scholarly journals Anti-tumor efficacy of an MMAE conjugated antibody targeting cell surface TACE/ADAM17-cleaved Amphiregulin in breast cancer

2021 ◽  
Author(s):  
Kristopher A Lofgren ◽  
Sreeja Sreekumar ◽  
E Charles Jenkins ◽  
Kyle J Ernzen ◽  
Paraic A Kenny
Keyword(s):  
Breast Cancer ◽  
Cell Surface ◽  
Transmembrane Protein ◽  
Growth Factor Receptor ◽  
Antibody Drug Conjugate ◽  
Estrogen Receptor Signaling ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded ◽  
Immunocompromised Mice

The Epidermal Growth Factor Receptor ligand, Amphiregulin, is a key proliferative effector of estrogen receptor signaling in breast cancer and also plays a role in other malignancies. Amphiregulin is a single-pass transmembrane protein proteolytically processed by TACE/ADAM17 to release the soluble EGFR ligand, leaving a residual transmembrane stalk that is subsequently internalized. Here, we report the development of an antibody drug conjugate, GMF-1A3-MMAE, targeting an AREG neo-epitope revealed following ADAM17-mediated cleavage. The antibody does not interact with uncleaved Amphiregulin, providing a novel means of targeting cells with high rates of Amphiregulin shedding. Using fluorescent dye conjugation, we demonstrated that the antibody is internalized by cancer cells in a manner dependent on the presence of cell surface cleaved Amphiregulin. Antibodies conjugated with monomethyl auristatin E (MMAE) were cytotoxic in vitro and induced rapid regression of established breast tumor xenografts in immunocompromised mice. We further demonstrate that these antibodies recognize the Amphiregulin neo-epitope in formalin fixed paraffin embedded tumor tissue, suggesting their utility as a companion diagnostic for patient selection.

scholarly journals Anti-tumor efficacy of an MMAE conjugated antibody targeting cell surface TACE/ADAM17-cleaved Amphiregulin in breast cancer

2021 ◽  
Author(s):  
Kristopher A Lofgren ◽  
Sreeja Sreekumar ◽  
E Charles Jenkins Jr ◽  
Kyle J Ernzen ◽  
Paraic A Kenny
Keyword(s):  
Breast Cancer ◽  
Cell Surface ◽  
Transmembrane Protein ◽  
Growth Factor Receptor ◽  
Antibody Drug Conjugate ◽  
Estrogen Receptor Signaling ◽  
Frequent Event ◽  
Formalin Fixed Paraffin Embedded ◽  
Immunocompromised Mice

Abstract Background The Epidermal Growth Factor Receptor ligand, Amphiregulin, is a key proliferative effector of estrogen receptor signaling in breast cancer and also plays a role in other malignancies. Amphiregulin is a single-pass transmembrane protein proteolytically processed by TACE/ADAM17 to release the soluble EGFR ligand, leaving a residual transmembrane stalk that is subsequently internalized. Methods Using phage display we identified antibodies that selectively recognize the residual transmembrane stalk of cleaved Amphiregulin. Conjugation with fluorescence labels and monomethyl auristatin E (MMAE) was used to study their intracellular trafficking and anti-cancer effects, respectively. Results We report the development of an antibody drug conjugate, GMF-1A3-MMAE, targeting an AREG neo-epitope revealed following ADAM17-mediated cleavage. The antibody does not interact with uncleaved Amphiregulin, providing a novel means of targeting cells with high rates of Amphiregulin shedding. Using fluorescent dye conjugation, we demonstrated that the antibody is internalized by cancer cells in a manner dependent on the presence of cell surface cleaved Amphiregulin. Antibodies conjugated with MMAE were cytotoxic in vitro and induced rapid regression of established breast tumor xenografts in immunocompromised mice. We further demonstrate that these antibodies recognize the Amphiregulin neo-epitope in formalin fixed paraffin embedded tumor tissue, suggesting their utility as a companion diagnostic for patient selection. Conclusions This ADC targeting Amphiregulin has potential utility in the treatment of breast and other tumors in which proteolytic Amphiregulin shedding is a frequent event.

scholarly journals HER2 antibody-drug conjugate controls growth of breast cancer brain metastases in hematogenous xenograft models, with heterogeneous blood–tumor barrier penetration unlinked to a passive marker

