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1.
PLoS One ; 15(10): e0239814, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33002048

RESUMO

BACKGROUND AND STUDY AIMS: Despite major technical advancements, endoscopic surveillance for detecting premalignant lesions in Barrett's esophagus is challenging because of their flat appearance with only subtle morphological changes. Molecular endoscopic imaging (MEI) using nanoparticles (NPs), coupled with fluorescently labeled antibody permits visualization of disease-specific molecular alterations. The aim of this ex vivo study was to assess the diagnostic applicability of MEI with NPs to detect Barrett's metaplasia. PATIENTS AND METHODS: Seven patients undergoing endoscopic surveillance of known Barrett's esophagus were recruited. Freshly resected biopsy specimens were incubated with NPs coupled with FITC labeled Muc-2 antibodies and examined with MEI. Fluorescence intensity from Barrett's mucosa and control specimens were compared, followed by histological confirmation. RESULTS: Fluorescence signals, indicating the presence of goblet cells, were noted for traditional MEI using Muc-2 antibodies in Barrett's intestinal metaplasia. Significantly stronger fluorescence signals were achieved with NPs coupled with FITC-conjugated Muc-2 antibodies. The results of MEI with NPs for the prediction of Barrett's metaplasia correlated with the final histopathological examination in all the cases. CONCLUSIONS: Highly-specific NPs detected Barrett's metaplasia more efficiently than conventional MEI in this first feasibility study. MEI was as effective as standard histopathology for identifying Muc-2 containing goblet cells for diagnosis of Barrett's metaplasia. (DRKS-ID: DRKS00017747).


Assuntos
Esôfago de Barrett/diagnóstico por imagem , Endoscopia/métodos , Nanoconjugados/química , Imagem Óptica/métodos , Idoso , Anticorpos/química , Anticorpos/imunologia , Esôfago de Barrett/patologia , Fluoresceína-5-Isotiocianato/química , Humanos , Pessoa de Meia-Idade , Mucinas/imunologia
2.
PLoS One ; 15(10): e0239282, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33095778

RESUMO

OBJECTIVES: To determine if the URO-MCP-1 mouse model for bladder IC/BPS is associated with in vivo bladder hyper-permeability, as measured by contrast-enhanced MRI (CE-MRI), and assess whether molecular-targeted MRI (mt-MRI) can visualize in vivo claudin-2 expression as a result of bladder hyper-permeability. Interstitial cystitis/bladder pain syndrome (IC/BPS) is a chronic, painful condition of the bladder that affects primarily women. It is known that permeability plays a substantial role in IC/BPS. Claudins are tight junction membrane proteins that are expressed in epithelia and endothelia and form paracellular barriers and pores that determine tight junction permeability. Claudin-2 is a molecular marker that is associated with increased hyperpermeability in the urothelium. MATERIALS AND METHODS: CE-MRI was used to measure bladder hyper-permeability in the URO-MCP-1 mice. A claudin-2-specific mt-MRI probe was used to assess in vivo levels of claudin-2. The mt-MRI probe consists of an antibody against claudin-2 conjugated to albumin that had Gd-DTPA (gadolinium diethylenetriamine pentaacetate) and biotin attached. Verification of the presence of the mt-MRI probe was done by targeting the biotin moiety for the probe with streptavidin-horse radish peroxidase (SA-HRP). Trans-epithelial electrical resistance (TEER) was also used to assess bladder permeability. RESULTS: The URO-MCP-1 mouse model for IC/BPS was found to have a significant increase in bladder permeability, following liposaccharide (LPS) exposure, compared to saline-treated controls. mt-MRI- and histologically-detectable levels of the claudin-2 probe were found to increase with LPS -induced bladder urothelial hyper-permeability in the URO-MCP-1 IC mouse model. Levels of protein expression for claudin-2 were confirmed with immunohistochemistry and immunofluorescence imaging. Claudin-2 was also found to highly co-localize with zonula occlidens-1 (ZO-1), a tight junction protein. CONCLUSION: The combination of CE-MRI and TEER approaches were able to demonstrate hyper-permeability, a known feature associated with some IC/BPS patients, in the LPS-exposed URO-MCP-1 mouse model. This MRI approach could be clinically translated to establish which IC/BPS patients have bladder hyper-permeability and help determine therapeutic options. In addition, the in vivo molecular-targeted imaging approach can provide invaluable information to enhance our understanding associated with bladder urothelium hyper-permeability in IC/BPS patients, and perhaps be used to assist in developing further therapeutic strategies.


