Site-specific conjugation to antibody lysine residues with solid-phase immobilized microbial transglutaminase mtg and mtg in solution

Abstract

Site-specific modification of proteins with microbial transglutaminase (MTG) is a powerful and versatile strategy for a controlled modification of proteins under physiological conditions. We present evidence that solid-phase microbead-immobilization can be used to site-specifically and efficiently attach different functional molecules important for further downstream applications to proteins of therapeutic relevance including scFV, Fab-fragment and antibodies. We demonstrate that MTG remained firmly immobilized with no detectable column bleeding and that enzyme activity was sustained during continuous operation, which allowed for a convenient recycling of the enzyme, thus going beyond solution-phase MTG conjugation. In addition it is showed that immobilized MTG shows enhanced selectivity towards a certain residue in the presence of several reactive residues which are all targeted if the conjugation was carried out in solution. It is also reported on the site-specific lysine conjugation of antibodies using potent glutamine containing peptides with immobilized and MTG in solution. In addition, the generation of dual site-specifically conjugated IgG1 with immobilized and MTG in solution is reported, i.e. site-specific conjugation to glutamine and lysine residues of IgG1 antibody. Site-specific glutamine conjugation with small peptides containing a lysine residue and a functional moiety is also described.

What is claimed is: 
     
         1 . A method for conjugating a peptide linker comprising a lysine and/or a glutamine residue to an antibody, or an antigen-binding fragment thereof, by means of a microbial transglutaminase (MTG) the method comprising:
 a) mixing the antibody, or the antigen-binding fragment thereof, the peptide linker and the MTG within a fluid under determined conditions, thereby conjugating the peptide linker to the antibody, or the antigen-binding fragment thereof, under the catalyzing effect of the MTG, wherein the peptide linker is mixed with the antibody, or the antigen-binding fragment thereof, at a 0.5x to 50x molar ratio; and   b) extracting the conjugate obtained in step (a) from the fluid.   
     
     
         2 . The method according to  claim 1 , wherein the MTG concentration in the fluid is 0.01 mg/mL to 10 mg/mL. 
     
     
         3 . The method according to  claim 1 , wherein a conjugation efficiency to the antibody, or the antigen-binding fragment thereof, of at least 30% is achieved. 
     
     
         4 . The method according to  claim 1 , wherein the peptide linker is a linker comprising a lysine residue. 
     
     
         5 . The method according to  claim 1 , wherein the fluid is an aqueous buffer solution. 
     
     
         6 . The method according to  claim 5 , wherein the aqueous buffer solution comprises Tris and NaCl. 
     
     
         7 . The method according to  claim 1 , wherein the antibody, or the antigen-binding fragment thereof, the peptide linker and the MTG are mixed at a pH of 7.6. 
     
     
         8 . The method according to  claim 1 , wherein the antibody is an antibody of IgG, IgM, IgA or IgE format, or a fragment thereof. 
     
     
         9 . The method according to  claim 1 , wherein the antigen-binding fragment is a Fab, a Fab′, a F(ab′) 2 , a F(ab′) 3 , a Dab, an Fv fragment, a single chain Fv (scFv) fragment or a scFv-Fc (scFv)2. 
     
     
         10 . The method according to  claim 1 , wherein the MTG modifies either one or more reactive glutamine residue or one or more reactive lysine residue on the antibody, or the antigen-binding fragment thereof, with the peptide linker; wherein the one or more reactive glutamine or lysine residue
 a) is an endogenous glutamine or lysine residue;   b) has been artificially introduced into the antibody, or the antigen-binding fragment thereof, by genetic means; or   c) a combination of (a) or (b).   
     
     
         11 . The method according to  claim 1 , wherein the peptide linker further comprises a fluorescent dye/label, a cell-cytotoxic or influencing moiety, a metal-chelator a functional peptide, a chemical moiety and/or a spacer moiety with C n , >20. 
     
     
         12 . The method according to  claim 1 , wherein the peptide linker further comprises an enzymatically cleavable peptide sequence. 
     
     
         13 . The method according to  claim 1 , wherein the peptide linker comprises the sequence KNAA, KAYA, KNAAGGG KDAAGGG, KAYAGGG, AKETAA, FGLQPRY or SLLQGR. 
     
     
         14 . The method according to  claim 1  wherein the peptide linker further comprises a self-immolative group. 
     
     
         15 . The method according to  claim 14 , wherein the self-immolative group is p-aminobenzyloxycarbonyl (PAB). 
     
     
         16 . The method according to  claim 1 , wherein the antibody is a monoclonal antibody, a chimeric antibody, a humanized antibody, a human antibody or a bispecific antibody, and/or wherein the antibody is deglycosylated or non-glycosylated containing a mutation at residue N297 in the EU numbering scheme. 
     
