Engineered polypeptide conjugates using transglutaminase

Abstract

The present invention provides engineered polypeptide conjugates (e.g., antibody-drug-conjugates) comprising specific acyl donor glutamine-containing tags and amine donor agents. The invention also provides methods of making such engineered polypeptide conjugates using transglutaminase and methods of using thereof.

What is claimed is: 
     
         1 - 28 . (canceled) 
     
     
         29 . A method of treating a cancer in a subject in need thereof, comprising administering to the subject an effective amount of an engineered polypeptide conjugate comprising the formula: polypeptide-T-A, wherein T is an acyl donor glutamine-containing tag engineered at a specific site, wherein A is an amine donor agent, wherein the amine donor agent is site-specifically conjugated to the acyl donor glutamine-containing tag at a carboxyl terminus, an amino terminus, or at an another site in the polypeptide, and wherein the acyl donor glutamine-containing tag comprises an amino acid sequence GGLLQGPP (SEQ ID NO:13). 
     
     
         30 . A method of treating a cancer in a subject in need thereof, comprising administering to the subject an effective amount of 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, wherein A is an amine donor agent, wherein the amine donor agent is site-specifically conjugated to the acyl donor glutamine-containing tag at a carboxyl terminus, an amino terminus, or at an another site in the Fc-containing polypeptide, wherein the acyl donor glutamine-containing tag comprises an amino acid sequence XXQX (SEQ ID NO:35), LLQ, or GLLQ (SEQ ID NO: 24), wherein X is any amino acid, and wherein the engineered Fc-containing polypeptide conjugate comprises an amino acid substitution from glutamine to asparagine at position 295 (Q295N; EU numbering scheme). 
     
     
         31 . The method of  claim 30 , wherein the acyl donor glutamine-containing tag comprises an amino acid sequence selected from the group consisting of LLQGG (SEQ ID NO:16), LLQG (SEQ ID NO:17), LSLSQG (SEQ ID NO:18), GGGLLQGG (SEQ ID NO:19), GLLQG (SEQ ID NO:20), LLQ, GSPLAQSHGG (SEQ ID NO:21), GLLQGGG (SEQ ID NO:22), GLLQGG (SEQ ID NO:23), GLLQ (SEQ ID NO:24), LLQLLQGA (SEQ ID NO:25), LLQGA (SEQ ID NO:26), LLQYQGA (SEQ ID NO:27), LLQGSG (SEQ ID NO:28), LLQYQG (SEQ ID NO:29), LLQLLQG (SEQ ID NO:30), SLLQG (SEQ ID NO:31), LLQLQ (SEQ ID NO:32), LLQLLQ (SEQ ID NO:33), LLQGR (SEQ ID NO:34), LLQGPP (SEQ ID NO:11), LLQGPA (SEQ ID NO:4), GGLLQGPP (SEQ ID NO:13), GGLLQGA (SEQ ID NO:12), LLQGA (SEQ ID NO:1), LLQGPGK (SEQ ID NO:2), LLQGPG (SEQ ID NO:3), LLQGP (SEQ ID NO:5), LLQP (SEQ ID NO:6), LLQPGK (SEQ ID NO:7), LLQAPGK (SEQ ID NO:8), LLQGAPG (SEQ ID NO:9), and LLQGAP (SEQ ID NO:10). 
     
     
         32 . The method of any one of  claims 29  to  31 , wherein the acyl donor glutamine-containing tag is not spatially adjacent to a reactive Lys in the polypeptide or the Fc-containing polypeptide. 
     
     
         33 . The method of  claim 29 , wherein the acyl donor glutamine-containing tag is not spatially adjacent to a reactive Lys in the polypeptide, and wherein the polypeptide comprises an amino acid modification at the last amino acid position in the carboxyl terminus relative to a wild-type polypeptide at the same position. 
     
     
         34 . The method of  claim 29 , 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. 
     
     
         35 . The method of  claim 29 , wherein the 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, a diabody, or an antibody fragment. 
     
     
         36 . The method of  claim 35 , wherein the antibody is an IgG. 
     
     
         37 . The method of  claim 36 , 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. 
     
     
         38 . The method of  claim 37 , 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, [(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, 2-aminoethoxy-PEG6, and Ac-Lys-putrescine. 
     
     
         39 . The method of  claim 37 , wherein the agent moiety is a cytotoxic agent. 
     
     
         40 . The method of  claim 39 , wherein the cytotoxic agent is selected from the group consisting of 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. 
     
     
         41 . The method of  claim 39 , wherein the cytotoxic agent is MMAD (Monomethyl Auristatin D) or 0101 (2-methylalanyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(1S)-2-phenyl-1-(1,3-thiazol-2-yl)ethyl]amino}propyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide). 
     
     
         42 . The method of  claim 37 , wherein the agent moiety 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-PEG-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, 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, and 2-aminoethoxy-PEG6-NODAGA. 
     
     
         43 . The method of  claim 37 , wherein the amine donor unit-linker (X-Y) is a branched unit and the agent moiety comprises at least about 2 agent moieties. 
     
     
         44 . The method of  claim 29 , wherein the engineered polypeptide conjugate comprises an amino acid substitution from glutamine to asparagine at position 295 (Q295N; EU numbering scheme).