ANTIBODY-DRUG CONJUGATES WITH HIGH DRUG LOADING

• US 20170043033A1
• Filed: 04/21/2015
• Published: 02/16/2017
• Est. Priority Date: 04/25/2014
• Status: Active Grant

• Current Assignee: Pfizer Inc., Rinat Neuroscience Corp. (Pfizer Inc.)
• Original Assignee: Pfizer Inc., Rinat Neuroscience Corp. (Pfizer Inc.)
• Inventors: Strop, Pavel, DELARIA, Katherine Anne, DORYWALSKA, Magdalena, FOLETTI, Davide Luciano, Dushin, Russell George, Shelton, David Louis, Rajpal, Arvind
• Granted Patent: US 11,602,525 B2
• Time in Patent Office: Days
• CPC Class Codes: A61K 2039/505 comprising antibodies
• CPC Class Codes: A61K 31/427 not condensed and containing further heterocyclic rings
• CPC Class Codes: A61K 47/65 Peptidic linkers, binders or spacers, e.g. peptidic enzyme-labile linkers
• CPC Class Codes: A61K 47/6803 Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
• CPC Class Codes: A61K 47/68031 the drug being an auristatin
• CPC Class Codes: A61K 47/6849 the antibody targeting a receptor, a cell surface antigen or a cell surface determinant
• CPC Class Codes: A61K 47/6851 the antibody targeting a determinant of a tumour cell
• CPC Class Codes: A61K 47/6889 Conjugates wherein the antibody being the modifying agent and wherein the linker, binder or spacer confers particular properties to the conjugates, e.g. peptidic enzyme-labile linkers or acid-labile linkers, providing for an acid-labile immuno conjugate wherein the drug may be released from its antibody conjugated part in an acidic, e.g. tumoural or environment
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: C07K 16/3076 against structure-related tumour-associated moieties
• CPC Class Codes: G01N 2333/705 Assays involving receptors, cell surface antigens or cell surface determinants
• CPC Class Codes: G01N 33/57492 involving compounds localized on the membrane of tumor or cancer cells

Abstract

The present invention provides transglutaminase-mediated antibody-drug conjugates with high anti-body-drug ratio (DAR) comprising 1) glutamine-containing tags, endogenous glutamines, and/or endogenous glutamines made reactive by antibody engineering or an engineered transglutaminase (e.g., with altered substrate specifity); and 2) amine donor agents comprising amine donor units, linkers, and agent moieties, wherein the DAR is at least about 5. The invention also provides methods of making and methods of using such higher drug loaded antibody-drug conjugates.

Claims

1. An antibody-drug conjugate comprising the formula:

antibody-(T-(X-Y-Z_a)_b)_c, wherein;

T is

     1) a glutamine-containing tag engineered at a specific site,

     2) an endogenous glutamine, and/or

     3) an endogenous glutamine made reactive by antibody engineering or an engineered transglutaminase;

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, the endogenous glutamine, and/or the reactive endogenous glutamine;

a is an integer from 1 to 6;

b is an integer from 1 to 6;

c is an integer from 1 to 20; and

wherein the product (drug-antibody ratio) of a, b, and c is at least about 5.

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 T comprises at least 1 endogenous glutamine or reactive endogenous glutamine.

4. The conjugate of claim 1, wherein the reactive endogenous glutamine is made reactive by deglycosylation or by an amino acid modification of another amino acid in the antibody.

5. The conjugate of claim 4, wherein the another amino acid modification is a substitution at position 297 (EU numbering scheme).

6. The conjugate of claim 5, wherein the substitution is from asparagine (N) to glutamine (Q) or from N to alanine (A) at position 297.

7. The conjugate of claim 5, wherein the antibody further comprises a second amino acid modification at positions K222, K340, and/or K370.

8. The conjugate of claim 7, wherein the second amino acid modification is a substitution from lysine to arginine.

9. The conjugate of claim 1, wherein the amine donor agent is site-specifically conjugated to the glutamine-containing tag at at least one or more positions of the antibody selecting from the group consisting of 1) carboxyl terminus of any of a light chain, a heavy chain, or both the light chain and the heavy chain;

2) amino terminus of any of a light chain, a heavy chain, or both the light chain and the heavy chain; and

     3) at position(s) S60-R61, R108, T135, S160, S168, S190-S192, P189-S192, G200-S202, K222-T225, K222-T223, T223, L251-S254, M252-I253, E294-N297, E293-N297, N297, and/or G385, wherein the glutamine-containing tag is inserted in the antibody or replaces one or more endogenous amino acids in the antibody.

