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. We found more than 300 phase II/III clinical trials on antibody combinations, with/without chemotherapy, radiotherapy, small molecules or vaccines, in the ClinicalTrials.gov database. Such combinations enhance the biological responses and bypass the resistance mechanisms observed with antibody monotherapy. Usually, such antibody combinations are administered sequentially as separate formulations. Combined formulations have also been developed in which separately produced antibodies are mixed before administration or are produced simultaneously in a single cell line or a single batch of different cell lines as a polyclonal master cell bank. The regulation, toxicity and injection sequence of these oligoclonal antibody mixtures still need to be addressed in order to optimize their delivery and their therapeutic effects.
We investigated whether radiation-induced nontargeted effects are involved in the cytotoxic effects of anticell surface monoclonal antibodies labeled with Auger electron emitters, such as iodine 125 ...(monoclonal antibodies labeled with (125)I (125)I-mAbs).
We showed that the cytotoxicity of (125)I-mAbs targeting the cell membrane of p53(+/+) HCT116 colon cancer cells is mainly due to nontargeted effects. Targeted and nontargeted cytotoxicities were inhibited in vitro following lipid raft disruption with Methyl-β-cyclodextrin (MBCD) or filipin or use of radical oxygen species scavengers. (125)I-mAb efficacy was associated with acid sphingomyelinase activation and modulated through activation of the AKT, extracellular signal-related kinase ½ (ERK1/2), p38 kinase, c-Jun N-terminal kinase (JNK) signaling pathways, and also of phospholipase C-γ (PLC-γ), proline-rich tyrosine kinase 2 (PYK-2), and paxillin, involved in Ca(2+) fluxes. Moreover, the nontargeted response induced by directing 5-(125)Iiodo-2'-deoxyuridine to the nucleus was comparable to that of (125)I-mAb against cell surface receptors. In vivo, we found that the statistical significance of tumor growth delay induced by (125)I-mAb was removed after MBCD treatment and observed oxidative DNA damage beyond the expected Auger electron range. These results suggest the involvement of nontargeted effects in vivo also.
Low-energy Auger electrons, such as those emitted by (125)I, have a short tissue range and are usually targeted to the nucleus to maximize their cytotoxicity. In this study, we show that targeting the cancer cell surface with (125)I-mAbs produces a lipid raft-mediated nontargeted response that compensates for the inferior efficacy of non-nuclear targeting.
Our findings describe the mechanisms involved in the efficacy of (125)I-mAbs targeting the cancer cell surface. Antioxid. Redox Signal. 25, 467-484.
In ovarian carcinoma, anti-Müllerian hormone (AMH) type II receptor (AMHRII) and the AMH/AMHRII signaling pathway are potential therapeutic targets. Here, AMH dose-dependent effect on signaling and ...proliferation was analyzed in four ovarian cancer cell lines, including sex cord stromal/granulosa cell tumors and high grade serous adenocarcinomas (COV434-AMHRII, SKOV3-AMHRII, OVCAR8 and KGN). As previously shown, incubation with exogenous AMH at concentrations above the physiological range (12.5-25 nM) decreased cell viability. Conversely, physiological concentrations of endogenous AMH improved cancer cell viability. Partial AMH depletion by siRNAs was sufficient to reduce cell viability in all four cell lines, by 20% (OVCAR8 cells) to 40% (COV434-AMHRII cells). In the presence of AMH concentrations within the physiological range (5 to 15 pM), the newly developed anti-AMH B10 antibody decreased by 25% (OVCAR8) to 50% (KGN) cell viability at concentrations ranging between 3 and 333 nM. At 70 nM, B10 reduced clonogenic survival by 57.5%, 57.1%, 64.7% and 37.5% in COV434-AMHRII, SKOV3-AMHRII, OVCAR8 and KGN cells, respectively. In the four cell lines, B10 reduced AKT phosphorylation, and increased PARP and caspase 3 cleavage. These results were confirmed in ovarian cancer cells isolated from patients' ascites, demonstrating the translational potential of these results. Furthermore, B10 reduced COV434-MISRII tumor growth in vivo and significantly enhanced the median survival time compared with vehicle (69 vs 60 days; p = 0.0173). Our data provide evidence for a novel pro-survival autocrine role of AMH in the context of ovarian cancer, which was targeted therapeutically using an anti-AMH antibody to successfully repress tumor growth.
