Paul Scherrer Institut

Philipp Rene Spycher

Roger Schibli

Martin Behe

Olivier Kreis

Jori Wehrmuller

David Hurwitz

The present invention relates to a method for generating an antibody-payload conjugate by means of a microbial transglutaminase (MTG). The method comprises a step of conjugating a linker having a primary amine residue, said linker having the peptide structure (shown in N->C direction) (Aax)-(Aax)(NH)-(Aax)-B-(Aax), or (Aax)-B-(Aax)-(Aax)(NH2)-(Aax), to a Gln residue comprised in the heavy or light chain of an antibody. Aax(NH) is an amino acid, amino acid derivative or amino acid mimetic comprising a side chain having a primary amine group.

Transglutaminase conjugation method and linker

Site-specific modification of proteins with microbial transglutaminase (MTG) is a powerful and versatile strategy for a controlled modification of proteins under physiological conditions. Solid-phase microbead-immobilization is 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. MTG remained firmly immobilized with no detectable column bleeding and enzyme activity was sustained during continuous operation. Immobilized MTG shows enhanced selectivity towards a certain residue in the presence of several reactive residues which are all targeted when the conjugation was carried out in solution. 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.

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Zurich, Switzerland

Philipp Rene Spycher