10. The conjugate of claim 8, comprising

a) amino acid substitutions at positions N297Q and K222R, wherein the amine donor agent is site-specifically conjugated to the endogenous glutamine at position 295 and the substituted glutamine at position 297;

and

b) one or more glutamine-containing tags, wherein the amine donor agent is site-specifically conjugated to the glutamine-containing tag(s) at a carboxyl terminus of a light chain of the antibody,

wherein the drug-antibody ratio is about 5-7.

11. The conjugate of claim 10, wherein the amine donor agent is further site-specifically conjugated to the glutamine-containing tag at one or more positions selected from the group consisting of S60-R61, R108, T135, S160, S168, S190-S192, P189-S192, G200-S202, K222-T225, K222-T223, T223, L251-S254, M252-I253, E294-N297, E293-N297, N297, and G385, wherein the glutamine-containing tag is inserted in the antibody or replaces one or more endogenous amino acids in the antibody, and wherein the drug-antibody ratio is at least about 6.

12. The conjugate of claim 10, wherein the amine donor agent is further site-specifically conjugated to the glutamine-containing tag at a carboxyl terminus of a heavy chain of the antibody, and wherein the drug-antibody ratio is about 6-9.

13. The conjugate of claim 10, wherein the amine donor agent is further site-specifically conjugated to the glutamine-containing tag inserted after amino acid position T135 in the antibody heavy chain, and wherein the drug-antibody ratio is about 6-9.

14. The conjugate of claim 10, wherein the amine donor agent is further 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, and wherein the drug-antibody ratio is about 6-11.

15. The conjugate of claim 9, 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 the drug-antibody ratio is about 5-7.

16. 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).

17. The conjugate of claim 16, 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).

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

19. The conjugate of claim 18, 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-p-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, [(3S,5S)-1-{3-[2-(2-aminoethoxy)ethoxy]propanoyl}piperidine-3,5-diyl]bis-Val-Cit-PABC, putrescine, and Ac-Lys-putrescine.

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

21. The conjugate of claim 20, 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.

22. 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-PABC-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,5S,7R,8R)-8-hydroxy-7-{(1E,3E)-5-[(2S,3S,5R,6R)-5-{[(2Z,4S)-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.

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

24. A pharmaceutical composition comprising a plurality of conjugates of claim 1, wherein an average drug-antibody ratio is at least about 5.0.

25. A pharmaceutical composition comprising a plurality of antibody-drug conjugates, wherein at least one antibody-drug conjugate is the conjugate of claim 1 and wherein an average drug-antibody ratio is at least about 4.1.

26. A method of treating a cancer in a subject in need thereof, comprising administering to the subject an effective amount of the pharmaceutical composition of claim 23.

27. A method of inhibiting tumor growth or progression in a subject in need thereof, comprising administering to the subject an effective amount of the pharmaceutical composition of claim 23.

28. A method of diagnosing cancer in a subject suspected of suffering from cancer, comprising a) contacting a sample of the subject with the conjugate of claim 1 under conditions that result in binding of the conjugate with a cancer-related protein, and b) determining binding of the conjugate to the cancer-related protein.

29. A method for preparing the conjugate of claim 1, comprising the steps of:

a) providing an antibody-T molecule comprising the antibody and the glutamine-containing tag; and

/or the antibody with the endogenous and/or reactive endogenous glutamine;

b) contacting the amine donor agent with the antibody-T molecule in the presence of a transglutaminase; and

c) allowing the antibody-T to covalently link to the amine donor agent to form the antibody-drug conjugate.

30. The method of claim 29, wherein the conjugate has conjugation efficiency of at least about 51%.

31. The method of claim 29, wherein the transglutaminase is a microbial, purified, or engineered transglutaminase.

32. The method of claim 29, further comprising a purification step, wherein the conjugate is purified by a chromatography step.