A general approach to reduce off-target radioactivity in vivo via Tetrazine-Knock-Out (TKO)

Monoclonal antibodies have had a remarkable impact on cancer therapy due to their high target specificity. However, their large molecular weight results in slow blood clearance, which can take weeks to clear from circulation. As companion nuclear imaging and diagnostic tools, these characteristics force delayed imaging and the use of isotopes with long half-lives such as 89Zr. For optimal clinical application, it is desirable that radioimmunoconjugates remain in the blood for just enough time to accumulate adequately in target tissues, while non-targeted or circulating radioactivity is ideally rapidly excreted from the body to maximize imaging contrast and minimize radiation dose to healthy tissues. We addressed the current challenges of antibody-based imaging by developing rituximab radioimmunoconjugates that accumulate sufficient activity for tumor imaging within 24 h of administration, while clearing circulating radioactivity via administration of a small molecule clearing agent. Rituximab, an anti-CD20 monoclonal antibody, is used as standard first-line therapy for diffuse large B-cell lymphoma. CD20 is expressed by 95% of B-lymphocytes and their malignant counterparts, making it a therapeutic target for B-cell malignancies. We attached 125I, 68Ga, and 89Zr to rituximab using a "clickable" linker containing trans-cyclooctene and tested the ability of tetrazines to induce the inverse electron demand Diels-Alder reaction (iEDDA) after antibody administration. This "tetrazine-knock-out" (TKO) approach liberates the radioactivity from rituximab in the bloodstream, resulting in its rapid renal excretion which enhances target-to-background ratios, and minimizes off-target radiation exposure. Due to the internalization of the radioimmunoconjugate in CD20+ tumor cells, no substantial clearance was observed from Raji xenografts. We characterized different leaving groups, several cellular models and antibodies with distinct internalizaing properties. The TKO approach opens opportunities to use radiolabeled antibodies for low-abundance or heterogeneously expressed biologic targets and may allow radioimmunotherapy (RIT) for targets traditionally untenable due to dose-limiting toxicities. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=94 SRC="FIGDIR/small/596510v1_ufig1.gif" ALT="Figure 1"> View larger version (27K): org.highwire.dtl.DTLVardef@11bc2f0org.highwire.dtl.DTLVardef@19b4f72org.highwire.dtl.DTLVardef@17cee32org.highwire.dtl.DTLVardef@fa4491_HPS_FORMAT_FIGEXP M_FIG C_FIG