Design and selection of optimal ErbB-targeting bispecific antibodies in pancreatic cancer.

The ErbB family of receptor tyrosine kinases is a primary target for small molecules and antibodies for pancreatic cancer treatment. Nonetheless, the current treatments for this tumor are not optimal due to lack of efficacy, resistance, or toxicity. Here, using the novel BiXAb™ tetravalent format platform, we generated bispecific antibodies against EGFR, HER2, or HER3 by considering rational epitope combinations.

Improving Biologics' Effectiveness in Clinical Oncology: From the Combination of Two Monoclonal Antibodies to Oligoclonal Antibody Mixtures.

Monoclonal antibodies have revolutionized the treatment of many diseases, but their clinical efficacy remains limited in some other cases. Pre-clinical and clinical trials have shown that combinations of antibodies that bind to the same target (homo-combinations) or to different targets (hetero-combinations) to mimic the polyclonal humoral immune response improve their therapeutic effects in cancer. The approval of the trastuzumab/pertuzumab combination for breast cancer and then of the ipilimumab/nivolumab combination for melanoma opened the way to novel antibody combinations or oligoclonal antibody mixtures as more effective biologics for cancer management.

Anti-tumoral activity of the Pan-HER (Sym013) antibody mixture in gemcitabine-resistant pancreatic cancer models.

Chemoresistance, particularly to gemcitabine, is a major challenge in pancreatic cancer. The epidermal growth factor receptor (EGFR) and human epidermal growth factor receptors 2 and 3 (HER2, HER3) are expressed in many tumors, and they are relevant therapeutic targets due to their synergistic interaction to promote tumor aggressiveness and therapeutic resistance. Cocktails of antibodies directed against different targets are a promising strategy to overcome these processes.

Biasing human epidermal growth factor receptor 4 (HER4) tyrosine kinase signaling with antibodies: Induction of cell death by antibody-dependent HER4 intracellular domain trafficking.

Human epidermal growth factor receptor 4 (HER4) isoforms have oncogenic or tumor suppressor functions depending on their susceptibility to proteolytic cleavage and HER4 intracellular domain (4ICD) translocation. Here, we report that the neuregulin 1 (NRG1) tumor suppressor mechanism through the HER4 JMa/CYT1 isoform can be mimicked by the agonist anti-HER4 Ab C6. Neuregulin 1 induced cleavage of poly(ADP-ribose) polymerase (PARP) and sub-G DNA fragmentation, and also reduced the metabolic activity of HER3 /HER4 cervical (C-33A) and ovarian (COV318) cancer cells.

Higher Anti-Tumor Efficacy of the Dual HER3-EGFR Antibody MEHD7945a Combined with Ionizing Irradiation in Cervical Cancer Cells.

Purpose: The outcome of locally advanced cervical cancer (LACC) is dismal. Biomarkers are needed to individualize treatments and to improve patient outcomes. Here, we investigated whether coexpression of epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor 3 (HER3) could be an outcome prognostic biomarker, and whether targeting both EGFR and HER3 with a dual antibody (MEHD7945A) enhanced ionizing radiation (IR) efficacy.

[Mimicking polyclonal immune response in therapy: from combination of two monoclonal antibodies to oligoclonal antibody-based mixtures].

Monoclonal antibodies have revolutionized the treatment of many diseases, but their clinical effectiveness remains limited in some cases. Associations of antibodies binding to the same target (homo-combination) or to several different targets (hetero-combination), thereby mimicking a polyclonal humoral immune response, have demonstrated a therapeutic improvement in pre-clinical and clinical trials, mainly in the field of oncology and infectious diseases. The combinations increase the efficacy of the biological responses and override resistance mechanisms observed with antibody monotherapy.

ITCH-dependent proteasomal degradation of c-FLIP induced by the anti-HER3 antibody 9F7-F11 promotes DR5/caspase 8-mediated apoptosis of tumor cells.

Background: HER3/ErbB3 receptor deletion or blockade leads to tumor cell apoptosis, whereas its overexpression confers anti-cancer drug resistance through upregulation of protective mechanisms against apoptosis. We produced the anti-HER3 antibody 9F7-F11 that promotes HER3 ubiquitination and degradation via JNK1/2-dependent activation of the E3 ubiquitin ligase ITCH, and that induces apoptosis of cancer cells. Cellular FLICE-like inhibitory protein (c-FLIP) is a key regulator of apoptotic pathways.

