Scission-Enhanced Molecular Imaging (SEMI)

Abstract

Positron emission tomography (PET) imaging methods have advanced our understanding of human biology, while targeted radiotherapeutic drug treatments are now routinely used clinically. The field is expected to grow considerably based on an expanding repertoire of available affinity ligands, radionuclides, conjugation chemistries, and their FDA approvals. With this increasing use, strategies for dose reduction have become of high interest to protect patients from unnecessary and off-target toxicity. Here, we describe a simple and powerful method, scission-enhanced molecular imaging (SEMI). The technique allows for rapid corporeal elimination of radionuclides once imaging or theranostic treatment is completed and relies on "click-to-release" bioorthogonal linkers.

Figure 1

Concept of SEMI. Affinity labels (antibodies, nanobodies, aptamers, proteins, peptides, oligo, nanoparticles) are labeled with a SEMI linker incorporating an immolative TCO moiety such as C₂TCO. This stable construct is used for in vivo imaging and therapy. When a dose reduction is desired (long circulation time of affinity ligand), a second IV injection of Tz is performed which cleaves the radiolabel-chelator complex in seconds. The latter is rapidly cleared by the kidneys, resulting in dose reduction and favorable imaging/therapy kinetics. Right: example of systemic radiation reduction and on-target specificity increase through SEMI. This strategy is conceptually comparable to bioorthogonal pretargeting, as seen in Figure S1, though SEMI has greater potential to be used with rapidly internalized PET agents.

Figure 2

Synthesis of C₂TCO probes and HK-Tz. SAFE647 was synthesized as described previously via NHS amide coupling followed by activation of the free carboxylate using TSTU. The resulting NHS-ester was validated via LC–MS and used without further purification. HK-Tz was prepared as previously described to yield the HCl salt. SEMI tracer was prepared from a symmetrical bis-amino-C₂TCO, which was reacted sequentially with NHS-DOTA and bis-PEG4-NHS under basic conditions to provide the desired compound.

Figure 3

Proof-of-principle in vivo experiments. To determine the kinetics and release of the linker, we used fluorescently labeled HER2 and EGFR constructs and then subjected them to Tz treatment. (A) AF647-C₂TCO was synthesized as described in Figure 2 and conjugated to antibodies. (B) AF647SEMI-mAb was injected via the tail vein, followed later by tail vein injection of the HK-Tz. 2 h later, the mice were sacrificed, and the urine was collected directly from the bladder. Collected urine was found to be green (C) and was successfully extracted from mice injected with SEMI-cetuximab (EGFR) and SEMI-trastuzumab (HER2); (D) percentage of dye recovered from the bladders of nu/nu mice treated with SEMI antibody after HK-Tz administration (n = 2 mice, measurements made in triplicate). (E) After intravenous injection of AF647SEMI-HER2, the ear vasculature was monitored via fluorescence microscopy, and the relative fluorescence within a region of interest was quantified. HK-Tz was injected intravenously after 15 min, and the fluorescence was observed to return to initial levels within 60 min. Fluorescence images were collected every 6 min. (F) Relative fluorescence in mice treated with AF647SEMI-HER2, followed by varying quantities of HK-Tz to determine the optimal dose for in vivo clearance. Measurements were collected in triplicate and reported as means ± s.e.m.

Figure 4

Kinetics of immolative linker. (A) DOTA-C₂TCO-NHS was synthesized as described in Figure 2, characterized by NMR and conjugated to trastuzumab (anti-HER2). (B) LCMS analysis of DOTA-C₂TCO-NHS with varying ratios of HK-Tz added in PBS (pH 7.4). (C) Stability of Trastuzumab-C₂TCO-DOTA loaded with ⁶⁴Cu ([64Cu]Cu-SEMI) over 72 h at 37 °C in 10% fetal bovine serum (in PBS). (D) [64Cu]Cu-SEMI was injected into nontumor-bearing mice, and blood was collected retroorbitally over 24 h. Radiation in the plasma was quantified via gamma counting (corrected for radionuclide decay) to determine the circulation half-life of the SEMI probe in vivo. Data are n = 3, means ± s.e.m.

Figure 5

Example of SEMI tracer distribution and PET imaging. (A) Schematic of [64Cu]Cu-SEMI and HK-Tz administration in mice bearing HT1080 (±HER2) tumors. (B) Two mice were injected with [64Cu]Cu-SEMI anti-HER2 antibody. The mouse on the left was given HK-Tz before sacrifice, whereas the mouse on the right was not. After IV injection of HK-Tz, the radioactivity is rapidly cleared, leading to rapid renal clearance. The mouse on the left received HK-Tz, whereas the one on the right did not. Note the higher activity in circulation, liver and rest of the body in the non-Hk-Tz animal. HK-Tz injection leads to lower background and dose reduction (see Figure 7 for modeling of effects). (C) After PET-CT imaging, tissues were harvested, weighed, analyzed via gamma counting, and incubated on a phosphor imaging for 48 h. The phosphor imaging plate was imaged on an Azure Sapphire Biomolecular Imager. (D) SUV measurements of relevant tissues determined from PET imaging.

Figure 6

Click-to-release enhances tumor to background. (A) CT and PET images (SUV map) of tumor bearing mice. Tumors are denoted by white arrows. (B) Tissue radiation intensities determined via PET imaging. Tumor to background ratio (TBR) is improved via administration of HK-Tz (2) scissors.

Figure 7

Modeling of SEMI PET imaging. (A) Multicompartment SEMI model to interpret tissue concentrations over time (see Figure S11 for details). (B) Relative blood radioactivity following IV administration of SEMI-antibody. The injection of Hk-Tz is comparable to a k_act of ∼10 M^–1 s^–1 at concentrations used in this study. The “k_act” is the bimolecular rate constant for the click reaction. At 30× higher concentrations, the k_act can be increased to ∼300 with near complete and immediate clearance of blood activity. (C) Effect of Hk-Tz on tumor/lung imaging ratio at different k_act. (D) Modeling the effects of HK-Tz administration on total body radiation, which may have applications in radiotheranostics.