Flow cytometry MFI and % positive cells data for fluorescently labelled trastuzumab and pertuzumab in SKBR3, HCC1954, T47D and MCF-7 treated with TKIs

Supplementary Figures S1-S4 from Trastuzumab and Pertuzumab Produce Changes in Morphology and Estrogen Receptor Signaling in Ovarian Cancer Xenografts Revealing New Treatment Strategies

1. Supplementary Figure 1. Illustration of tubes area detection as rectangles based on the bright field channel. 2. Supplementary Figure 2. Diarrhea severity during combination treatment with lumretuzumab (RO5479599), pertuzumab and paclitaxel in patients. 3. Full Unedited western blots of figures 2c and 2d

Supplementary Figure Legends 1-2 from Strongly Enhanced Antitumor Activity of Trastuzumab and Pertuzumab Combination Treatment on HER2-Positive Human Xenograft Tumor Models

Additional supporting information for methods outlined in main manuscript

Flow cytometry control scatter plots for trastuzumab-488 and pertuzumab-550

MFI comparison at T0 and T6 for T47D/MCF-7

Densitometry analysis for Figure 6D/E

Supplementary Figure 1 from Strongly Enhanced Antitumor Activity of Trastuzumab and Pertuzumab Combination Treatment on HER2-Positive Human Xenograft Tumor Models

Densitometry analysis for Figure 6B

Rituximab ADCC negative control

All ADCC ratios from Figure 3 and trastuzumab/pertuzumab ADCC comparison

Targeted therapies have shown striking success in the treatment of cancer over the last years. However, their specific effects on an individual tumor appear to be varying and difficult to predict. Using an integrative modeling approach that combines mechanistic and regression modeling, we gained insights into the response mechanisms of breast cancer cells due to different ligand–drug combinations. The multi-pathway model, capturing ERBB receptor signaling as well as downstream MAPK and PI3K pathways was calibrated on time-resolved data of the luminal breast cancer cell lines MCF7 and T47D across an array of four ligands and five drugs. The same model was then successfully applied to triple negative and HER2-positive breast cancer cell lines, requiring adjustments mostly for the respective receptor compositions within these cell lines. The additional relevance of cell-line-specific mutations in the MAPK and PI3K pathway components was identified via L1 regularization, where the impact of these mutations on pathway activation was uncovered. Finally, we predicted and experimentally validated the proliferation response of cells to drug co-treatments. We developed a unified mathematical model that can describe the ERBB receptor and downstream signaling in response to therapeutic drugs targeting this clinically relevant signaling network in cell line that represent three major subtypes of breast cancer. Our data and model suggest that alterations in this network could render anti-HER therapies relevant beyond the HER2-positive subtype.

HER3 is highly expressed in luminal breast cancer subtypes. Its activation by NRG1 promotes activation of AKT and ERK1/2, contributing to tumour progression and therapy resistance. HER3-targeting agents that block this activation, are currently under phase 1/2 clinical studies, and although they have shown favorable tolerability, their activity as a single agent has proven to be limited. Here we show that phosphorylation and activation of HER3 in luminal breast cancer cells occurs in a paracrine manner and is mediated by NRG1 expressed by cancer-associated fibroblasts (CAFs). Moreover, we uncover a HER3-independent NRG1 signaling in CAFs that results in the induction of a strong migratory and pro-fibrotic phenotype, describing a subtype of CAFs with elevated expression of NRG1 and an associated transcriptomic profile that determines their functional properties. Finally, we identified Hyaluronan Synthase 2 (HAS2), a targetable molecule strongly correlated with NRG1, as an attractive player supporting NRG1 signaling in CAFs.

Purpose This study investigated the safety and clinical activity of lumretuzumab, a humanised antihuman epidermal growth factor receptor 3 (HER3) monoclonal antibody, in combination with carboplatin and paclitaxel in first-line treatment of patients with squamous non-small cell lung cancer (sqNSCLC). HER3 ligand heregulin and HER3 protein expression were evaluated as potential biomarkers of clinical activity. Patients and methods This open-label, phase Ib/II study enrolled patients receiving lumretuzumab at 800 mg (flat) in combination with carboplatin (area under the curve (AUC) 6 mg/mL×min) and paclitaxel (200 mg/m2) administered intravenously on a every 3-week schedule. Adverse event (AE) rates and tumour responses were determined. Heregulin messenger RNA (mRNA) and HER3 protein expression were investigated in archival tumour biopsies. Results Altogether, 12 patients received lumretuzumab in combination with carboplatin and paclitaxel. The most frequent AEs were gastrointestinal, haematological and nervous system toxicities, which were generally mild and manageable. Partial responses were observed in 3 of 12 patients lasting 81, 177 and 207 days. All responses were achieved in tumours expressing higher heregulin mRNA levels. Conclusion Lumretuzumab in combination with carboplatin and paclitaxel was well tolerated. Objective responses were enriched in tumours expressing higher heregulin mRNA levels.

The quality control testing of chemical degradations in the bio-pharmaceutical industry is currently under controversial debate. Here we have systematically applied in vitro and in vivo stress conditions to investigate the influence of protein degradation on structure-function. Extensive purification and characterization enabled identification and functional assessment of the physiological degradation of chemical modification sites in the variable complementarity-determining regions (CDRs) and conserved region of trastuzumab. We demonstrate that the degradation of the solvent-accessible residues located in the CDR and the conserved fragment crystallizable region (Fc) occurs faster in vivo (within days) compared to the levels observed for bio-process and real-time storage conditions. These results hence question the rationality of extreme monitoring of low level alterations in such chemical modifications as critical patient safety parameters in product quality control testing, given that these modifications merely mirror the natural/physiological aging process of endogenous antibodies.

