Simultaneous molecular imaging and biodistribution of two radiolabeled domain-specific anti-HER2 antibody drug conjugates in HER2-positive xenografts

Introduction: Human epidermal growth factor receptor 2 positive breast cancer (HER2+ BC) is characterized by poor prognosis, especially in advanced stage disease despite the availability of anti-HER2 targeted therapeutics. HER2 is overexpressed in 25 – 30% of BC. HER2 receptors have an extracellular domain made of four binding domains (I-IV). Monoclonal antibodies (mAbs) against HER2 including trastuzumab (domain IV-specific) and pertuzumab (domain II-specific), and antibody drug conjugates (ADC) trastuzumab emtansine and trastuzumab deruxtecan are some of the FDA approved agents against HER2-positive BC. Despite their promise, response rates are not high enough and resistance is widespread. Recent preclinical and clinical data show that simultaneously targeting multiple domains of a receptor can be synergistic compared with monotherapy using the mAb or ADC. Here, for the first time, we propose to use molecular imaging to understand the biodistribution of two domain-specific ADCs that bind to HER2. Our objective, was to develop ⁸⁹Zr-trastuzumab-PEG₆-DM1 (domain IV) and ⁶⁷Cu pertuzumab-PEG₆-DM1 (domain II) ADCs and evaluate their in vivo properties in mouse models of HER2 positive BC using imaging and ex vivo biodistribution studies.

Methods: We developed trastuzumab and pertuzumab ADCs by conjugating the antibodies with drug linker NHS-PEG₆-DM1 to form trastuzumab-PEG₆-DM1 and pertuzumab-PEG₆-DM1 with an average drug-to-antibody ratio (DAR) of 4. Trastuzumab-PEG₆-DM1 and pertuzumab-PEG₆-DM1 were radiolabeled with ⁸⁹Zr and ⁶⁷Cu, respectively via a desferrioxamine (DFO) and DOTA chelator, respectively. Quality control of the immunoconjugates was done using size exclusion chromatography (SEC)-HPLC, bioanalyzer and flow cytometry using high (BT-474), medium (JIMT-1) and low (MCF7) density HER2 expressing cell lines. Radioligand and competitive binding assays were performed using BT-474 cells. In vitro stability was evaluated in human serum and in PBS over a period of 5 days. Pharmacokinetic and in vivo stability studies were carried out in healthy Balb/C mice (n = 3/group). ImmunoPET/SPECT imaging and biodistribution of ⁸⁹Zr-trastuzumab-PEG₆-DM1 and ⁶⁷Cu-pertuzumab-PEG₆-DM1 were done in tumor bearing mice (BT-474 and JIMT-1 xenografts) at different time points (24 h, 48 h and 120 h) to understand their in vivo characteristics.

Results: Conjugation produced trastuzumab-PEG₆-DM1 and pertuzumab-PEG₆-DM1 ADCs with a DAR of 3.0-3.6 that were pure and homogenous as shown using SEC-HPLC and bioanalyzer. Flow cytometry showed low K_D values of the DFO-trastuzumab-PEG₆-DM1 (71.6 nM) and DOTA-pertuzumab-PEG₆-DM1 (36.7 nM). Radiochemical purities were >= 95% at specific activities of 0.5 MBq/µg (⁸⁹Zr) and 1 MBq/µg (⁶⁷Cu). K_D of ⁶⁷Cu-pertuzumab-PEG₆-DM1 using BT-474 was 11.5 nM. Trastuzumab did not compete with ⁶⁷Cu-pertuzumab-PEG₆-DM1 for binding to HER2. Both radiotracers were stable in human serum and PBS after incubation at 37 °C for 5 days. Pharmacokinetics showed biphasic half-lives with fast distribution (t_1/2α: 8.9 ± 5.2 h [⁸⁹Zr]and 10.0 ± 3.4 h [⁶⁷Cu]) and a slow elimination (t_1/2β: 282.4 ± 301.7 h [⁸⁹Zr]and 121.6 ± 31.5 h [⁶⁷Cu]) of the tracers. Tumor uptake of ⁸⁹Zr-trastuzumab-PEG₆-DM1 was 16.6 ± 4.04% IA/g (BT-474) and 13.12 ± 5.5% IA/g (JIMT-1) at 120 h post injection (p.i.). Similarly, tumor uptake of ⁶⁷Cu-pertuzumab-PEG₆-DM1 was 10.9 ± 4.9% IA/g (BT-474) and 10.03 ± 2.6% IA/g (JIMT-1) at 120 h p.i. Tumor uptake and biodistribution of the individual tracers were similar when the tracers were injected solo or simultaneously in same mouse model.

Conclusions: We were able for the first time, to develop both trastuzumab-PEG₆-DM1 and pertuzumab-PEG₆-DM1 ADCs and studied their in vivo characteristics using PET and SPECT following simultaneous administration in tumor bearing mice. This study is important because of the potential to use these biologics simultaneously as biparatopic therapeutic/theranostic agents.