Recognition tags for tgase-mediated conjugation

Abstract

The present application relates to methods for the functionalization of antibodies using transglutaminase, in particular antibodies lacking Fc regions. Also disclosed herein are peptide tags for transglutaminase, linking reagents, functionalized antibodies, multi-specific antibodies, pharmaceutical compositions, and method of treating disease and/or conditions.

1 - 46 . (canceled) 
     
     
         47 . A polypeptide comprising a TGase recognition tag comprising a c-myc tag peptide sequence, wherein said TGase recognition tag comprises an acceptor glutamine residue functionalized with a moiety-of-interest via a lysine based linker. 
     
     
         48 . The polypeptide of  claim 47 , wherein the TGase recognition tag comprises an amino acid sequence of SEQ ID NO: 1. 
     
     
         49 . The polypeptide of  claim 47 , wherein the moiety-of-interest is an amino acid or polypeptide bound to the acceptor glutamine via an isopeptide bond. 
     
     
         50 . The polypeptide of  claim 47 , wherein the polypeptide is an antibody, optionally an antibody lacking an Fc domain. 
     
     
         51 . The polypeptide of  claim 47 , wherein the antibody is a F(ab), a F(ab′) 2 , an scFv, an affibody, a V H  domain, a V L  domain, a V-NAR domain or a V H H domain. 
     
     
         52 . The polypeptide of  claim 47 , wherein the functionalized acceptor glutamine residue (Q) of Formula IV, below,
 (Q)-NH—(C) n —X-L-(V-(Y-(M)z)q)r Formula IV where:   Q is glutamine residue present in the TGase recognition tag;   (C) n  is a substituted or unsubstituted alkyl or heteroalkyl chain, optionally wherein any carbon of the chain is substituted with an alkoxy, hydroxyl, alkylcarbonyloxy, alkyl-S—, thiol, alkyl-C(O)S—, amine, alkylamine, amide, or alkylamide;   n is an integer selected from among the range of 2 to 20;   X is NH, O, S, absent, or a bond;   L is independently absent, a bond or a continuation of a bond, or a carbon comprising framework of 5 to 200 atoms substituted at one or more atoms, optionally, wherein the carbon comprising framework comprises a linear framework of 5 to 30 carbon atoms optionally substituted at one or more atoms, optionally wherein the carbon comprising framework is a linear hydrocarbon, a symmetrically or asymmetrically branched hydrocarbon, monosaccharide, disaccharide, linear or branched oligosaccharide (asymmetrically branched or symmetrically branched), other natural linear or branched oligomers (asymmetrically branched or symmetrically branched), or a dimer, trimer, or higher oligomer (linear, asymmetrically branched or symmetrically branched) resulting from any chain-growth or step-growth polymerization process;   r is an integer selected from among 1, 2, 3 or 4;   q is an integer selected from among 1, 2, 3 or 4;   z is an integer selected from among 1, 2, 3 or 4; and   V is independently absent, a non-cleavable moiety or a conditionally-cleavable moiety;   Y is independently absent, a bond or a continuation of a bond, or a spacer system which is comprised of 1 or more spacers; and   M is ((RR′)-L′-(V′-(Y′-(Z)z′)q′)r′),   wherein Z is a moiety-of-interest, optionally a moiety that improves the pharmacokinetic properties, or a therapeutic moiety or a diagnostic moiety,   wherein each of L′, V′, Y′, z′, q′, and r′ are as defined as L, V, Y, z, q and r, and   wherein RR′ is an addition product between a reactive moiety R and a complementary reactive moiety R′, wherein R and R′ are moieties capable of undergoing a Huisgen 1,3-cycloaddition reaction, wherein one of R and R′ is an azide and the other is a strained cycloalkyne.   
     
     
         53 . A method for making a polypeptide conjugate, the method comprising reacting a polypeptide comprising a TGase recognition tag comprising a c-myc tag peptide sequence, with a lysine-based linker or polypeptide comprising a lysine residue, in the presence of a TGase and under conditions sufficient such that a polypeptide conjugated to a lysine-based linker or polypeptide comprising a lysine residue is obtained. 
     
     
         54 . The method of  claim 53 , wherein said lysine-based linker is a linker comprising a NH—(C) n — moiety, where (C) n  is a substituted or unsubstituted alkyl or heteroalkyl chain, optionally wherein any carbon of the chain is substituted with an alkoxy, hydroxyl, alkylcarbonyloxy, alkyl-S—, thiol, alkyl-C(O)S—amine, alkylamine, amide, or alkylamide. 
     
     
         55 . The method of  claim 53 , wherein the TGase recognition tag comprises an amino acid sequence of SEQ ID NO: 1.