Assuntos
Claudina-2/metabolismo , Cistite Intersticial/patologia , Imagem por Ressonância Magnética/métodos , Sondas Moleculares/química , Bexiga Urinária/fisiopatologia , Animais , Anticorpos/química , Anticorpos/imunologia , Claudina-2/imunologia , Cistite Intersticial/metabolismo , Modelos Animais de Doenças , Gadolínio DTPA/química , Imuno-Histoquímica , Lipopolissacarídeos/toxicidade , Camundongos , Permeabilidade/efeitos dos fármacos , Albumina Sérica/química
3.
PLoS Biol ; 18(9): e3000821, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32886672

RESUMO

As a novel alternative to established surface display or combinatorial chemistry approaches for the discovery of therapeutic peptides, we present a method for the isolation of small, cysteine-rich domains from bovine antibody ultralong complementarity-determining regions (CDRs). We show for the first time that isolated bovine antibody knob domains can function as autonomous entities by binding antigen outside the confines of the antibody scaffold. This yields antibody fragments so small as to be considered peptides, each stabilised by an intricate, bespoke arrangement of disulphide bonds. For drug discovery, cow immunisations harness the immune system to generate knob domains with affinities in the picomolar to low nanomolar range, orders of magnitude higher than unoptimized peptides from naïve library screening. Using this approach, knob domain peptides that tightly bound Complement component C5 were obtained, at scale, using conventional antibody discovery and peptide purification techniques.


Assuntos
Anticorpos/química , Dissulfetos/isolamento & purificação , Domínios de Imunoglobulina , Fragmentos de Peptídeos/isolamento & purificação , Domínios e Motivos de Interação entre Proteínas , Animais , Anticorpos/imunologia , Anticorpos/metabolismo , Afinidade de Anticorpos , Formação de Anticorpos , Especificidade de Anticorpos , Antígenos/genética , Antígenos/imunologia , Linfócitos B/fisiologia , Bovinos , Complemento C5/química , Complemento C5/genética , Complemento C5/imunologia , Regiões Determinantes de Complementaridade/química , Regiões Determinantes de Complementaridade/genética , Regiões Determinantes de Complementaridade/imunologia , Dissulfetos/química , Dissulfetos/imunologia , Mapeamento de Epitopos/métodos , Humanos , Imunização , Domínios de Imunoglobulina/genética , Modelos Moleculares , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/imunologia , Domínios e Motivos de Interação entre Proteínas/genética
4.
Proc Natl Acad Sci U S A ; 117(36): 22341-22350, 2020 09 08.
Artigo em Inglês | MEDLINE | ID: mdl-32855302

RESUMO

Conformational diversity and self-cross-reactivity of antigens have been correlated with evasion from neutralizing antibody responses. We utilized single cell B cell sequencing, biolayer interferometry and X-ray crystallography to trace mutation selection pathways where the antibody response must resolve cross-reactivity between foreign and self-proteins bearing near-identical contact surfaces, but differing in conformational flexibility. Recurring antibody mutation trajectories mediate long-range rearrangements of framework (FW) and complementarity determining regions (CDRs) that increase binding site conformational diversity. These antibody mutations decrease affinity for self-antigen 19-fold and increase foreign affinity 67-fold, to yield a more than 1,250-fold increase in binding discrimination. These results demonstrate how conformational diversity in antigen and antibody does not act as a barrier, as previously suggested, but rather facilitates high affinity and high discrimination between foreign and self.


Assuntos
Anticorpos , Diversidade de Anticorpos/genética , Autoantígenos , Rearranjo Gênico do Linfócito B/genética , Mutação/genética , Animais , Anticorpos/química , Anticorpos/genética , Anticorpos/metabolismo , Afinidade de Anticorpos/genética , Autoanticorpos/química , Autoanticorpos/genética , Autoanticorpos/metabolismo , Autoantígenos/química , Autoantígenos/metabolismo , Regiões Determinantes de Complementaridade/genética , Imunidade Humoral/genética , Camundongos , Modelos Moleculares , Conformação Proteica , Hipermutação Somática de Imunoglobulina/genética
5.
Nat Commun ; 11(1): 3859, 2020 07 31.
Artigo em Inglês | MEDLINE | ID: mdl-32737298

RESUMO

Non-enzymatic proteins including antibodies function as biomarkers and are used as biopharmaceuticals in several diseases. Protein-responsive soft materials capable of the controlled release of drugs and proteins have potential for use in next-generation diagnosis and therapies. Here, we describe a supramolecular/agarose hydrogel composite that can release a protein in response to a non-enzymatic protein. A non-enzymatic protein-responsive system is developed by hybridization of an enzyme-sensitive supramolecular hydrogel with a protein-triggered enzyme activation set. In situ imaging shows that the supramolecular/agarose hydrogel composite consists of orthogonal domains of supramolecular fibers and agarose, which play distinct roles in protein entrapment and mechanical stiffness, respectively. Integrating the enzyme activation set with the composite allows for controlled release of the embedded RNase in response to an antibody. Such composite hydrogels would be promising as a matrix embedded in a body, which can autonomously release biopharmaceuticals by sensing biomarker proteins.


