Development and Evaluation of Novel Anti-HER2 Theranostics Against HER2-Positive Breast Cancer

Breast cancer (BC) is the deadliest cancer type in women worldwide with high incidence (11.6%) and mortality (6.9%) rates. Human epidermal growth factor receptor 2 (HER2) is overexpressed in 20-30% of BC. HER2 overexpression is also observed in other cancers such as gastric/gastroesophageal, ovarian, lung, prostate, bladder, colon and head and neck. HER2-positive BC is characterized by aggressive disease and poor prognosis and has one of the worst 4-year survival rates. HER2 can homo- or heterodimerize with itself or other HER family members (HER1/EGFR, HER3 and HER4), initiating downstream signalling pathways that regulate the proliferation, differentiation and growth of cancer cells. In the past 20 years, remarkable advancements have been achieved in developing treatments against HER2-positive BC. One of such early and remarkable success stories was the development and approval of anti-HER2 monoclonal antibody trastuzumab which is now the standard of care treatment. However, the disease relapses in almost all patients. As a result, alternative HER2-targeted therapies, such as pertuzumab, margetuximab, T-DM1, T-DXd, lapatinib, neratinib, pyrotinib, gefitinib, and tucatinib, have been developed. Nonetheless, despite of their improved effectiveness, de novo and acquired resistance to current treatments are very common in BC patients; almost all develop resistance with these is still common and most patients advance to metastatic BC (MBC). Newer approaches such as radioimmunotherapies (using beta and alpha particles-based agents), immune checkpoint inhibitors, and vaccines and combinations of these with standards of care are active areas of research. The overall hypothesis of the thesis is that safe and more effective anti-HER2 theranostics can improve the management of HER2-positive BC. This thesis aimed to develop more potent trastuzumab radioimmunoconjugates (RICs) and novel trastuzumab/pertuzumab antibody-drug radioconjugates (ADRs) radiolabeled with beta and alpha particles, against HER2-positive BC. These agents were evaluated as potential theranostics for SPECT/PET imaging and for radioimmunotherapy. We developed domain-specific anti-HER2 antibody-drug conjugates (ADCs) trastuzumab-PEG6-DM1 and pertuzumab-PEG6-DM1. Conjugation using bifunctional chelators p-SCN-DFO and p-SCN-DOTA afforded DFO-trastuzumab-PEG6-DM1 and DOTA-pertuzumab-PEG6-DM1, respectively for radiolabeling with 89Zr and 67Cu for microPET/SPECT/CT imaging. We also radiolabeled these ADCs with [225Ac]Ac via a stable eighteen-membered macrocyclic chelator p-SCN-Macropa for alpha particle therapy. All immunoconjugates were characterized using size exclusion high performance liquid chromatography (SEC-HPLC), flow cytometry, and antibody internalization. The binding affinities and specificities of the RICs were evaluated by saturation radioligand binding assays, and the results showed high specific HER2 binding. In Chapter 2 (Ketchemen et al. Br J Cancer. 2023; 129(1): 153–162), we developed and evaluated [89Zr]Zr-trastuzumab-PEG6-DM1 and [67Cu]Cu-pertuzumab-PEG6-DM1 as biparatopic anti-HER2 theranostics since they are domain-specific. We showed in vitro that internalization of the biparatopic combination of trastuzumab-PEG6-DM1 + pertuzumab-PEG6-DM1 was several folds higher in all HER2-positive cell lines compared with the individual ADCs. This higher internalization resulted in significantly lower IC50 values for trastuzumab-PEG6-DM1 + pertuzumab-PEG6-DM1 combination compared with the individual ADCs, and