Introduction: Measuring drug payload biodistribution in preclinical models can provide mechanistic insights into antibody drug conjugate (ADC) efficacy and tolerability but often requires the use of ³H/¹⁴C labels or mass spectrometric methods to maintain drug structure and function.¹ Noninvasive imaging of radioimmunoconjugates by PET or SPECT is seldom a faithful surrogate for ADC biodistribution as the fate of the radiolabeled probe does not necessarily mirror that of the drug payload.² Such efforts are further confounded by the risk that radiolabeling drug-modified antibodies can lead to over-modification and altered disposition.³ Inspired by the concept of theranostics, we designed an antibody-conjugatable ADC payload that serves as a conventional SPECT probe in one state but with the potential to be converted on demand to a conventional cytotoxic ADC payload – a therapeutic state – by administration of a bioorthogonal switch, effectively enabling imaging and therapy from a single molecular entity we term an antibody radio-drug conjugate (ArDC). Importantly, this work is distinct from previous click-to-release approaches for ADCs^4,5 in that payload activation can be achieved for intracellular ArDC catabolites through use of cell permeable tetrazines.

Methods: An ArDC was designed to incorporate a potent DNA-damaging agent, pyrrolobenzodiazepine (PBD) dimer, masked by a novel transcyclooctene-para-aminobenzyl-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (TCO-PAB-DOTA), which served as a site of radiolabeling and to attenuate drug activity (Figure 1a). All in vivo studies were conducted in mice bearing HCC1569X2 xenografts expressing the internalizing human lung cancer antigen, Ly6E.⁶ After confirming stability, an in vivo SPECT imaging study with ¹¹¹In-labeled ArDC – alongside the ¹¹¹In-DOTA-labeled parent antibody as a control – confirmed the ability to noninvasively image the in vivo disposition of the ArDC payload. In a separate non-radioactive study, tumor growth inhibition was measured for a single 0.5 mg/kg dose of the parent ADC, the ArDC alone, or the ArDC followed 6 days later by a single dose (10, 30 or 100 umol/kg) of tetrazine. Traceless, on-demand conversion from the TCO-PAB-DOTA-masked state to the cytotoxic state was accomplished via the bond-breaking variant of the tetrazine/trans-cyclooctene (Tz/TCO) inverse-electron demand Diels-Alder (IEDDA) click reaction.

Results: Live animal SPECT-CT imaging of tumor-bearing mice dosed with the radiolabeled conjugate showed steady accumulation of ¹¹¹In signal in the tumor over 6 days for [¹¹¹In]ArDC, which was comparable to that of the control, [¹¹¹In]αLy6E (Figure 1b). The tetrazine ultimately proved highly effective at switching the ArDC to a cytotoxic form in vivo, inducing tumor growth inhibition in mice whereas the tetrazine or ArDC alone was inactive (Figure 1c).

Conclusions: The efforts herein lay the groundwork for directly characterizing ADC payload distribution/efficacy and distribution/toxicity relationships in living subjects, which may accelerate engineering efforts to achieve more tumor-selective delivery of ADCs. The ArDC approach may eventually offer the possibility of selecting and treating patients with a single active pharmaceutical agent while gating payload activation on maximization of delivery – measured by noninvasive imaging – to tumor versus normal tissues. Despite this potential, several challenges lie ahead of implementing such an effort including the overall complexity of the approach and optimizing payload biodistribution.

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Figure 1. A) Structural diagram of antibody-radio-drug conjugate (ArDC). The payload is a pyrrolobenzodiazepine (PBD) dimer (red) labeled and attenuated in potency by a TCO-PAB-DOTA group (yellow-black-green) attached to the N10 nitrogen; the DOTA can chelate a radioactive metal isotope to enable tracking in vivo of both conjugated and released payload. Reaction of the TCO with a tetrazine prompts click/release of the TCO-PAB-DOTA and free drug payload from the ArDC or downstream catabolites (even intracellular), effectively resulting in the fully-cytotoxic and unlabeled ADC or free PBD dimer. B) SPECT-CT imaging (¹¹¹In) of the anti-Ly6E targeted antibody-radio-drug conjugate labeled through DOTA within the radio-drug (ArDC, upper) versus conventional antibody-drug conjugate labeled through DOTA conjugated via lysines (ADC, lower). C) Tumor growth inhibition observed following tetrazine-induced switching of ArDC in the HCC1569X2 mouse xenograft model. SCID mice were inoculated with HCC1569X2 tumor cells and tumors were allowed to grow for ~7 days to a volume of 200-300 mm³ (Day 0), at which time the ArDC was administered intravenously in the tail vein at a dose of 0.5 mg/kg. Six days later, a single dose of tetrazine was administered IV in the tail vein at 100, 30 or 10 mmol/kg. Controls with ArDC only (1 mg/kg), ADC only (0.5 mg/kg) or tetrazine only (100 mmol/kg) are also shown.

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