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1.
Pharm Res ; 36(11): 154, 2019 Sep 03.
Artigo em Inglês | MEDLINE | ID: mdl-31482205

RESUMO

PURPOSE: Conjugation of nanocarriers with antibodies that bind to specific membrane receptors that are overexpressed in cancer cells enables targeted delivery. In the present study, we developed and synthesised two PAMAM dendrimer-trastuzumab conjugates that carried docetaxel or paclitaxel, specifically targeted to cells which overexpressed HER-2. METHODS: The 1H NMR, 13C NMR, FTIR and RP-HPLC were used to analyse the characteristics of the products and assess their purity. The toxicity of PAMAM-trastuzumab, PAMAM-doc-trastuzumab and PAMAM-ptx-trastuzumab conjugates was determined using MTT assay and compared with free trastuzumab, docetaxel and paclitaxel toward HER-2-positive (SKBR-3) and negative (MCF-7) human breast cancer cell lines. The cellular uptake and internal localisation were studied using flow cytometry and confocal microscopy, respectively. RESULTS: The PAMAM-drug-trastuzumab conjugates in particular showed extremely high toxicity toward the HER-2-positive SKBR-3 cells and very low toxicity towards to HER-2-negative MCF-7 cells. As expected, the HER-2-positive SKBR-3 cell line accumulated trastuzumab from both conjugates rapidly; but surprisingly, although a large amount of PAMAM-ptx-trastuzumab conjugate was observed in the HER-2-negative MCF-7 cells. Confocal microscopy confirmed the intracellular localisation of analysed compounds. The key result of fluorescent imaging was the identification of strong selective binding of the PAMAM-doc-trastuzumab conjugate with HER-2-positive SKBR-3 cells only. CONCLUSIONS: Our results confirm the high selectivity of PAMAM-doc-trastuzumab and PAMAM-ptx-trastuzumab conjugates for HER-2-positive cells, and demonstrate the utility of trastuzumab as a targeting agent. Therefore, the analysed conjugates present an promising approach for the improvement of efficacy of targeted delivery of anticancer drugs such as docetaxel or paclitaxel.


Assuntos
Antineoplásicos/química , Neoplasias da Mama/tratamento farmacológico , Dendrímeros/química , Docetaxel/química , Paclitaxel/química , Receptor ErbB-2/metabolismo , Trastuzumab/química , Antineoplásicos/farmacologia , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Docetaxel/farmacologia , Interações de Medicamentos , Liberação Controlada de Fármacos , Corantes Fluorescentes/química , Humanos , Paclitaxel/farmacologia , Propriedades de Superfície , Trastuzumab/farmacologia , Resultado do Tratamento
2.
Expert Opin Investig Drugs ; 28(7): 617-627, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31230485

RESUMO

INTRODUCTION: Despite improvements in the management of HER2+ breast cancer, metastatic disease is still fatal. Usually, these patients receive several lines of chemotherapy associated with HER2 targeted treatments. Most of the trials using innovative approaches are positioning themselves in disease that is resistant to pertuzumab and trastuzumab emtansine (TDM1). AREAS COVERED: We describe the recent advances in clinical development of anti-HER2 treatments. To this aim, we used literature search via Pubmed and made an inventory of abstracts published during the last two years in major oncology conferences. EXPERT OPINION: Further changes will probably occur during the next decade in the management of metastatic HER2-positive breast cancer. This is mainly driven by the fact that the two mainstay drugs (pertuzumab and TDM-1) that confer prolonged survival (56 months) to these patients are currently being used in the treatment of early-stage disease in a subset of patients. Thus, there is an urgent need to develop new, innovative approaches in those patients whose disease has become resistant to these highly potent drugs. Several new antibody-drug conjugates, bispecific antibodies or new generation tyrosine kinase inhibitor (TKIs) hold promise and should be assessed and compared with drugs currently used.


Assuntos
Antineoplásicos/farmacologia , Neoplasias da Mama/tratamento farmacológico , Receptor ErbB-2/metabolismo , Animais , Anticorpos Monoclonais Humanizados/administração & dosagem , Anticorpos Monoclonais Humanizados/farmacologia , Antineoplásicos/administração & dosagem , Neoplasias da Mama/patologia , Desenho de Drogas , Resistencia a Medicamentos Antineoplásicos , Drogas em Investigação/administração & dosagem , Drogas em Investigação/farmacologia , Feminino , Humanos , Imunoconjugados/administração & dosagem , Imunoconjugados/farmacologia , Maitansina/administração & dosagem , Maitansina/análogos & derivados , Maitansina/farmacologia , Terapia de Alvo Molecular , Estadiamento de Neoplasias , Trastuzumab/administração & dosagem , Trastuzumab/farmacologia
3.
Nat Commun ; 10(1): 2139, 2019 05 13.
Artigo em Inglês | MEDLINE | ID: mdl-31086176

