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1.
Mol Ther ; 31(2): 362-373, 2023 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-36114671

RESUMO

The uneven worldwide vaccination coverage against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and emergence of variants escaping immunity call for broadly effective and easily deployable therapeutic agents. We have previously described the human single-chain scFv76 antibody, which recognizes SARS-CoV-2 Alpha, Beta, Gamma and Delta variants. We now show that scFv76 also neutralizes the infectivity and fusogenic activity of the Omicron BA.1 and BA.2 variants. Cryoelectron microscopy (cryo-EM) analysis reveals that scFv76 binds to a well-conserved SARS-CoV-2 spike epitope, providing the structural basis for its broad-spectrum activity. We demonstrate that nebulized scFv76 has therapeutic efficacy in a severe hACE2 transgenic mouse model of coronavirus disease 2019 (COVID-19) pneumonia, as shown by body weight and pulmonary viral load data. Counteraction of infection correlates with inhibition of lung inflammation, as observed by histopathology and expression of inflammatory cytokines and chemokines. Biomarkers of pulmonary endothelial damage were also significantly reduced in scFv76-treated mice. The results support use of nebulized scFv76 for COVID-19 induced by any SARS-CoV-2 variants that have emerged so far.


Assuntos
COVID-19 , SARS-CoV-2 , Humanos , Animais , Camundongos , SARS-CoV-2/genética , Microscopia Crioeletrônica , Aerossóis e Gotículas Respiratórios , Anticorpos , Camundongos Transgênicos , Pulmão , Anticorpos Antivirais , Anticorpos Neutralizantes
2.
Mol Ther ; 30(5): 1979-1993, 2022 05 04.
Artigo em Inglês | MEDLINE | ID: mdl-35167974

RESUMO

As of December 2021, coronavirus disease 2019 (COVID-19), caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), remains a global emergency, and novel therapeutics are urgently needed. Here we describe human single-chain variable fragment (scFv) antibodies (76clAbs) that block an epitope of the SARS-CoV-2 spike protein essential for ACE2-mediated entry into cells. 76clAbs neutralize the Delta variant and other variants being monitored (VBMs) and inhibit spike-mediated pulmonary cell-cell fusion, a critical feature of COVID-19 pathology. In two independent animal models, intranasal administration counteracted the infection. Because of their high efficiency, remarkable stability, resilience to nebulization, and low cost of production, 76clAbs may become a relevant tool for rapid, self-administrable early intervention in SARS-CoV-2-infected subjects independently of their immune status.


Assuntos
COVID-19 , SARS-CoV-2 , Animais , Anticorpos Neutralizantes/uso terapêutico , Anticorpos Antivirais/uso terapêutico , Humanos , Fragmentos de Imunoglobulinas , SARS-CoV-2/genética , Glicoproteína da Espícula de Coronavírus
3.
Chem Commun (Camb) ; 57(7): 867-870, 2021 Jan 28.
Artigo em Inglês | MEDLINE | ID: mdl-33433550

RESUMO

Antitumor hydroxamates SAHA and Dacinostat have been linked to cetuximab and trastuzumab through a non-cleavable linker based on the p-mercaptobenzyl alcohol structure. These antibody drug conjugates (ADCs) were able to inhibit HDAC in several tumour cell lines. The cetuximab based ADCs block human lung adenocarcinoma cell proliferation, demonstrating that bioconjugation with antibodies is a suitable approach for targeted therapy based on hydroxamic acid-containing drugs. This work also shows that ADC-based delivery might be used to overcome the classical pharmacokinetic problems of hydroxamic acids.


Assuntos
Inibidores de Histona Desacetilases/química , Histona Desacetilases/metabolismo , Ácidos Hidroxâmicos/química , Imunoconjugados/química , Células A549 , Proliferação de Células/efeitos dos fármacos , Cetuximab/química , Inibidores de Histona Desacetilases/metabolismo , Inibidores de Histona Desacetilases/farmacologia , Histona Desacetilases/química , Humanos , Ácidos Hidroxâmicos/metabolismo , Ácidos Hidroxâmicos/farmacologia , Imunoconjugados/metabolismo , Trastuzumab/química
4.
Front Oncol ; 9: 1534, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-32039017

