SPYCHER, Philipp René,BEHE, Martin,SCHIBLI, Roger,HURWITZ, David
申请号：
EPEP2017/067403
公开号：
WO2018/015213A1
申请日:
2017.07.11
申请国别(地区):
WO
年份:
2018
代理人:
摘要：
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.La modification site-spécifique de protéines à laide dune transglutaminase microbienne (MTG) est une stratégie puissante et polyvalente pour la modification contrôlée de protéines dans des conditions physiologiques. Nous avons apporté des preuves montrant que limmobilisation de microbilles en phase solide peut être utilisée pour lier efficacement et de façon spécifique au site diverses molécules fonctionnelles, importantes pour des applications supplémentaires en aval, à des protéines présentant une pertinence thérapeutique, lesquelles comprennent le scFV, les fragment