RESUMO

Trastuzumab is integral to HER2+ cancer treatment, but its therapeutic index is narrowed by the development of resistance. Phosphorylation of the translation initiation factor eIF2α (eIF2α-P) is the nodal point of the integrated stress response, which promotes survival or death in a context-dependent manner. Here, we show an anti-tumor function of the protein kinase PKR and its substrate eIF2α in a mouse HER2+ breast cancer model. The anti-tumor function depends on the transcription factor ATF4, which upregulates the CDK inhibitor P21CIP1 and activates JNK1/2. The PKR/eIF2α-P arm is induced by Trastuzumab in sensitive but not resistant HER2+ breast tumors. Also, eIF2α-P stimulation by the phosphatase inhibitor SAL003 substantially increases Trastuzumab potency in resistant HER2+ breast and gastric tumors. Increased eIF2α-P prognosticates a better response of HER2+ metastatic breast cancer patients to Trastuzumab therapy. Hence, the PKR/eIF2α-P arm antagonizes HER2 tumorigenesis whereas its pharmacological stimulation improves the efficacy of Trastuzumab therapy.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Neoplasias da Mama/patologia , Fator de Iniciação 2 em Eucariotos/metabolismo , Neoplasias Gástricas/patologia , Trastuzumab/farmacologia , eIF-2 Quinase/metabolismo , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Biomarcadores Tumorais/metabolismo , Mama/patologia , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/mortalidade , Linhagem Celular Tumoral , Progressão da Doença , Resistencia a Medicamentos Antineoplásicos , Feminino , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Pessoa de Meia-Idade , Fosforilação , Prognóstico , Receptor ErbB-2/metabolismo , Neoplasias Gástricas/tratamento farmacológico , Análise de Sobrevida , Análise Serial de Tecidos , Trastuzumab/uso terapêutico , Regulação para Cima , Ensaios Antitumorais Modelo de Xenoenxerto , eIF-2 Quinase/antagonistas & inibidores
4.
Pharm Res ; 36(7): 103, 2019 May 17.
Artigo em Inglês | MEDLINE | ID: mdl-31101998

RESUMO

PURPOSE: A rapid and broadly applicable method to assess relevant oxidative damage in biopharmaceuticals is important for lifecycle management of product quality. Multiple methods are currently employed as stress tests to induce oxidative damage for assessment of stability, safety, and efficacy. We compared two common methods for inducing oxidative damage to assess differences in impact on bioactivity and structure of the biopharmaceuticals. METHODS: Biopharmaceuticals were treated with either metal-catalyzed oxidation (MCO) conditions or the reactive-oxygen species (ROS) inducer 2,2'-Azobis(2-amidinopropane) dihydrochloride (AAPH), then analyzed for changes in structure and bioactivity. RESULTS: We demonstrate that commonly used chemical methods for assessing oxidation yield distinct oxidation profiles for each of the biotechnology products analyzed, including monoclonal antibodies. We further report oxidant- and product-specific changes in bioactivity under oxidizing conditions, along with differential oxidation on the molecular subunits of monoclonal antibodies. CONCLUSION: Our results highlight the need for product-specific optimization and selection of orthogonal, relevant oxidizers when characterizing stress responses in biopharmaceuticals.


Assuntos
Produtos Biológicos/química , Estresse Oxidativo , Anticorpos Monoclonais Humanizados/química , Anticorpos Monoclonais Humanizados/farmacologia , Antineoplásicos Imunológicos/química , Antineoplásicos Imunológicos/farmacologia , Produtos Biológicos/farmacologia , Linhagem Celular Tumoral , Estabilidade de Medicamentos , Humanos , Indicadores e Reagentes/química , Metionina/química , Oxidantes/química , Oxirredução , Espécies Reativas de Oxigênio/química , Rituximab/química , Rituximab/farmacologia , Trastuzumab/química , Trastuzumab/farmacologia
6.
Biomed Res Int ; 2019: 9205851, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31019975

