Site-specific conjugation allows modulation of click reaction stoichiometry for pretargeted SPECT imaging

Abstract

Antibody pretargeting is a promising strategy for improving molecular imaging, wherein the separation in time of antibody targeting and radiolabeling can lead to rapid attainment of high contrast, potentially increased sensitivity, and reduced patient radiation exposure. The inverse electron demand Diels-Alder ‘click’ reaction between trans-cyclooctene (TCO) conjugated antibodies and radiolabeled tetrazines presents an ideal platform for pretargeted imaging due to rapid reaction kinetics, bioorthogonality, and potential for optimization of both slow and fast clearing components. Herein, we evaluated a series of anti-human epidermal growth factor receptor 2 (HER2) pretargeting antibodies containing distinct molar ratios of site-specifically incorporated TCO. The effect of stoichiometry on tissue distribution was assessed for pretargeting TCO-modified antibodies (monitored by 125I) and subsequent accumulation of an 111In-labeled tetrazine in a therapeutically relevant HER2+tumor-bearing mouse model. Single photon emission computed tomography (SPECT) imaging was also employed to assess tumor imaging at various TCO-to-monoclonal antibody (mAb) ratios. Increasing TCO-to-mAb molar ratios correlated with increased in vivo click reaction efficiency evident by increased tumor distribution and systemic exposure of 111In-labeled tetrazines. The pharmacokinetics of TCO-modified antibodies did not vary with stoichiometry. Pretargeted SPECT imaging of HER2-expressing tumors using 111In-labeled tetrazine demonstrated robust click reaction with circulating antibody at ~2 hours and good tumor delineation for both the 2 and 6 TCO-to-mAb ratio variants at 24 hours, consistent with a limited cell-surface pool of pretargeted antibody and benefit from further distribution and internalization. To our knowledge, this represents the first reported systematic analysis of how pretargeted imaging is affected solely by variation in click reaction stoichiometry through site-specific conjugation chemistry.

Effect of Modulating FcRn Binding on Direct and Pretargeted Tumor Uptake of Full-length Antibodies

“…Anti-HER2 THIOMAB™ antibodies (clone 7C2, which binds a different epitope than trastuzumab or pertuzumab; see Supplementary Table 1 for 7C2 sequence as previously reported in (23)) were engineered to introduce cysteine residues at three positions (24) for conjugation to the click-reactive TCO-PEG 3 -maleimide (PEG = polyethylene glycol) linker (25,26), the metal chelator DOTA-maleimide (DOTA = 1,4,7,10-tetraazacyclododecane-1,4,7triyl)triacetic acid) for imaging via 111 In-DOTA 6 -mAbs, and the capping reagent N-ethyl maleimide (NEM) as previously described (15) to give final conjugates with ~6 TCO, DOTA, or NEM groups per antibody (Supplementary Table 2). These cysteine mutations were engineered alone or in combination with additional mutations -H310A/H435Qto ablate binding to FcRn (20).…”

“…7C2-NEM 6 , 7C2-HAHQ-NEM 6 , 7C2-TCO 6 , and 7C2-HAHQ-TCO 6 antibodies were radioiodinated ( 125 I) for invasive biodistribution assessment through tyrosine residues, (Supplement S1) (15,28). direct imaging was achieved via engineered thiols (Supplement S2) (15); additionally, 111 In-DOTA-Tz was synthesized (Supplement S3) for pretargeted imaging (15,29).…”

“…The KPL-4 tumor-bearing C.B-17 SCID.bg mouse model (mean tumor volume at least 250 mm 3 ) was utilized as previously described (15). An overview of the SPECT-CT imaging study design is presented in…”

“…Tissue samples collected from the terminal harvests included tumor, kidney, liver, spleen, muscle, fat pad, small intestine, skin, heart, lungs, and brain. Tissues collection, analysis, and calculations were performed as previously described (15).…”

“…Tumor-to-background ratios in pretargeted imaging are typically better for slow/non-internalizing targets than for internalizing targets. For instance, we previously demonstrated that same-day imaging of pretargeted anti-HER2 (internalizing) antibodies was inferior to imaging with directly labeled antibodies, as substantial levels of systemic click reaction limited delineation of tumors from blood pool background (15). Furthermore, antibody pretargeting often benefits from clearing/blocking agents to reduce systemic reaction/labeling (16), but this adds another step that could be disadvantageous from an operational viewpoint.…”