Antibody-drug conjugates with high drug loading

1. An antibody-drug conjugate comprising the formula: antibody-(T—(X—Y—Z)) c , wherein:

T is a glutamine-containing tag engineered at a specific site;

X is an amine donor unit; Y is a linker; and Z is an agent moiety;

X—Y—Z is an amine donor agent site-specifically conjugated to the glutamine-containing tag;

c is an integer from 6 to 20;

wherein the antibody comprises an amino acid modification at positions K222, K340, and/or K370; and

wherein the amine donor agent is site-specifically conjugated to the glutamine-containing tag on the antibody at positions according to EU number selected from the group consisting of S60-R61, R108, T135, S160, S168, S190-192, P189-S192, G200-S202, T223-T225, T223, L251-S254, M252-I253, E294-N297, E293-N297, and/or G385; and wherein the glutamine-containing tag is inserted in the antibody or replaces one or more endogenous amino acids in the antibody.

2. The conjugate of claim 1 , wherein the antibody is a monoclonal antibody, a polyclonal antibody, a human antibody, a humanized antibody, a chimeric antibody, a bispecific antibody, a minibody, a diabody, or an antibody fragment.

3. The conjugate of claim 1 , wherein the amino acid modification is a substitution from lysine (K) to arginine (R).

4. The conjugate of claim 1 , wherein c is an integer from 6 to 8.

5. The conjugate of claim 4 , wherein c is 6.

6. The conjugate of claim 4 , wherein c is 8.

7. The conjugate of claim 4 , wherein the amine donor agent is site-specifically conjugated to the glutamine-containing tag inserted after amino acid position T135 in the antibody heavy chain.

8. The conjugate of claim 4 , wherein the amine donor agent is site-specifically conjugated to the glutamine-containing tag at amino acid positions G200-S202 in the antibody light chain, wherein the endogenous amino acid residues are replaced with the glutamine-containing tag.

9. The conjugate of claim 1 , comprising the amine donor agent site-specifically conjugated to the glutamine-containing tag

a) at a carboxyl terminus of a light chain of the antibody;

b) after amino acid position T135 in the antibody heavy chain; and

c) at amino acid positions G200-S202 in the antibody light chain, wherein the endogenous amino acid residues are replaced with the glutamine-containing tag;

wherein c is an integer from 6 to 8.

10. The conjugate of claim 1 , wherein the glutamine-containing tag comprises an amino acid sequence selected from the group consisting of Q, LQG, LLQGG (SEQ ID NO:1), LLQG (SEQ ID NO:2), LSLSQG (SEQ ID NO:3), GGGLLQGG (SEQ ID NO:4), GLLQG (SEQ ID NO:5), LLQ, GSPLAQSHGG (SEQ ID NO:6), GLLQGGG (SEQ ID NO:7), GLLQGG (SEQ ID NO:8), GLLQ (SEQ ID NO:9), LLQLLQGA (SEQ ID NO:10), LLQGA (SEQ ID NO:11), LLQYQGA (SEQ ID NO:12), LLQGSG (SEQ ID NO:13), LLQYQG (SEQ ID NO:14), LLQLLQG (SEQ ID NO:15), SLLQG (SEQ ID NO:16), LLQLQ (SEQ ID NO:17), LLQLLQ (SEQ ID NO:18), LLQGR (SEQ ID NO:19), LLQGPP (SEQ ID NO:20), LLQGPA (SEQ ID NO:21), GGLLQGPP (SEQ ID NO:22), GGLLQGA (SEQ ID NO:23), LLQGPGK (SEQ ID NO:25), LLQGPG (SEQ ID NO:26), LLQGP (SEQ ID NO:27), LLQP (SEQ ID NO:28), LLQPGK (SEQ ID NO:29), LLQAPGK (SEQ ID NO:30), LLQGAPG (SEQ ID NO:31), LLQGAP (SEQ ID NO:32), and LLQLQG (SEQ ID NO:36).

11. The conjugate of claim 10 , wherein the glutamine-containing tag is LLQGA (SEQ ID NO: 11), LQG, GGLLQGA (SEQ ID NO:23), LLQGPA (SEQ ID NO:21), LLQGPP (SEQ ID NO:20), GGLLQGPP (SEQ ID NO:22), LLQGSG (SEQ ID NO:13), LLQG (SEQ ID NO:2), LLQYQG (SEQ ID NO:14), LLQLLQG (SEQ ID NO:15), LLQLQG (SEQ ID NO:36), LLQLLQ (SEQ ID NO:18), LLQLQ (SEQ ID NO:17), LLQGR (SEQ ID NO:19), LLQYQGA (SEQ ID NO:12), SLLQG (SEQ ID NO:16), or LLQLLQGA (SEQ ID NO:10).