RESUMO

Targeted therapy using monoclonal antibodies conjugated to toxins is gaining space in the treatment of cancer. Here, we report the anti-tumor effect of a new antibody drug conjugate (ADC) delivering a HDAC inhibitor to ErbB2+ solid tumors. Trastuzumab was partially reduced with tris [2-carboxyethyl] phosphine (TCEP) and conjugated to ST7464AA1, the active form of the prodrug HDAC inhibitor ST7612AA1, through a maleimide-thiol linker to obtain the Antibody Drug Conjugate (ADC) ST8176AA1. The average drug/antibody ratio (DAR) was 4.5 as measured by hydrophobic interaction chromatography (HIC). Binding of ST8176AA1 to ErbB2 receptor and internalization in tumor cells were investigated by enzyme-linked immunosorbent assay (ELISA), surface plasmon resonance (SPR), cytofluorimetry, and High Content Screening (HCS) Imaging. The biological activity of the ADC was evaluated in vitro and in vivo by measuring cell proliferation/cell cycle, apoptosis/DNA damage, tubulin, and histone acetylation and modulation of Epithelial/Mesenchymal Transition (EMT) markers. Receptor binding and internalization of ST8176AA1 were confirmed to be similar to trastuzumab. Higher anti-tumor activity of ST8176AA1 compared to trastuzumab was observed in vitro in tumor cell lines. Such higher activity correlated with increased acetylation of histones and alfa-tubulin as a consequence of HDAC inhibitor-mediated epigenetic modulation that also induced increased expression of ErbB2 and estrogen receptor in triple negative breast cancer cells. Consistently with in vitro data, ST8176AA1 exhibited higher tumor growth inhibition than trastuzumab in xenograft models of ovary and colon carcinoma and in two patient-derived xenograft (PDX) models of pancreatic carcinoma. Immunohistochemistry analysis of tumor masses showed lower expression of the proliferation marker Ki67 and higher expression of cleaved caspase-3 in mice treated with the ADC compared to those treated with trastuzumab and results correlated with increased acetylation of both histones and tubulin. Collectively, present data indicate that ADC ST8176AA1 can target epigenetic modulation to ErbB2+ tumors. Interestingly, the amount of HDACi estimated to be delivered at the ST8176AA1 effective dose would correspond to ~1/1,000 of ST7612AA1 effective dose. Therefore, ST8176AA1 is an attractive new therapeutic candidate because it exhibits increased anti-tumor potency compared to trastuzumab by exerting epigenetic modulation at a much safer dose compared to standard HDACi-based therapeutic protocols.

5.
Oncotarget ; 8(14): 22590-22605, 2017 Apr 04.
Artigo em Inglês | MEDLINE | ID: mdl-28186982

RESUMO

The oxidized version of Avidin, known as AvidinOX, was previously shown to link to tissue proteins upon injection or nebulization, thus becoming a stable receptor for biotinylated therapeutics. AvidinOX is currently under clinical investigation to target radioactive biotin to inoperable tumor lesions (ClinicalTrials.gov NCT02053324). Presently, we show that the anti-ErbB2 monoclonal antibodies Trastuzumab and Pertuzumab can be chemically biotinylated while maintaining their biochemical and biological properties. By using several and diverse experimental conditions, we show that when AvidinOX is conjugated to tumor cells, low antibody concentrations of biotinylated Trastuzumab (bTrast) or Pertuzumab (bPert) prevent internalization of ErbB2, induce endoplasmic reticulum stress, cell cycle arrest and apoptosis leading to inhibition of proliferation and ErbB2 signaling. Moreover, we found that the treatment is able to induce down-modulation of ErbB2 thus bypassing the known resistance of this receptor to degradation. Interestingly, we show that AvidinOX anchorage is a way to counteract agonistic activities of Trastuzumab and Pertuzumab. Present data are in agreement with previous observations from our group indicating that the engagement of the Epidermal Growth Factor Receptor (EGFR) by AvidinOX-bound biotinylated Cetuximab or Panitumumab, leads to potent tumor inhibition both in vitro and in animal models. All results taken together encourage further investigation of AvidinOX-based treatments with biotinylated antibodies directed to the members of the EGFR family.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Apoptose/efeitos dos fármacos , Avidina/metabolismo , Neoplasias da Mama/tratamento farmacológico , Receptor ErbB-2/antagonistas & inibidores , Anticorpos Monoclonais Humanizados/administração & dosagem , Avidina/química , Biomarcadores Tumorais/metabolismo , Biotinilação , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Proliferação de Células/efeitos dos fármacos , Feminino , Humanos , Receptor ErbB-2/imunologia , Transdução de Sinais , Trastuzumab/administração & dosagem , Células Tumorais Cultivadas
6.
Oncotarget ; 7(1): 914-28, 2016 Jan 05.
Artigo em Inglês | MEDLINE | ID: mdl-26575422

