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1.
Methods Mol Biol ; 2217: 3-15, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33215372

RESUMO

Soluble ligand and conformation-dependent antibody binding assay of integrins on the cell surface is an effective approach to evaluate the activation status of integrins in live cells. The ligands or antibodies are usually labeled with biotin or a fluorescent dye and incubated with integrin-expressing cells in suspension. The cell-bound ligands and antibodies are then detected by flow cytometry. Here we describe the detailed protocols of soluble ligand or antibody binding assay for αIIbß3, αVß3, α5ß1, and αLß2 integrins that are transiently or stably expressed in the model cell lines such as HEK293 or CHO-k1 cells.


Assuntos
Bioensaio , Integrina alfaVbeta3/química , Antígeno-1 Associado à Função Linfocitária/química , Glicoproteína IIb da Membrana de Plaquetas/química , Receptores de Vitronectina/química , Coloração e Rotulagem/métodos , Animais , Anticorpos/química , Anticorpos/metabolismo , Células CHO , Adesão Celular , Cricetulus , Citometria de Fluxo , Corantes Fluorescentes/química , Células HEK293 , Humanos , Imunoconjugados/química , Imunoconjugados/metabolismo , Integrina alfaVbeta3/genética , Integrina alfaVbeta3/metabolismo , Molécula 1 de Adesão Intercelular , Ligantes , Antígeno-1 Associado à Função Linfocitária/genética , Antígeno-1 Associado à Função Linfocitária/metabolismo , Camundongos , Plasmídeos/química , Plasmídeos/metabolismo , Glicoproteína IIb da Membrana de Plaquetas/genética , Glicoproteína IIb da Membrana de Plaquetas/metabolismo , Ligação Proteica , Receptores de Vitronectina/genética , Receptores de Vitronectina/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transfecção
2.
World J Microbiol Biotechnol ; 36(4): 53, 2020 Mar 14.
Artigo em Inglês | MEDLINE | ID: mdl-32172335

RESUMO

The recent scientific progresses on the use of enzyme-mediated reactions in organic, non-aqueous and aqueous media have significantly supported the growing demand of new biotechnological and/or pharmacological products. Today, a plethora of microbial enzymes, used as biocatalysts, are available. Among these, microbial transglutaminases (MTGs) are broadly used for their ability to catalyse the formation of an isopeptide bond between the γ-amide group of glutamines and the ε-amino group of lysine. Due to their promiscuity towards primary amine-containing substrates and the more stringent specificity for glutamine-containing peptide sequences, several combined approaches can be tailored for different settings, making MTGs very attractive catalysts for generating protein-protein and protein small molecule's conjugates. The present review offers a recent update on the modifications attainable by MTG-catalysed bioreactions as reported between 2014 and 2019. In particular, we present a detailed and comparative overview on the MTG-based methods for proteins and antibodies engineering, with a particular outlook on the synthesis of homogeneous antibody-drug conjugates.


Assuntos
Bactérias/enzimologia , Fungos/enzimologia , Engenharia de Proteínas/métodos , Transglutaminases/metabolismo , Proteínas de Bactérias/metabolismo , Biocatálise , Biotecnologia , Proteínas Fúngicas/metabolismo , Imunoconjugados/metabolismo , Especificidade por Substrato
3.
Nature ; 579(7799): 421-426, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32188939

RESUMO

Bioorthogonal chemistry capable of operating in live animals is needed to investigate biological processes such as cell death and immunity. Recent studies have identified a gasdermin family of pore-forming proteins that executes inflammasome-dependent and -independent pyroptosis1-5. Pyroptosis is proinflammatory, but its effect on antitumour immunity is unknown. Here we establish a bioorthogonal chemical system, in which a cancer-imaging probe phenylalanine trifluoroborate (Phe-BF3) that can enter cells desilylates and 'cleaves' a designed linker that contains a silyl ether. This system enabled the controlled release of a drug from an antibody-drug conjugate in mice. When combined with nanoparticle-mediated delivery, desilylation catalysed by Phe-BF3 could release a client protein-including an active gasdermin-from a nanoparticle conjugate, selectively into tumour cells in mice. We applied this bioorthogonal system to gasdermin, which revealed that pyroptosis of less than 15% of tumour cells was sufficient to clear the entire 4T1 mammary tumour graft. The tumour regression was absent in immune-deficient mice or upon T cell depletion, and was correlated with augmented antitumour immune responses. The injection of a reduced, ineffective dose of nanoparticle-conjugated gasdermin along with Phe-BF3 sensitized 4T1 tumours to anti-PD1 therapy. Our bioorthogonal system based on Phe-BF3 desilylation is therefore a powerful tool for chemical biology; our application of this system suggests that pyroptosis-induced inflammation triggers robust antitumour immunity and can synergize with checkpoint blockade.