Assuntos
Anidrase Carbônica II/química , Preparações de Ação Retardada/síntese química , Hidrogéis/química , Ribonucleases/química , Sefarose/química , Animais , Anticorpos/química , Avidina/química , Biotina/química , Anidrase Carbônica II/antagonistas & inibidores , Inibidores da Anidrase Carbônica/química , Bovinos , Ativação Enzimática , Transição de Fase , Reologia , Ribonucleases/antagonistas & inibidores , Sulfonamidas/química
6.
Nature ; 584(7820): 291-297, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32728216

RESUMO

The majority of therapies that target individual proteins rely on specific activity-modulating interactions with the target protein-for example, enzyme inhibition or ligand blocking. However, several major classes of therapeutically relevant proteins have unknown or inaccessible activity profiles and so cannot be targeted by such strategies. Protein-degradation platforms such as proteolysis-targeting chimaeras (PROTACs)1,2 and others (for example, dTAGs3, Trim-Away4, chaperone-mediated autophagy targeting5 and SNIPERs6) have been developed for proteins that are typically difficult to target; however, these methods involve the manipulation of intracellular protein degradation machinery and are therefore fundamentally limited to proteins that contain cytosolic domains to which ligands can bind and recruit the requisite cellular components. Extracellular and membrane-associated proteins-the products of 40% of all protein-encoding genes7-are key agents in cancer, ageing-related diseases and autoimmune disorders8, and so a general strategy to selectively degrade these proteins has the potential to improve human health. Here we establish the targeted degradation of extracellular and membrane-associated proteins using conjugates that bind both a cell-surface lysosome-shuttling receptor and the extracellular domain of a target protein. These initial lysosome-targeting chimaeras, which we term LYTACs, consist of a small molecule or antibody fused to chemically synthesized glycopeptide ligands that are agonists of the cation-independent mannose-6-phosphate receptor (CI-M6PR). We use LYTACs to develop a CRISPR interference screen that reveals the biochemical pathway for CI-M6PR-mediated cargo internalization in cell lines, and uncover the exocyst complex as a previously unidentified-but essential-component of this pathway. We demonstrate the scope of this platform through the degradation of therapeutically relevant proteins, including apolipoprotein E4, epidermal growth factor receptor, CD71 and programmed death-ligand 1. Our results establish a modular strategy for directing secreted and membrane proteins for lysosomal degradation, with broad implications for biochemical research and for therapeutics.


Assuntos
Espaço Extracelular/metabolismo , Lisossomos/metabolismo , Proteínas de Membrana/metabolismo , Proteólise , Proteínas Recombinantes de Fusão/metabolismo , Animais , Anticorpos/química , Anticorpos/metabolismo , Antígenos CD/metabolismo , Apolipoproteína E4/metabolismo , Antígeno B7-H1/metabolismo , Sistemas CRISPR-Cas , Linhagem Celular , Receptores ErbB/metabolismo , Feminino , Glicopeptídeos/síntese química , Glicopeptídeos/metabolismo , Humanos , Ligantes , Proteínas de Membrana/química , Camundongos , Domínios Proteicos , Transporte Proteico , Receptor IGF Tipo 2/metabolismo , Receptores da Transferrina/metabolismo , Proteínas Recombinantes de Fusão/síntese química , Proteínas Recombinantes de Fusão/química , Solubilidade , Especificidade por Substrato
7.
Proc Natl Acad Sci U S A ; 117(29): 16949-16960, 2020 07 21.
Artigo em Inglês | MEDLINE | ID: mdl-32616569

RESUMO

Affinity maturation is a powerful technique in antibody engineering for the in vitro evolution of antigen binding interactions. Key to the success of this process is the expansion of sequence and combinatorial diversity to increase the structural repertoire from which superior binding variants may be selected. However, conventional strategies are often restrictive and only focus on small regions of the antibody at a time. In this study, we used a method that combined antibody chain shuffling and a staggered-extension process to produce unbiased libraries, which recombined beneficial mutations from all six complementarity-determining regions (CDRs) in the affinity maturation of an inhibitory antibody to Arginase 2 (ARG2). We made use of the vast display capacity of ribosome display to accommodate the sequence space required for the diverse library builds. Further diversity was introduced through pool maturation to optimize seven leads of interest simultaneously. This resulted in antibodies with substantial improvements in binding properties and inhibition potency. The extensive sequence changes resulting from this approach were translated into striking structural changes for parent and affinity-matured antibodies bound to ARG2, with a large reorientation of the binding paratope facilitating increases in contact surface and shape complementarity to the antigen. The considerable gains in therapeutic properties seen from extensive sequence and structural evolution of the parent ARG2 inhibitory antibody clearly illustrate the advantages of the unbiased approach developed, which was key to the identification of high-affinity antibodies with the desired inhibitory potency and specificity.


