title: Specific Conjugation of the Hinge Region for Homogeneous Preparation of Antibody Fragment-Drug Conjugate: A Case Study for Doxorubicin-PEG-anti-CD20 Fab' Synthesis.
abstract: Conventional preparation strategies for antibody-drug conjugates (ADCs) result in heterogeneous products with various molecular sizes and species. In this study, we developed a homogeneous preparation strategy by site-specific conjugation of the anticancer drug with an antibody fragment. The model drug doxorubicin (DOX) was coupled to the Fab' fragment of anti-CD20 IgG at its permissive sites through a heterotelechelic PEG linker, generating an antibody fragment-drug conjugate (AFDC). Anti-CD20 IgG was digested and reduced specifically with β-mercaptoethylamine to generate the Fab' fragment with two free mercapto groups in its hinge region. Meanwhile, DOX was conjugated with α-succinimidylsuccinate ω-maleimide polyethylene glycol (NHS-PEG-MAL) to form MAL-PEG-DOX, which was subsequently linked to the free mercapto containing Fab' fragment to form a Fab'-PEG-DOX conjugate. The dual site-specific bioconjugation was achieved through the combination of highly selective reduction of IgG and introduction of heterotelechelic PEG linker. The resulting AFDC provides an utterly homogeneous product, with a definite ratio of one fragment to two drugs. Laser confocal microscopy and cell ELISA revealed that the AFDC could accumulate in the antigen-positive Daudi tumor cell. In addition, the Fab'-PEG-DOX retained appreciable targeting ability and improved antitumor activity, demonstrating an excellent therapeutic effect on the lymphoma mice model for better cure rate and significantly reduced side effects.
authors.affiliation: National Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences , No.1 Beierjie Street, Zhongguancun, Haidian District, Beijing 100190, China.|University of Chinese Academy of Sciences , Beijing 100049, China.
authors.forename: Zhan
authors.initials: Z
authors.lastname: Zhou
authors.affiliation: National Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences , No.1 Beierjie Street, Zhongguancun, Haidian District, Beijing 100190, China.
authors.forename: Jing
authors.initials: J
authors.lastname: Zhang
authors.affiliation: National Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences , No.1 Beierjie Street, Zhongguancun, Haidian District, Beijing 100190, China.
authors.forename: Yan
authors.initials: Y
authors.lastname: Zhang
authors.affiliation: National Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences , No.1 Beierjie Street, Zhongguancun, Haidian District, Beijing 100190, China.
authors.forename: Guanghui
authors.initials: G
authors.lastname: Ma
authors.affiliation: National Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences , No.1 Beierjie Street, Zhongguancun, Haidian District, Beijing 100190, China.
authors.forename: Zhiguo
authors.initials: Z
authors.lastname: Su
mesh_terms: D000818:Animals; D000903:Antibiotics, Antineoplastic / Q000737:chemistry / Q000493:pharmacokinetics / Q000494:pharmacology; D000906:Antibodies / Q000737:chemistry*; D018951:Antigens, CD20 / Q000276:immunology; D004317:Doxorubicin / Q000737:chemistry* / Q000493:pharmacokinetics / Q000494:pharmacology*; D016503:Drug Delivery Systems; D005455:Fluorescent Antibody Technique; D006801:Humans; D018796:Immunoconjugates / Q000737:chemistry* / Q000493:pharmacokinetics; D007140:Immunoglobulin Fab Fragments / Q000737:chemistry* / Q000276:immunology; D007074:Immunoglobulin G / Q000737:chemistry; D020014:K562 Cells; D008297:Male; D016513:Mice, SCID; D018613:Microscopy, Confocal; D011092:Polyethylene Glycols / Q000737:chemistry; D023041:Xenograft Model Antitumor Assays
chemical_list: D000903:Antibiotics, Antineoplastic; D000906:Antibodies; D018951:Antigens, CD20; D018796:Immunoconjugates; D007140:Immunoglobulin Fab Fragments; D007074:Immunoglobulin G; D011092:Polyethylene Glycols; D004317:Doxorubicin