12. The conjugate of claim 1 , wherein the amine donor unit-linker (X—Y) is linear or branched.

13. The conjugate of claim 12 , wherein the amine donor unit-linker (X—Y) is selected from the group consisting of Ac-Lys-Gly (acetyl-lysine-glycine), aminocaproic acid, Ac-Lys-β-Ala (acetyl-lysine-β-alanine), amino-PEG2 (polyethylene glycol)-C2, amino-PEG3-C2, amino-PEG6-C2, Ac-Lys-Val-Cit-PABC (acetyl-lysine-valine-citrulline-p-aminobenzyloxycarbonyl), amino-PEG6-C2-Val-Cit-PABC, amino-PEG3-C2-Val-Cit-PABC, aminocaproyl-Val-Cit-PABC, [(3R,5R)-1- {3-[2-(2-aminoethoxy)ethoxy]propanoyl}piperidine-3,5-diyl}bis-Val-Cit-PABC, [(3 S,5 S)-1- {3-[2-(2-aminoethoxy)ethoxy]propanoyl}piperidine-3,5-diyl}bis-Val-Cit-PAB C, putrescine, and Ac-Lys-putrescine.

14. The conjugate of claim 1 , wherein the agent moiety is a cytotoxic agent.

15. The conjugate of claim 14 , wherein the cytotoxic agent is selected from the group consisting of an anthracycline, an auristatin, a camptothecin, a combretastatin, a dolastatin, a duocarmycin, an enediyne, a geldanamycin, an indolino-benzodiazepine dimer, a maytansine, a puromycin, a pyrrolobenzodiazepine dimer, a taxane, a vinca alkaloid, a tubulysin, a hemiasterlin, a spliceostatin, a pladienolide, and stereoisomers, isosteres, analogs, or derivatives thereof.

16. The conjugate of claim 1 , wherein the amine donor agent is selected from the group consisting of Alexa 488 cadaverine, 5-FITC cadaverine, Alexa 647 cadaverine, Alexa 350 cadaverine, 5-TAMRA cadaverine, 5-FAM cadaverine, SR101 cadaverine, 5,6-TAMRA cadaverine, 5-FAM lysine, Ac-LysGly-MMAD, amino-PEG3-C2-MMAD, amino-PEG6-C2-MMAD, amino-PEG3-C2-amino-nonanoyl-MMAD, aminocaproyl-Val-Cit-PABC-MMAD, amino-PEG3-C2-Val-Cit-PABC-MMAD, amino-PEG6-C2-Val-Cit-PABC-MMAD, Ac-Lys-Val-Cit-PABC-MMAD, aminocaproyl-MMAD, Ac-Lys-β-Ala-MMAD, amino-PEG2-C2-MMAE, aminocaproyl-MMAE, amino-PEG3-C2-MMAE, aminocaproyl-MMAF, aminocaproyl-Val-Cit-PABC-MMAE, amino-PEG-6-C2-Val-Cit-PABC-MMAE, Ac-Lys-Val-Cit-PABC-MMAE, aminocaproyl-Val-Cit-PABC-MMAF, amino-PEG-6-C2-Val-Cit-PABC-MMAF, Ac-Lys-Val-Cit-PABC-MMAF, amino-PEG6-C2-Val-Cit-PAB C-0101, Ac-Lys-Val-Cit-PABC-0101, putrescinyl-geldanamycin, Ac-Lys-putrescinyl-geldanamycin, aminocaproyl-3377, amino-PEG6-C2-3377, aminocaproyl-0131, amino-PEG6-C2-0131, aminocaproyl-0121, amino-PEG6-C2-0121, [(3R,5R)-1-{3-[2-(2-aminoethoxy)ethoxy]propanoyl}piperidine-3,5-diyl]bis-Val-Cit-PABC-MMAD, [(3R,5R)-1-{3-[2-(2-aminoethoxy)ethoxy]propanoyl}piperidine-3,5-diyl]bis-Val-Cit-PABC-MMAe, 2-aminoethoxy-PEG6-NODAGA, and N-2-acetyl-L-lysyl-L-valyl-N˜5˜-carbamoyl-N-[4-({[(2-{[(3R,5 S,7R,8R)-8-hydroxy-7-{(1E,3E)-5-[(2S,3 S,5R,6R)-5-{[(2Z,4 S)-4-hydroxypent-2-enoyl]amino}-3,6-dimethyltetrahydro-2H-pyran-2-yl]-3-methylpenta-1,3-dien-1-yl}-1,6-dioxaspiro[2.5]oct-5-yl]acetyl}hydrazinyl)carbonyl]oxy}methyl)phenyl}-L-ornithinamide.

17. A pharmaceutical composition comprising the conjugate of claim 1 , and a pharmaceutically acceptable excipient.