Acta Pharmacologica Sinica 2006 February; 27 (2): 237-241; doi: 10.1111/j.1745-7254.2006.00242.x

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. ⁶⁷Cu 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.

Keywords: conjugation; BAT; chelator; monoclonal antibody