RESUMO

The effects of standard clinical therapies including surgery and chemotherapy are poor in advanced gallbladder cancer (GBC). There are a few reported cases of human epidermal growth factor receptor 2 (HER2)-positive GBC that responded well to trastuzumab. But trastuzumab has not yet been used to treat HER2-negative GBC. In this study, we investigated the cytotoxic effects of different combined therapies with trastuzumab and gemcitabine and/or 5-fluorouracil on HER2-negative GBC cell lines in vitro and in vivo. Trastuzumab alone showed almost no cytotoxicity to GBC cells with originally low HER2 gene amplification. Sequential therapy with chemotherapy followed by trastuzumab showed superiority over reverse sequential chemotherapy (P<0.05), concurrent combined chemotherapy (P<0.05), chemotherapy alone (P<0.05), and trastuzumab alone (P<0.05) in terms of cytotoxicity. Sequential therapy with chemotherapy followed by trastuzumab nearly completely inhibited cell viability in HER2-negative GBC cells. Similar results were observed with regard to apoptosis. Western blot analysis showed that gemcitabine/5-fluorouracil increased the expressions of total and phosphorylated forms of HER2, thus enhancing the cytotoxicity of trastuzumab. In vivo study verified the results of in vitro study by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling assay and immunohistochemical analysis. Moreover, not only the lightest tumor bearing but also the best survival state was detected in sequential therapy with chemotherapy followed by trastuzumab group compared with other groups. Our in vivo and in vitro data suggest that sequential therapy with gemcitabine/5-fluorouracil followed by trastuzumab represents a novel and promising therapeutic strategy against HER2-negative GBC. The upregulation of phosphorylated HER2 and phosphorylated-AKT induced by gemcitabine/5-fluorouracil treatment shows that HER2/AKT pathway is triggered.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Neoplasias da Vesícula Biliar , Animais , Apoptose/efeitos dos fármacos , Desoxicitidina/análogos & derivados , Desoxicitidina/farmacologia , Feminino , Fluoruracila/farmacologia , Neoplasias da Vesícula Biliar/tratamento farmacológico , Neoplasias da Vesícula Biliar/metabolismo , Neoplasias da Vesícula Biliar/patologia , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Receptor ErbB-2/metabolismo , Trastuzumab/farmacologia , Ensaios Antitumorais Modelo de Xenoenxerto
7.
Prep Biochem Biotechnol ; 49(8): 759-766, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31032734

RESUMO

In recent decades, immunotoxins have attracted significant attention in treatment of a wide range of diseases including cancers due to their natural origins and their role in blocking crucial pathways within the cells. Ribosome inactivating proteins (RIPs) are efficient molecules in blocking protein synthesis through interactions with ribosomal rRNA molecules. cDNA molecule encoding HER2 scFv antibody fragment originated from trastuzumab attached to the mature alpha luffin gene fragment was subcloned into pET28a expression vector and expressed in different E. coli expression hosts. Identity of the expressed recombinant protein was investigated through western blotting and the fusion protein was purified using Ni-NTA affinity chromatography. The biological activity (toxicity) of the protein was investigated on DNA and RNA samples. A 58 kDa protein was expressed in E. coli. The best protein expression level was achieved in 0.2 mM IPTG at 30 °C in TB medium using E. coli BL21 (DE3) host strain. The fusion protein showed RNase and DNA glycosylase activity on tested RNA and DNA samples. DNA glycosylase activity of the recombinant fusion protein showed that alpha luffin part of this protein is active in conjugation to the scFv molecule and the expressed protein can be further studied in targeted biological in vitro assays.


Assuntos
Clonagem Molecular/métodos , Escherichia coli/genética , Imunotoxinas/genética , Proteínas Inativadoras de Ribossomos Tipo 1/genética , Anticorpos de Cadeia Única/genética , Trastuzumab/genética , Linhagem Celular , Vetores Genéticos/genética , Humanos , Imunotoxinas/farmacologia , Receptor ErbB-2/antagonistas & inibidores , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/farmacologia , Proteínas Inativadoras de Ribossomos Tipo 1/farmacologia , Anticorpos de Cadeia Única/farmacologia , Trastuzumab/farmacologia
8.
Med Sci Monit ; 25: 2211-2220, 2019 Mar 26.
Artigo em Inglês | MEDLINE | ID: mdl-30910994