Assuntos
Anticorpos/química , Afinidade de Anticorpos , Arginase/imunologia , Regiões Determinantes de Complementaridade/química , Anticorpos/genética , Anticorpos/imunologia , Sítios de Ligação de Anticorpos , Regiões Determinantes de Complementaridade/imunologia , Humanos
8.
Proc Natl Acad Sci U S A ; 117(24): 13509-13518, 2020 06 16.
Artigo em Inglês | MEDLINE | ID: mdl-32493749

RESUMO

Protein misfolding and aggregation is the hallmark of numerous human disorders, including Alzheimer's disease. This process involves the formation of transient and heterogeneous soluble oligomers, some of which are highly cytotoxic. A major challenge for the development of effective diagnostic and therapeutic tools is thus the detection and quantification of these elusive oligomers. Here, to address this problem, we develop a two-step rational design method for the discovery of oligomer-specific antibodies. The first step consists of an "antigen scanning" phase in which an initial panel of antibodies is designed to bind different epitopes covering the entire sequence of a target protein. This procedure enables the determination through in vitro assays of the regions exposed in the oligomers but not in the fibrillar deposits. The second step involves an "epitope mining" phase, in which a second panel of antibodies is designed to specifically target the regions identified during the scanning step. We illustrate this method in the case of the amyloid ß (Aß) peptide, whose oligomers are associated with Alzheimer's disease. Our results show that this approach enables the accurate detection and quantification of Aß oligomers in vitro, and in Caenorhabditis elegans and mouse hippocampal tissues.


Assuntos
Peptídeos beta-Amiloides/metabolismo , Anticorpos/imunologia , Agregados Proteicos , Doença de Alzheimer/diagnóstico , Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/química , Animais , Anticorpos/química , Anticorpos/metabolismo , Especificidade de Anticorpos , Caenorhabditis elegans , Modelos Animais de Doenças , Epitopos , Hipocampo/metabolismo , Camundongos , Ligação Proteica , Conformação Proteica , Anticorpos de Domínio Único
9.
Proc Natl Acad Sci U S A ; 117(21): 11624-11635, 2020 05 26.
Artigo em Inglês | MEDLINE | ID: mdl-32385154

RESUMO

Activation-induced cytidine deaminase (AID) is the key enzyme for class switch recombination (CSR) and somatic hypermutation (SHM) to generate antibody memory. Previously, heterogeneous nuclear ribonucleoprotein K (hnRNP K) was shown to be required for AID-dependent DNA breaks. Here, we defined the function of major RNA-binding motifs of hnRNP K, GXXGs and RGGs in the K-homology (KH) and the K-protein-interaction (KI) domains, respectively. Mutation of GXXG, RGG, or both impaired CSR, SHM, and cMyc/IgH translocation equally, showing that these motifs were necessary for AID-dependent DNA breaks. AID-hnRNP K interaction is dependent on RNA; hence, mutation of these RNA-binding motifs abolished the interaction with AID, as expected. Some of the polypyrimidine sequence-carrying prototypical hnRNP K-binding RNAs, which participate in DNA breaks or repair bound to hnRNP K in a GXXG and RGG motif-dependent manner. Mutation of the GXXG and RGG motifs decreased nuclear retention of hnRNP K. Together with the previous finding that nuclear localization of AID is necessary for its function, lower nuclear retention of these mutants may worsen their functional deficiency, which is also caused by their decreased RNA-binding capacity. In summary, hnRNP K contributed to AID-dependent DNA breaks with all of its major RNA-binding motifs.


Assuntos
Anticorpos , Citidina Desaminase , Quebras de DNA , Ribonucleoproteínas Nucleares Heterogêneas Grupo K , Motivos de Ligação ao RNA/genética , Animais , Anticorpos/química , Anticorpos/genética , Anticorpos/metabolismo , Citidina Desaminase/genética , Citidina Desaminase/metabolismo , Ribonucleoproteínas Nucleares Heterogêneas Grupo K/química , Ribonucleoproteínas Nucleares Heterogêneas Grupo K/genética , Ribonucleoproteínas Nucleares Heterogêneas Grupo K/metabolismo , Humanos , Switching de Imunoglobulina/genética , Cadeias Pesadas de Imunoglobulinas/química , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias Pesadas de Imunoglobulinas/metabolismo , Camundongos , Hipermutação Somática de Imunoglobulina/genética
10.
Nucleic Acids Res ; 48(W1): W125-W131, 2020 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-32432715

RESUMO

While antibodies are becoming an increasingly important therapeutic class, especially in personalized medicine, their development and optimization has been largely through experimental exploration. While there have been many efforts to develop computational tools to guide rational antibody engineering, most approaches are of limited accuracy when applied to antibody design, and have largely been limited to analysing a single point mutation at a time. To overcome this gap, we have curated a dataset of 242 experimentally determined changes in binding affinity upon multiple point mutations in antibody-target complexes (89 increasing and 153 decreasing binding affinity). Here, we have shown that by using our graph-based signatures and atomic interaction information, we can accurately analyse the consequence of multi-point mutations on antigen binding affinity. Our approach outperformed other available tools across cross-validation and two independent blind tests, achieving Pearson's correlations of up to 0.95. We have implemented our new approach, mmCSM-AB, as a web-server that can help guide the process of affinity maturation in antibody design. mmCSM-AB is freely available at http://biosig.unimelb.edu.au/mmcsm_ab/.