Assuntos
Preparações de Ação Retardada/administração & dosagem , Neoplasias Mamárias Experimentais/imunologia , Piroptose/imunologia , Animais , Cumarínicos/administração & dosagem , Cumarínicos/química , Cumarínicos/metabolismo , Cumarínicos/farmacocinética , Preparações de Ação Retardada/química , Preparações de Ação Retardada/metabolismo , Preparações de Ação Retardada/farmacocinética , Feminino , Proteínas de Fluorescência Verde/administração & dosagem , Proteínas de Fluorescência Verde/química , Proteínas de Fluorescência Verde/metabolismo , Proteínas de Fluorescência Verde/farmacocinética , Células HeLa , Humanos , Imunoconjugados/administração & dosagem , Imunoconjugados/química , Imunoconjugados/metabolismo , Imunoconjugados/farmacocinética , Inflamassomos/imunologia , Inflamação/imunologia , Inflamação/metabolismo , Inflamação/patologia , Neoplasias Mamárias Experimentais/metabolismo , Neoplasias Mamárias Experimentais/patologia , Camundongos , Camundongos Endogâmicos BALB C , Oligopeptídeos/administração & dosagem , Oligopeptídeos/química , Oligopeptídeos/metabolismo , Oligopeptídeos/farmacocinética , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Proteínas/administração & dosagem , Proteínas/química , Proteínas/metabolismo , Proteínas/farmacocinética , Silanos/administração & dosagem , Silanos/química , Silanos/metabolismo , Silanos/farmacocinética , Linfócitos T/imunologia , Trastuzumab/administração & dosagem , Trastuzumab/química , Trastuzumab/metabolismo , Trastuzumab/farmacocinética , Ensaios Antitumorais Modelo de Xenoenxerto
4.
Int J Mol Sci ; 21(4)2020 Feb 22.
Artigo em Inglês | MEDLINE | ID: mdl-32098299

RESUMO

Pretargeting is widely explored in immunoPET as a strategy to reduce radiation exposure of non-target organs and allow the use of short-lived radionuclides that would not otherwise be compatible with the slow pharmacokinetic profiles of antibodies. Here we investigate a pretargeting strategy based on gallium-68 and the chelator THPMe as a high-affinity pair capable of combining in vivo. After confirming the ability of THPMe to bind 68Ga in vivo at low concentrations, the bifunctional THPMe-NCS was conjugated to a humanised huA33 antibody targeting the A33 glycoprotein. Imaging experiments performed in nude mice bearing A33-positive SW1222 colorectal cancer xenografts compared pretargeting (100 µg of THPMe-NCS-huA33, followed after 24 h by 8-10 MBq of 68Ga3+) with both a directly labelled radioimmunoconjugate (89Zr-DFO-NCS-huA33, 88 µg, 7 MBq) and a 68Ga-only negative control (8-10 MBq of 68Ga3+). Imaging was performed 25 h after antibody administration (1 h after 68Ga3+ administration for negative control). No difference between pretargeting and the negative control was observed, suggesting that pretargeting via metal chelation is not feasible using this model. However, significant accumulation of "unchelated" 68Ga3+ in the tumour was found (12.9 %ID/g) even without prior administration of THPMe-NCS-huA33, though tumour-to-background contrast was impaired by residual activity in the blood. Therefore, the 68Ga-only experiment was repeated using THPMe (20 µg, 1 h after 68Ga3+ administration) to clear circulating 68Ga3+, producing a three-fold improvement of the tumour-to-blood activity concentration ratio. Although preliminary, these results highlight the potential of THPMe as a 68Ga clearing agent in imaging applications with gallium citrate.


Assuntos
Anticorpos/metabolismo , Quelantes/farmacocinética , Imunoconjugados/farmacocinética , Compostos Radiofarmacêuticos/farmacocinética , Animais , Anticorpos/química , Linhagem Celular Tumoral , Quelantes/química , Feminino , Radioisótopos de Gálio/química , Radioisótopos de Gálio/metabolismo , Radioisótopos de Gálio/farmacocinética , Xenoenxertos , Humanos , Imunoconjugados/química , Imunoconjugados/metabolismo , Taxa de Depuração Metabólica , Camundongos Endogâmicos BALB C , Camundongos Nus , Estrutura Molecular , Tomografia Computadorizada com Tomografia por Emissão de Pósitrons , Compostos Radiofarmacêuticos/química , Compostos Radiofarmacêuticos/metabolismo , Distribuição Tecidual
5.
Drug Metab Rev ; 52(1): 66-124, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-32045530

RESUMO

Bioconjugation of therapeutic agents has been used as a selective drug delivery platform for many therapeutic areas. Bioconjugates are prepared by the covalent linkage of active compounds (small or large molecule) to a carrier molecule (lipids, proteins, peptides, carbohydrates, and polymers) through a chemical linker. The linkage of the active component to a carrier molecule enhances the therapeutic window through a targeted delivery and by reducing toxicity. Bioconjugates also possess improved pharmacokinetic properties such as a long half-life, increased stability, and cleavage by intracellular enzymes/environment. However, premature cleavage of the bioconjugates and the resulting metabolites/catabolites may produce undesirable toxic effects and, hence, it is critical to understand cleavage mechanisms, metabolism of bioconjugates, and translatability to human in the discovery stages. This article provides a comprehensive overview of linker cleavage pathways and catabolism/metabolism of antibody-drug conjugates, glycoconjugates, polymer-drug conjugates, lipid-drug conjugates, folate-targeted small molecule-drug conjugates, and drug-drug conjugates.


