Site-specific conjugation of bifunctional chelator BAT to mouse IgG1 Fab1 fragment

Abstract

Aim: To perform a site-specific conjugation of Fab' fragments of a mouse monoclonal antibody(MoAb) B43(of IgG 1 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 antigenbinding 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 sitespecific 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.

Site-Specifically Labeled Immunoconjugates for Molecular Imaging—Part 1: Cysteine Residues and Glycans

“…After radiolabeling the Fab′-EDTA construct with In-111, the radioimmunoconjugate was successfully employed for in vivo imaging in a murine model of myocarditis. Using a very similar strategy, another laboratory created a CA125-targeting Fab′ fragment from the mouse IgG B43 [ 49 ]. In this case, however, instead of a maleimide-bearing construct, the researchers employed a bifunctional variant of 1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid (TETA) (Fig.…”

Thiol-Reactive Bifunctional Chelators for the Creation of Site-Selectively Modified Radioimmunoconjugates with Improved Stability

“…This “leakage” causes a higher rate of uptake in nontarget tissues and a lower rate of uptake in target tissues, ultimately resulting in larger radiation doses to healthy tissues, reduced imaging contrast, and lower therapeutic ratios. Several alternative reagents that create more stable linkages with thiols have been used for the bioconjugation and radiolabeling of antibodies, most notably tosylates, bromo- and iodo-acetyls, and vinyl sulfones. ,− However, each of these options has drawbacks as well, including lower reaction rates with thiols as well as reactivity with other amino acids …”

Synthesis and Bioconjugation of Thiol-Reactive Reagents for the Creation of Site-Selectively Modified Immunoconjugates

“…After the lyophilization of the aqueous phase, our product -PODS -was obtained as a white powder in 98% yield. The progress of the reaction was followed via thin layer chromatography, and the identity of each product was confirmed via 1 H-NMR, 13 C-NMR, and HRMS-ESI ( Table 1).…”

“…The resultant bifunctional chelator was then purified via reverse phase C 18 HPLC and isolated in ~75% yield. As with the other precursors, the progress of the reaction was followed via thin layer chromatography, and the identity of the product was confirmed via 1 H-NMR, 13 C-NMR, and HRMS-ESI (Table 1).…”

“…Several alternative thiol-reactive reagents have been developed in an effort to circumvent these issues, including tosylates, bromo-and iodo-acetyls, and vinyl sulfones. [11][12][13][14][15][16][17] However, all of these approaches have limitations that have hampered their widespread application.…”