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

Abstract

Maleimide-bearing bifunctional probes have been employed for decades for the site-selective modification of thiols in biomolecules, especially antibodies. Yet maleimide-based conjugates display limited stability in vivo because the succinimidyl thioether linkage can undergo a retro-Michael reaction. This, of course, can lead to the release of the radioactive payload or its exchange with thiol-bearing biomolecules in circulation. Both of these processes can produce elevated activity concentrations in healthy organs as well as decreased activity concentrations in target tissues, resulting in reduced imaging contrast and lower therapeutic ratios. In 2018, we reported the creation of a modular, stable, and easily accessible phenyloxadiazolyl methyl sulfone reagentdubbed 'PODS'-as a platform for thiol-based bioconjugations. We have clearly demonstrated that PODS-based site-selective bioconjugations reproducibly and robustly create homogenous, well-defined, highly immunoreactive, and highly stable radioimmunoconjugates. Furthermore, preclinical experiments in murine models of colorectal cancer have shown that these siteselectively labeled radioimmunoconjugates exhibit far superior in vivo performance compared to radiolabeled antibodies synthesized via maleimide-based conjugations. In this protocol, we will describe the four-step synthesis of PODS, the creation of a bifunctional PODS-bearing variant of the ubiquitous chelator DOTA (PODS-DOTA), and the conjugation of PODS-DOTA to the HER2targeting antibody trastuzumab.

Synthesis and Comparative In Vivo Evaluation of Site-Specifically Labeled Radioimmunoconjugates for DLL3-Targeted ImmunoPET

“…In this investigation, we have harnessed an emergent, thiol-reactive bioconjugation reagent based on a p henyl o xa d iazolyl methyl s ulfone (PODS) core to create a site-specifically modified 89 Zr-radioimmunoconjugate as a companion diagnostic for a DLL3-targeted antibody–drug conjugate (ADC). PODS-based reagents react quickly, cleanly, and (unlike maleimides) irreversibly with thiols (Figure A). − Even more importantly, we have previously demonstrated that the site-selective modification of wild-type antibodies with PODS-bearing chelators produces 177 Lu- and 89 Zr-labeled radioimmunoconjugates with high stability and excellent in vivo performance (Figure B) …”

Site-Specific Radiohalogenation of a HER2-Targeted Single-Domain Antibody Fragment Using a Novel Residualizing Prosthetic Agent

“…The radiosynthesis of the novel reagents is summarized in Schemes – , while the synthesis of Boc 2 - iso -SGMIB, Boc 2 - iso -SGMTB, and PODS has been reported before. ,, The synthesis of DOTA-PODS followed a modified method using p -SCN-Bn-DOTA (Macrocyclics, Inc, Texas). The conjugations of Boc 2 - iso -SGMIB and Boc 2 - iso -SGMTB to PODS were performed under basic conditions using a common protocol.…”

Recent developments in chemical conjugation strategies targeting native amino acids in proteins and their applications in antibody–drug conjugates

“…In 2019, Zeglis and co-workers adopted this linker to generate a conjugate of trastuzumab and a radionuclide chelator 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA). 118 The antibody was incubated with 10 equiv. of PODS–DOTA for 2 h at room temperature and yielded the conjugate with 80% yield.…”