Assuntos
Imunoconjugados/metabolismo , Animais , Anticorpos Monoclonais/metabolismo , Anticorpos Monoclonais/farmacocinética , Anticorpos Monoclonais/farmacologia , Antineoplásicos/metabolismo , Antineoplásicos/farmacocinética , Antineoplásicos/farmacologia , Reagentes para Ligações Cruzadas/metabolismo , Reagentes para Ligações Cruzadas/farmacocinética , Humanos , Imunoconjugados/farmacocinética , Imunoconjugados/farmacologia
6.
Nat Biotechnol ; 38(4): 420-425, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32042168

RESUMO

Several cancer immunotherapy approaches, such as immune checkpoint blockade and adoptive T-cell therapy, boost T-cell activity against the tumor, but these strategies are not effective in the absence of T cells specific for displayed tumor antigens. Here we outline an immunotherapy in which endogenous T cells specific for a noncancer antigen are retargeted to attack tumors. The approach relies on the use of antibody-peptide epitope conjugates (APECs) to deliver suitable antigens to the tumor surface for presention by HLA-I. To retarget cytomegalovirus (CMV)-specific CD8+ T cells against tumors, we used APECs containing CMV-derived epitopes conjugated to tumor-targeting antibodies via metalloprotease-sensitive linkers. These APECs redirect pre-existing CMV immunity against tumor cells in vitro and in mouse cancer models. In vitro, APECs activated specifically CMV-reactive effector T cells whereas a bispecific T-cell engager activated both effector and regulatory T cells. Our approach may provide an effective alternative in cancers that are not amenable to checkpoint inhibitors or other immunotherapies.


Assuntos
Anticorpos/imunologia , Linfócitos T CD8-Positivos/transplante , Citomegalovirus/imunologia , Epitopos de Linfócito T/imunologia , Imunoconjugados/uso terapêutico , Neoplasias/terapia , Animais , Anticorpos/química , Linfócitos T CD8-Positivos/imunologia , Linhagem Celular Tumoral , Epitopos de Linfócito T/química , Antígenos de Histocompatibilidade Classe I/imunologia , Humanos , Imunoconjugados/química , Imunoconjugados/imunologia , Imunoconjugados/metabolismo , Imunomodulação , Imunoterapia Adotiva , Ativação Linfocitária , Metaloproteinases da Matriz/metabolismo , Camundongos , Neoplasias/imunologia
7.
J Nucl Med ; 61(4): 512-519, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-31586002

RESUMO

Immunotherapy is becoming the mainstay for treatment of a variety of malignancies, but only a subset of patients responds to treatment. Tumor-infiltrating CD8-positive (CD8+) T lymphocytes play a central role in antitumor immune responses. Noninvasive imaging of CD8+ T cells may provide new insights into the mechanisms of immunotherapy and potentially predict treatment response. We are studying the safety and utility of 89Zr-IAB22M2C, a radiolabeled minibody against CD8+ T cells, for targeted imaging of CD8+ T cells in patients with cancer. Methods: The initial dose escalation phase of this first-in-humans prospective study included 6 patients (melanoma, 1; lung, 4; hepatocellular carcinoma, 1). Patients received approximately 111 MBq (3 mCi) of 89Zr-IAB22M2C (at minibody mass doses of 0.2, 0.5, 1.0, 1.5, 5, or 10 mg) as a single dose, followed by PET/CT scans at approximately 1-2, 6-8, 24, 48, and 96-144 h after injection. Biodistribution in normal organs, lymph nodes, and lesions was evaluated. In addition, serum samples were obtained at approximately 5, 30, and 60 min and later at the times of imaging. Patients were monitored for safety during infusion and up to the last imaging time point. Results: 89Zr-IAB22M2C infusion was well tolerated, with no immediate or delayed side effects observed after injection. Serum clearance was typically biexponential and dependent on the mass of minibody administered. Areas under the serum time-activity curve, normalized to administered activity, ranged from 1.3 h/L for 0.2 mg to 8.9 h/L for 10 mg. Biodistribution was dependent on the minibody mass administered. The highest uptake was always in spleen, followed by bone marrow. Liver uptake was more pronounced with higher minibody masses. Kidney uptake was typically low. Prominent uptake was seen in multiple normal lymph nodes as early as 2 h after injection, peaking by 24-48 h after injection. Uptake in tumor lesions was seen on imaging as early as 2 h after injection, with most 89Zr-IAB22M2C-positive lesions detectable by 24 h. Lesions were visualized early in patients receiving treatment, with SUV ranging from 5.85 to 22.8 in 6 target lesions. Conclusion: 89Zr-IAB22M2C imaging is safe and has favorable kinetics for early imaging. Biodistribution suggests successful targeting of CD8+ T-cell-rich tissues. The observed targeting of tumor lesions suggests this may be informative for CD8+ T-cell accumulation within tumors. Further evaluation is under way.


