ENGINEERED POLYPEPTIDE CONJUGATES AND METHODS FOR MAKING THEREOF USING TRANSGLUTAMINASE

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; and

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.

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 acyl donor glutamine-containing tag comprises an amino acid sequence XXQX (SEQ ID NO:1), wherein X is any amino acid.

4 . The engineered Fc-containing polypeptide conjugate of claim 3 , wherein the acyl donor glutamine-containing tag comprises an amino acid sequence selected from the group consisting of LLQGG (SEQ ID NO:2), LLQG (SEQ ID NO:3), 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).

5 . 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.

6 . 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.

7 . The engineered Fc-containing polypeptide conjugate of claim 6 , 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.

8 . The engineered Fc-containing polypeptide conjugate of claim 7 , 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.

9 . The engineered Fc-containing polypeptide conjugate of claim 6 , 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.

10 . 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.

11 . The engineered Fc-containing polypeptide conjugate of claim 10 , wherein the antibody is an IgG.

12 . The engineered Fc-containing polypeptide conjugate of claim 11 , wherein effector function of the IgG decreases no greater than about 2-fold relative to a wild type IgG.

13 . 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.

14 . The engineered Fc-containing polypeptide conjugate of claim 13 , 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.

15 . The engineered Fc-containing polypeptide conjugate of claim 13 , wherein the agent moiety is a cytotoxic agent.

16 . The engineered Fc-containing polypeptide conjugate of claim 15 , 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.

17 . The engineered Fc-containing polypeptide conjugate of claim 13 , 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, 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, aminocaproyl-Val-Cit-PABC-MMAF, putrescinyl-geldanamycin, and Ac-Lys-putrescinyl-geldanamycin.

18 . The engineered Fc-containing polypeptide conjugate of claim 17 , wherein the amine donor agent is amino-PEG6-C2-MMAD or aminocaproyl-Val-Cit-PABC-MMAD.

19 . The engineered Fc-containing polypeptide conjugate of claim 13 , wherein the amine donor unit-linker (X—Y) is a branched unit and the agent moiety comprises at least about 2 agent moieties.

20 . An engineered Fc-containing polypeptide conjugate comprising the formula:

(Fc-containing polypeptide)-T-A,

wherein the Fc-containing polypeptide comprises a first Fc-containing polypeptide;

wherein T is an acyl donor glutamine-containing tag engineered at a specific site or comprises an endogenous glutamine made reactive by the first Fc-containing polypeptide engineering;

wherein A is an amine donor agent;

wherein the amine donor agent comprises a second Fc-containing polypeptide and a tag and does not comprise a reactive Gln; and

wherein the acyl donor glutamine-containing tag or the endogenous glutamine is site-specifically crosslinked to the first Fc-containing polypeptide and the second Fc-containing polypeptide.

21 . An engineered Fc-containing polypeptide conjugate comprising the formula:

(Fc-containing polypeptide)-T-A,

wherein the Fc-containing polypeptide comprises a first Fc-containing polypeptide;

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 comprises a second Fc-containing polypeptide and does not comprise a reactive Gln; and

wherein the acyl donor glutamine-containing tag is site-specifically crosslinked to the first Fc-containing polypeptide and the second Fc-containing polypeptide.

22 . The engineered Fc-containing polypeptide conjugate of claim 20 or 21 , wherein the acyl donor glutamine-containing tag is not spatially adjacent to a reactive Lys in the first Fc-containing polypeptide.

23 . The engineered Fc-containing polypeptide conjugate of claim 20 , wherein the tag comprises a G or GG and wherein the tag is spatially adjacent to a reactive Lys in the second Fc-containing polypeptide.

24 . The engineered Fc-containing polypeptide conjugate of claim 20 , wherein the tag is an amine donor tag comprising a Lys.

25 . The engineered Fc-containing polypeptide conjugate of claim 24 , wherein the amine donor tag comprises an amino acid sequence KG.

26 . The engineered Fc-containing polypeptide conjugate of claim 24 , wherein the amine donor tag comprises an amino acid sequence selected from the group consisting of KGG, GKGG (SEQ ID NO:11), GSKGG (SEQ ID NO:12), GSGKGG (SEQ ID NO:13), and GSGGKGG (SEQ ID NO:14).

27 . The engineered Fc-containing polypeptide conjugate of claim 20 or 21 , wherein the first Fc-containing polypeptide comprises an amino acid modification at the last amino acid position in the carboxyl terminus of the first Fc-containing polypeptide relative to a wild-type Fc-containing polypeptide at the same position.

28 . The engineered Fc-containing polypeptide conjugate of claim 20 or 21 , wherein the engineered Fc-containing polypeptide conjugate is a bispecific Fc-containing polypeptide.

29 . The Fc-containing polypeptide conjugate of claim 20 or 21 , wherein the acyl donor glutamine-containing tag is located at the carboxyl terminus of the Fc-containing polypeptide.

