Enzymatic conjugation of antibodies

1. A human or humanized antibody comprising one or more solvent-exposed glutamine residues in a variable region and at least one acceptor glutamine residue in its constant region or in a sequence fused to a variable or constant region, wherein said one or more solvent-exposed glutamine residues are present in a light chain variable domain (VL) CDR at a Kabat position selected from the group consisting of 27, 55, and a combination thereof,

wherein said antibody is conjugated via said acceptor glutamine residue to one or more moieties-of-interest (Z) through a linker that comprises a NH—(C) n — moiety,

wherein

(C) n is a substituted or unsubstituted alkyl or heteroalkyl chain, optionally wherein any carbon of the chain is substituted with an alkoxy, hydroxyl, alkylcarbonyloxy, alkyl-S—, thiol, alkyl-C(O)S—, amine, alkylamine, amide, or alkylamide, and n is an integer selected from among the range of 2 to 20;

Z is a reactive moiety or a moiety that improves the pharmacokinetic properties, a therapeutic moiety or a diagnostic moiety, and

wherein the antibody comprises heavy chain variable region sequence selected from the group consisting of SEQ ID NOs: 3-239 and light chain variable region sequence selected from group consisting of SEQ ID NO: 240-299.

2. The antibody of claim 1 , wherein the antibody is a tetrameric antibody and said constant region is a heavy chain constant region.

3. The antibody of claim 1 , wherein the antibody comprises:

a heavy chain framework region 1 (FR-H1) comprising a glutamine residue at Kabat position 1, 3, 5, 6, 10, 11, 12, 13 and/or 16;

a heavy chain framework region 2 (FR-H2) comprising a glutamine residue at Kabat position 38, 39, 43 and/or 45;

a heavy chain framework region 3 (FR-H3) comprising a glutamine residue at Kabat position 66, 75, 77, 81 and/or 85;

a heavy chain framework region 1 (CDR-H1) comprising a glutamine residue at Kabat position 26 and/or 35; and/or

a heavy chain framework region 2 (CDR-H2) comprising a glutamine residue at Kabat position 50, 52, 56, 61 and/or 64, wherein said glutamine residue is not an acceptor glutamine.

4. A composition comprising a plurality of human or humanized antibodies of claim 1 , wherein each of said human or humanized antibodies comprises one acceptor glutamine on each heavy chain, wherein at least 80% of the antibodies in the composition comprise on each heavy chain one functionalized acceptor glutamine residue (Q) having Formula IVa,

(Q)-NH—(C) n —X-L- (V—(Y—(Z) z ) q ) r   Formula IVa,

or a pharmaceutically acceptable salt or solvate thereof,

wherein:

Q is a glutamine residue present in the human or humanized antibody;

(C) n is a substituted or unsubstituted alkyl or heteroalkyl chain, optionally wherein any carbon of the chain is substituted with an alkoxy, hydroxyl, alkylcarbonyloxy, alkyl-S—, thiol, alkyl-C(O)S—, amine, alkylamine, amide, or alkylamide;

n is an integer selected from among the range of 2 to 20;

X is NH, O, S, absent, or a bond;

L is independently absent, a bond or a continuation of a bond, or a carbon comprising framework of 5 to 200 atoms substituted at one or more atoms;

r is an integer selected from among 1, 2, 3 or 4;

q is an integer selected from among 1 , 2, 3 or 4;

z is an integer selected from among 1, 2, 3 or 4; and

V is independently absent, a bond or a continuation of a bond, a non-cleavable moiety or a conditionally-cleavable moiety;

Y is independently absent, a bond or a continuation of a bond, or a spacer system which is comprised of 1 or more spacers; and

Z is a moiety that improves pharmacokinetic properties, a therapeutic moiety or a diagnostic moiety.