Assuntos
Antígenos CD8/imunologia , Imunoconjugados/química , Imunoconjugados/farmacocinética , Neoplasias/diagnóstico por imagem , Tomografia Computadorizada com Tomografia por Emissão de Pósitrons/métodos , Radioisótopos , Zircônio , Adulto , Idoso , Idoso de 80 Anos ou mais , Transporte Biológico , Feminino , Humanos , Imunoconjugados/sangue , Imunoconjugados/metabolismo , Masculino , Pessoa de Meia-Idade , Metástase Neoplásica , Neoplasias/metabolismo , Neoplasias/patologia , Distribuição Tecidual
8.
AAPS J ; 22(1): 12, 2019 12 11.
Artigo em Inglês | MEDLINE | ID: mdl-31828446

RESUMO

Antibody-drug conjugates (ADCs) are cancer drugs composed of a humanized antibody linked to a cytotoxic payload, allowing preferential release of payload in cancer cells expressing the antibody-targeted antigen. Here, a systems pharmacology model is used to simulate ADC transport from blood to tumor tissue and ADC uptake by tumor cells. The model includes effects of spatial gradients in drug concentration in a three-dimensional network of tumor blood vessels with realistic geometry and accounts for diffusion of ADC in the tumor extracellular space, binding to antigen, internalization, intracellular processing, and payload efflux from cells. Cells that process an internalized ADC-antigen complex may release payload that can be taken up by other "bystander" cells. Such bystander effects are included in the model. The model is used to simulate conditions in previous experiments, showing good agreement with experimental results. Simulations are used to analyze the relationship of bystander effects to payload properties and single-dose administrations. The model indicates that exposure of payload to cells distant from vessels is sensitive to the free payload diffusivity in the extracellular space. When antigen expression is heterogeneous, the model indicates that the amount of payload accumulating in non-antigen-expressing cells increases linearly with dose but depends only weakly on the percentage of antigen-expressing cells. The model provides an integrated mechanistic framework for understanding the effects of spatial gradients on drug distribution using ADCs and for designing ADCs to achieve more effective payload distribution in solid tumors, thereby increasing the therapeutic index of the ADC.


Assuntos
Efeito Espectador/efeitos dos fármacos , Sistemas de Liberação de Medicamentos/métodos , Imunoconjugados/administração & dosagem , Modelos Biológicos , Neoplasias/irrigação sanguínea , Neoplasias/tratamento farmacológico , Antineoplásicos , Efeito Espectador/fisiologia , Linhagem Celular Tumoral , Sistemas de Liberação de Medicamentos/tendências , Humanos , Imunoconjugados/metabolismo , Microvasos/efeitos dos fármacos , Microvasos/metabolismo , Neoplasias/metabolismo
9.
Arthritis Res Ther ; 21(1): 298, 2019 12 23.
Artigo em Inglês | MEDLINE | ID: mdl-31870429

RESUMO

BACKGROUND: Although disease in a majority of rheumatoid arthritis (RA) patients is often initially limited to one or a few joints, currently approved medications including anti-tumor necrosis factor-α antibody (α-TNF) are injected systemically. Given that α-TNF systemic injection typically does not cure RA and involves risk of treatment-related adverse events, one possible approach to enhance therapeutic efficacy and reduce α-TNF systemic exposure is to retain the antibodies in arthritic joints after local administration. The aim of this study was to evaluate the approach of conferring extracellular matrix (ECM) binding affinity to α-TNF antibodies in a RA model. METHODS: α-TNF was chemically conjugated with a promiscuous ECM-binding peptide derived from placenta growth factor 2 (PlGF-2123-144). The binding activity of PlGF-2123-144-conjugated α-TNF (PlGF-2123-144-α-TNF) against ECM proteins was assessed by ELISA and by immunostaining on human cartilage specimens. The effect of conjugation on antibody function was assessed as a neutralizing activity against osteoclast differentiation. Retention at the injection site and therapeutic efficacy of PlGF-2123-144-α-TNF were tested in a collagen antibody-induced arthritis (CAIA) model in the mouse. RESULTS: PlGF-2123-144 peptide conjugation conferred α-TNF with affinity to ECM proteins without impairment of antigen recognition. PlGF-2123-144-α-TNF locally injected at a paw in the CAIA model was retained for at least 96 h at the injection site, whereas unmodified α-TNF was dispersed rapidly after injection. Local treatment with unmodified α-TNF did not suppress the arthritis score relative to isotype controls. By contrast, local administration of PlGF-2123-144-α-TNF suppressed arthritis development almost completely in the treated paw even at a 1000× lower dose. CONCLUSION: These data demonstrate that retention of α-TNF in arthritic joints can suppress arthritis development and enhance therapeutic efficacy. This simple bioengineering approach of ECM-binding peptide conjugation offers a powerful and clinically translational approach to treat RA.