30 . An engineered Fc-containing polypeptide conjugate comprising the formula:

(Fc-containing polypeptide)-T-A,

wherein the Fc-containing polypeptide comprises a first Fc-containing polypeptide and a second Fc-containing polypeptide;

wherein T is an acyl donor glutamine-containing tag comprising a first acyl donor glutamine-containing tag and a second acyl donor glutamine-containing tag crosslinked to the first Fc-containing polypeptide and the second Fc-containing polypeptide, respectively;

wherein A is an amine donor agent; and

wherein the first and the second acyl donor glutamine-containing tags are site-specifically crosslinked to each other.

31 . The engineered Fc-containing polypeptide conjugate of claim 31 , wherein the first acyl donor glutamine-containing tag and the second acyl donor glutamine-containing tag are not spatially adjacent to a reactive Lys in the first Fc-containing polypeptide and the second Fc-containing polypeptide, respectively.

32 . The engineered Fc-containing polypeptide conjugate of claim 30 , wherein the amine donor agent is a compound comprising a diamine.

33 . The engineered Fc-containing polypeptide conjugate of claim 32 , wherein the compound is selected from the group consisting of putrescine (butane-1,4-diamine), ethylenediamine, cadaverine (pentane-1,5-diamine), spermidine, spermine, hydrazine, 1,3-diaminopropane, hexamethylenediamine, phenylenediamine, xylylenediamine, diphenylethylenediamine, 1,8-diaminonapthalene, and stereoisomers, isosteres, analogs or derivatives thereof.

34 . The engineered Fc-containing polypeptide conjugate of claim 30 , wherein the amine donor agent does not comprise a reactive Gln.

35 . An engineered Fab-containing polypeptide conjugate comprising the formula:

(Fab-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 Fab-containing polypeptide engineering;

wherein A is an amine donor agent;

wherein the amine donor agent is a biocompatible polymer comprising a reactive amine;

and wherein the biocompatible polymer 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 Fab-containing polypeptide.

36 . An engineered toxin polypeptide conjugate comprising the formula: (toxin 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 toxin polypeptide engineering;

wherein A is an amine donor agent;

wherein the amine donor agent is a biocompatible polymer comprising a reactive amine;

and wherein the biocompatible polymer 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 toxin polypeptide.

37 . An engineered toxin polypeptide conjugate comprising the formula: (toxin polypeptide)-T-B,

wherein T is an acyl donor glutamine-containing tag engineered at a specific site;

wherein B is a biocompatible polymer;

wherein the toxin polypeptide is site-specifically conjugated to the acyl donor glutamine-containing tag at any site in the biocompatible polymer.

38 . A composition comprising the engineered Fc-containing polypeptide conjugate, the engineered Fab-containing polypeptide conjugate, or the engineered toxin polypeptide conjugate of any one of claims 1 - 37 .

39 . A pharmaceutical composition comprising the engineered Fc-containing polypeptide conjugate, the engineered Fab-containing polypeptide conjugate, or the engineered toxin polypeptide conjugate of any one of claims 1 - 37 , and a pharmaceutically acceptable excipient.

40 . A method for preparing the engineered Fc-containing polypeptide conjugate of claim 1 , comprising the steps of:

a) providing an engineered (Fc-containing polypeptide)-T molecule comprising the Fc-containing polypeptide located at the acyl donor glutamine-containing tag or the endogenous glutamine;

b) contacting the amine donor agent with the engineered (Fc-containing polypeptide)-T molecule in the presence of a transglutaminase; and

c) allowing the engineered (Fc-containing polypeptide)-T to covalently link to the amine donor agent to form the engineered Fc-containing polypeptide conjugate.

41 . The method of claim 40 , wherein the engineered Fc-containing polypeptide is an antibody.

42 . The method of claim 41 , wherein the acyl donor glutamine-containing tag is located at the antibody at the carboxyl terminus of a heavy chain, a light chain, or both the heavy chain and the light chain.

43 . The method of claim 42 , 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 another site of the antibody.

44 . The method of claim 43 , wherein the first acyl donor glutamine-containing tag comprises an amino acid sequence LLQGG (SEQ ID NO:2) or LLQGA (SEQ ID NO:48) and the second acyl donor glutamine-containing tag comprises an amino acid sequence LLQGG (SEQ ID NO:2) or LLQGA (SEQ ID NO:48).

45 . The method of claim 40 , wherein the engineered Fc-containing polypeptide conjugate has conjugation efficiency of at least about 51%.

46 . The method of claim 40 , wherein the amine donor agent has the formula: X—Y—Z, wherein X is an amine donor unit, Y is a linker, and Z is an agent moiety.

