Radioimmunotherapy of HER2-positive breast cancer using 225Ac-labeled trastuzumab and 67Cu-labeled pertuzumab biparatopic radioimmunoconjugates

Introduction: Radioimmunotherapeutics use monoclonal antibodies (mAbs) to selectively deliver highly potent radioisotopes to target sites. Despite recent advancement in oncology therapeutics, the challenges of resistance and reoccurrence in HER2 positive breast cancer (BC) necessitate improved therapeutic innovations against the disease. Recent data from our laboratory show favorable internalization of a combination of domain IV- specific antibody trastuzumab with domain ll-specific antibody pertuzumab compared with the individual antibodies. Here, we propose to develop and evaluate anti-HER2 biparatopic radioimmunoconjugates (RICs) consisting of [²²⁵Ac]Ac-trastuzumab and [⁶⁷Cu]Cu-pertuzumab. We hypothesize that enhanced internalization of the combination of the domain-specific RICs would be more tumoricidal than monotherapy of these RICs against resistant and aggressive BC.

Methods: Trastuzumab and pertuzumab were conjugated to chelators macropa-NCS and p-SCN-Bn-NOTA respectively and the uptake of the antibodies singly and in combination in vitro on HER2 positive JIMT-1 cell line was evaluated. P-Bn-NOTA-pertuzumab was radiolabeled with [⁶⁷Cu]CuCl₂ and macropa-trastuzumab radiolabeled with [²²⁵Ac]AcNO₃. Saturation binding assay of radiolabeled pertuzumab was conducted on JIMT-1 cells. Internalization and binding assays of the antibodies and the RICs were carried out in murine 4T1 cells transfected with HER2 (4T1_HER2) and trastuzumab-resistant human JIMT-1 cells using live-cell imaging.

For monotherapy, therapeutic efficacy was studied after administration of two doses at 10 days apart of the RICs [²²⁵Ac]-macropa-trastuzumab (13 KBq/dose) or [⁶⁷Cu]Cu-NOTA-pertuzumab (18 MBq/dose) to athymic nude mice bearing JIMT-1 xenograft. Therapeutic effectiveness of the combination of the RICs at half the doses used in the monotherapy will be evaluated in JIMT-1 xenograft bearing mice. Saline, unlabeled pertuzumab and trastuzumab, and ²²⁵Ac/⁶⁷Cu-labeled control IgG serve as controls for in vivo therapy studies. Mice bearing HER2-positive 4T1_HER2 isograft will similarly be evaluated.

Results: The extent of internalization (represented as AUC) of the combined antibody conjugates NOTA-pertuzumab + macropa-trastuzumab (598672 µm²h ± 3592.2) was higher than for unconjugated antibodies pertuzumab + trastuzumab (386259 µm²h ± 7191.8) in JIMT-1 cells. In general, biparatopic immunoconjugates NOTA-pertuzumab + macropa-trastuzumab internalized better than the single treatments of the unconjugated and conjugated antibodies. Higher internalization can cause enhanced DNA double strand break and cell death. The low K_D of [⁶⁷Cu]Cu-NOTA-pertuzumab (14.19 nM) on JIMT-1 indicates high affinity for HER2 receptor. After 90 days, monotherapy using [²²⁵Ac]Ac-macropa-trastuzumab was more efficacious with 75 % complete remission (6/8 mice in comparison to 50% complete remission (3/6 mice) for [⁶⁷Cu]Cu-NOTA-pertuzumab treatment. Treatment study using [225Ac]Ac-macropa-trastuzumab + [67Cu]Cu-NOTA-pertuzumab in mice bearing JIMT-1 xenograft is ongoing.

Conclusions: Biparatopic combination of macropa-trastuzumab and NOTA-pertuzumab had a better internalization profile in vitro compared with either antibody used singly. Monotherapy of [²²⁵Ac]Ac-macropa-trastuzumab or [⁶⁷Cu]Cu-NOTA-pertuzumab was more effective than the unlabeled antibodies and we anticipate better therapeutic effects using combination of both radiolabeled antibodies in ongoing experiments.

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