Assuntos
Anticorpos/genética , Afinidade de Anticorpos/genética , Mutação Puntual , Software , Anticorpos/química , Complexo Antígeno-Anticorpo/química , Complexo Antígeno-Anticorpo/genética , Engenharia de Proteínas
11.
Biochem Biophys Res Commun ; 526(4): 941-946, 2020 06 11.
Artigo em Inglês | MEDLINE | ID: mdl-32284170

RESUMO

Human serum albumin (HSA) has been used to extend the serum half-lives of various protein therapeutics through genetic fusion because HSA exhibits an exceptionally long circulation time as a result of neonatal Fc receptor (FcRn)-mediated recycling. As another serum half-life extender, the human antibody Fab SL335 that strongly binds HSA was developed. When SL335 was fused to a protein therapeutic, SL335 was shown to prolong the half-life of the drug. Despite the significance of SL335-HSA binding in the extension of drug circulation time, it remains unclear how SL335 interacts with HSA at a molecular structural level. To reveal the structural basis of HSA recognition by SL335, we determined the crystal structure of the SL335-HSA complex at a resolution of 2.95 Å. SL335 binds HSA at a 1:1 stoichiometry. SL335 uses the exposed loops of its heavy and light chains to specifically recognize the IIa and IIb subdomains of HSA. The SL335 epitope is located on the opposite side of the FcRn-binding site and does not overlap with it, suggesting that SL335 extends the serum half-lives of itself and its fusion partner through an FcRn-dependent recycling mechanism.


Assuntos
Anticorpos/imunologia , Fragmentos Fab das Imunoglobulinas/imunologia , Proteínas Recombinantes/sangue , Proteínas Recombinantes/uso terapêutico , Albumina Sérica Humana/química , Albumina Sérica Humana/imunologia , Anticorpos/química , Reações Cruzadas , Meia-Vida , Antígenos de Histocompatibilidade Classe I/metabolismo , Humanos , Fragmentos Fab das Imunoglobulinas/química , Modelos Moleculares , Ligação Proteica , Receptores Fc/metabolismo
12.
Nucleic Acids Res ; 48(10): 5281-5293, 2020 06 04.
Artigo em Inglês | MEDLINE | ID: mdl-32347936

RESUMO

Gene silencing by RNA interference (RNAi) has emerged as a powerful treatment strategy across a potentially broad range of diseases. Tailoring siRNAs to silence genes vital for cancer cell growth and function could be an effective treatment, but there are several challenges which must be overcome to enable their use as a therapeutic modality, among which efficient and selective delivery to cancer cells remains paramount. Attempts to use antibodies for siRNA delivery have been reported but these strategies use either nonspecific conjugation resulting in mixtures, or site-specific methods that require multiple steps, introduction of mutations, or use of enzymes. Here, we report a method to generate antibody-siRNA (1:2) conjugates (ARCs) that are structurally defined and easy to assemble. This ARC platform is based on engineered dual variable domain (DVD) antibodies containing a natural uniquely reactive lysine residue for site-specific conjugation to ß-lactam linker-functionalized siRNA. The conjugation is efficient, does not compromise the affinity of the parental antibody, and utilizes chemically stabilized siRNA. For proof-of-concept, we generated DVD-ARCs targeting various cell surface antigens on multiple myeloma cells for the selective delivery of siRNA targeting ß-catenin (CTNNB1). A set of BCMA-targeting DVD-ARCs at concentrations as low as 10 nM revealed significant CTNNB1 mRNA and protein knockdown.


Assuntos
Região Variável de Imunoglobulina/química , Interferência de RNA , RNA Interferente Pequeno/química , Anticorpos/química , Linhagem Celular Tumoral , Humanos , RNA Interferente Pequeno/farmacocinética , beta Catenina/genética
13.
PLoS Comput Biol ; 16(4): e1007779, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32339164