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. We found that the MMAE‐based HER3 antibody‐drug conjugate (HER3‐ADC) was efficiently internalized in tumor cells, increased the fraction of cells arrested in G2/M, which is the most radiosensitive phase of the cell cycle, and promoted programmed cell death of irradiated HER3‐positive pancreatic cancer cells (BxPC3 and HPAC cell lines). HER3‐ADC decreased the clonogenic survival of irradiated cells by increasing DNA double‐strand break formation (based on γH2AX level), and by modulating DNA damage repair. Tumor radiosensitization with HER3‐ADC favored the inhibition of the AKT‐induced survival pathway, together with more efficient caspase 3/PARP‐mediated apoptosis. Incubation with HER3‐ADC before irradiation synergistically reduced the phosphorylation of STAT3, which is involved in chemoradiation resistance. In vivo, the combination of HER3‐ADC with radiation therapy increased the overall survival of mice harboring BxPC3, HPAC cell xenografts or patient‐derived xenografts, and reduced proliferation (KI67‐positive cells). Combining auristatin radiosensitizer delivery via an HER3‐ADC with radiotherapy is a new promising therapeutic strategy in PDAC.
What's new?
In pancreatic ductal adenocarcinoma (PDAC), chemoradiation is prescribed to patients with borderline resectable lesions to make surgery possible. The HER3 receptor is a key signaling hub in PDAC. Here, the authors developed a novel antibody‐drug conjugate targeting HER3 (HER3‐ADC) that enhanced radiosensitivity of PDAC by arresting cells in G2/M. HER3‐ADC increased radiation response in mice xenografted with PDAC cells, through inhibition of cell survival and induction of DNA break formation and apoptosis. Combining auristatin radiosensitizer delivery via an HER3‐ADC with radiotherapy could help increase the rate of resection for patients with borderline resectable pancreatic cancer.
Triple-negative breast cancer (TNBC) has a worse prognosis compared with other breast cancer subtypes, and biomarkers to identify patients at high risk of recurrence are needed. Here, we investigated ...the expression of human epidermal receptor (HER) family members in TNBC and evaluated their potential as biomarkers of recurrence.
We developed Time Resolved-Förster Resonance Energy Transfer (TR-FRET) assays to quantify HER1, HER2 and HER3 in formalin-fixed paraffin-embedded (FFPE) tumour tissues. After assessing the performance and precision of our assays, we quantified HER protein expression in 51 TNBC specimens, and investigated the association of their expression with relapse-free survival.
The assays were quantitative, accurate, and robust. In TNBC specimens, HER1 levels ranged from ≈4000 to more than 2 million receptors per cell, whereas HER2 levels varied from ≈1000 to 60,000 receptors per cell. HER3 expression was very low (less than 5500 receptors per cell in all samples). Moderate HER2 expression was significantly associated with higher risk of recurrence (HR = 3.93; P = 0.003).
Our TR-FRET assays accurately quantify HER1, HER2 and HER3 in FFPE breast tumour specimens. Moderate HER2 expression may represent a novel prognostic marker in patients with TNBC.
