Engineered polypeptide conjugates and methods for making thereof using transglutaminase

Patent metadata

Field	Value
Publication number	US-9676871-B2
Application number	US-201113883535-A
Country	US
Kind code	B2
Filing date	Nov 3, 2011
Priority date	Nov 5, 2010
Publication date	Jun 13, 2017
Grant date	Jun 13, 2017

Abstract

The present invention provides engineered polypeptide conjugates (e.g., antibody-drug-conjugates, toxin-(biocompatible polymer) conjugates, antibody-(biocompatible polymer) conjugates, and bispecific antibodies) comprising acyl donor glutamine-containing tags and amine donor agents. In one aspect, the invention provides an engineered Fc-containing polypeptide conjugate comprising the formula (Fc-containing polypeptide)-T-A, wherein T is an acyl donor glutamine-containing tag engineered at a specific site or comprises an endogenous glutamine made reactive by the Fc-containing polypeptide engineering, wherein A is an amine donor agent, and wherein the amine donor agent is site-specifically conjugated to the acyl donor glutamine-containing tag or the endogenous glutamine. The invention also provides methods of making engineered polypeptide conjugates using transglutaminase.

First claim

What is claimed is:

1. An engineered Fc-containing polypeptide conjugate comprising the formula: (Fc-containing polypeptide)-T-A, wherein T is an acyl donor glutamine-containing tag engineered at a specific site or comprises an endogenous glutamine (Q) made reactive by the Fc-containing polypeptide engineering; wherein A is an amine donor agent; wherein the amine donor agent is site-specifically conjugated to the acyl donor glutamine-containing tag or the endogenous glutamine at a carboxyl terminus, an amino terminus, or at an another site in the Fc-containing polypeptide, wherein the acyl donor glutamine-containing tag consists of an amino acid sequence selected from the group consisting of LLQGG (SEQ ID NO:2), LSLSQG (SEQ ID NO:4), GGGLLQGG (SEQ ID NO:5), GLLQG (SEQ ID NO:6), LLQ, GSPLAQSHGG (SEQ ID NO:7), GLLQGGG (SEQ ID NO:8), GLLQGG (SEQ ID NO:9), GLLQ (SEQ ID NO:10), LLQLLQGA (SEQ ID NO:47), LLQGA (SEQ ID NO:48), LLQYQGA (SEQ ID NO:49), LLQGSG (SEQ ID NO:50), LLQYQG (SEQ ID NO:51), LLQLLQG (SEQ ID NO:52), SLLQG (SEQ ID NO:53), LLQLQ (SEQ ID NO:54), LLQLLQ (SEQ ID NO:55), and LLQGR (SEQ ID NO:56).

2. The engineered Fc-containing polypeptide conjugate of claim 1 , wherein the acyl donor glutamine-containing tag is not spatially adjacent to a reactive Lys in the Fc-containing polypeptide.

3. The engineered Fc-containing polypeptide conjugate of claim 1 , wherein the Fc-containing polypeptide comprises an amino acid modification at the last amino acid position in the carboxyl terminus relative to a wild-type Fc-containing polypeptide at the same position.

4. The engineered Fc-containing polypeptide conjugate of claim 1 , wherein the Fc-containing polypeptide conjugate comprises a full length antibody heavy chain and an antibody light chain.

5. The engineered Fc-containing polypeptide conjugate of claim 4 , wherein the acyl donor glutamine-containing tag is located at the carboxyl terminus of a heavy chain, a light chain, or both the heavy chain and the light chain.

6. The engineered Fc-containing polypeptide conjugate of claim 5 , wherein the acyl donor glutamine-containing tag comprises a first acyl donor glutamine-containing tag and a second acyl donor glutamine-containing tag, wherein the first acyl donor glutamine-containing tag is located at the carboxyl terminus of the heavy chain, and the second acyl donor glutamine-containing tag is located elsewhere at the another site on the Fc-containing polypeptide.

7. The engineered Fc-containing polypeptide conjugate of claim 4 , wherein the acyl donor glutamine-containing tag is located at the Fc-containing polypeptide at the amino terminus of a heavy chain, a light chain, or both the heavy chain and the light chain.

8. The engineered Fc-containing polypeptide conjugate of claim 1 , wherein the Fc-containing polypeptide comprises an antibody, wherein the antibody is a monoclonal antibody, a polyclonal antibody, a human antibody, a humanized antibody, a chimeric antibody, a bispecific antibody, a minibody, or an antibody fragment.