5. A composition comprising a plurality of human or humanized antibodies of claim 1 , wherein each of said human or humanized antibodies comprises one acceptor glutamine on each heavy chain, wherein at least 80% of the antibodies in the composition comprise on each heavy chain two functionalized acceptor glutamine residues (Q) having Formula IVb,

(Q)-NH—(C) n —X-L-(V—(Y-(M) z ) q ) r   Formula IVb

or a pharmaceutically acceptable salt or solvate thereof,

wherein:

Q is a glutamine residue present in the human or humanized antibody;

(C) n is a substituted or unsubstituted alkyl or heteroalkyl chain, optionally wherein any carbon of the chain is substituted with an alkoxy, hydroxyl, alkylcarbonyloxy, alkyl-S—, thiol, alkyl-C(O)S—, amine, alkylamine, amide, or alkylamide;

n is an integer selected from among the range of 2 to 20;

X is NH, O, S, absent, or a bond;

L is independently absent, a bond or a continuation of a bond, or a carbon comprising framework of 5 to 200 atoms substituted at one or more atoms;

r is an integer selected from among 1, 2, 3 or 4;

q is an integer selected from among 1 , 2, 3 or 4;

z is an integer selected from among 1, 2, 3 or 4; and

V is independently absent, a bond or a continuation of a bond, a non-cleavable moiety or a conditionally-cleavable moiety;

Y is independently absent, a bond or a continuation of a bond, or a spacer system which is comprised of 1 or more spacers; and

M is independently: R or (RR′) -L′-(V′—(Y′—(Z)z′)q′)r′), wherein R is a reactive moiety, wherein each of L′, V′, Y′, z′, q′, and r′ are as defined for L, V, Y, z, q, and r, wherein RR′ is an addition product between a reactive moiety R and a complementary reactive moiety R′, and wherein Z is a moiety that improves pharmacokinetic properties, a therapeutic moiety or a diagnostic moiety.

6. The composition of claim 5 , wherein the composition has a mean Z:antibody ratio of at least 1.5, wherein less than 10% of the antibodies comprise more than two moieties of interest (Z) per antibody and less than 25% comprise less than two moieties of interest (Z) per antibody.

7. A composition comprising a plurality of human or humanized antibodies of claim 1 , wherein each of said human or humanized antibodies is linked to a moiety of interest (Z), wherein the composition has a mean Z: antibody ratio of at least 1.5 wherein less than 10% of the antibodies comprise more than two moieties of interest (Z) per antibody.

8. The composition of claim 7 , wherein less than 25% comprise less than two moieties of interest (Z) per antibody.

9. The composition of claim 7 , wherein the antibodies are linked to said moiety of interest (Z) via one functionalized acceptor glutamine on each heavy chain of the antibody.

10. A composition comprising a plurality of human or humanized antibodies of claim 1 , wherein at least 80% of the antibodies in the composition comprise on each heavy chain one functionalized acceptor glutamine residue (Q) having Formula IVa or Formula IVb,

(Q)-NH—(C) n —X-L-(V—(Y—(Z) z ) q ) r   Formula IVa;

(Q)-NH—(C) n —X-L-(V—(Y-(M) z ) q ) r   Formula IVb,

or a pharmaceutically acceptable salt or solvate thereof,

wherein:

Q is a glutamine residue present in the human or humanized antibody;

(C) n is a substituted or unsubstituted alkyl or heteroalkyl chain, optionally wherein any carbon of the chain is substituted with an alkoxy, hydroxyl, alkylcarbonyloxy, alkyl-S—, thiol, alkyl-C(O)S—, amine, alkylamine, amide, or alkylamide;

n is an integer selected from among the range of 2 to 20;

X is NH, O, S, absent, or a bond;

L is independently absent, a bond or a continuation of a bond, or a carbon comprising framework of 5 to 200 atoms substituted at one or more atoms;

r is an integer selected from among 1, 2, 3 or 4;

q is an integer selected from among 1 , 2, 3 or 4;

z is an integer selected from among 1, 2, 3 or 4; and

V is independently absent, a bond or a continuation of a bond, a non-cleavable moiety or a conditionally-cleavable moiety;