RESUMO

Antibodies are capable of potently and specifically binding individual antigens and, in some cases, disrupting their functions. The key challenge in generating antibody-based inhibitors is the lack of fundamental information relating sequences of antibodies to their unique properties as inhibitors. We develop a pipeline, Antibody Sequence Analysis Pipeline using Statistical testing and Machine Learning (ASAP-SML), to identify features that distinguish one set of antibody sequences from antibody sequences in a reference set. The pipeline extracts feature fingerprints from sequences. The fingerprints represent germline, CDR canonical structure, isoelectric point and frequent positional motifs. Machine learning and statistical significance testing techniques are applied to antibody sequences and extracted feature fingerprints to identify distinguishing feature values and combinations thereof. To demonstrate how it works, we applied the pipeline on sets of antibody sequences known to bind or inhibit the activities of matrix metalloproteinases (MMPs), a family of zinc-dependent enzymes that promote cancer progression and undesired inflammation under pathological conditions, against reference datasets that do not bind or inhibit MMPs. ASAP-SML identifies features and combinations of feature values found in the MMP-targeting sets that are distinct from those in the reference sets.


Assuntos
Anticorpos , Biologia Computacional/métodos , Aprendizado de Máquina , Análise de Sequência de Proteína/métodos , Software , Algoritmos , Anticorpos/química , Anticorpos/metabolismo , Bases de Dados de Proteínas , Humanos , Inibidores de Metaloproteinases de Matriz/química , Inibidores de Metaloproteinases de Matriz/metabolismo , Metaloproteinases da Matriz/química , Metaloproteinases da Matriz/metabolismo
14.
J Nanobiotechnology ; 18(1): 65, 2020 Apr 28.
Artigo em Inglês | MEDLINE | ID: mdl-32345308

RESUMO

Nanoparticle based gene delivery systems holds great promise. Superparamagnetic iron oxide nanoparticles (SPIONs) are being heavily investigated due to good biocompatibility and added diagnostic potential, rendering such nanoparticles theranostic. Yet, commonly used cationic coatings for efficient delivery of such anionic cargos, results in significant toxicity limiting translation of the technology to the clinic. Here, we describe a highly biocompatible, small and non-cationic SPION-based theranostic nanoparticles as novel gene therapy agents. We propose for the first-time, the usage of the microRNA machinery RISC complex component Argonaute 2 (AGO2) protein as a microRNA stabilizing agent and a delivery vehicle. In this study, AGO2 protein-conjugated, anti-HER2 antibody-linked and fluorophore-tagged SPION nanoparticles were developed (SP-AH nanoparticles) and used as a carrier for an autophagy inhibitory microRNA, MIR376B. These functionalized nanoparticles selectively delivered an effective amount of the microRNA into HER2-positive breast cancer cell lines in vitro and in a xenograft nude mice model of breast cancer in vivo, and successfully blocked autophagy. Furthermore, combination of the chemotherapy agent cisplatin with MIR376B-loaded SP-AH nanoparticles increased the efficacy of the anti-cancer treatment both in vitro in cells and in vivo in the nude mice. Therefore, we propose that AGO2 protein conjugated SPIONs are a new class of theranostic nanoparticles and can be efficiently used as innovative, non-cationic, non-toxic gene therapy tools for targeted therapy of cancer.


Assuntos
Proteínas Argonauta/química , Autofagia , Materiais Biocompatíveis/uso terapêutico , Neoplasias da Mama/tratamento farmacológico , Nanopartículas de Magnetita/química , MicroRNAs/metabolismo , Animais , Anticorpos/química , Anticorpos/imunologia , Antineoplásicos/química , Antineoplásicos/uso terapêutico , Autofagia/efeitos dos fármacos , Proteína Beclina-1/genética , Proteína Beclina-1/metabolismo , Materiais Biocompatíveis/química , Materiais Biocompatíveis/farmacologia , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Cisplatino/química , Cisplatino/uso terapêutico , Feminino , Humanos , Camundongos , Camundongos Nus , MicroRNAs/química , Receptor ErbB-2/imunologia , Transplante Heterólogo
15.
Chemistry ; 26(38): 8400-8406, 2020 Jul 08.
Artigo em Inglês | MEDLINE | ID: mdl-32240571

RESUMO

One of the main problems in the development of immunosensors is to overcome the complexity of binding antibodies to the sensor surface. Most immobilizing methods lead to a random orientation of antibodies with a lower binding site density and immunoaffinity. In order to control the orientation of antibody immobilization, several resorc[4]arene derivatives were designed and synthesized. After the spectroscopic characterization of resorc[4]arene self-assembled monolayers (SAMs) onto gold films, the surface coverage and the orientation of insulin antibody (Ab-Ins) were assessed by a surface plasmon resonance (SPR) technique and compared with a random immobilization method. Experimental results combined with theoretical studies confirmed the dipole-dipole interaction as an important factor in antibody orientation and demonstrated the importance of the upper rim functionalization of resorcarenes. Accordingly, resorcarene 5 showed a major binding force towards Ab-Ins thanks to the H-bond interactions with the amine protein groups. Based on these findings, the resorcarene-based immunosensor is a powerful system with improved sensitivity providing new insight into sensor development.