•HER3 is associated with tumor development, resistance and poor clinical prognosis in different cancers.•HER3-targeting agents have been undergoing clinical evaluation for the last 10 years in clinical phase 1 and 2 studies.•Combinations of HER3-binding antibodies with other HER-targeting therapies or chemotherapies have been pursued in various solid tumors.•HER3 and heregulin have been identified as potential response prediction markers.•Inhibition of HER3 has not yet resulted in any meaningful clinical efficacy in the treatment of solid tumors.

Abstract Combination of targeted therapies is expected to provide superior efficacy in the treatment of cancer either by enhanced antitumor activity or by preventing or delaying the development of resistance. Common challenges in developing combination therapies include the potential of additive and aggravated toxicities associated with pharmacologically related adverse effects. We have recently reported that combination of anti-HER2 and anti-HER3 antibodies, pertuzumab and lumretuzumab, along with paclitaxel chemotherapy in metastatic breast cancer, resulted in a high incidence of diarrhea that ultimately limited further clinical development of this combination. Here, we further dissected the diarrhea profile of the various patient dose cohorts and carried out in vitro investigations in human colon cell lines and explants to decipher the contribution and the mechanism of anti-HER2/3 therapeutic antibodies to intestinal epithelium malfunction. Our clinical investigations in patients revealed that while dose reduction of lumretuzumab, omission of pertuzumab loading dose, and introduction of a prophylactic antidiarrheal treatment reduced most severe adverse events, patients still suffered from persistent diarrhea during the treatment. Our in vitro investigations showed that pertuzumab and lumretuzumab combination treatment resulted in upregulation of chloride channel activity without indication of intestinal barrier disruption. Overall, our findings provide a mechanistic rationale to explore alternative of conventional antigut motility using medication targeting chloride channel activity to mitigate diarrhea of HER combination therapies. Mol Cancer Ther; 17(7); 1464–74. ©2018 AACR.

Summary Purpose To investigate the safety and clinical activity of comprehensive human epidermal growth factor receptor (HER) family receptor inhibition using lumretuzumab (anti-HER3) and pertuzumab (anti-HER2) in combination with paclitaxel in patients with metastatic breast cancer (MBC). Methods This phase Ib study enrolled 35 MBC patients (first line or higher) with HER3-positive and HER2-low (immunohistochemistry 1+ to 2+ and in-situ hybridization negative) tumors. Patients received lumretuzumab (1000 mg in Cohort 1; 500 mg in Cohorts 2 and 3) plus pertuzumab (840 mg loading dose [LD] followed by 420 mg in Cohorts 1 and 2; 420 mg without LD in Cohort 3) every 3 weeks, plus paclitaxel (80 mg/m 2 weekly in all cohorts). Patients in Cohort 3 received prophylactic loperamide treatment. Results Diarrhea grade 3 was a dose-limiting toxicity of Cohort 1 defining the maximum tolerated dose of lumretuzumab when given in combination with pertuzumab and paclitaxel at 500 mg every three weeks. Grade 3 diarrhea decreased from 50% (Cohort 2) to 30.8% (Cohort 3) with prophylactic loperamide administration and omission of the pertuzumab LD, nonetheless, all patients still experienced diarrhea. In first-line MBC patients, the objective response rate in Cohorts 2 and 3 was 55% and 38.5%, respectively. No relationship between HER2 and HER3 expression or somatic mutations and clinical response was observed. Conclusions Combination treatment with lumretuzumab, pertuzumab and paclitaxel was associated with a high incidence of diarrhea. Despite the efforts to alter dosing, the therapeutic window remained too narrow to warrant further clinical development. Trial registration: on ClinicalTrials.gov with the identifier NCT01918254 first registered on 3rd July 2013.

Bidirectional cross talk between members of the human epidermal growth factor family of receptors (HER) and the estrogen receptor (ER) is believed to underlie resistance mechanisms that develop in response to treatment with anti-HER agents and endocrine therapy. We investigated the interaction between HER2, HER3 and the ER in vitro using human embryonic kidney cells transfected with human HER2, HER3, and ERα. We also investigated the additive efficacy of combination regimens consisting of anti-HER3 (lumretuzumab), anti-HER2 (pertuzumab), and endocrine (fulvestrant) therapy in vivo. Our data show that both HER2 and HER3 can directly complex with the ER and can mediate phosphorylation of the ER. Phosphorylation of the ER was only observed in cells that expressed both HER2 and ERα or in heregulin-stimulated cells that expressed both HER3 and ERα. Using a mouse xenograft model of ER+/HER2-low (HER2 immunohistochemistry 1+ or 2+ without gene amplification) human breast cancer we show that the combination of lumretuzumab and pertuzumab is highly efficacious and induces long-lasting tumor regression in vivo and adding endocrine therapy (fulvestrant) to this combination further improved efficacy. In addition, a prolonged clinical response was observed with the combination of lumretuzumab and pertuzumab in a patient with ER+/HER2-low breast cancer who had failed endocrine therapy. These preclinical data confirm that direct cross talk exists between HER2/HER3 and ER which may explain the resistance mechanisms to endocrine therapy and monoclonal antibodies that target HER2 and HER3. Our data also indicate that the triplet of anti-HER2, anti-HER3, and endocrine therapy might be an efficacious combination for treating patients with ER+/HER2-low breast cancer, which is an area of significant unmet medical need.