Assuntos
Anticorpos/imunologia , Artrite Reumatoide/imunologia , Matriz Extracelular/imunologia , Imunoconjugados/imunologia , Fator de Crescimento Placentário/imunologia , Fator de Necrose Tumoral alfa/imunologia , Sequência de Aminoácidos , Animais , Anticorpos/metabolismo , Artrite Experimental/imunologia , Artrite Experimental/metabolismo , Artrite Experimental/prevenção & controle , Artrite Reumatoide/metabolismo , Artrite Reumatoide/prevenção & controle , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/imunologia , Modelos Animais de Doenças , Matriz Extracelular/metabolismo , Humanos , Imunoconjugados/metabolismo , Imunoconjugados/farmacologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Osteoclastos/efeitos dos fármacos , Osteoclastos/metabolismo , Fragmentos de Peptídeos/imunologia , Fragmentos de Peptídeos/metabolismo , Fator de Crescimento Placentário/química , Fator de Crescimento Placentário/metabolismo , Células RAW 264.7 , Fator de Necrose Tumoral alfa/metabolismo
10.
J Am Chem Soc ; 141(43): 17133-17141, 2019 10 30.
Artigo em Inglês | MEDLINE | ID: mdl-31580665

RESUMO

Temporal and reversible control over protein and cell conjugations holds great potential for traceless release of antibody-drug conjugates (ADCs) on tumor sites as well as on-demand altering or removal of targeting elements on cell surface. We herein developed a bioorthogonal and traceless releasable reaction on proteins and intact cells to fulfill such purposes. A systematic survey of transition metals in catalyzing the bioorthogonal cleavage reactions revealed that copper complexes such as Cu(I)-BTTAA and dual-substituted propargyl (dsPra) or propargyloxycarbonyl (dsProc) moieties offered a bioorthogonal releasable pair for reversible blockage and rescue of primary amines and phenol alcohols on small molecule drugs, protein side chains, as well as intact cell surface. For proof-of-concept, we employed such Cu(I)-BTTAA/dsProc and Cu(I)-BTTAA/dsPra pairs as a "traceless linker" strategy to construct cleavable ADCs to unleash cytotoxic compounds on cancer cells in situ and as a "reversible modification" strategy for cell surface engineering. Furthermore, by coupling with the genetic code expansion strategy, we site-specifically modulated ligand-receptor interactions on live cell membranes. Together, our work expanded the transition-metal-mediated bioorthogonal cleavage tool kit from terminal decaging to internal-linker breakage, which offered a temporal and reversible conjugation strategy on therapeutic proteins and cells.


Assuntos
Membrana Celular/química , Cobre/química , Imunoconjugados/química , Compostos Organometálicos/química , Pró-Fármacos/química , Mapas de Interação de Proteínas/genética , Aminas/química , Cumarínicos/química , Doxorrubicina/química , Doxorrubicina/farmacocinética , Liberação Controlada de Fármacos , Etoposídeo/química , Etoposídeo/farmacocinética , Proteínas de Fluorescência Verde/genética , Células HeLa , Humanos , Imunoconjugados/metabolismo , Ligantes , Lisina-tRNA Ligase/genética , Mutagênese , Fenóis/química , Pró-Fármacos/farmacocinética , Estudo de Prova de Conceito , Receptor ErbB-2/química , Receptor ErbB-2/metabolismo
11.
Cell Chem Biol ; 26(12): 1643-1651.e4, 2019 Dec 19.
Artigo em Inglês | MEDLINE | ID: mdl-31604616

RESUMO

Degradable crosslinkers that respond to intracellular biological stimuli are a critical component of many drug delivery systems. With numerous stimuli-responsive drug delivery systems in development, it is important to quantitatively study their intracellular processing. Herein we report a framework for quantifying the rate of intracellular bond degradation in the endocytic pathway. Toward this end, we devised and synthesized a reduction-sensitive FRET-based crosslinker that can be readily conjugated to a variety of targeting ligands. This crosslinker was conjugated to trastuzumab, a humanized monoclonal antibody against the HER2 receptor. We developed a model based on mass-action kinetics to describe the intracellular processing of this conjugate. The kinetic model was developed in conjunction with live-cell experiments to extract the rate constant for intracellular disulfide bond degradation. This framework may be applied to other endocytosis pathways, bond types, and cell types to quantify this fundamental degradation rate parameter.


Assuntos
Imunoconjugados/metabolismo , Trastuzumab/metabolismo , Compostos de Boro/química , Compostos de Boro/metabolismo , Linhagem Celular Tumoral , Dissulfetos/química , Dissulfetos/metabolismo , Transferência Ressonante de Energia de Fluorescência , Glutationa/química , Meia-Vida , Humanos , Imunoconjugados/imunologia , Cinética , Microscopia Confocal , Modelos Teóricos , Receptor ErbB-2/imunologia , Rodaminas/química , Rodaminas/metabolismo , Transglutaminases/metabolismo , Trastuzumab/imunologia
12.
Mol Pharm ; 16(10): 4416-4421, 2019 10 07.
Artigo em Inglês | MEDLINE | ID: mdl-31483993