Assuntos
Anticorpos Imobilizados/química , Anticorpos/química , Ouro/química , Ressonância de Plasmônio de Superfície/métodos , Sítios de Ligação
16.
Org Biomol Chem ; 18(15): 2886-2892, 2020 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-32236230

RESUMO

Terminal α-2,6-sialylation of N-glycans is a humanized glycosylation that affects the properties and efficacy of therapeutic glycoproteins. Fc di-sialylation (a biantennary N-glycan with two α-2,6-linked sialic acids) of IgG antibodies imparts them with enhanced anti-inflammatory activity and other roles. However, the microheterogeneity of N-glycoforms presents a challenge for therapeutic development. Therefore, controlled sialylation has drawn considerable attention, but direct access to well-defined di-sialylated antibodies remains limited. Herein, a one-pot three-enzyme protocol was developed by engineering a bacterial sialyltransferase to facilitate the modification of therapeutic antibodies with N-acetylneuraminic acid or its derivatives towards optimized glycosylation. To overcome the low proficiency of bacterial sialyltransferase in antibody remodeling, the Photobacterium sp. JT-ISH-224 α-2,6-sialyltransferase (Psp2,6ST) was genetically engineered by terminal truncation and site-directed mutagenesis based on its protein crystal structure. With the optimized reaction conditions and using activity-based screening of various Psp2,6ST variants, a truncated mutant Psp2,6ST (111-511)-His6 A235M/A366G was shown to effectively improve the catalytic efficiency of antibody di-sialylation. Herceptin and the donor substrate promiscuity allow the introduction of bioorthogonal modifications of N-acetylneuraminic acid into antibodies for site-specific conjugation. 2-AB hydrophilic interaction chromatography analysis of the released N-glycans and intact mass characterization confirmed the high di-sialylation of Herceptin via the optimized one-pot three-enzyme reaction. This study established a versatile enzymatic approach for producing highly di-sialylated IgG antibodies. It provides new insights into engineering bacterial sialyltransferase for adaptation to the enzymatic glycoengineering of therapeutic antibodies and the glycosite-specific conjugation of antibodies.


Assuntos
Anticorpos/metabolismo , Photobacterium/enzimologia , Engenharia de Proteínas , Ácidos Siálicos/metabolismo , Sialiltransferases/metabolismo , Anticorpos/química , Sialiltransferases/genética
17.
PLoS One ; 15(4): e0231781, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32302363

RESUMO

The mushroom poison that causes the most deaths is the class of toxins known as amatoxins. Current methods to sensitively and selectively detect these toxins are limited by the need for expensive equipment, or they lack accuracy due to cross-reactivity with other chemicals found in mushrooms. In this work, we report the development of a competition-based lateral flow immunoassay (LFIA) for the rapid, portable, selective, and sensitive detection of amatoxins. Our assay clearly indicates the presence of 10 ng/mL of α-AMA or γ-AMA and the method including extraction and detection can be completed in approximately 10 minutes. The test can be easily read by eye and has a presumed shelf-life of at least 1 year. From testing 110 wild mushrooms, the LFIA identified 6 out of 6 species that were known to contain amatoxins. Other poisonous mushrooms known not to contain amatoxins tested negative by LFIA. This LFIA can be used to quickly identify amatoxin-containing mushrooms.


Assuntos
Amanita/química , Amanitinas/análise , Imunoensaio/métodos , Amanitinas/química , Anticorpos/química , Ouro/química , Peptídeos/toxicidade , Padrões de Referência
18.
Int J Mol Sci ; 21(6)2020 Mar 13.
Artigo em Inglês | MEDLINE | ID: mdl-32183096

RESUMO

HER3-binding affibody molecules are a promising format for visualization of HER3 expression. Cobalt-55, a positron-emitting isotope, with a half-life of 17.5 h, allows for next-day imaging. We investigated the influence of the charge of the radiocobalt-chelator complex on the biodistribution of anti-HER3 affibody molecule (HE)3-ZHER3 and compared the best radiocobalt-labeled variant with a recently optimized gallium-labeled variant. Affibody conjugates (HE)3-ZHER3-X (X = NOTA, NODAGA, DOTA, DOTAGA) were labeled with [57Co]Co (surrogate for 55Co). Affinity measurements, binding specificity and cellular processing were studied in two HER3-expressing cancer cell lines. Biodistribution was studied 3 and 24 h post-injection (pi) in mice with HER3-expressing BxPC-3 xenografts and compared to [68Ga]Ga-(HE)3-ZHER3-NODAGA. Micro-single-photon emission tomography/computed tomography (microSPECT/CT) and micro-positron emission tomography/computed tomography (microPET/CT) imaging was performed 3 and 24 h pi. Stably labeled conjugates bound to HER3 with subnanomolar affinity. [57Co]Co-(HE)3-ZHER3-DOTA had the best tumor retention and a significantly lower concentration in blood than other conjugates, leading to superior tumor-to-blood and tumor-to-liver ratios 24 h pi. Compared to [68Ga]Ga-(HE)3-ZHER3-NODAGA 3 h pi, [57Co]Co-(HE)3-ZHER3-DOTA provided superior imaging contrast in liver 24 h pi. Concluding, the composition and charge of the [57Co]Co-chelator complex influenced the uptake in tumors and normal tissue. [57Co]Co-(HE)3-ZHER3-DOTA provided the best imaging properties among the cobalt-labeled conjugates. Delayed imaging of HER3 expression with [57Co]Co-(HE)3-ZHER3-DOTA improved imaging contrast compared to early-time-point imaging with [68Ga]Ga-(HE)3-ZHER3-NODAGA.