2020 ◽  
Vol 22 (11) ◽  
pp. 1625-1636 ◽  
Author(s):  
Brunilde Gril ◽  
Debbie Wei ◽  
Alexandra S Zimmer ◽  
Christina Robinson ◽  
Imran Khan ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Brain Metastasis ◽  
Brain Metastases ◽  
Statistical Significance ◽  
Growth Factor Receptor ◽  
Antibody Drug Conjugate ◽  
Brain Penetration ◽  
Her2 Breast Cancer ◽  
Antibody Drug

Abstract Background Brain metastases of HER2+ breast cancer persist as a clinical challenge. Many therapeutics directed at human epidermal growth factor receptor 2 (HER2) are antibodies or antibody-drug conjugates (ADCs), and their permeability through the blood–tumor barrier (BTB) is poorly understood. We investigated the efficacy of a biparatopic anti-HER2 antibody-tubulysin conjugate (bHER2-ATC) in preclinical models of brain metastases. Methods The compound was evaluated in 2 hematogenous HER2+ brain metastasis mouse models, SUM190-BR and JIMT-1-BR. Endpoints included metastasis count, compound brain penetration, cancer cell proliferation, and apoptosis. Results Biparatopic HER2-ATC 3 mg/kg prevented metastasis outgrowth in the JIMT-1-BR model. At 1 mg/kg bHER2-ATC, a 70% and 92% reduction in large and micrometastases was observed. For the SUM190-BR model, an 85% and 53% reduction, respectively, in large and micrometastases was observed at 3 mg/kg, without statistical significance. Proliferation was reduced in both models at the highest dose. At the endpoint, bHER2-ATC uptake covered a median of 4–6% and 7–17% of metastasis area in the JIMT-1-BR and SUM190-BR models, respectively. Maximal compound uptake in the models was 19% and 86% in JIMT-1-BR and SUM190-BR, respectively. Multiple lesions in both models demonstrated ADC uptake in the absence or low diffusion of Texas Red Dextran, a marker of paracellular permeability. Using in vitro BTB assays, the ADC was endocytosed into brain endothelial cells, identifying a potentially new mechanism of antibody permeability. Conclusions Biparatopic HER2-ATC significantly prevented JIMT-1-BR brain metastasis outgrowth and showed activity in the SUM190-BR model. The bHER2-ATC penetration into metastases that are impermeable to fluorescent dye suggested an endocytic mechanism of brain penetration.

Fibroblast growth factor receptor 1 (FGFR1) and SRY-related HMG-box (SOX2) amplification in squamous cell carcinoma (SCC) of the lung.

2013 ◽  
Vol 31 (15_suppl) ◽  
pp. 11025-11025
Author(s):  
Amaya Gasco ◽  
Pedro Mendez ◽  
Jose Luis Ramirez ◽  
Santiago Viteri Ramirez ◽  
Teresa Moran ◽  
...  
Keyword(s):  
Gene Copy Number ◽  
Growth Factor Receptor ◽  
Gene Copy ◽  
Blood Leukocytes ◽  
Formalin Fixed Paraffin ◽  
Ffpe Tumor ◽  
Formalin Fixed Paraffin Embedded ◽  
Tumor Biopsies