9. The engineered Fc-containing polypeptide conjugate of claim 8 , wherein the antibody is an IgG.

10. The engineered Fc-containing polypeptide conjugate of claim 9 , wherein effector function of the IgG decreases no greater than about 2-fold relative to a wild type IgG.

11. The engineered Fc-containing polypeptide conjugate of claim 1 , wherein the amine donor agent comprises the formula: X—Y—Z, wherein X is an amine donor unit; Y is a linker; and Z is an agent moiety.

12. The engineered Fc-containing polypeptide conjugate of claim 11 , wherein the amine donor unit-linker (X—Y) is selected from the group consisting of Ac-Lys-Gly, aminocaproic acid, Ac-Lys-β-Ala, amino-PEG2-C2, amino-PEG3-C2, amino-PEG6-C2, Ac-Lys-Val-Cit-PABC, aminocaproyl-Val-Cit-PABC, putrescine, and Ac-Lys-putrescine.

13. The engineered Fc-containing polypeptide conjugate of claim 11 , where in the agent moiety is a cytotoxic agent.

14. The engineered Fc-containing polypeptide conjugate of claim 13 , wherein the cytotoxic agent is selected from the group consisting of an anthracycline, an auristatin, a dolastatin, a duocarmycin, an enediyne, a geldanamycin, a maytansine, a puromycin, a taxane, a vinca alkaloid, SN-38, a tubulysin, a hemiasterlin, and stereoisomers, isosteres, analogs, or derivatives thereof.

15. The engineered Fc-containing polypeptide conjugate of claim 11 , 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-carboxytetramethylrhodamine (5-TAMRA) cadaverine, 5-fluorescein amidite (5-FAM) cadaverine, sulforhodamine (SR) 101 cadaverine, 5,6-TAMRA cadaverine, 5-FAM lysine, Ac-LysGly-monomethyl auristatin D (MMAD), amino-PEG3-C2-MMAD, amino-PEG6-C2-MMAD, amino-PEG3-C2-amino-nonanoyl-MMAD, aminocaproyl-Val-Cit-p-aminobenzyloxycarbonyl (PABC)-MMAD, Ac-Lys-Val-Cit-PABC-MMAD, aminocaproyl-MMAD, Ac-Lys-β-Ala-MMAD, amino-PEG2-C2-monomethyl auristatin E (MMAE), aminocaproyl-MMAE, amino-PEG3-C2-MMAE, aminocaproyl-monomethyl auristatin F (MMAF), Aminocaproyl-Val-Cit-PABC-MMAE, aminocaproyl-Val-Cit-PABC-MMAF, putrescinyl-geldanamycin, and Ac-Lys-putrescinyl-geldanamycin.

16. The engineered Fc-containing polypeptide conjugate of claim 15 , wherein the amine donor agent is amino-PEG6-C2-MMAD or aminocaproyl-Val-Cit-PABC-MMAD.

17. The engineered Fc-containing polypeptide conjugate of claim 11 , wherein the amine donor unit-linker (X—Y) is a branched unit and the agent moiety comprises at least about 2 agent moieties, wherein the agent moiety can be the same or different.

18. A pharmaceutical composition comprising the engineered Fc-containing polypeptide conjugate of claim 1 , and a pharmaceutically acceptable excipient.

19. The engineered Fc-containing polypeptide conjugate of claim 1 , wherein the acyl donor glutamine-containing tag engineering or the Fc-containing polypeptide engineering is not an amino acid substitution from asparagine (Asn) to glutamine (Gln) at position 297 of a human IgG.

Assignees

• Strop Pavel
• Dorywalska Magdalena Grazyna
• Rajpal Arvind
• Shelton David
• Liu Shu-Hui
• Pons Jaume
• DUSHIN Russell
• Pfizer
• Rinat Neuroscience Corp

Inventors

• Strop Pavel
• Dorywalska Magdalena Grazyna
• Rajpal Arvind
• Shelton David
• Liu Shu-Hui
• Pons Jaume
• DUSHIN Russell

Classifications

• C07K2317/31

  multispecific · CPC title

• A61K47/6889

  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 title

• C07K2317/73

  Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation · CPC title

• A61P43/00

  Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00 · CPC title

• A61K47/6851

  the antibody targeting a determinant of a tumour cell · CPC title