An auristatin-based antibody-drug conjugate targeting HER3 enhances the radiation response in pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive cancer characterized by poor response to chemotherapy and radiotherapy due to the lack of efficient therapeutic tools and early diagnostic markers. We previously generated the nonligand competing anti-HER3 antibody 9F7-F11 that binds to pancreatic tumor cells and induces tumor regression in vivo in experimental models. Here, we asked whether coupling 9F7-F11 with a radiosensitizer, such as monomethylauristatin E (MMAE), by using the antibody-drug conjugate (ADC) technology could improve radiation therapy efficacy in PDAC.

Rational development of synergistic combinations of chemotherapy and molecular targeted agents for colorectal cancer treatment.

Background: The irinotecan-induced phosphokinome changes in colorectal cancer (CRC) cells were used to guide the selection of targeted agents to be tested in combination with irinotecan.

Methods: Phosphokinome profiling with peptide arrays of tumour samples from nude mice xenografted with HT29 cells and treated or not with an effective dose of irinotecan was used to identify signalling pathways activated by irinotecan treatment. Then, drugs targeting these pathways were combined in vitro with irinotecan to test potential synergistic effect.

Targeting the NRG1/HER3 pathway in tumor cells and cancer-associated fibroblasts with an anti-neuregulin 1 antibody inhibits tumor growth in pre-clinical models of pancreatic cancer.

Neuregulin 1 (NRG1), a ligand for HER3 and HER4 receptors, is secreted by both pancreatic tumor cells (PC) and cancer-associated fibroblasts (CAFs), the latter representing the most abundant compound of pancreatic stroma. This desmoplastic stroma contributes to Pancreatic Ductal Adenocarcinoma (PDAC) aggressiveness and therapeutic failure by promoting tumor progression, invasion and resistance to chemotherapies. In the present work, we aimed at disrupting the complex crosstalk between PC and CAF in order to prevent tumor cell proliferation.

Impact of cathepsin B-sensitive triggers and hydrophilic linkers on in vitro efficacy of novel site-specific antibody-drug conjugates.

Herein we describe the synthesis and evaluation of four novel HER2-targeting, cathepsin B-sensitive antibody-drug conjugates bearing a monomethylauristatin E (MMAE) cytotoxic payload, constructed via the conjugation of cleavable linkers to trastuzumab using a site-specific bioconjugation methodology. These linkers vary by both cleavable trigger motif and hydrophilicity, containing one of two cathepsin B sensitive dipeptides (Val-Cit and Val-Ala), and engendered with either hydrophilic or hydrophobic character via application of a PEG spacer. Through evaluation of physical properties, in vitro cytotoxicity, and receptor affinity of the resulting antibody-drug conjugates (ADCs), we have demonstrated that while both dipeptide triggers are effective, the increased hydrophobicity of the Val-Ala pair limits its utility within this type of linker.

Neuregulin 1 Allosterically Enhances the Antitumor Effects of the Noncompeting Anti-HER3 Antibody 9F7-F11 by Increasing Its Binding to HER3.

Exploratory clinical trials using therapeutic anti-HER3 antibodies strongly suggest that neuregulin (NRG1; HER3 ligand) expression at tumor sites is a predictive biomarker of anti-HER3 antibody efficacy in cancer. We hypothesized that in NRG1-expressing tumors, where the ligand is present before antibody treatment, anti-HER3 antibodies that do not compete with NRG1 for receptor binding have a higher receptor-neutralizing action than antibodies competing with the ligand for binding to HER3. Using time-resolved-fluorescence energy transfer (TR-FRET), we demonstrated that in the presence of recombinant NRG1, binding of 9F7-F11 (a nonligand-competing anti-HER3 antibody) to HER3 is increased, whereas that of ligand-competing anti-HER3 antibodies (H4B-121, U3-1287, Ab#6, Mab205.

Therapeutic Activity of Anti-AXL Antibody against Triple-Negative Breast Cancer Patient-Derived Xenografts and Metastasis.

AXL receptor tyrosine kinase has been described as a relevant molecular marker and a key player in invasiveness, especially in triple-negative breast cancer (TNBC). We evaluate the antitumor efficacy of the anti-AXL monoclonal antibody 20G7-D9 in several TNBC cell xenografts or patient-derived xenograft (PDX) models and decipher the underlying mechanisms. In a dataset of 254 basal-like breast cancer samples, genes correlated with expression are enriched in EMT, migration, and invasion signaling pathways.

FOXO3a and the MAPK p38 are activated by cetuximab to induce cell death and inhibit cell proliferation and their expression predicts cetuximab efficacy in colorectal cancer.