RESUMO

Recent years have played witness to the advent of nuclear theranostics: the synergistic use of "matched pair" radiopharmaceuticals for diagnostic imaging and targeted radiotherapy. In this investigation, we report the extension of this concept to in vivo pretargeting based on the rapid and bioorthogonal inverse electron demand Diels-Alder reaction between tetrazine (Tz) and trans-cyclooctene (TCO). We demonstrate that a single injection of a TCO-modified immunoconjugate can be used as a platform for pretargeted PET imaging and radiotherapy via the sequential administration of a pair of Tz-bearing radioligands labeled with the positron-emitting radiometal copper-64 (t1/2 ≈ 12.7 h) and the beta-emitting radiometal lutetium-177 (t1/2 ≈ 6.7 days). More specifically, a mouse model of human colorectal carcinoma received a dose of the A33 antigen-targeting immunoconjugate huA33-TCO, followed 24 and 48 h later by injections of [64Cu]Cu-SarAr-Tz and [177Lu]Lu-DOTA-PEG7-Tz, respectively. This approach produces high activity concentrations of both radioligands in tumor tissue (16.4 ± 2.7 %ID/g for [64Cu]Cu-SarAr-Tz at 48 h post-injection and 18.1 ± 2.1 %ID/g for [177Lu]Lu-DOTA-PEG7-Tz at 120 h post-injection) as well as promising tumor-to-healthy organ activity concentration ratios. Ultimately, we believe that this work could not only have important implications in nuclear theranostics-most excitingly with isotopologue-based radioligand pairs such as [64Cu]Cu-SarAr-Tz and [67Cu]Cu-SarAr-Tz-but also in the delivery of fractionated doses during pretargeted radioimmunotherapy.


Assuntos
Neoplasias Colorretais/diagnóstico por imagem , Neoplasias Colorretais/terapia , Imunoconjugados/metabolismo , Glicoproteínas de Membrana/imunologia , Radioimunoterapia/métodos , Compostos Radiofarmacêuticos/metabolismo , Nanomedicina Teranóstica , Animais , Neoplasias Colorretais/imunologia , Neoplasias Colorretais/patologia , Radioisótopos de Cobre/química , Ciclo-Octanos/química , Feminino , Compostos Heterocíclicos com 1 Anel/química , Humanos , Imunoconjugados/química , Lutécio/química , Lutécio/metabolismo , Camundongos , Camundongos Nus , Tomografia por Emissão de Pósitrons , Radioisótopos/química , Radioisótopos/metabolismo , Compostos Radiofarmacêuticos/química , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto
13.
Bioconjug Chem ; 30(10): 2614-2623, 2019 10 16.
Artigo em Inglês | MEDLINE | ID: mdl-31535847

RESUMO

Immunotherapy is considered the fourth major treatment mode for cancer following surgery, chemotherapy, and radiotherapy. In recent years, tumor immunotherapy has achieved breakthrough progress; therefore, it is important to screen patients to identify those who will respond to tumor immunotherapy. Here, we report the construction of a novel heavy chain-only antibody (HCAb) and its corresponding 124I-labeled probe. Using phage display technology, we generated a novel anti-hPD-L1-specific HCAb named Nb6 (selected from 95 monoclones) with high affinity for hPD-L1. The positron-emitting 124I-labeled hPD-L1-targeted HCAb probe was prepared for further evaluation, and nonradioactive natural iodine (natI)-labeled anti-hPD-L1 Nb6 was synthesized as a reference compound. 125I-anti-hPD-L1 Nb6 uptake in OS-732 cells in vitro can be blocked by the precursor. The binding affinity of 125I-anti-hPD-L1 Nb6 to OS-732 cell lines was 2.19 nM. For in vivo studies, an osteosarcoma OS-732 tumor-bearing mouse model was successfully constructed. Polymerase chain reaction (PCR) and Western blot analyses were performed to confirm the presence of the hPD-L1 gene and antigen in the tumor tissue of the OS-732 mouse model. Biodistribution showed that uptake of 124I-anti-hPD-L1 Nb6 probes at 24 h was 4.43 ± 0.33% ID/g in OS-732 tumor tissues. Tumor lesions can be clearly delineated on micro-PET (positron emission tomography)/CT (computed tomography) imaging 24 h after injection of 124I-anti-hPD-L1 Nb6, while the blocking group shows substantially decreased uptake on imaging. Pathological staining validated hPD-L1 expression on the surface of the tumor cell membrane; thus, 124I-anti-hPD-L1 Nb6 can be used for in vivo noninvasive PET imaging. When administered in tandem, Nb6 and 124I-anti-hPD-L1 Nb6 may provide a novel strategy to clinically screen patients for hPD-L1 to identify those who would benefit from immunotherapy of malignant tumors such as osteosarcoma.


Assuntos
Antígeno B7-H1/metabolismo , Neoplasias Ósseas/metabolismo , Regulação Neoplásica da Expressão Gênica , Imunoconjugados/química , Cadeias Pesadas de Imunoglobulinas/imunologia , Radioisótopos do Iodo , Osteossarcoma/metabolismo , Animais , Antígeno B7-H1/imunologia , Transporte Biológico , Neoplasias Ósseas/patologia , Linhagem Celular Tumoral , Humanos , Imunoconjugados/metabolismo , Imunoconjugados/farmacocinética , Marcação por Isótopo , Camundongos , Osteossarcoma/patologia , Biblioteca de Peptídeos , Distribuição Tecidual
14.
Biochemistry (Mosc) ; 84(7): 746-761, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31509726

RESUMO

Cysteine cathepsins are proteolytic enzymes involved in protein degradation in lysosomes and endosomes. Cysteine cathepsins have been also found in the tumor microenvironment during carcinogenesis, where they are implicated in proliferation, invasion and metastasis of tumor cells through the degradation of extracellular matrix, suppression of cell-cell interactions, and promotion of angiogenesis. In this regard, cathepsins can have a diagnostic value and represent promising targets for antitumor drugs aimed at inhibition of these proteases. Moreover, cysteine cathepsins can be used as activators of novel targeted therapeutic agents. This review summarizes recent discovered roles of cysteine cathepsins in carcinogenesis and discusses new trends in cancer therapy and diagnostics using cysteine cathepsins as markers, targets, or activators.