Y is independently absent, a bond or a continuation of a bond, or a spacer system which is comprised of 1 or more spacers;

Z is a moiety that improves pharmacokinetic properties, a therapeutic moiety or a diagnostic moiety; and

M is independently: R or (RR′)-L′-(V′—(Y′—(Z)z′)q′)r′), wherein R is a reactive moiety, wherein each of L′, V′, Y′, z′, q′, and r′ are as defined for L, V, Y, z, q, and r, and wherein RR′ is an addition product between a reactive moiety R and a complementary reactive moiety R′.

11. A composition comprising a plurality of human or humanized antibodies of claim 1 , wherein each of the human or humanized antibodies comprises in a constant region or in a sequence fused to a variable region a functionalized acceptor glutamine linked to a moiety of interest (Z), wherein the composition is characterized by a mean Z: antibody ratio of at least 3.2, wherein less than 10% of the antibodies comprise more than two moieties of interest (Z) per antibody.

12. The composition of claim 11 , wherein the antibodies are linked to said moiety of interest (Z) via two functionalized acceptor glutamines on each heavy chain of the antibody.

13. A composition comprising a plurality of human or humanized antibodies of claim 1 , wherein at least 80% of the antibodies in the composition comprise on each heavy chain two functionalized acceptor glutamine residues (Q) having Formula IVa or Formula IVb,

(Q)-NH—(C) n —X-L-(V—(Y—(Z) z ) q ) r   Formula IVa;

(Q)-NH—(C) n —X-L-(V—(Y-(M) z ) q ) r   Formula IVb,

or a pharmaceutically acceptable salt or solvate thereof,

wherein:

Q is a glutamine residue present in the human or humanized antibody;

(C) n is a substituted or unsubstituted alkyl or heteroalkyl chain, optionally wherein any carbon of the chain is substituted with an alkoxy, hydroxyl, alkylcarbonyloxy, alkyl-S—, thiol, alkyl-C(O)S—, amine, alkylamine, amide, or alkylamide;

n is an integer selected from among the range of 2 to 20;

X is NH, O, S, absent, or a bond;

L is independently absent, a bond or a continuation of a bond, or a carbon comprising framework of 5 to 200 atoms substituted at one or more atoms;

r is an integer selected from among 1, 2, 3 or 4;

q is an integer selected from among 1 , 2, 3 or 4;

z is an integer selected from among 1, 2, 3 or 4; and

V is independently absent, a bond or a continuation of a bond, a non-cleavable moiety or a conditionally-cleavable moiety;

Y is independently absent, a bond or a continuation of a bond, or a spacer system which is comprised of 1 or more spacers;

Z is a moiety that improves pharmacokinetic properties, a therapeutic moiety or a diagnostic moiety; and

M is independently: R or (RR′)-L′-(V′—(Y′—(Z)z′)q′)r′), wherein R is a reactive moiety, wherein each of L′, V′, Y′, z′, q′, and r′ are as defined for L, V, Y, z, q, and r, and wherein RR′ is an addition product between a reactive moiety R and a complementary reactive moiety R′.

14. The antibody of claim 1 , wherein Z is a hydrophobic compound.

15. The antibody of claim 1 , wherein Z is an organic compound having a molecular weight of at least 400 g/mol.

16. The antibody of claim 1 , wherein Z is an organic compound having a molecular weight of at least 500 g/mol.

17. The antibody of claim 1 , wherein Z is a negatively charged compound.

18. The antibody of claim 1 , wherein said constant region is a human constant region.

19. The antibody of claim 1 , wherein said functionalized or conjugated acceptor glutamine residue(s) (Q) has a structure of Formula IVa,

(Q)-NH—(C) n -X-L-(V—(Y—(Z) z ) q ) r   Formula Iva

or a pharmaceutically acceptable salt or solvate thereof,

wherein:

Q is a glutamine residue present in the human or humanized antibody;

(C) n is a substituted or unsubstituted alkyl or heteroalkyl chain, optionally wherein any carbon of the chain is substituted with an alkoxy, hydroxyl, alkylcarbonyloxy, alkyl-S—, thiol, alkyl-C(O)S—, amine, alkylamine, amide, or alkylamide;

n is an integer selected from among the range of 2 to 20;

X is NH, O, S, absent, or a bond;

L is independently absent, a bond or a continuation of a bond, or a carbon comprising framework of 5 to 200 atoms substituted at one or more atoms;

r is an integer selected from among 1, 2, 3 or 4;

q is an integer selected from among 1 , 2, 3 or 4;

z is an integer selected from among 1, 2, 3 or 4; and

V is independently absent, a bond or a continuation of a bond, a non-cleavable moiety or a conditionally-cleavable moiety;

Y is independently absent, a bond or a continuation of a bond, or a spacer system which is comprised of 1 or more spacers; and

Z is a moiety that improves pharmacokinetic properties, a therapeutic moiety or a diagnostic moiety.

20. The antibody of claim 1 , wherein said functionalized or conjugated acceptor glutamine residue(s) (Q) has a structure of Formula II:

(Q)-NH—(C) n —X-L-(V—(Y—(R) z ) q ) r   Formula II

or a pharmaceutically acceptable salt or solvate thereof,

wherein:

Q is a glutamine residue present in the human or humanized antibody;

(C) n is a substituted or unsubstituted alkyl or heteroalkyl chain, optionally wherein any carbon of the chain is optionally substituted with alkoxy, hydroxyl, alkylcarbonyloxy, alkyl-S—, thiol, alkyl- C(O)S—, amine, alkylamine, amide, or alkylamide;

n is an integer from among the range of 2 to 20;

X is NH, O, S, absent or a bond;

L is independently absent, a bond or a continuation of a bond, or a carbon comprising framework of 1 to 200 atoms substituted at one or more atoms, optionally wherein the carbon comprising framework comprises a linear framework of 3 to 30 carbon atoms optionally substituted at one or more atoms, optionally wherein the carbon comprising framework is a linear hydrocarbon, a symmetrically or asymmetrically branched hydrocarbon, monosaccharide, disaccharide, linear or branched oligosaccharide (asymmetrically branched or symmetrically branched), other natural linear or branched oligomers (asymmetrically branched or symmetrically branched), or a dimer, trimer, or higher oligomer (linear, asymmetrically branched or symmetrically branched) resulting from any chain-growth or step-growth polymerization process;

r is an integer selected from among 1, 2, 3 or 4;

q is an integer selected from among 1 , 2, 3 or 4;

z is an integer selected from among 1, 2, 3 or 4; and

V is independently absent, a bond or a continuation of a bond, a non-cleavable moiety or a conditionally-cleavable moiety;

Y is independently absent, a bond or a continuation of a bond, or a spacer system which is comprised of 1 or more spacers; and

R is a reactive moiety.

21. The antibody of claim 1 , wherein V is a conditionally-cleavable moiety following prior conditional transformation, which can be cleaved or transformed by a chemical, photochemical, physical, biological, or enzymatic process, such as a di-, tri-, tetra-, or oligopeptide.

22. The antibody of claim 1 , wherein Z is a cytotoxic anti-cancer agent.

23. The antibody of claim 22 , wherein Z is selected from the group consisting of taxanes, anthracyclines, camptothecins, epothilones, mytomycins, combretastatins, vinca alkaloids, nitrogen mustards, maytansinoids, calicheamycins, duocarmycins, tubulysins, amatoxins, dolastatins and auristatins, enediynes, radioisotopes, therapeutic proteins and peptides, and toxins or fragments thereof.

24. A pharmaceutical composition comprising an antibody of claim 1 , and a pharmaceutically acceptable carrier.