RESUMO

BACKGROUND Trastuzumab therapy is important for patients with HER2-positive breast cancer, but more and more patients have experienced trastuzumab resistance during recent years. Accumulating evidence from recent studies showed that long non-coding RNAs (lncRNAs) play essential roles in chemoresistance of various cancer types, but the precise role of lncRNAs in trastuzumab resistance is unclear. In the present study, we aimed to identify the biofunction of lncRNA APAP2-AS1 in tranastuzumab resistance and to reveal the underlying regulatory mechanism. MATERIAL AND METHODS By culturing HER2-positive SKBR-3 and BT474 cells with transtuzumab-containing medium, we built trastuzumab-resistant cells. Quantitative real-time PCR was used to test the expression of AGAP2-AS1 in the built trastuzumab-resistant cells. Cell viability assay and TUNEL assay were used to test the cell viability and apoptosis in each group. Exosomes were purified from cells cultured in exosomes-depleted FBS and identified by transmission electron microscopy. RESULTS qRT-PCR assay suggested that AGAP2-AS1 was upregulated in the built trastuzumab-resistant cells when compared with parental sensitive cells. Cell viability assay showed that silencing of AGAP2-AS1 enhanced the cytotoxicity induced by trastuzumab treatment. Mechanistically, we revealed that AGAP2-AS1 was secreted outside cells by incorporation into exosomes in an hnRNPA2B1-dependent manner. More importantly, co-culture AGAP2-AS1-containing exosomes with sensitive cells reduced the trastuzumab-induced cell death, and silencing of AGAP2-AS1 from exosomes reversed this effect. In summary, AGAP2-AS1 promotes trastuzumab resistance of breast cancer cells through packaging into exosomes. CONCLUSIONS Knockdown of AGAP2-AS1 may be helpful for improving the clinical outcome for HER2+ breast cancer patients and could serve as a therapeutic target.


Assuntos
Neoplasias da Mama/tratamento farmacológico , RNA Longo não Codificante/biossíntese , Trastuzumab/farmacologia , Apoptose/efeitos dos fármacos , Apoptose/genética , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Resistencia a Medicamentos Antineoplásicos , Exossomos/genética , Exossomos/metabolismo , Feminino , Humanos , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Ativação Transcricional
9.
Iran J Immunol ; 16(1): 26-42, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30864553

RESUMO

BACKGROUND: We have recently produced an inhibitory mouse anti-human HER2 mAb (2A8) which displayed potent anti-tumor activity in combination with trastuzumab. OBJECTIVE: To describe chimerization and functional characterization of 2A8 mAb. METHODS: The VH and VL genes of 2A8 mAb were amplified from cDNA of the mouse hybridoma, ligated to constant regions of human immunoglobulin, and expressed in CHO cell line. Reactivity with four members of human HER family, the inhibitory effects and antibody-dependent cell cytotoxicity (ADCC) of purified chimeric mAb (c2A8) were assessed by ELISA, XTT, H3-tymidine incorporation and lactate dehydrogenase assays. Inhibition of ERK and AKT downstream signaling pathways by the chimeric antibody were analyzed by Western blotting. RESULTS: Chimeric 2A8 mAb bound to recombinant human HER2 and did not cross-react with the other members of HER family. Moreover, c2A8 was able to recognize HER2-overexpressing cancer cell line and inhibited growth and proliferation of these cells. The binding affinity of c2A8 was comparable to the mouse parental mAb. ADCC and Western blotting results showed that the mouse 2A8 mAb was successfully chimerized and could significantly inhibit phosphorylation of AKT in combination with trastuzumab. CONCLUSION: The c2A8 mAb is potentially a valuable tool for targeted immunotherapy of HER2 positive cancers.


Assuntos
Anticorpos Monoclonais Humanizados/farmacologia , Antineoplásicos Imunológicos/farmacologia , Receptor ErbB-2/antagonistas & inibidores , Animais , Anticorpos Monoclonais Humanizados/genética , Anticorpos Monoclonais Humanizados/imunologia , Especificidade de Anticorpos/genética , Especificidade de Anticorpos/imunologia , Citotoxicidade Celular Dependente de Anticorpos/imunologia , Células CHO , Linhagem Celular Tumoral , Cricetulus , Reações Cruzadas/imunologia , Mapeamento de Epitopos , Epitopos/imunologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Expressão Gênica , Vetores Genéticos/genética , Humanos , Imunoglobulina G/genética , Imunoglobulina G/imunologia , Imunoglobulina G/farmacologia , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias Pesadas de Imunoglobulinas/imunologia , Cadeias Leves de Imunoglobulina/genética , Cadeias Leves de Imunoglobulina/imunologia , Macaca fascicularis , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Transfecção , Trastuzumab/farmacologia
10.
MAbs ; 11(3): 500-515, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30835621

RESUMO

Most strategies used to prepare homogeneous site-specific antibody-drug conjugates (ADCs) result in ADCs with a drug-to-antibody ratio (DAR) of two. Here, we report a disulfide re-bridging strategy to prepare homogeneous ADCs with DAR of one using a dual-maleimide pyrrolobenzodiazepine (PBD) dimer (SG3710) and an engineered antibody (Flexmab), which has only one intrachain disulfide bridge at the hinge. We demonstrate that SG3710 efficiently re-bridge a Flexmab targeting human epidermal growth factor receptor 2 (HER2), and the resulting ADC was highly resistant to payload loss in serum and exhibited potent anti-tumor activity in a HER2-positive gastric carcinoma xenograft model. Moreover, this ADC was tolerated in rats at twice the dose compared to a site-specific ADC with DAR of two prepared using a single-maleimide PBD dimer (SG3249). Flexmab technologies, in combination with SG3710, provide a platform for generating site-specific homogenous PBD-based ADCs with DAR of one, which have improved biophysical properties and tolerability compared to conventional site-specific PBD-based ADCs with DAR of two.


