein the groups are optionally substituted with an oxo group and/or interrupted by one or more heteroatoms selected from N and 0;

F 4 is selected from the group consisting of —[C(R 5 ) 2 C(R 5 ) 2 O] q —, —X—, —NR 5 —, —C(X)NR 5 —, —C(R 5 ) 2 X—, —C(X)—, —C(X)X—, —S(O)—, —S(O) 2 —, —S(O) 2 O—, —S(O)NR 5 —, —S(O) 2 NR 5 —, —OS(O)—, —OS(O) 2 —, —OS(O)O—, —OS(O) 2 O—, —P(O)(R 5 )(O)—, —P(O)(OR 5 )—, —OP(O)(OR 5 )—, —Si(R 5 ) 2 —, —XC(X)—, —XC(X)X—, —XC(X)N(R 5 )—, —N(R 5 )C(X)—, —N(R 5 )C(X)X—, —N(R 5 )C(X)N(R 5 )—, cleavable linker, and combinations of two or more thereof;

wherein:

q is in the range of 1 to 200;

X is independently oxygen or sulphur; and

R 5 is independently selected from the group consisting of hydrogen, 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;

provided that together F 3 -L-F 4 comprise at least one —[C(R 5 ) 2 C(R 5 ) 2 O] q — group;

R 1a , R 1 , and R 3 are each hydrogen;

R 4 is selected from the group consisting of hydrogen, —F 3 -L-(F 4 -D) r , —(CH 2 ) n —F 3 -L-(F 4 -D) r , and halogen;

D is an active substance, wherein an active substance is a substance that is biologically and/or pharmaceutically active;

a is 0, 1, or 2;

a′ is 0, 1, or 2

r is an integer from 1 to 8; and

n is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

14. The antibody-conjugate according to claim 13 , wherein the antibody-conjugate has the Formula (21):

15. The antibody-conjugate according to claim 14 , wherein n is 1, 2, 3, 4, or 5.

16. The antibody-conjugate according to claim 13 , wherein the antibody-conjugate is of the Formula (15b), (15c), (22), or (22b):

17. The antibody-conjugate according to claim 13 , wherein the antibody-conjugate is of the Formula (16b) or (16c):

18. The antibody-conjugate according to claim 13 , wherein the molecule of interest (D) is selected from the group consisting of an active substance and a reporter molecule.

19. The antibody-conjugate according to claim 13 , wherein GlcNAc is present at a N297 N-glycosylation site of the antibody.

20. The antibody-conjugate according to claim 13 , wherein F 4 comprise at least one vc-PABC group.

21. The antibody-conjugate according to claim 13 , wherein n is 1, 2, 3, 4, or 5.

22. The antibody-conjugate according to claim 14 , wherein r is an integer from 1 to 4.

23. The antibody-conjugate according to claim 15 , wherein r is an integer from 1 to 4.

24. The antibody-conjugate according to claim 16 , wherein r is an integer from 1 to 4.

25. The antibody-conjugate according to claim 17 , wherein r is an integer from 1 to 4.

26. A process for the preparation of an antibody-conjugate comprising reacting a modified antibody with a modified active substance,

wherein:

the modified antibody has the Formula (4):

wherein:

the GlcNAc-S(A) x substituent is bonded to the antibody via C1 of the N-acetylglucosamine of the GlcNAc-S(A)x substituent;

S is selected from the group consisting of galactose, mannose, N-acetylglucosamine, glucose, N-acetylgalactosamine, glucuronic acid, fucose, and N-acetylneuraminic acid, comprising x functional groups A,

A is an azido group,

x is 1 or 2,

AB represents an antibody;

GlcNAc is N-acetylglucosamine and is present at an N-glycosylation site of the antibody;

Fuc is fucose;

b is 0 or 1; and

y is 1 to 4;

the modified active substance has the Formula (15), (16), (17), or (19):

wherein:

F 3 is selected from the group consisting of —[C(R 5 ) 2 C(R 5 ) 2 O] q —, —X—, —NR 5 —, —C(X)NR 5 —, —C(R 5 ) 2 X—, —C(X)—, —C(X)X—, —S(O)—, —S(O) 2 —, —S(O) 2 O—, —S(O)NR 5 —, —S(O) 2 NR 5 —, —OS(O)—, —OS(O) 2 —, —OS(O)O—, —OS(O) 2 O—, —P(O)(R 5 )(O)—, —P(O)(OR 5 )—, —OP(O)(OR 5 )—, —Si(R 5 ) 2 —, —XC(X)—, —XC(X)X—, —XC(X)N(R 5 )—, —N(R 5 )C(X)—, —N(R 5 )C(X)X—, —N(R 5 )C(X)N(R 5 )—, and combinations of two or more thereof;

