Endosomal acidic pH-induced conformational changes of a cytosol-penetrating antibody mediate endosomal escape.
Endosomal escape after endocytosis is a critical step for protein-based agents to exhibit their effects in the cytosol of cells. However, antibodies internalized into cells by endocytosis cannot reach the cytosol due to their inability to escape from endosomes. Here, we report a unique endosomal escape mechanism of the IgG-format TMab4 antibody, which can reach the cytosol of living cells after internalization. Dissociation of TMab4 from its cell surface receptor heparan sulfate proteoglycan by activated heparanase in acidified early endosomes and then local structural changes of the endosomal escape motif of TMab4 in response to the acidified endosomal pH were critical for the formation of membrane pores through which TMab4 escaped into the cytosol. Identification of structural determinants of endosomal escape led us to generate a TMab4 variant with ~3-fold improved endosomal escape efficiency. Our finding of the endosomal escape mechanism of the cytosol-penetrating antibody and its improvement will establish a platform technology that enables a full-length IgG antibody to directly target cytosolic proteins.
Department of Molecular Science and Technology, Ajou University, Suwon, 16499, Korea.
Ji-Sun
JS
Kim
Department of Molecular Science and Technology, Ajou University, Suwon, 16499, Korea.
Dong-Ki
DK
Choi
Department of Molecular Science and Technology, Ajou University, Suwon, 16499, Korea.
Ju-Yeon
JY
Shin
Department of Molecular Science and Technology, Ajou University, Suwon, 16499, Korea.
Seung-Min
SM
Shin
Department of Molecular Science and Technology, Ajou University, Suwon, 16499, Korea.
Seong-Wook
SW
Park
Department of Systems Biology, Yonsei University, Seoul 03722, Korea.
Hyun-Soo
HS
Cho
Department of Molecular Science and Technology, Ajou University, Suwon, 16499, Korea. Electronic address: kimys@ajou.ac.kr.
Yong-Sung
YS
Kim
D000818:Animals; D016466:CHO Cells; D003412:Cricetulus; D003600:Cytosol / Q000378:metabolism*; D011992:Endosomes / Q000737:chemistry*; D005966:Glucuronidase / Q000235:genetics / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007074:Immunoglobulin G / Q000008:administration & dosage* / Q000737:chemistry / Q000494:pharmacology; D011487:Protein Conformation; D014404:Tubulin / Q000276:immunology; D000099175:Heparanase
D005966:Glucuronidase; D007074:Immunoglobulin G; D014404:Tubulin; D000099175:Heparanase
Antibody intracellular trafficking; Cytosol-penetrating antibody; Endosomal escape; Membrane pore formation; pH-dependent conformational change