Background: Cetuximab, a monoclonal antibody against EGFR used for the treatment of colorectal cancer (CRC), is ineffective in many patients. The aim of this study was to identify the signalling pathways activated by cetuximab in CRC cells and define new biomarker of response.

Methods: We used in vitro, in vivo models and clinical CRC samples to assess the role of p38 and FOXO3a in cetuximab mechanism of action.

The anti-HER3 (ErbB3) therapeutic antibody 9F7-F11 induces HER3 ubiquitination and degradation in tumors through JNK1/2- dependent ITCH/AIP4 activation.

We characterized the mechanism of action of the neuregulin-non-competitive anti-HER3 therapeutic antibody 9F7-F11 that blocks the PI3K/AKT pathway, leading to cell cycle arrest and apoptosis in vitro and regression of pancreatic and breast cancer in vivo. We found that 9F7-F11 induces rapid HER3 down-regulation. Specifically, 9F7-F11-induced HER3 ubiquitination and degradation in pancreatic, breast and prostate cancer cell lines was driven mainly by the itchy E3 ubiquitin ligase (ITCH/AIP4).

Dual targeting of HER1/EGFR and HER2 with cetuximab and trastuzumab in patients with metastatic pancreatic cancer after gemcitabine failure: results of the "THERAPY"phase 1-2 trial.

To improve treatment efficacy, we decided to simultaneously target HER1 and HER2 with trastuzumab and cetuximab. Following promising preclinical results, we conducted a phase 1-2 trial in advanced pancreatic cancer patients after first-line gemcitabine-based chemotherapy failure. In this single-arm, non-randomized, multicenter trial, patients received weekly cetuximab (400mg/m², then 250mg/m²).

Examination of HER3 targeting in cancer using monoclonal antibodies.

The human EGF receptor (HER/EGFR) family of receptor tyrosine kinases serves as a key target for cancer therapy. Specifically, EGFR and HER2 have been repeatedly targeted because of their genetic aberrations in tumors. The therapeutic potential of targeting HER3 has long been underestimated, due to relatively low expression in tumors and impaired kinase activity.

HER3 as biomarker and therapeutic target in pancreatic cancer: new insights in pertuzumab therapy in preclinical models.

The anti-HER2 antibody pertuzumab inhibits HER2 dimerization and affects HER2/HER3 dimer formation and signaling. As HER3 and its ligand neuregulin are implicated in pancreatic tumorigenesis, we investigated whether HER3 expression could be a predictive biomarker of pertuzumab efficacy in HER2low-expressing pancreatic cancer. We correlated in vitro and in vivo HER3 expression and neuregulin dependency with the inhibitory effect of pertuzumab on cell viability and tumor progression.

Preclinical validation of AXL receptor as a target for antibody-based pancreatic cancer immunotherapy.

AXL receptor tyrosine kinase (RTK) is implicated in proliferation and invasion of many cancers, particularly in pancreatic ductal adenocarcinoma (PDAC), for which new therapeutic options are urgently required. We investigated whether inhibition of AXL activity by specific monoclonal antibodies (mAbs) is efficient in limiting proliferation and migration of pancreatic cancer cells. Expression of AXL was evaluated by immunohistochemistry in 42 PDAC.

Anti-HER3 domain 1 and 3 antibodies reduce tumor growth by hindering HER2/HER3 dimerization and AKT-induced MDM2, XIAP, and FoxO1 phosphorylation.

Blockade of the human epidermal growth factor receptor 3 (HER3) and of the downstream phosphatidylinositide 3-kinase (PI3K)/AKT pathway is a prerequisite for overcoming drug resistance and to develop novel treatments for cancers that are not eligible for the currently approved targeted therapies. To this end, we generated specific antibodies (Abs) against domain 1 (D1) and domain 3 (D3) of HER3 that recognize epitopes that do not overlap with the neuregulin-binding site. The fully human H4B-121 Ab and the mouse monoclonal Abs 16D3-C1 and 9F7-F11 inhibited tumor growth in nude mice xenografted with epidermoid, pancreatic, or triple-negative breast cancer cells.

Quantification of HER expression and dimerization in patients' tumor samples using time-resolved Förster resonance energy transfer.

Following the development of targeted therapies against EGFR and HER2, two members of the human epidermal receptor (HER) family of receptor tyrosine kinases, much interest has been focused on their expression in tumors. However, knowing the expression levels of individual receptors may not be sufficient to predict drug response. Here, we describe the development of antibody-based time-resolved Förster resonance energy transfer (TR-FRET) assays for the comprehensive analysis not only of EGFR and HER2 expression in tumor cryosections, but also of their activation through quantification of HER homo- or heterodimers.