Highly potent [225Ac]Ac-macropa-trastuzumab-PEG6-DM1 antibody drug radioconjugate against HER2-positive breast cancer xenografts

Introduction: Actinium-225 (²²⁵Ac) is a potent alpha emitter with excellent pharmacological characteristics due to its short range, and high liner energy transfer. There is overwhelming interest in using ²²⁵Ac to develop targeted alpha therapies (TAT). Antibody drug conjugates (ADCs) are highly cytotoxic with improved therapeutic index, antitumor activity, and decreased off-target toxicity over naked antibodies. Combining ²²⁵Ac with ADCs to develop antibody drug radiocongates (ADRs) is expected to be more effective than ADCs against primary and metastatic breast cancers (BC). Poor prognosis and outcomes are characteristics of human epidermal growth factor receptor 2 (HER2) positive BC, which is overexpressed in 25 – 30% of BC. Trastuzumab is the first anti-HER2 monoclonal antibody and its two ADCs (trastuzumab emtansine – T-DM1, and trastuzumab deruxtecan-T-DXd) are FDA-approved. Despite these treatments, response rates are not high enough and resistance is common. We propose for the first time to develop an ADR [²²⁵Ac]Ac-macropa-trastuzumab-PEG₆-DM1 and evaluate its effectiveness in HER2-positive BC xenografts.

Aim: To develop and evaluate ADRs [⁸⁹Zr]Zr-DFO-trastuzumab-PEG₆-DM1 and [²²⁵Ac]Ac-macropa-trastuzumab-PEG₆-DM1 for immunoPET imaging and radiotherapy of HER2-positive BC using mouse models.

Methods: [⁸⁹Zr]Zr-DFO-trastuzumab-PEG₆-DM1 and [²²⁵Ac]Ac-macropa-trastuzumab-PEG₆-DM1 were developed with drug linker NHS-PEG₆-DM1 and bifunctional chelators p-SCN-DFO and p-SCN-macropa respectively. Quality control was done using size exclusion chromatography (SEC)-HPLC, flow cytometry, antibody internalization, in vitro stability and radioligand binding assay. Biodistribution and safety evaluation of [²²⁵Ac]Ac-macropa-trastuzumab-PEG₆-DM1 were carried out in healthy Balb/C mice. ImmunoPET imaging and biodistribution using [⁸⁹Zr]Zr-DFO-trastuzumab-PEG₆-DM1 and radiotherapy using [²²⁵Ac]Ac-macropa-trastuzumab-PEG₆-DM1 were done in athymic nude mice bearing trastuzumab-sensitive HCC1954 (high HER2 density) and T-DM1/trastuzumab resistant JIMT-1 (medium HER2 density) tumor bearing mice.

Results: Pure trastuzumab ADCs and ADRs were obtained as confirmed using SEC-HPLC. [⁸⁹Zr]Zr-DFO-trastuzumab-PEG₆-DM1 and [²²⁵Ac]Ac-macropa-trastuzumab-PEG₆-DM1 had a radiochemical purity >95% at specific activity of 1 MBq/2 µg, and 1MBq/300µg, respectively. Low K_D values of trastuzumab-PEG₆-DM1 in HCC1954 (36.98 nM) and JIMT-1 (20.16 nM) were obtained. After 7 days of incubation at 37ºC, [²²⁵Ac]Ac-macropa-trastuzumab-PEG₆-DM1 was stable in both human serum (89.2 ± 0.9%) and PBS (82.8 ± 0.4%). Internalization in HCC1954 and JIMT-1 cells was HER2 density-dependent with trastuzumab-PEG₆-DM1 (13418997 ± 657149 vs 2716687 ± 86280) significantly higher than trastuzumab (5319405 ± 365855 vs 243078 ± 3087), respectively (p<0.0001). Trastuzumab-PEG₆-DM1 (8 mg/Kg) and [²²⁵Ac]Ac-trastuzumab-PEG₆-DM1(3 X 18 KBq) administered separately in healthy Balb/C mice, 10 days apart was well tolerated biochemically and haematologically for 30 days. Imaging and biodistribution of [⁸⁹Zr]Zr-DFO-trastuzumab-PEG₆-DM1 in athymic nude mice showed tumor uptakes of 38.1 ± 2.8% IA/g (HCC1954) and 14.6 ± 1% IA/g (JIMT-1) at 120 h post injection. In trastuzumab-sensitive HCC1954-tumor bearing mice, all treatment groups had complete remission (CR), while in trastuzumab/T-DM1-resistant JIMT-1 xenografts at 23 days post treatment, tumor volumes were 332.1 ± 77.5 vs 244.6 ± 63 vs 417.9 ± 62.1 vs 102.4 ± 18.5 for the saline (negative control), T-DM1 (positive control), trastuzumab-PEG₆-DM1 and [²²⁵Ac]Ac-macropa-trastuzumab-PEG₆-DM1 respectively. (Figure 1)

Conclusions: The ADR [²²⁵Ac]Ac-macropa-trastuzumab-PEG₆-DM1 is more potent than its ADC trastuzumab-PEG₆-DM1 against T-DM1-resistant BC necessitating clinical translation.

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