Assuntos
Catepsinas/metabolismo , Cisteína/metabolismo , Neoplasias/diagnóstico , Neoplasias/tratamento farmacológico , Animais , Biomarcadores Tumorais , Carcinogênese/metabolismo , Catepsinas/antagonistas & inibidores , Catepsinas/classificação , Resistencia a Medicamentos Antineoplásicos , Matriz Extracelular/metabolismo , Transferência Ressonante de Energia de Fluorescência , Humanos , Imunoconjugados/metabolismo , Camundongos , Terapia de Alvo Molecular , Nanopartículas/metabolismo , Neoplasias/metabolismo , Proteólise , Microambiente Tumoral
15.
Bioconjug Chem ; 30(9): 2452-2457, 2019 09 18.
Artigo em Inglês | MEDLINE | ID: mdl-31409067

RESUMO

Site-specific modification of native antibodies has proven advantageous, as it enhances the properties of antibody-based bioconjugates without the need to manipulate the genetic code. However, native antibody modification is typically limited to strategies that introduce a single functional handle. In this work, we addressed this limitation by designing heterobifunctional substrates for microbial transglutaminase (MTG) that contain both azide and methyltetrazine "click" handles. Structure-conjugation relationships for these substrates were evaluated using the Her2-targeted antibody trastuzumab. Förster resonance energy transfer (FRET) was used to demonstrate that these chemical handles are mutually orthogonal. This orthogonality was leveraged for the one-pot synthesis of a bifunctional antibody-drug conjugate (ADC). This ADC, containing a maytansine-derived payload and a hydrophobicity-masking polyethylene glycol (PEG) side chain, demonstrated potent in vitro activity in SKOV3 cells. These studies establish the dual "click" approach as a powerful technique in the toolbox for native antibody modification.


Assuntos
Imunoconjugados/química , Imunoconjugados/metabolismo , Transglutaminases/metabolismo , Linhagem Celular Tumoral , Química Click , Cisteína/química , Células HEK293 , Humanos , Microbiologia , Oxirredução , Triptofano/química
16.
Anal Chim Acta ; 1081: 138-145, 2019 Nov 12.
Artigo em Inglês | MEDLINE | ID: mdl-31446951

RESUMO

Lot release and stability testing of biologics are essential parts of the quality control strategy for ensuring therapeutic material dosed to patients is safe and efficacious, and consistent with previous clinical and toxicological experience. Characterization of protein aggregation is of particular significance, as aggregates may lose the intrinsic pharmaceutical properties as well as engage with the immune system instigating undesirable downstream immunogenicity. While important, real-time identification and quantification of subvisible particles in the monoclonal antibody (mAb) drug products remains inaccessible with existing techniques due to limitations in measurement time, sensitivity or experimental conditions. Here, owing to its exquisite molecular specificity, non-perturbative nature and lack of sample preparation requirements, we propose label-free Raman spectroscopy in conjunction with multivariate analysis as a solution to this unmet need. By leveraging subtle, but consistent, differences in vibrational modes of the biologics, we have developed a support vector machine-based regression model that provides fast, accurate prediction for a wide range of protein aggregations. Moreover, in blinded experiments, the model shows the ability to precisely differentiate between aggregation levels in mAb like product samples pre- and post-isothermal incubation, where an increase in aggregate levels was experimentally determined. In addition to offering fresh insights into mAb like product-specific aggregation mechanisms that can improve engineering of new protein therapeutics, our results highlight the potential of Raman spectroscopy as an in-line analytical tool for monitoring protein particle formation.


Assuntos
Anticorpos Monoclonais/análise , Imunoconjugados/análise , Agregados Proteicos , Algoritmos , Anticorpos Monoclonais/metabolismo , Imunoconjugados/metabolismo , Análise Multivariada , Análise de Componente Principal , Multimerização Proteica , Análise de Regressão , Análise Espectral Raman/métodos , Máquina de Vetores de Suporte
17.
Nat Commun ; 10(1): 3854, 2019 08 26.
Artigo em Inglês | MEDLINE | ID: mdl-31451692

RESUMO

Exosomes have been implicated in numerous biological processes, and they may serve as important disease markers. Surface proteins on exosomes carry information about their tissues of origin. Because of the heterogeneity of exosomes it is desirable to investigate them individually, but this has so far remained impractical. Here, we demonstrate a proximity-dependent barcoding assay to profile surface proteins of individual exosomes using antibody-DNA conjugates and next-generation sequencing. We first validate the method using artificial streptavidin-oligonucleotide complexes, followed by analysis of the variable composition of surface proteins on individual exosomes, derived from human body fluids or cell culture media. Exosomes from different sources are characterized by the presence of specific combinations of surface proteins and their abundance, allowing exosomes to be separately quantified in mixed samples to serve as markers for tissue-specific engagement in disease.


