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Quantification of Trastuzumab-HER2 Engagement In Vitro and In Vivo.

ABSTRACT: Human EGF Receptor 2 (HER2) is an important oncogene driving aggressive metastatic growth in up to 20% of breast cancer tumors. At the same time, it presents a target for passive immunotherapy such as trastuzumab (TZM). Although TZM has been widely used clinically since 1998, not all eligible patients benefit from this therapy due to primary and acquired drug resistance as well as potentially lack of drug exposure. Hence, it is critical to directly quantify TZM-HER2 binding dynamics, also known as cellular target engagement, in undisturbed tumor environments in live, intact tumor xenograft models. Herein, we report the direct measurement of TZM-HER2 binding in HER2-positive human breast cancer cells and tumor xenografts using fluorescence lifetime Forster Resonance Energy Transfer (FLI-FRET) via near-infrared (NIR) microscopy (FLIM-FRET) as well as macroscopy (MFLI-FRET) approaches. By sensing the reduction of fluorescence lifetime of donor-labeled TZM in the presence of acceptor-labeled TZM, we successfully quantified the fraction of HER2-bound and internalized TZM immunoconjugate both in cell culture and tumor xenografts in live animals. Ex vivo immunohistological analysis of tumors confirmed the binding and internalization of TZM-HER2 complex in breast cancer cells. Thus, FLI-FRET imaging presents a powerful analytical tool to monitor and quantify cellular target engagement and subsequent intracellular drug delivery in live HER2-positive tumor xenografts.

SUBMITTER: Rudkouskaya A

PROVIDER: S-EPMC7767145 | biostudies-literature | 2020 Dec

REPOSITORIES: biostudies-literature

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Quantification of Trastuzumab-HER2 Engagement In Vitro and In Vivo.

Rudkouskaya Alena A Smith Jason T JT Intes Xavier X Barroso Margarida M

Molecules (Basel, Switzerland) 20201217 24

Human EGF Receptor 2 (HER2) is an important oncogene driving aggressive metastatic growth in up to 20% of breast cancer tumors. At the same time, it presents a target for passive immunotherapy such as trastuzumab (TZM). Although TZM has been widely used clinically since 1998, not all eligible patients benefit from this therapy due to primary and acquired drug resistance as well as potentially lack of drug exposure. Hence, it is critical to directly quantify TZM-HER2 binding dynamics, also known ...[more]

PMID: 33348564

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Project description:INTRODUCTION: Trastuzumab has been widely used for the treatment of human epidermal growth factor receptor 2 (HER2) overexpressing breast cancer for more than a decade. However, reports on the involvement of HER2 downregulation in trastuzumab's mechanism of action are inconsistent. The aim of this study is to investigate if the dependence of trastuzumab-mediated cancer cell HER2 downregulation on immune effector cells represents a novel mechanism of action for trastuzumab. METHODS: HER2 expression was evaluated by Western blotting, flow cytometry, and real-time polymerase chain reaction (PCR) in cell lysates from co-cultures of multiple cancer cell lines with peripheral blood mononuclear cells (PBMCs) in the presence or absence of trastuzumab. The engagement of immune cells by trastuzumab through Fc gamma receptors (Fc?Rs) was tested using three trastuzumab variants with compromised or no Fc (fragment crystallizable) functions and Fc?Rs blocking experiments. The engagement of immune cells by trastuzumab in HER2 downregulation was also evaluated in in vivo mouse xenograft tumor models. RESULTS: HER2 downregulation of cancer cells by trastuzumab occurred only when trastuzumab was actively engaged with immune cells and cancer cells, as demonstrated consistently in co-cultures of cancer cell lines with PBMCs and in vivo mouse xenograft tumor models. We further demonstrated that HER2 downregulation in cancer cells by immune-cell-engaged trastuzumab was at the transcriptional level, not through the HER2 degradation pathway. Activation of signal transducer and activator of transcription 1 (STAT1) in cancer cells by the increased interferon gamma (IFN-?) production in immune cells played an important role in downregulating HER2 in cancer cells upon engagement of immune cells by trastuzumab. Furthermore, HER2 downregulation in cancer cells induced by trastuzumab engagement of immune cells was correlated with the antibody's antitumor efficacy in vivo. CONCLUSIONS: This study reveals that engagement of immune effector cells by trastuzumab induces HER2 downregulation in HER2-expressing cancer cells, which represents a new function of immune cells in trastuzumab-mediated antitumor efficacy and serves as a novel mechanism of action for trastuzumab. Our results imply that HER2 downregulation in cancer cells treated by trastuzumab may predict active engagement of immune effector cells in tumor microenvironment.