Assuntos
Antineoplásicos , Benzodiazepinas/química , Imunoconjugados , Pirróis/química , Receptor ErbB-2/antagonistas & inibidores , Neoplasias Gástricas/tratamento farmacológico , Trastuzumab , Animais , Antineoplásicos/química , Antineoplásicos/farmacologia , Feminino , Humanos , Imunoconjugados/química , Imunoconjugados/farmacologia , Células MCF-7 , Camundongos Nus , Ratos , Receptor ErbB-2/metabolismo , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/patologia , Trastuzumab/química , Trastuzumab/farmacologia , Ensaios Antitumorais Modelo de Xenoenxerto
11.
Int J Mol Sci ; 20(5)2019 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-30832287

RESUMO

The combination of hydrodynamic and electrophoretic experiments and computer simulations is a powerful approach to study the interaction between proteins. In this work, we present hydrodynamic and electrophoretic experiments in an aqueous solution along with molecular dynamics and hydrodynamic modeling to monitor and compute biophysical properties of the interactions between the extracellular domain of the HER2 protein (eHER2) and the monoclonal antibody trastuzumab (TZM). The importance of this system relies on the fact that the overexpression of HER2 protein is related with the poor prognosis breast cancers (HER2++ positives), while the TZM is a monoclonal antibody for the treatment of this cancer. We have found and characterized two different complexes between the TZM and eHER2 proteins (1:1 and 1:2 TZM:eHER2 complexes). The conformational features of these complexes regulate their hydrodynamic and electrostatic properties. Thus, the results indicate a high degree of molecular flexibility in the systems that ultimately leads to higher values of the intrinsic viscosity, as well as lower values of diffusion coefficient than those expected for simple globular proteins. A highly asymmetric charge distribution is detected for the monovalent complex (1:1 complex), which has strong implications in correlations between the experimental electrophoretic mobility and the modeled net charge. In order to understand the dynamics of these systems and the role of the specific domains involved, it is essential to find biophysical correlations between dynamics, macroscopic transport and electrostatic properties. The results should be of general interest for researchers working in this area.


Assuntos
Antineoplásicos Imunológicos/química , Simulação de Acoplamento Molecular , Receptor ErbB-2/química , Trastuzumab/química , Antineoplásicos Imunológicos/farmacologia , Sítios de Ligação , Humanos , Hidrodinâmica , Ligação Proteica , Receptor ErbB-2/metabolismo , Eletricidade Estática , Trastuzumab/farmacologia
12.
DNA Cell Biol ; 38(2): 184-192, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30702337

RESUMO

Resistance to trastuzumab has become a limiting factor for therapeutic efficacy of human epidermal growth factor 2 (HER2)-positive breast cancer. Different expression levels of miRNAs in cancer cells have been associated with poor prognosis and response to chemotherapy. The aim of this study was to evaluate miRNAs that were thought to be associated with HER2-positive breast cancer chemoresistance. In this study, the relative expression of candidate miRNAs to U6 RNA was evaluated in trastuzumab-resistant and trastuzumab-sensitive cells using relative real-time PCR. Our results demonstrated that miR-23b-3p, miR-195-5p, miR-656-5p, and miR-340-5p were significantly dysregulated. For the first time in this study, these miRNAs were identified to be involved in trastuzumab resistance. TargetScan and miRDB were then used for predicting the potential targets of the candidate miRNAs. Our results also revealed that the predicted potential targets of these miRNAs were strongly associated with drug resistance pathways. As a relative expression of candidate miRNAs was statistically different in trastuzumab-resistant and trastuzumab-sensitive cells, their potential targets were involved in drug resistance pathways. We strongly hypothesized the dysregulation of miRNAs as a possible mechanism of trastuzumab resistance. We also assumed that the strategic manipulation of these regulatory networks might be a possible therapeutic strategy to improve the results of chemotherapy for this resistance. However, more research is needed to evaluate the role of these miRNAs in the acquisition of trastuzumab resistance.