Assuntos
Exossomos/metabolismo , Perfilação da Expressão Gênica/métodos , Proteínas de Membrana/metabolismo , Líquidos Corporais/citologia , Linhagem Celular Tumoral , DNA de Cadeia Simples/genética , DNA de Cadeia Simples/metabolismo , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Imunoconjugados/genética , Imunoconjugados/metabolismo , Proteínas de Membrana/genética , Sondas Moleculares/genética , Sondas Moleculares/metabolismo , Oligonucleotídeos/genética , Oligonucleotídeos/metabolismo , Análise de Sequência de DNA/métodos
18.
J Am Soc Mass Spectrom ; 30(11): 2419-2429, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31429052

RESUMO

Middle-down mass spectrometry (MD MS) has emerged as a promising alternative to classical bottom-up approaches for protein characterization. Middle-level experiments after enzymatic digestion are routinely used for subunit analysis of monoclonal antibody (mAb)-related compounds, providing information on drug load distribution and average drug-to-antibody ratio (DAR). However, peptide mapping is still the gold standard for primary amino acid sequence assessment, post-translational modifications (PTM), and drug conjugation identification and localization. However, peptide mapping strategies can be challenging when dealing with more complex and heterogeneous mAb formats, like antibody-drug conjugates (ADCs). We report here, for the first time, MD MS analysis of a third-generation site-specific DAR4 ADC using different fragmentation techniques, including higher-energy collisional- (HCD), electron-transfer (ETD) dissociation and 213 nm ultraviolet photodissociation (UVPD). UVPD used as a standalone technique for ADC subunit analysis afforded, within the same liquid chromatography-MS/MS run, enhanced performance in terms of primary sequence coverage compared to HCD- or ETD-based MD approaches, and generated substantially more MS/MS fragments containing either drug conjugation or glycosylation site information, leading to confident drug/glycosylation site identification. In addition, our results highlight the complementarity of ETD and UVPD for both primary sequence validation and drug conjugation/glycosylation site assessment. Altogether, our results highlight the potential of UVPD for ADC MD MS analysis for drug conjugation/glycosylation site assessment, and indicate that MD MS strategies can improve structural characterization of empowered next-generation mAb-based formats, especially for PTMs and drug conjugation sites validation.


Assuntos
Imunoconjugados/química , Imunoconjugados/metabolismo , Espectrometria de Massas/métodos , Mapeamento de Peptídeos/métodos , Sítios de Ligação , Humanos
19.
Chem Soc Rev ; 48(16): 4361-4374, 2019 Aug 12.
Artigo em Inglês | MEDLINE | ID: mdl-31294429

RESUMO

Antibody-Drug Conjugates (ADCs) are now established as a major class of therapeutics for the clinical treatment of cancer. The properties of the linker between the antibody and the payload are proven to be critical to the success of an ADC. Although ADC linkers can be 'non-cleavable', the vast majority of ADCs in clinical development have specific release mechanisms to allow controlled linker cleavage at the target site and are thus termed 'cleavable'. In recent years, the development of new methods of drug release from ADCs has continued in parallel to the deepening understanding of the biological processes underlying the mechanisms of action of pre-existing technologies. This review summarises the advances in the field of cleavable linker technologies for ADCs.


Assuntos
Anticorpos Monoclonais/química , Imunoconjugados/química , Ácidos/química , Animais , Antineoplásicos/química , Antineoplásicos/metabolismo , Catepsina B/metabolismo , Dissulfetos/química , Estabilidade de Medicamentos , Humanos , Imunoconjugados/sangue , Imunoconjugados/metabolismo
20.
Bioconjug Chem ; 30(10): 2483-2501, 2019 10 16.
Artigo em Inglês | MEDLINE | ID: mdl-31339691

RESUMO

Antibody-oligonucleotide conjugates (AOCs) are a novel class of synthetic chimeric biomolecules that has been continually gaining traction in different fields of modern biotechnology. This is mainly due to the unique combination of the properties of their two constituents, exceptional targeting abilities and antibody biodistribution profiles, in addition to an extensive scope of oligonucleotide functional and structural roles. Combining these two classes of biomolecules in one chimeric construct has therefore become an important milestone in the development of numerous biotechnological applications, including imaging (DNA-PAINT), detection (PLA, PEA), and therapeutics (targeted siRNA/antisense delivery). Numerous synthetic approaches have been developed to access AOCs ranging from stochastic chemical bioconjugation to site-specific conjugation with reactive handles, introduced into antibody sequences through protein engineering. This Review gives a general overview of the current status of AOC applications with a specific emphasis on the synthetic methods used for their preparation. The reported synthetic techniques are discussed in terms of their practical aspects and limitations. The importance of the development of novel methods for the facile generation of AOCs possessing a defined constitution is highlighted as a priority in AOC research to ensure the advance of their new applications.


Assuntos
Anticorpos/metabolismo , Imunoconjugados/uso terapêutico , Imagem Molecular/métodos , Oligonucleotídeos/metabolismo , Animais , Humanos , Imunoconjugados/química , Imunoconjugados/metabolismo
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