Project description:BackgroundTrastuzumab improves survival in the adjuvant treatment of HER-positive breast cancer, although combined therapy with anthracycline-based regimens has been associated with cardiac toxicity. We wanted to evaluate the efficacy and safety of a new nonanthracycline regimen with trastuzumab.MethodsWe randomly assigned 3222 women with HER2-positive early-stage breast cancer to receive doxorubicin and cyclophosphamide followed by docetaxel every 3 weeks (AC-T), the same regimen plus 52 weeks of trastuzumab (AC-T plus trastuzumab), or docetaxel and carboplatin plus 52 weeks of trastuzumab (TCH). The primary study end point was disease-free survival. Secondary end points were overall survival and safety.ResultsAt a median follow-up of 65 months, 656 events triggered this protocol-specified analysis. The estimated disease-free survival rates at 5 years were 75% among patients receiving AC-T, 84% among those receiving AC-T plus trastuzumab, and 81% among those receiving TCH. Estimated rates of overall survival were 87%, 92%, and 91%, respectively. No significant differences in efficacy (disease-free or overall survival) were found between the two trastuzumab regimens, whereas both were superior to AC-T. The rates of congestive heart failure and cardiac dysfunction were significantly higher in the group receiving AC-T plus trastuzumab than in the TCH group (P<0.001). Eight cases of acute leukemia were reported: seven in the groups receiving the anthracycline-based regimens and one in the TCH group subsequent to receiving an anthracycline outside the study.ConclusionsThe addition of 1 year of adjuvant trastuzumab significantly improved disease-free and overall survival among women with HER2-positive breast cancer. The risk-benefit ratio favored the nonanthracycline TCH regimen over AC-T plus trastuzumab, given its similar efficacy, fewer acute toxic effects, and lower risks of cardiotoxicity and leukemia. (Funded by Sanofi-Aventis and Genentech; BCIRG-006 ClinicalTrials.gov number, NCT00021255.).

Project description:Trastuzumab emtansine (T-DM1) is an antibody-drug conjugate incorporating the human epidermal growth factor receptor 2 (HER2)-targeted antitumor properties of trastuzumab with the cytotoxic activity of the microtubule-inhibitory agent DM1. The antibody and the cytotoxic agent are conjugated by means of a stable linker.We randomly assigned patients with HER2-positive advanced breast cancer, who had previously been treated with trastuzumab and a taxane, to T-DM1 or lapatinib plus capecitabine. The primary end points were progression-free survival (as assessed by independent review), overall survival, and safety. Secondary end points included progression-free survival (investigator-assessed), the objective response rate, and the time to symptom progression. Two interim analyses of overall survival were conducted.Among 991 randomly assigned patients, median progression-free survival as assessed by independent review was 9.6 months with T-DM1 versus 6.4 months with lapatinib plus capecitabine (hazard ratio for progression or death from any cause, 0.65; 95% confidence interval [CI], 0.55 to 0.77; P<0.001), and median overall survival at the second interim analysis crossed the stopping boundary for efficacy (30.9 months vs. 25.1 months; hazard ratio for death from any cause, 0.68; 95% CI, 0.55 to 0.85; P<0.001). The objective response rate was higher with T-DM1 (43.6%, vs. 30.8% with lapatinib plus capecitabine; P<0.001); results for all additional secondary end points favored T-DM1. Rates of grade 3 or 4 adverse events were higher with lapatinib plus capecitabine than with T-DM1 (57% vs. 41%). The incidences of thrombocytopenia and increased serum aminotransferase levels were higher with T-DM1, whereas the incidences of diarrhea, nausea, vomiting, and palmar-plantar erythrodysesthesia were higher with lapatinib plus capecitabine.T-DM1 significantly prolonged progression-free and overall survival with less toxicity than lapatinib plus capecitabine in patients with HER2-positive advanced breast cancer previously treated with trastuzumab and a taxane. (Funded by F. Hoffmann-La Roche/Genentech; EMILIA ClinicalTrials.gov number, NCT00829166.).

Dataset Information

Quantification of Trastuzumab-HER2 Engagement In Vitro and In Vivo.

Publications

Quantification of Trastuzumab-HER2 Engagement In Vitro and In Vivo.

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