11025 Background: SOX2 is a key transcription factor that is amplified in lung SCC. FGFR1 is a receptor tyrosine kinase that promotes cell proliferation, survival and apoptotic resistance through the PLCγ/PKC, RAS/MAPK and PI3K-AKT pathways, respectively. FGFR1 is amplified in 15-25% of lung SCC. Pre-clinical studies of targeted inhibitors showed a growth dependency on FGFR1 amplification both in vitro and in vivo. A European, multicenter clinical trial of second-line treatment with BIBF1120, an FGFR1 inhibitor, will be performed in p with lung SCC with FGFR1 amplification. Methods: We have examined FGFR1 and SOX2 gene copy number (GCN) in 76 lung SCC p by multiplex ligation-dependent probe amplification (MLPA). Genomic DNA (gDNA) was isolated from enriched tumor cells by laser capture microdissection from formalin-fixed paraffin embedded (FFPE) tumor tissue. 50-100 ng of gDNA was analyzed in each of three independent replicates per tumor sample. Two independent probe sets were used for each gene analyzed. For inter-patient GCN comparisons, the results from each patient were normalized against the GCN values derived from FFPE peripheral blood leukocytes. In order to study intra-tumor heterogeneity (TH), we examined FGFR1 and SOX2 GCN in different areas of 4 tumors. In 2 p, TH was examined in serial tumor biopsies and/or resections obtained at different points of disease progression. Results: High FGFR1 amplification was detected in 13/76 (17.10%) and low amplification in 7/76 (9.21%) p. High SOX2 amplification was observed in 38/63 (60.32%) and low amplification in 9/63 (14.28%) p. 46.15% of the FGFR1-amplified tumors were also co-amplified for SOX2. Intra-TH was observed in 2/4 tumors. Survival according to FGFR1 and SOX2 GCN will be presented. In addition, GCN changes in FGFR1, SOX2, PIK3CA, PDGFRA, KDR, EGFR and MET over 10 years of follow-up will be presented for one surgically resected SCC lung p. Conclusions: FGFR1 and SOX2 co-amplification could represent a novel therapeutic target and warrants further research.

scholarly journals 3D Visualization of the Dynamic Bidirectional Talk Between ER/PR and Her2 Pathways

2021 ◽  
Vol 20 ◽  
pp. 153303382110656
Author(s):  
Jiahong Lyv ◽  
Guohua Yu ◽  
Yunyun Zhang ◽  
Yan Lyv ◽  
Wenfeng Zhang ◽  
...  
Keyword(s):  
Breast Cancer ◽  
3D Visualization ◽  
Growth Factor Receptor ◽  
Human Tumor ◽  
Clinical Intervention ◽  
Breast Cancer Patients ◽  
Dot Blot ◽  
Protein Levels ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded

Background: Extensive amounts of archived formalin fixed paraffin embedded (FFPE) human tumor tissues are the ultimate resource to investigate signaling network underlying tumorigenesis in human. Yet, their usage is severely limited for lacking of suitable protein techniques. In this study, a quantitative, objective, absolute, and high throughput immunoblot method, quantitative dot blot (QDB), was explored to address this issue by investigating the putative relationship between estrogen receptor (ER)/progesterone receptor (PR) and human epidermal growth factor receptor 2 (Her2) pathways in breast cancer tumorigenesis. Methods: In this descriptive observational retrospective study, ER, PR, Her2, and Ki67 protein levels were measured absolutely and quantitatively in 852 FFPE breast cancer tissues using the QDB method. ER, PR, and Her2 levels were charted on the X, Y, and Z-axes to observe samples distribution in a 3D scatterplot. Results: A “seesaw” relationship between ER/PR and Her2 pathways was observed in ER–PR–Her2 space, characterized by the expression levels of these 3 proteins. Specimens with strong expressions of ER/PR proteins were found spreading along the ER/PR floor while those with strong Her2 expression were found wrapping around the Her2 axis. Those lacking strong expressions of all 3 proteins were found accumulating at the intersection of the ER, PR, and Her2 axes. Few specimens floated in the ER–PR–Her2 space to suggest the lack of co-expression of all 3 proteins simultaneously. Ki67 levels were found to be significantly reduced in specimens spreading in the ER–PR space. Conclusions: The unique distribution of specimens in ER–PR–Her2 space prior to any clinical intervention provided visual support of bidirectional talk between ER/PR and Her2 pathways in breast cancer specimens. Clinical interventions to suppress these 2 pathways alternatively warrant further exploration for breast cancer patients accordingly. Our study also demonstrated that the QDB method is an effective tool to analyze archived FFPE cancer specimens in biomedical research.

scholarly journals Overexpression of circulating MiR-30b-5p identifies advanced breast cancer

2019 ◽  
Vol 17 (1) ◽  
Author(s):  
Helena Estevão-Pereira ◽  
João Lobo ◽  
Sofia Salta ◽  
Maria Amorim ◽  
Paula Lopes ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Validation Cohort ◽  
Advanced Disease ◽  
Clinical Tool ◽  
Liquid Biopsies ◽  
Primary Tumors ◽  
Bodily Fluids ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded ◽  
Formalin Fixed