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‐G1 DNA fragmentation, and also reduced the metabolic activity of HER3−/HER4+ cervical (C‐33A) and ovarian (COV318) cancer cells. This effect was confirmed in HER4 JMa/CYT1‐, but not JMa/CYT2‐transfected BT549 triple‐negative breast cancer cells. Neuregulin 1 favored 4ICD cleavage and retention in mitochondria in JMa/CYT1‐transfected BT549 cells, leading to reactive oxygen species (ROS) production through mitochondrial depolarization. Similarly, the anti‐HER4 Ab C6, which binds to a conformational epitope located on a.a. 575‐592 and 605‐620 of HER4 domain IV, induced 4ICD cleavage and retention in mitochondria, and mimicked NRG1‐mediated effects on PARP cleavage, ROS production, and mitochondrial membrane depolarization in cancer cells. In vivo, C6 reduced growth of COV434 and HCC1187 tumor cell xenografts in nude mice. Biasing 4ICD trafficking to mitochondria with anti‐HER4 Abs to mimic NRG1 suppressor functions could be an alternative anticancer strategy.
Neuregulin 1 (NRG1) induced cleavage of poly(ADP‐ribose) polymerase (PARP) in human epidermal growth factor receptor 4 (HER4) JMa/CYT1‐expressing cancer cells. NRG1 favored HER4 intracellular domain (4ICD) cleavage and retention into mitochondria leading to reactive oxygen species (ROS) production through mitochondrial depolarization in HER4 JMa/CYT1‐expressing cancer cells. Phage‐displayed selected anti‐HER4 Ab C6 induced 4ICD cleavage and retention into mitochondria, mimicking NRG1‐mediated effects on PARP cleavage, ROS production, and mitochondrial membrane depolarization. C6 Ab reduced in vivo growth of ovarian COV434 and breast HCC1187 tumor cell xenografts in nude mice.
Significance The human EGF receptor (EGFR/HER) family plays critical roles in tumor progression. Therefore, several therapies intercepting these receptors were developed and clinically approved. ...Importantly, patients treated with such therapeutics often develop resistance, and in some cases this resistance has been associated with activation of HER3. Potentially, HER3 blockade might overcome patient resistance. Hence, antibodies to HER3 have been developed by us and other researchers. However, it has remained unclear which antibody attributes are required for effective tumor inhibition. To address this issue, we generated several monoclonal antibodies, which were tested in vitro and in tumor-bearing animals. Our results suggest that anti-HER3 antibodies able to intercept stroma–tumor interactions, as well as accelerate HER3 degradation, might inhibit tumor growth better than other 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. Nevertheless, in addition to serving as a dimerization partner of EGFR and HER2, HER3 acts as a key player in tumor cells’ ability to acquire resistance to cancer drugs. In this study, we generated several monoclonal antibodies to HER3. Comparisons of their ability to degrade HER3, decrease downstream signaling, and inhibit growth of cultured cells, as well as recruit immune effector cells, selected an antibody that later emerged as the most potent inhibitor of pancreatic cancer cells grown as tumors in animals. Our data predict that anti-HER3 antibodies able to intercept autocrine and stroma–tumor interactions might strongly inhibit tumor growth, in analogy to the mechanism of action of anti-EGFR antibodies routinely used now to treat colorectal cancer patients.
Triple-negative breast cancer (TNBC) has poorer outcomes than other breast cancers (BC), including HER2
BC. Cathepsin D (CathD) is a poor prognosis marker overproduced by BC cells, hypersecreted in ...the tumour microenvironment with tumour-promoting activity. Here, we characterized the immunomodulatory activity of the anti-CathD antibody F1 and its improved Fab-aglycosylated version (F1M1) in immunocompetent mouse models of TNBC (C57BL/6 mice harbouring E0771 cell grafts) and HER2-amplified BC (BALB/c mice harbouring TUBO cell grafts).
CathD expression was evaluated by western blotting and immunofluorescence, and antibody binding to CathD by ELISA. Antibody anti-tumour efficacy was investigated in mouse models. Immune cell recruitment and activation were assessed by immunohistochemistry, immunophenotyping, and RT-qPCR.
