Jun Li1, Xue-hao Wang, Xiao-ming Wang, Zhao-lai Chen. 1. Division of Hepatic Surgery, Department of Surgery, the First Affiliated Hospital of Nanjing Medical University and Jiangsu Province Hospital, Nanjing 210029, China. jun-li62@yeah.net
Abstract
AIM: To perform a site-specific conjugation of Fab' fragments of a mouse monoclonal antibody(MoAb) B43(of IgG1 subtype) to a bifunctional chelator 6-[p-(bromoacetamido)benzyl]-1,4,8,11-tetraazacyclotetradecane-N,N',N' 'N' ' '-tetraacetic acid (BAT) via the thiol groups in the hinge distal to the antigen-binding site of the Fab'. METHODS: B43 was cleaved using a simple 2-step method. First, stable F(ab')(2) was produced by pepsin treatment. Fab' with free thiol in the hinge region was then obtained by cysteine reduction of F(ab')2. Second, a site-specific conjugation of Fab' to thiol-specific BAT was performed in a one-step reaction. RESULTS: The Fab' fragment had approximately 1.8 free thiol groups per molecule after cysteine reduction. The conjugation efficiency and the chemical yield were approximately 1.28 moles chelator/Fab' and 74% of the initial concentration of Fab', respectively. The F(ab')2, Fab' and Fab'-BAT all maintained reasonable antigen-binding properties. (67)Cu labeling of the conjugate under standard conditions did not impair the immunoreactivity of Fab'-BAT. CONCLUSION: This is a simple and efficient method for producing immunoreactive conjugates of Fab'-BAT, which can be used to make radiometal-labeled conjugates for further diagnostic and therapeutic applications.
AIM: To perform a site-specific conjugation of Fab' fragments of a mouse monoclonal antibody(MoAb) B43(of IgG1 subtype) to a bifunctional chelator 6-[p-(bromoacetamido)benzyl]-1,4,8,11-tetraazacyclotetradecane-N,N',N' 'N' ' '-tetraacetic acid (BAT) via the thiol groups in the hinge distal to the antigen-binding site of the Fab'. METHODS: B43 was cleaved using a simple 2-step method. First, stable F(ab')(2) was produced by pepsin treatment. Fab' with free thiol in the hinge region was then obtained by cysteine reduction of F(ab')2. Second, a site-specific conjugation of Fab' to thiol-specific BAT was performed in a one-step reaction. RESULTS: The Fab' fragment had approximately 1.8 free thiol groups per molecule after cysteine reduction. The conjugation efficiency and the chemical yield were approximately 1.28 moles chelator/Fab' and 74% of the initial concentration of Fab', respectively. The F(ab')2, Fab' and Fab'-BAT all maintained reasonable antigen-binding properties. (67)Cu labeling of the conjugate under standard conditions did not impair the immunoreactivity of Fab'-BAT. CONCLUSION: This is a simple and efficient method for producing immunoreactive conjugates of Fab'-BAT, which can be used to make radiometal-labeled conjugates for further diagnostic and therapeutic applications.
Authors: Sai Kiran Sharma; Pierre Adumeau; Outi Keinänen; Vikram Sisodiya; Hetal Sarvaiya; Robert Tchelepi; Joshua A Korsen; Jacob Pourat; Kimberly J Edwards; Ashwin Ragupathi; Omar Hamdy; Laura R Saunders; Charles M Rudin; John T Poirier; Jason S Lewis; Brian M Zeglis Journal: Bioconjug Chem Date: 2021-04-09 Impact factor: 4.774