L is selected from the group consisting of selected from the group consisting of linear or branched C 1 -C 200 alkylene groups, C 2 -C 200 alkenylene groups, C 2 -C 200 alkynylene groups, C 3 -C 200 cycloalkylene groups, C 5 -C 200 cycloalkenylene groups, C 8 -C 200 cycloalkynylene groups, C 7 -C 200 alkylarylene groups, C 7 -C 200 arylalkylene groups, C 8 -C 200 arylalkenylene groups, and C 9 -C 200 arylalkynylene groups, wherein the groups are optionally substituted with an oxo group and/or interrupted by one or more heteroatoms selected from N and O;

F 4 is selected from the group consisting of —[C(R 5 ) 2 C(R 5 ) 2 O] q —, —X—, —NR 5 —, —C(X)NR 5 —, —C(R 5 ) 2 X—, —C(X)—, —C(X)X—, —S(O)—, —S(O) 2 —, —S(O) 2 O—, —S(O)NR 5 —, —S(O) 2 NR 5 —, —OS(O)—, —OS(O) 2 —, —OS(O)O—, —OS(O) 2 O—, —P(O)(R 5 )(O)—, —P(O)(OR 5 )—, —OP(O)(OR 5 )—, —Si(R 5 ) 2 —, —XC(X)—, —XC(X)X—, —XC(X)N(R 5 )—, —N(R 5 )C(X)—, —N(R 5 )C(X)X—, —N(R 5 )C(X)N(R 5 )—, cleavable linker, and combinations of two or more thereof;

wherein:

 q is in the range of 1 to 200;

 X is independently oxygen or sulphur; and

 R 5 is independently selected from the group consisting of hydrogen, 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;

provided that together F 3 -L-F 4 comprise at least one —[C(R 5 ) 2 C(R 5 ) 2 O] q — group;

R 1a , R 1 , and R 3 are each hydrogen;

R 4 is selected from the group consisting of hydrogen, —F 3 -L-(F 4 -D) r , —(CH 2 ) n —F 3 -L-(F 4 -D) r , and halogen;

D is an active substance, wherein an active substance is a substance that is biologically and/or pharmaceutically active;

a is 0, 1, or 2;

a′ is 0, 1, or 2;

r is an integer from 1 to 8; and

n is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; and

the antibody-conjugate has the Formula (15b), (15c), (16b), (16c), (21), (22), or (22b):

wherein:

p is 0 or 1; and

Q is —N(H)C(O)CH 2 — or —CH 2 —.

27. The process according to claim 26 further comprising:

reacting an antibody comprising a core N-acetylglucosamine (GlcNAc) substituent with a compound of the formula S(A) x -P in the presence of a catalyst,

wherein:

the core N-acetylglucosamine substituent is present at an N-glycosylation site of the antibody and is optionally fucosylated,

S(A) x -P is a substrate for the catalyst,

wherein P is selected from the group consisting of uridine diphosphate (UDP), guanosine diphosphate (GDP), and cytidine diphosphate (CDP); and

the catalyst is selected from the group consisting of GalT Y289L, GalT Y289N, GalT Y289I, GalT Y289F, GalT Y289M, GalT Y289V, GalT Y289G, GalT Y289I, and GalT Y289A;

to obtain the modified antibody.

28. The process according to claim 26 , wherein the modified active substance has the Formula (17):

29. The process according to claim 26 , wherein the modified active substance has the Formula (15) or (19):

30. The process according to claim 9 , wherein the modified active substance has the Formula (16)

31. The antibody-conjugate according to claim 26 , wherein F 4 comprise at least one vc-PABC group.

32. The antibody-conjugate according to claim 26 , wherein n is 1, 2, 3, 4, or 5.

33. The antibody-conjugate according to claim 28 , wherein r is an integer from 1 to 4.

34. The antibody-conjugate according to claim 29 , wherein r is an integer from 1 to 4.

35. The antibody-conjugate according to claim 30 , wherein r is an integer from 1 to 4.