F1 and F1M1 antibodies remodelled the tumour immune landscape. Both antibodies promoted innate antitumour immunity by preventing the recruitment of immunosuppressive M2-polarized tumour-associated macrophages (TAMs) and by activating natural killer cells in the tumour microenvironment of both models. This translated into a reduction of T-cell exhaustion markers in the tumour microenvironment that could be locally supported by enhanced activation of anti-tumour antigen-presenting cell (M1-polarized TAMs and cDC1 cells) functions. Both antibodies inhibited tumour growth in the highly-immunogenic E0771 model, but only marginally in the immune-excluded TUBO model, indicating that anti-CathD immunotherapy is more relevant for BC with a high immune cell infiltrate, as often observed in TNBC.
Anti-CathD antibody-based therapy triggers the anti-tumour innate and adaptive immunity in preclinical models of BC and is a promising immunotherapy for immunogenic TNBC.
Triple‐negative breast cancer (TNBC) is the most aggressive breast cancer subtype and lacks specific targeted therapeutic agents. The current mechanistic evidence from cell‐based studies suggests ...that the matricellular protein SPARC has a tumor‐promoting role in TNBC; however, data on the clinical relevance of SPARC expression/secretion by tumor and stromal cells in TNBC are limited. Here, we analyzed by immunohistochemistry the prognostic value of tumor and stromal cell SPARC expression in 148 patients with non‐metastatic TNBC and long follow‐up (median: 5.4 years). We also quantified PD‐L1 and PD‐1 expression. We detected SPARC expression in tumor cells (42.4%), cancer‐associated fibroblasts (CAFs; 88.1%), tumor‐associated macrophages (77.1%), endothelial cells (75.2%) and tumor‐infiltrating lymphocytes (9.8%). Recurrence‐free survival was significantly lower in patients with SPARC‐expressing CAFs. Multivariate analysis showed that SPARC expression in CAFs was an independent prognostic factor. We also detected tumor and stromal cell SPARC expression in TNBC cytosols, and in patient‐derived xenografts and cell lines. Furthermore, we analyzed publicly available single‐cell mRNA sequencing data and found that in TNBC, SPARC is expressed by different CAF subpopulations, including myofibroblasts and inflammatory fibroblasts that are involved in tumor‐related processes. We then showed that fibroblast‐secreted SPARC had a tumor‐promoting role by inhibiting TNBC cell adhesion and stimulating their motility and invasiveness. Overall, our study demonstrates that SPARC expression in CAFs is an independent prognostic marker of poor outcome in TNBC. Patients with SPARC‐expressing CAFs could be eligible for anti‐SPARC targeted therapy.
What's new?
In vitro evidence suggests that the matricellular protein SPARC has a tumor‐promoting role in triple‐negative breast cancer (TNBC). However, the clinical relevance of SPARC in triple‐negative breast cancer remains unclear. Here, the authors analyzed the prognostic value of tumor and stromal SPARC in 148 patients with non‐metastatic TNBC. SPARC was most often expressed by myofibroblasts and inflammatory cancer‐associated fibroblasts (CAFs), and fibroblast‐secreted SPARC exhibited a tumor‐promoting role in TNBC. Moreover, SPARC expression in CAFs was an independent prognostic marker of poor outcome. This study points to CAF‐derived SPARC as a potential novel therapeutic target in triple‐negative breast 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. To do so, we demonstrated the promising tumor growth inhibitory effect of the 7E3, an original antibody directed to NRG1. This antibody promotes antibody dependent cellular cytotoxicity in NRG1-positive PC and CAFs and inhibits NRG1-associated signaling pathway induction, by blocking NRG1-mediated HER3 activation. Moreover, 7E3 inhibits migration and growth of pancreatic cancer cells co-cultured with CAFs, both in vitro and in vivo using orthotopic pancreatic tumor xenografts. Our preclinical results demonstrate that the anti-NRG1 antibody 7E3 could represent a promising approach to target pancreatic stroma and cancer cells, thereby providing novel therapeutic options for PDAC.
•NRG1 is expressed in human pancreatic tumors and cancer-associated fibroblasts.•NRG1 promotes pancreatic cell growth.•NRG1 trapping by 7E3 antibody inhibits HER3 activation and downstream signaling.•Targeting NRG1 inhibits growth of orthotopic pancreatic tumor xenografts.
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