the IC50 value was several folds lower than approved T-DM1 (Kadcyla®). In vivo imaging studies confirmed that biparatopic combination of trastuzumab-PEG6-DM1 + pertuzumab-PEG6-DM1 was synergistic which has profound implications for therapy. In Chapter 3 (Ketchemen et al. Eur J Nucl Med Mol Imaging 2024;51(7):2070-2084), we developed and evaluated the effectiveness of [67Cu]Cu-trastuzumab as a theranostic single. We started off by evaluating the most suitable chelator for [67Cu]Cu complexation from a set of bifunctional chelators p-SCN-Bn-NOTA, 3p-C-NETA-NCS, or p-SCN-Bn-DOTA. p-SCN-Bn-NOTA emerged as the best chelator for [67Cu]Cu. We, therefore, evaluated [67Cu]Cu-NOTA-trastuzumab in vitro and in vivo. [67Cu]Cu-NOTA-trastuzumab was more effective at inhibiting the growth of trastuzumab-sensitive and trastuzumab-resistant/T-DM1-resistant xenografts in vivo including complete remissions. In Chapter 4 (Ketchemen et al. Clin Cancer Res, accepted for publication), we developed and evaluated the effectiveness of antibody-drug radioconjugate [225Ac]Ac-Macropa-trastuzumab-PEG6-DM1 against HER2-positive BC. [89Zr]Zr-DFO-trastuzumab-PEG6-DM1 was developed as the imaging pair. We compared the effectiveness [225Ac]Ac-Macropa-trastuzumab-PEG6-DM1 in that of the ADC trastuzumab-PEG6-DM1 in HER2-positive xenografts. [225Ac]Ac-Macropa-trastuzumab-PEG6-DM1 was safe and more effective compared with trastuzumab and ADC trastuzumab-PEG6-DM1 at inhibiting tumor growth including complete remissions of trastuzumab resistant/T-DM1 sensitive HCC1954 and trastuzumab-resistant/T-DM1 resistant JIMT-1 xenografts. In Chapter 5 (Ketchemen et al. Submitted to Eur J Nucl Med Mol Imaging), we developed and evaluated the effectiveness of [225Ac]Ac-Macropa-pertuzumab-PEG6-DM1 against HER2-positive BC xenografts. We developed [67Cu]Cu-DOTA-pertuzumab-PEG6-DM1 as the imaging pair for the therapeutic. Pertuzumab has unique binding characteristics to HER2, and radioimmunoconjugates potentially have the added advantage of inhibiting homo- and hetero-dimerization with other HER family receptors leading to additional therapeutic effects. We compared the effectiveness of [225Ac]Ac-Macropa-pertuzumab-PEG6-DM1 in that of the ADC pertuzumab-PEG6-DM1 in HER2-positive xenografts. [225Ac]Ac-Macropa-pertuzumab-PEG6-DM1 was safe and more effective compared with pertuzumab and ADC pertuzumab-PEG6-DM1 at inhibiting tumor growth including complete remissions of trastuzumab-resistant/T-DM1 sensitive HCC1954 and trastuzumab-resistant/T-DM1 resistant JIMT-1 xenografts. In Chapter 6 (Ketchemen et al. manuscript in preparation), we ]evaluated the effectiveness of biparatopic combination of [225Ac]Ac-Macropa-trastuzumab-PEG6-DM1 and [225Ac]Ac-Macropa-pertuzumab-PEG6-DM1 against HER2-positive BC xenografts. We compared with the single agents and their ADCs. [225Ac]Ac-Macropa-trastuzumab-PEG6-DM1 + [225Ac]Ac-Macropa-pertuzumab-PEG6-DM1 was safe and, more effective than the combined ADCs against trastuzumab resistant/T-DM1-sensitive HCC1954 and trastuzumab-resistant/T-DM1-resistant JIMT-1 xenografts. In conclusion, we developed effective imaging and therapeutic agents using trastuzumab and pertuzumab. Antibody-drug radioconjugates [225Ac]Ac-Macropa-trastuzumab-PEG6-DM1 and [225Ac]Ac-Macropa-pertuzumab-PEG6-DM1 were the most effective and they were safe. [225Ac]Ac-Macropa-trastuzumab-PEG6-DM1, [225Ac]Ac-Macropa-pertuzumab-PEG6-DM1 or their biparatopic combination would drastically improve outcomes in patients with HER2-positive BC and should be translated in phase 1 trials.

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