Assuntos
Radioisótopos de Cobalto/química , Neoplasias Experimentais/diagnóstico por imagem , Tomografia por Emissão de Pósitrons/métodos , Compostos Radiofarmacêuticos/farmacocinética , Receptor ErbB-3/genética , Acetatos/química , Animais , Anticorpos/química , Anticorpos/imunologia , Linhagem Celular Tumoral , Feminino , Compostos Heterocíclicos com 1 Anel/química , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Ligação Proteica , Compostos Radiofarmacêuticos/química , Receptor ErbB-3/imunologia , Receptor ErbB-3/metabolismo , Distribuição Tecidual
19.
Biochemistry ; 59(14): 1420-1427, 2020 04 14.
Artigo em Inglês | MEDLINE | ID: mdl-32212642

RESUMO

Cathepsin B (CTSB) is an abundant cysteine protease that functions in both endolysosomal compartments and extracellular regions. A considerable number of preclinical and clinical studies indicate that CTSB is implicated in many human diseases. Expression levels and activity of CTSB significantly correlate with disease progression and severity. Current inhibitors of CTSB are lack of adequate specificity and pharmacological activities. Through structure-guided rational design, we hereby designed and generated a humanized antibody inhibitor targeting human CTSB. This was achieved by genetically fusing the propeptide of procathepsin B, a naturally occurring inhibitor of CTSB, into heavy chain complementarity-determining region 3 (CDR3H) of Herceptin that is used in the clinic for the treatment of breast cancer. The resulting antibody-propeptide fusion displayed high specificity for inhibiting CTSB proteolytic activity at nanomolar levels. Pharmacokinetic studies in mice revealed a plasma half-life of approximately 42 h for this anti-CTSB antibody inhibitor, comparable to that of the parental Herceptin scaffold. This study demonstrates a new approach for the efficient generation of humanized antibody inhibitors with high potency and specificity for human CTSB, which may be extended to develop antibody inhibitors against other disease relevant cathepsin proteases.


Assuntos
Anticorpos/química , Catepsina B/antagonistas & inibidores , Inibidores Enzimáticos/química , Animais , Anticorpos/administração & dosagem , Anticorpos/genética , Anticorpos/metabolismo , Catepsina B/química , Catepsina B/genética , Catepsina B/metabolismo , Desenho de Fármacos , Inibidores Enzimáticos/administração & dosagem , Inibidores Enzimáticos/metabolismo , Feminino , Humanos , Camundongos , Neoplasias/tratamento farmacológico , Neoplasias/enzimologia , Domínios Proteicos
20.
Nat Commun ; 11(1): 1598, 2020 03 27.
Artigo em Inglês | MEDLINE | ID: mdl-32221310

RESUMO

We propose the concept of universal fiducials based on a set of pre-made semi-synthetic antibodies (sABs) generated by customized phage display selections against the fusion protein BRIL, an engineered variant of apocytochrome b562a. These sABs can bind to BRIL fused either into the loops or termini of different GPCRs, ion channels, receptors and transporters without disrupting their structure. A crystal structure of BRIL in complex with an affinity-matured sAB (BAG2) that bound to all systems tested delineates the footprint of interaction. Negative stain and cryoEM data of several examples of BRIL-membrane protein chimera highlight the effectiveness of the sABs as universal fiducial marks. Taken together with a cryoEM structure of sAB bound human nicotinic acetylcholine receptor, this work demonstrates that these anti-BRIL sABs can greatly enhance the particle properties leading to improved cryoEM outcomes, especially for challenging membrane proteins.


Assuntos
Anticorpos/farmacologia , Microscopia Crioeletrônica/métodos , Proteínas de Membrana/química , Anticorpos/química , Membrana Celular/metabolismo , Técnicas de Visualização da Superfície Celular , Cristalografia por Raios X , Humanos , Proteínas de Membrana/efeitos dos fármacos , Proteínas de Membrana/metabolismo , Modelos Moleculares , Polímeros , Propilaminas , Ligação Proteica , Conformação Proteica
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