Assuntos
Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Resistencia a Medicamentos Antineoplásicos/genética , MicroRNAs/genética , Receptor ErbB-2/genética , Trastuzumab/farmacologia , Linhagem Celular Tumoral , Feminino , Perfilação da Expressão Gênica/métodos , Regulação Neoplásica da Expressão Gênica/genética , Humanos
13.
Chem Commun (Camb) ; 55(10): 1394-1397, 2019 Jan 29.
Artigo em Inglês | MEDLINE | ID: mdl-30632546

RESUMO

We describe the preparation of gold(i)-compounds that are amenable to efficient bioconjugation with monoclonal antibodies via activated ester or maleimide linkers. New Trastuzumab-gold conjugates were synthesized and fully characterized. These bioconjugates are significantly more cytotoxic (sub-micromolar range) to HER2-positive breast cancer cells than the gold complexes and Trastuzumab.


Assuntos
Antineoplásicos Imunológicos/química , Ouro/química , Trastuzumab/química , Antineoplásicos Imunológicos/farmacologia , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Feminino , Humanos , Células MCF-7 , Espectroscopia de Ressonância Magnética , Receptor ErbB-2/metabolismo , Trastuzumab/farmacologia
14.
Transl Res ; 207: 44-55, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30639369

RESUMO

CD151/Tspan24 (SFS-1, PETA3) is one of the best characterized members of the tetraspanin family, whose involvement in breast cancer (BCa) progression was demonstrated both in vitro and in vivo. We have recently reported that in ErbB2-overexpressing BCa cells grown in 3D laminin-rich extracellular matrix, CD151 regulated basal phosphorylation and homodimerization of ErbB2 and sensitized the cells to Herceptin (trastuzumab). Following from these data, we have here analyzed an involvement of CD151 in regulation of ErbB2/ErbB3 heterodimerization and its impact on cell response to Herceptin. CD151 was found to: (1) impair ErbB2/ErbB3 heterodimerization, (2) inhibit heregulin-dependent cell growth in 3D and signaling, and (3) counteract the protective effect of heregulin on Herceptin-mediated growth inhibition. Analysis of tissue samples demonstrated for the first time clinical significance of CD151 in patients with ErbB2-overexpressing BCa undergone trastuzumab-based therapy. Consistent with in vitro results, CD151 impact on disease outcome was ErbB3-dependent. In patients with ErbB3-negative tumors, CD151 significantly improved both overall survival (OS) (hazard ratio [HR] = 0.19, P = 0.034) and progression-free survival (PFS) (HR = 0.36, P = 0.043), while in ErbB3-positive cases it had no significant effect on patient survival (OS: HR = 3.33, P = 0.283; PFS: HR = 2.40, P = 0.208). These results support previous findings and show that CD151 acts as an important component of ErbB2 signaling axis in BCa cells, affecting their sensitivity to ErbB2-targeting therapy.


Assuntos
Neoplasias da Mama/metabolismo , Multimerização Proteica , Receptor ErbB-2/metabolismo , Receptor ErbB-3/metabolismo , Tetraspanina 24/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Intervalo Livre de Doença , Feminino , Humanos , Pessoa de Meia-Idade , Neuregulina-1/farmacologia , Multimerização Proteica/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Trastuzumab/farmacologia
15.
Methods Mol Biol ; 1913: 167-179, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30666606

RESUMO

Antibody-dependent cell-mediated cytotoxicity (ADCC) is a mechanism in which immune cell activation is induced by the cross-linking of CD16 with the Fc region of antibodies that at the same time bind specifically to cell surface antigens. ADCC stimulates the secretion of perforin, granzymes, and cytokines leading to lysis of the malignant cells. Natural killer (NK) cells express the CD16 receptor and can therefore be activated by ADCC to kill tumor cells. To study the cytotoxicity of NK cells against cancer cells, an ADCC-based assay is described: the chromium release assay. In this method, the antibody trastuzumab, which binds specifically to HER2-positive malignant cells, is used to trigger ADCC.


Assuntos
Radioisótopos de Cromo/metabolismo , Testes Imunológicos de Citotoxicidade/métodos , Células Matadoras Naturais/imunologia , Citotoxicidade Celular Dependente de Anticorpos/efeitos dos fármacos , Citotoxicidade Celular Dependente de Anticorpos/imunologia , Neoplasias da Mama/sangue , Neoplasias da Mama/imunologia , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Separação Celular/instrumentação , Separação Celular/métodos , Testes Imunológicos de Citotoxicidade/instrumentação , Feminino , Citometria de Fluxo/instrumentação , Citometria de Fluxo/métodos , Proteínas Ligadas por GPI/imunologia , Proteínas Ligadas por GPI/metabolismo , Humanos , Células Matadoras Naturais/metabolismo , Receptor ErbB-2/antagonistas & inibidores , Receptor ErbB-2/metabolismo , Receptores de IgG/imunologia , Receptores de IgG/metabolismo , Trastuzumab/farmacologia
16.
Methods Mol Biol ; 1913: 181-194, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30666607

RESUMO

Antibody-dependent cell-mediated cytotoxicity (ADCC) is a mechanism in which immune cell activation is induced by the cross-linking of CD16 with the Fc region of antibodies that at the same time bind specifically to cell surface antigens. ADCC stimulates the secretion of perforin, granzymes, and cytokines leading to lysis of the malignant cells. Natural killer (NK) cells express the CD16 receptor and can therefore be activated by ADCC to kill tumor cells. To study the cytotoxicity of NK cells against cancer cells, an ADCC-based assay is described: the flow cytometry-based cytotoxicity assay. In this method, the antibody trastuzumab, which binds specifically to HER2-positive malignant cells, is used to trigger ADCC.