47 . A method for preparing the engineered Fc-containing polypeptide conjugate of claim 20 , comprising the steps of:

a) providing an engineered (Fc-containing polypeptide)-T molecule comprising the first Fc-containing polypeptide located at the acyl donor glutamine-containing tag or the endogenous glutamine;

b) providing an engineered (Fc-containing polypeptide)-tag comprising the second Fc-containing polypeptide located at the tag;

c) contacting the engineered (Fc-containing polypeptide)-T molecule with the engineered (Fc-containing polypeptide)-tag molecule in reducing environment; and

d) allowing the engineered (Fc-containing polypeptide)-T molecule to site-specifically and covalently link to the engineered (Fc-containing polypeptide)-tag molecule to form the Fc-containing polypeptide conjugate in the presence of a transglutaminase.

48 . A method for preparing the engineered Fc-containing polypeptide conjugate of claim 21 , comprising the steps of:

a) providing an engineered (Fc-containing polypeptide)-T molecule comprising the first Fc-containing polypeptide located at the acyl donor glutamine-containing tag;

b) providing the second Fc-containing polypeptide;

c) contacting the engineered (Fc-containing polypeptide)-T molecule with the second Fc-containing polypeptide in reducing environment; and

d) allowing the engineered (Fc-containing polypeptide)-T molecule to site-specifically and covalently link to the second Fc-containing polypeptide to form the Fc-containing polypeptide conjugate in the presence of a transglutaminase.

49 . The method of claim 20 or 21 , wherein the acyl donor glutamine-containing tag is not spatially adjacent to a reactive Lys in the first Fc-containing polypeptide.

50 . The method of claim 20 or 21 , wherein the acyl donor glutamine-containing tag comprises an amino acid sequence GSPLAQSHGG (SEQ ID NO:7) and wherein the amine donor tag comprises an amino acid sequence GSGGKGG (SEQ ID NO:13).

51 . The method of claim 50 , wherein crosslinking efficiency of the engineered (Fc-containing polypeptide)-T molecule to the engineered (Fc-containing polypeptide)-tag molecule is at least about 30%.

52 . A method for preparing the engineered Fc-containing polypeptide conjugate of claim 30 , comprising the steps of:

a) providing a first engineered (Fc-containing polypeptide)-T molecule comprising the first Fc-containing polypeptide located at the first acyl donor glutamine-containing tag;

b) providing a second engineered (Fc-containing polypeptide)-T molecule comprising the second Fc-containing polypeptide located at the second acyl donor glutamine-containing tag;

c) contacting the first engineered (Fc-containing polypeptide)-T molecule with the second engineered (Fc-containing polypeptide)-T molecule and the amine donor agent in reducing environment; and

d) allowing the first engineered (Fc-containing polypeptide)-T molecule to site-specifically and covalently link to the second engineered (Fc-containing polypeptide)-T molecule to form the engineered Fc-containing polypeptide conjugate in the presence of a transglutaminase.

53 . A method for preparing an engineered Fab-containing polypeptide conjugate of claim 35 , comprising the steps of:

a) providing an engineered (Fab-containing polypeptide)-T molecule comprising the Fab-containing polypeptide located at the acyl donor glutamine-containing tag or the endogenous glutamine;

b) contacting the biocompatible polymer with the engineered (Fab-containing polypeptide)-T molecule in the presence of a transglutaminase; and

c) allowing the engineered (Fab-containing polypeptide)-T to covalently link to the biocompatible polymer to form the engineered Fab-containing polypeptide conjugate.

54 . A method for preparing an engineered toxin polypeptide conjugate of claim 36 , comprising the steps of:

a) providing an engineered (toxin polypeptide)-T molecule comprising the toxin polypeptide located at the acyl donor glutamine-containing tag or the endogenous glutamine;

b) contacting the biocompatible polymer with the engineered (toxin polypeptide)-T molecule in the presence of a transglutaminase; and

c) allowing the engineered (toxin polypeptide)-T to covalently link to the biocompatible polymer to form the engineered toxin polypeptide conjugate.

55 . A method for preparing an engineered toxin polypeptide conjugate of claim 37 , comprising the steps of:

a) providing an engineered T-B molecule comprising the acyl donor glutamine-containing tag located at the biocompatible polymer;

b) contacting the toxin polypeptide with the engineered T-B molecule in the presence of a transglutaminase; and

c) allowing the engineered T-B molecule to covalently link to the toxin polypeptide to form the engineered toxin polypeptide conjugate.

56 . The method of any one of claims 40 , 47 - 48 , and 52 - 55 , wherein the transglutaminase is a microbial protein.

57 . The method of any one of claims 40 , 47 - 48 , and 52 - 55 , wherein the transglutaminase is a purified transglutaminase.

58 . The method of any one of claims 40 , 47 - 48 , and 52 - 55 , further comprising a purification step, wherein the engineered Fc-containing polypeptide conjugate, Fab-containing polypeptide, or toxin polypeptide conjugate is purified by an affinity chromatography step.