Abstract Background Breast cancer (BrC) remains the leading cause of cancer-related death in women, mainly due to recurrent and/or metastatic events, entailing the need for biomarkers predictive of progression to advanced disease. MicroRNAs hold promise as noninvasive cancer biomarkers due to their inherent stability and resilience in tissues and bodily fluids. There is increasing evidence that specific microRNAs play a functional role at different steps of the metastatic cascade, behaving as signaling mediators to enable the colonization of a specific organ. Herein, we aimed to evaluate the biomarker performance of microRNAs previously reported as associated with prognosis for predicting BrC progression in liquid biopsies. Methods Selected microRNAs were assessed using a quantitative reverse transcription-polymerase chain reaction in a testing cohort of formalin-fixed paraffin-embedded primary (n = 16) and metastatic BrC tissues (n = 22). Then, miR-30b-5p and miR-200b-3p were assessed in a validation cohort #1 of formalin-fixed paraffin-embedded primary (n = 82) and metastatic BrC tissues (n = 93), whereas only miR-30b-5p was validated on a validation cohort #2 of liquid biopsies from BrC patients with localized (n = 20) and advanced (n = 25) disease. ROC curve was constructed to evaluate prognostic performance. Results MiR-30b-5p was differentially expressed in primary tumors and paired metastatic lesions, with bone metastases displaying significantly higher miR-30b-5p expression levels, paralleling the corresponding primary tumors. Interestingly, patients with advanced disease disclosed increased circulating miR-30b-5p expression compared to patients with localized BrC. Conclusions MiR-30b-5p might identify BrC patients at higher risk of disease progression, thus, providing a useful clinical tool for patients’ monitoring, entailing earlier and more effective treatment. Nonetheless, validation in larger multicentric cohorts is mandatory to confirm these findings.

scholarly journals The impact of RNA extraction method on accurate RNA sequencing from formalin-fixed paraffin-embedded tissues

BMC Cancer ◽  
2019 ◽  
Vol 19 (1) ◽  
Author(s):  
Michal Marczyk ◽  
Chunxiao Fu ◽  
Rosanna Lau ◽  
Lili Du ◽  
Alexander J. Trevarton ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Gene Expression ◽  
Rna Sequencing ◽  
Rna Extraction ◽  
Formalin Fixed Paraffin ◽  
Ffpe Samples ◽  
Formalin Fixed Paraffin Embedded ◽  
The Impact ◽  
Formalin Fixed

Abstract Background Utilization of RNA sequencing methods to measure gene expression from archival formalin-fixed paraffin-embedded (FFPE) tumor samples in translational research and clinical trials requires reliable interpretation of the impact of pre-analytical variables on the data obtained, particularly the methods used to preserve samples and to purify RNA. Methods Matched tissue samples from 12 breast cancers were fresh frozen (FF) and preserved in RNAlater or fixed in formalin and processed as FFPE tissue. Total RNA was extracted and purified from FF samples using the Qiagen RNeasy kit, and in duplicate from FFPE tissue sections using three different kits (Norgen, Qiagen and Roche). All RNA samples underwent whole transcriptome RNA sequencing (wtRNAseq) and targeted RNA sequencing for 31 transcripts included in a signature of sensitivity to endocrine therapy. We assessed the effect of RNA extraction kit on the reliability of gene expression levels using linear mixed-effects model analysis, concordance correlation coefficient (CCC) and differential analysis. All protein-coding genes in the wtRNAseq and three gene expression signatures for breast cancer were assessed for concordance. Results Despite variable quality of the RNA extracted from FFPE samples by different kits, all had similar concordance of overall gene expression from wtRNAseq between matched FF and FFPE samples (median CCC 0.63–0.66) and between technical replicates (median expression difference 0.13–0.22). More than half of genes were differentially expressed between FF and FFPE, but with low fold change (median |LFC| 0.31–0.34). Two out of three breast cancer signatures studied were highly robust in all samples using any kit, whereas the third signature was similarly discordant irrespective of the kit used. The targeted RNAseq assay was concordant between FFPE and FF samples using any of the kits (CCC 0.91–0.96). Conclusions The selection of kit to purify RNA from FFPE did not influence the overall quality of results from wtRNAseq, thus variable reproducibility of gene signatures probably relates to the reliability of individual gene selected and possibly to the algorithm. Targeted RNAseq showed promising performance for clinical deployment of quantitative assays in breast cancer from FFPE samples, although numerical scores were not identical to those from wtRNAseq and would require calibration.