     
         17 . The method according to  claim 11 , wherein the cell-cytotoxic or influencing moiety is a toxin or an immune cell immunomodulatory/stimulating compound; and/or
 wherein the metal-chelator is suitable for SPECT/PET or MRI; and/or   wherein the chemical moiety comprises a reactive group suitable for a click reaction; and/or   wherein the spacer moiety comprises an alkyl or heteroalkyl chain, or a derivative thereof, or a polyethylene glycol moiety.   
     
     
         18 . The method according to  claim 17 , wherein the toxin is MMAE; and/or
 wherein the reactive group suitable for a click reaction comprises an azide moiety, a cyclooctyne moiety, a tertrazine moiety, a trans-cyclooctene moiety, or a derivative thereof.   
     
     
         19 . The method according to  claim 1 , wherein the fluid comprises up to 60% of glycerol and/or an organic solvent. 
     
     
         20 . The method according to  claim 1 , wherein the lysine peptide has a size of (C+N) n >20 and said glutamine peptide has a size of 1<(C+N) n <200. 
     
     
         21 . The method according to  claim 1 , wherein the MTG is conjugated to a polymer. 
     
     
         22 . The method according to  claim 21 , wherein the MTG polymer conjugate is immobilized on a microbead via a covalent and/or ionic bond. 
     
     
         23 . The method according to  claim 22 , wherein the microbeads are selected from the group consisting of: glass, nickel, polyethylene, polypropylene, poly(4-methulbutene), polystyrene, polyacrylate, polyethylene terephthalate, rayon, nylon, poly(vinyl butyrate), polyvinylidene difluoride (PCDF), silicones, polyformaldehyde, cellulose, cellulose acetate, nitrocellulose, gelatin, polysaccharides, polycaprolactone (PCL), polyacrylamide, polyacrolein, polydimethylsiloxane, polyvinyl alcohol, polymethylacrylate, perfluorocarbon, inorganic compounds, or copolymers consisting of any combination of two or more naturally occurring polymers, synthetic polymers or inorganic compounds and/or wherein the size of the microbead varies from 1 nm to 1000 μm. 
     
     
         24 . The method according to  claim 23 , wherein the polysaccharide is agarose, alginate, carrageenan, chitin, dextran or starch; and/or wherein the inorganic compound is silica, glass, kieselguhr, alumina, gold, iron oxide, graphene, graphene dioxide or another metal oxide. 
     
     
         25 . The method according to  claim 21 , wherein the polymer is selected from the group consisting of: polyethylene glycol, polypropylene glycol, polyethyleneoxide, poly(alkyloxazolines), polyvinylpyrrolidone, polylysine and polyglutamate, poly(ethyloxazoline), polymethacrylic acid and polypropacrylic acid or mixtures and dendrimeric structures thereof; also included are polymers based on sugar residues, poly-N-isopropylacrylamide (polyNIPAM), poly(glycidyl methacrylate), polytetrafluoroethylene (PTFE) and poly(ethylene-alt-tetrafluoroethylene) (ETFE), poly(oligoethylene glycol) meth-acrylate (POEGMA), poly(2-methyl-2-oxazoline) (PMOXA), poly(vinyl alcohol) (PVA) and poly(ethylene imine) and derivatives thereof and/or wherein the polymer is a second generation dendronized polymer (dePG2). 
     
     
         26 . The method according to  claim 21  wherein the MTG polymer conjugate is involving a linker (spacer) between the polymer and the MTG, said linker is a bifunctional linker system S-HyNic (succinimidyl-6-hydrazino-nicotinamide, S-4FB (4-formylbenzoate) or derivatives thereof, or SMCC (succinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate) or derivatives thereof, homo- or heterobifunctional spacers which have a structure like Y-S-Z (Y can also be Z and vice versa), whereas Y and Z are of the following group or derivatives thereof: tetrazines, trans-cyclooctenes, azides, cyclooctenes (e.g. dibenzylcyclooctyne or bicyclononynes), n-hydroxysuccinimide, maleimide, isothiocyanate, aldehyde, epoxides, alcohols, amines, thiols, phosphonates, alkynes, potassium acyltrifluoroborates, a-ketoacid-hydroxylamines, O-acylhydroxylamines, carboxylic acids, hydrazines, imines, norborenes, nitriles and cyclopropenes, and S is a spacer entity being a polymer or derivatives thereof, e.g. oligo or poly(ethylene glycol) (PEG), dextranes, made of an alkylmoieties, amino acids or peptide derivatives. 
     
     
         27 . The method according to  claim 21 , wherein the MTG polymer conjugate is retained in an active flow reactor.