RESUMO

For locally advanced and metastatic head and neck squamous cell carcinoma (HNSCC), the current clinical use of Cetuximab in chemo/radiotherapy protocols is often associated to severe systemic toxicity. Here we report in vitro data in human FaDu pharynx SCC cells, showing that inactive concentrations of biotinylated Cetuximab (bCet) become active upon anchorage to AvidinOX on the surface of tumor cells. AvidinOX-anchored bCet induces apoptosis and DNA damage as well as specific inhibition of signaling, degradation and abrogation of nuclear translocation of EGFR. In the mouse model of FaDu cancer, we show that intra-tumor injection of AvidinOX allows anti-tumor activity of an otherwise inactive, intraperitoneally delivered, low dose bCet. Consistently with in vitro data, in vivo tumor inhibition is associated to induction of apoptosis, DNA damage and reduced angiogenesis. AvidinOX is under clinical investigation for delivering radioactive biotin to inoperable tumors (ClinicalTrials.gov NCT02053324) and present data support its use for the local treatment of HNSCC in combination with systemic administration of low dose bCet.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Carcinoma de Células Escamosas/tratamento farmacológico , Neoplasias de Cabeça e Pescoço/tratamento farmacológico , Ensaios Antitumorais Modelo de Xenoenxerto , Animais , Antineoplásicos/administração & dosagem , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Avidina/administração & dosagem , Biotinilação , Carcinoma de Células Escamosas/irrigação sanguínea , Carcinoma de Células Escamosas/patologia , Linhagem Celular Tumoral , Cetuximab/administração & dosagem , Relação Dose-Resposta a Droga , Receptores ErbB/metabolismo , Feminino , Neoplasias de Cabeça e Pescoço/irrigação sanguínea , Neoplasias de Cabeça e Pescoço/patologia , Humanos , Immunoblotting , Imuno-Histoquímica , Camundongos Nus , Neovascularização Patológica/prevenção & controle , Resultado do Tratamento , Carga Tumoral/efeitos dos fármacos
7.
Oncotarget ; 5(19): 9239-55, 2014 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-25238453

RESUMO

Lung cancer, as well as lung metastases from distal primary tumors, could benefit from aerosol treatment. Unfortunately, because of lung physiology, clearance of nebulized drugs is fast, paralleled by unwanted systemic exposure. Here we report that nebulized AvidinOX can act as an artificial receptor for biotinylated drugs. In nude and SCID mice with advanced human KRAS-mutated A549 metastatic lung cancer, pre-nebulization with AvidinOX enables biotinylated Cetuximab to control tumor growth at a dose lower than 1/25,000 the intravenous effective dose. This result correlates with a striking, specific and unpredictable effect of AvidinOX-anchored biotinylated Cetuximab, as well as Panitumumab, observed on a panel of tumor cell lines, leading to inhibition of dimerization and signalling, blockade of endocytosis, induction of massive lysosomal degradation and abrogation of nuclear translocation of EGFR. Excellent tolerability, together with availability of pharmaceutical-grade AvidinOX and antibodies, will allow rapid clinical translation of the proposed therapy.


Assuntos
Anticorpos Monoclonais Humanizados/uso terapêutico , Avidina/uso terapêutico , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Receptores ErbB/antagonistas & inibidores , Neoplasias Pulmonares/tratamento farmacológico , Transporte Ativo do Núcleo Celular/efeitos dos fármacos , Administração por Inalação , Animais , Anticorpos Monoclonais/uso terapêutico , Antineoplásicos/uso terapêutico , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Cetuximab , Endocitose/efeitos dos fármacos , Receptores ErbB/metabolismo , Humanos , Lisossomos/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Camundongos SCID , Panitumumabe , Multimerização Proteica/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos
8.
J Biol Chem ; 283(15): 10147-61, 2008 Apr 11.
Artigo em Inglês | MEDLINE | ID: mdl-18223257