scholarly journals Enhanced Anticancer Activity of Nanoformulation of Dasatinib against Triple-Negative Breast Cancer by Reduced Metabolic Degradation

2021 ◽  
Author(s):  
Fatemah Bahman ◽  
Valeria Pittalà ◽  
Mohamed Haider ◽  
Khaled Greish
Keyword(s):  
Breast Cancer ◽  
Anticancer Activity ◽  
Triple Negative Breast Cancer ◽  
Tyrosine Kinases ◽  
Triple Negative ◽  
Growth Factor Receptor ◽  
Initial Response ◽  
Response To Chemotherapy

Triple negative breast cancer (TNBC) is the most aggressive breast cancer accounting for around 15% of identified breast cancer cases. TNBC, by lacking estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2), is unresponsive to current targeted therapies. Existing treatment relies on chemotherapeutic treatment but, despite an initial response to chemotherapy, the inception of resistance and relapse is unfortunately common. Dasatinib is an approved second-generation inhibitor of multiple tyrosine kinases and literature data strongly support its use in the management of TNBC. However, dasatinib binds to plasma proteins and undergoes extensive metabolism through oxidation and conjugation. To protect dasatinib from fast pharmacokinetic degradation and to prolong its activity, it was encapsulated on poly(styrene-co-maleic acid) (SMA) micelles. The obtained SMA-dasatinib nanoparticles (NPs) were evaluated for their physicochemical properties, in vitro antiproliferative activity in different TNBC cell lines, and in vivo anticancer activity in a syngeneic model of breast cancer. Obtained results showed that SMA-dasatinib is more potent against 4T1 TNBC tumor growth in vivo compared to free drug. This enhanced effect was ascribed to the encapsulation of the drug protecting it from a rapid metabolism. Our finding highlights the often-overlooked value of nanoformulations in protecting its cargo from degradation. Overall, results may provide an alternative therapeutic strategy for TNBC management.

scholarly journals Trastuzumab-Modified Gold Nanoparticles Labeled with 211At as a Prospective Tool for Local Treatment of HER2-Positive Breast Cancer

Nanomaterials ◽  
2019 ◽  
Vol 9 (4) ◽  
pp. 632 ◽  
Author(s):  
Łucja Dziawer ◽  
Agnieszka Majkowska-Pilip ◽  
Damian Gaweł ◽  
Marlena Godlewska ◽  
Marek Pruszyński ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Gold Nanoparticles ◽  
Local Treatment ◽  
Growth Factor Receptor ◽  
Dark Field ◽  
Her2 Positive ◽  
Her2 Positive Breast Cancer ◽  
Biological Studies ◽  
Positive Breast Cancer

Highly localized radiotherapy with radionuclides is a commonly used treatment modality for patients with unresectable solid tumors. Herein, we propose a novel α-nanobrachytherapy approach for selective therapy of human epidermal growth factor receptor 2 (HER2)-positive breast cancer. This uses local intratumoral injection of 5-nm-diameter gold nanoparticles (AuNPs) labeled with an α-emitter (211At), modified with polyethylene glycol (PEG) chains and attached to HER2-specific monoclonal antibody (trastuzumab). The size, shape, morphology, and zeta potential of the 5 nm synthesized AuNPs were characterized by TEM (Transmission Electron Microscopy) and DLS (Dynamic Light Scattering) techniques. The gold nanoparticle surface was modified by PEG and subsequently used for antibody immobilization. Utilizing the high affinity of gold for heavy halogens, the bioconjugate was labelled with 211At obtained by α irradiation of the bismuth target. The labeling yield of 211At was greater than 99%. 211At bioconjugates were stable in human serum. Additionally, in vitro biological studies indicated that 211At-AuNP-PEG-trastuzumab exhibited higher affinity and cytotoxicity towards the HER2-overexpressing human ovarian SKOV-3 cell line than unmodified nanoparticles. Confocal and dark field microscopy studies revealed that 211At-AuNP-PEG-trastuzumab was effectively internalized and deposited near the nucleus. These findings show promising potential for the 211At-AuNP-PEG-trastuzumab radiobioconjugate as a perspective therapeutic agent in the treatment of unresectable solid cancers expressing HER2 receptors.

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