Assuntos
Testes Imunológicos de Citotoxicidade/métodos , Citometria de Fluxo/métodos , Células Matadoras Naturais/imunologia , Citotoxicidade Celular Dependente de Anticorpos/efeitos dos fármacos , Citotoxicidade Celular Dependente de Anticorpos/imunologia , Neoplasias da Mama/sangue , Neoplasias da Mama/imunologia , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Separação Celular/instrumentação , Separação Celular/métodos , Testes Imunológicos de Citotoxicidade/instrumentação , Feminino , Citometria de Fluxo/instrumentação , Proteínas Ligadas por GPI/imunologia , Proteínas Ligadas por GPI/metabolismo , Humanos , Células Matadoras Naturais/metabolismo , Receptor ErbB-2/antagonistas & inibidores , Receptor ErbB-2/metabolismo , Receptores de IgG/imunologia , Receptores de IgG/metabolismo , Trastuzumab/farmacologia
17.
Biologicals ; 58: 16-21, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30655169

RESUMO

Therapeutic monoclonal antibodies (mAbs) are within the fastest growing group of pharmaceuticals on the global market. IgG1 subclass is the most potent effector in Fc-related functions. The N-linked glycosylation of mAbs Fc-domain significantly influences its therapeutic activity and the presence of this modification is largely dependent on producer cell and parameters of manufacturing process. Here we examined and characterized cell culture conditions that determine during cultivation selective changes in galactose content of a model therapeutic mAb IgG1, trastuzumab biosimilar. We demonstrated that such in cultivation process shift of galactosylation does not affect binding of the mAb to its antigen yet modifies interaction of the mAb with Fcγ receptors and therefore enhances antibody dependent cellular cytotoxicity (ADCC).


Assuntos
Citotoxicidade Celular Dependente de Anticorpos/efeitos dos fármacos , Medicamentos Biossimilares/farmacologia , Imunoglobulina G , Receptores de IgG/imunologia , Trastuzumab , Animais , Células CHO , Cricetulus , Glicosilação , Humanos , Imunoglobulina G/imunologia , Imunoglobulina G/farmacologia , Trastuzumab/imunologia , Trastuzumab/farmacologia
18.
Int J Clin Pharmacol Ther ; 57(3): 160-162, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30574866

RESUMO

OBJECTIVE: The European Medicines Agency (EMA) and the Food and Drug Administration (FDA) have recently approved four different trastuzumab biosimilars, marketed by different pharmaceutical companies. Biosimilars, unlike their originators, are typically supported by less clinical data about safety and efficacy. The objective of our analysis was to demonstrate that one of the approved trastuzumab biosimilars (MYL-1401O) is as effective as its originator in terms of hazard ratio (HR) of progression-free survival (PFS). MATERIALS AND METHODS: We carried out a network meta-analysis to compare the HR values for biosimilar, originator, and standard of care (SOC). Our analysis included a preliminary pairwise meta-analysis. The pooled HR for all pairwise comparisons was the output of the analysis (fixed-effect inverse variance method), along with 95% confidence intervals (CIs). The indirect comparison between biosimilar and SOC was performed using a network meta-analysis software. RESULTS: We estimated an HR of 0.59 (95% CI: 0.52 - 0.66) for the comparison between PFS values of originator and SOC, 0.97 (95% CI: 0.74 - 1.28) for biosimilar vs. originator, and 0.61 (95% CI: 0.44 - 0.83) for biosimilar vs. SOC. Our network meta-analysis increased the overall number of evaluated patients from 458 to 1,955. CONCLUSION: According to the network meta-analysis, MYL-1401O biosimilar is as effective as its originator in terms of HR of PFS.
.