RESUMO

PTX3 is an acute phase glycoprotein that plays key roles in resistance to certain pathogens and in female fertility. PTX3 exerts its functions by interacting with a number of structurally unrelated molecules, a capacity that is likely to rely on its complex multimeric structure stabilized by interchain disulfide bonds. In this study, PAGE analyses performed under both native and denaturing conditions indicated that human recombinant PTX3 is mainly composed of covalently linked octamers. The network of disulfide bonds supporting this octameric assembly was resolved by mass spectrometry and Cys to Ser site-directed mutagenesis. Here we report that cysteine residues at positions 47, 49, and 103 in the N-terminal domain form three symmetric interchain disulfide bonds stabilizing four protein subunits in a tetrameric arrangement. Additional interchain disulfide bonds formed by the C-terminal domain cysteines Cys(317) and Cys(318) are responsible for linking the PTX3 tetramers into octamers. We also identified three intrachain disulfide bonds within the C-terminal domain that we used as structural constraints to build a new three-dimensional model for this domain. Previously it has been shown that PTX3 is a key component of the cumulus oophorus extracellular matrix, which forms around the oocyte prior to ovulation, because cumuli from PTX3(-/-) mice show defective matrix organization. Recombinant PTX3 is able to restore the normal phenotype ex vivo in cumuli from PTX3(-/-) mice. Here we demonstrate that PTX3 Cys to Ser mutants, mainly assembled into tetramers, exhibited wild type rescue activity, whereas a mutant, predominantly composed of dimers, had impaired functionality. These findings indicate that protein oligomerization is essential for PTX3 activity within the cumulus matrix and implicate PTX3 tetramers as the functional molecular units required for cumulus matrix organization and stabilization.


Assuntos
Proteína C-Reativa/química , Dissulfetos/química , Matriz Extracelular/química , Componente Amiloide P Sérico/química , Substituição de Aminoácidos , Animais , Proteína C-Reativa/genética , Proteína C-Reativa/metabolismo , Células CHO , Cricetinae , Cricetulus , Células do Cúmulo/metabolismo , Dissulfetos/metabolismo , Matriz Extracelular/genética , Matriz Extracelular/metabolismo , Feminino , Fertilidade/fisiologia , Humanos , Camundongos , Camundongos Knockout , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Oócitos/química , Oócitos/metabolismo , Ovulação/fisiologia , Estrutura Quaternária de Proteína/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Componente Amiloide P Sérico/genética , Componente Amiloide P Sérico/metabolismo
9.
Cytokine ; 31(4): 314-23, 2005 Aug 21.
Artigo em Inglês | MEDLINE | ID: mdl-16009564

RESUMO

Interleukin-6 (IL-6) is a growth and survival factor in Epstein-Barr virus (EBV)-infected B lymphoma cells and IL-6 antagonists have been used in clinical practice for this pathology. We thus wanted to investigate the effect of the IL-6 receptor antagonist Sant7 on proliferative and anti-apoptotic signals in the IL-6-secreting LCL41 B lymphoid cells, taken from a patient with EBV-induced lymphoproliferative disorder. Results show efficient inhibition of constitutive Stat3 activation by Sant7. However, this inhibition is associated with marginal induction of apoptosis and with minor decrease of cell proliferation, contrary to the effect of the Jak kinase inhibitor AG490, which down-regulates both proliferation and Stat3 activation. Anti-apoptotic markers such as Bcl-xL or Mcl-1 are constitutively expressed in these cells, and their expression is not affected by Sant7 treatment. Inhibition of Stat3 activation is therefore not sufficient to prevent proliferation and to induce apoptosis in these cells. In addition, low cell density is a condition favouring inhibition of cell clustering and anti-proliferative Sant7 activity. A marked inhibition of cell cluster formation and proliferation is achieved by antibody treatment against the CD23 mature B cell surface marker expressed in LCL41 cells. These findings may thus contribute to the identification of possible resistance mechanisms to growth arrest in B cell lymphoproliferative conditions.


Assuntos
Apoptose/fisiologia , Divisão Celular/fisiologia , Interleucina-6/fisiologia , Linfoma de Células B/patologia , Receptores de IgE/fisiologia , Western Blotting , Linhagem Celular Tumoral , Citometria de Fluxo , Humanos
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