Assuntos
Medicamentos Biossimilares/farmacologia , Neoplasias da Mama/tratamento farmacológico , Trastuzumab/farmacologia , Humanos , Meta-Análise em Rede
19.
Nucl Med Commun ; 40(1): 22-29, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30418380

RESUMO

BACKGROUND: Trastuzumab (T) and anthracycline (A)-based chemotherapy is considered the standard of care in human epidermal growth factor receptor-2+ overexpressing breast cancer, but requires monitoring for known cardiotoxicity using left ventricular (LV) ejection fraction (EF) every 3-4 months during treatment. It is not conclusively established whether diastolic dysfunction (DD) precedes LVEF decrease in patients developing trastuzumab-induced cardiotoxicity (TIC). OBJECTIVE: The aim was to elucidate whether DD precedes LVEF decrease in trastuzumab-treated patients being monitored with radionuclide multigated acquisition for TIC. PATIENTS AND METHODS: Patients treated with T±A-based chemotherapy who had undergone multigated acquisition were selected by date range (January 2006-September 2015). Up to four scans were analyzed per patient: (a) pre-A therapy, (b) pre-T therapy, (c) 4 months into T therapy, and (d) at end of T therapy. Baseline referred to the first scan of each patient (i.e. pre-A or pre-T). LV systolic and DD were defined as follows: EF less than 50% or a 10-point decrease from baseline and LV peak filling rate (PFR) less than 2.5 end-diastolic volume/s and time to peak LV filling rates (TPFR) greater than 180 ms, respectively. RESULTS: A total of 202 patients were screened for this study, of whom 153 had received A therapy (5.1±4.1 months duration) before T, 192 had 4 months of follow-up data, and 146 had 4 months of follow-up data and beyond (10.5±5.0 months). LVEF decreased with A and T therapy (P<0.005), but remained stable between 4 months and the final exam (P=0.26). In patients with normal diastolic function at baseline (45.5%), PFR decreased with A and T, and DD preceded SD by 73 days on average. In the remaining patients, with abnormal diastolic function at baseline (54.5%), PFR did not change over the course of treatment (P>0.1), nor did TPFR (P>0.3). CONCLUSION: Patients with normal diastolic function at baseline receiving trastuzumab±anthracycline adjuvant therapy may develop DD before SD, therefore offering an opportunity for early referral to cardiologists to optimize cardiovascular risk factors and manage cardiotoxicity.


Assuntos
Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/fisiopatologia , Diástole/efeitos dos fármacos , Sístole/efeitos dos fármacos , Trastuzumab/uso terapêutico , Neoplasias da Mama/diagnóstico por imagem , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Estudos Retrospectivos , Trastuzumab/farmacologia
20.
Mol Biol Rep ; 46(1): 823-831, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30535550

RESUMO

Human epidermal growth factor receptor 2 positive (HER2+) breast cancer (BC) is an aggressive BC subtype characterized by HER2 overexpression/amplification. Genomic alterations of HER2 and others have been reported to be associated with, HER2 overexpression and prediction of trastuzumab-response. Here, we aimed at identifying germline and somatic alterations associated with HER2+ BC and evaluating their association with clinical outcome in response to trastuzumab therapy given to HER2+ BC patients. Global Sequencing Array (GSA) and polymerase chain reaction-restriction length polymorphism (PCR-RFLP) techniques were used to determine alterations in HER2 and other HER2-interacting as well as signaling-related genes in HER2+ BC. In addition, 20 formalin fixed paraffin-embedded tissue samples were also evaluated by GSA for identifying significant variations associated with HER + BC as well as response to trastuzumab therapy. A germline variant in HER2 (I655V) was found to be significantly associated with the risk of the disease (p < 0.01). A nonsense mutation in PTPN11 (K99X), a pathogenic CCND1 splice site variant (P241P), a hotspot missense mutation in PIK3CA (E542K) and a hotspot missense mutation in TP53 (R249S); were observed in 25%, 75%, 30% and 40% of the HER2+ BC tissue samples, respectively. Mutant CCND1 (P241P) and PIK3CA (E542K) were found to be significantly associated with reduced disease-free survival (DFS) in patients treated with trastuzumab (p: 0.018 and 0.005, respectively). These results indicate that HER2, PTPN11, CCND1 and PIK3CA genes are important biomarkers in HER2+ BC. Moreover, the patients harboring mutant CCND1 and PIK3CA exhibit a poorer clinical outcome as compared to those carrying wild-type CCND1 and PIK3CA.


Assuntos
Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Predisposição Genética para Doença , Genoma Humano , Mutação/genética , Receptor ErbB-2/genética , Trastuzumab/uso terapêutico , Estudos de Casos e Controles , Intervalo Livre de Doença , Feminino , Células Germinativas/metabolismo , Humanos , Estimativa de Kaplan-Meier , Pessoa de Meia-Idade , Modelos Biológicos , Especificidade de Órgãos , Fatores de Risco , Trastuzumab/farmacologia , Resultado do Tratamento
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