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1.
PLoS One ; 15(1): e0226928, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31914456

RESUMO

Secreted R-spondin1-4 proteins (RSPO1-4) orchestrate stem cell renewal and tissue homeostasis by potentiating Wnt/ß-catenin signaling. RSPOs induce the turnover of negative Wnt regulators RNF43 and ZNRF3 through a process that requires RSPO interactions with Leucine-rich repeat-containing G-protein coupled receptors (LGRs), or through an LGR-independent mechanism that is enhanced by RSPO binding to heparin sulfate proteoglycans (HSPGs). Here, we describe the engineering of 'surrogate RSPOs' that function independently of LGRs to potentiate Wnt signaling on cell types expressing a target surface marker. These bispecific proteins were generated by fusing an RNF43- or ZNRF3-specific single chain antibody variable fragment (scFv) to the immune cytokine IL-2. Surrogate RSPOs mimic the function of natural RSPOs by crosslinking the extracellular domain (ECD) of RNF43 or ZNRF3 to the ECD of the IL-2 receptor CD25, which sequesters the complex and results in highly selective amplification of Wnt signaling on CD25+ cells. Furthermore, surrogate RSPOs were able substitute for wild type RSPO in a colon organoid growth assay when intestinal stem cells were transduced to express CD25. Our results provide proof-of-concept for a technology that may be adapted for use on a broad range of cell- or tissue-types and will open new avenues for the development of Wnt-based therapeutics for regenerative medicine.


Assuntos
Colo/crescimento & desenvolvimento , Anticorpos de Cadeia Única/metabolismo , Trombospondinas/metabolismo , Via de Sinalização Wnt , Sítios de Ligação , Colo/metabolismo , Células HEK293 , Humanos , Subunidade alfa de Receptor de Interleucina-2/metabolismo , Técnicas de Cultura de Órgãos , Especificidade de Órgãos , Ubiquitina-Proteína Ligases/química , Ubiquitina-Proteína Ligases/imunologia
2.
Nat Commun ; 11(1): 283, 2020 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-31941907

RESUMO

Chimeric antigen receptor (CAR)-expressing T cells targeting B-cell maturation antigen (BCMA) have activity against multiple myeloma, but improvements in anti-BCMA CARs are needed. We demonstrated recipient anti-CAR T-cell responses against a murine single-chain variable fragment (scFv) used clinically in anti-BCMA CARs. To bypass potential anti-CAR immunogenicity and to reduce CAR binding domain size, here we designed CARs with antigen-recognition domains consisting of only a fully human heavy-chain variable domain without a light-chain domain. A CAR designated FHVH33-CD8BBZ contains a fully human heavy-chain variable domain (FHVH) plus 4-1BB and CD3ζ domains. T cells expressing FHVH33-CD8BBZ exhibit similar cytokine release, degranulation, and mouse tumor eradication as a CAR that is identical except for substitution of a scFv for FHVH33. Inclusion of 4-1BB is critical for reducing activation-induced cell death and promoting survival of T cells expressing FHVH33-containing CARs. Our results indicate that heavy-chain-only anti-BCMA CARs are suitable for evaluation in a clinical trial.


Assuntos
Cadeias Pesadas de Imunoglobulinas/metabolismo , Receptores de Antígenos Quiméricos/genética , Receptores de Antígenos Quiméricos/imunologia , Linfócitos T/imunologia , Animais , Antígeno de Maturação de Linfócitos B/imunologia , Linhagem Celular Tumoral , Sobrevivência Celular , Células Cultivadas , Humanos , Leucócitos Mononucleares/imunologia , Camundongos , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/imunologia , Domínios Proteicos/genética , Domínios Proteicos/imunologia , Engenharia de Proteínas , Receptores de Antígenos Quiméricos/metabolismo , Receptores de Fator de Crescimento Neural/genética , Receptores de Fator de Crescimento Neural/imunologia , Anticorpos de Cadeia Única/genética , Anticorpos de Cadeia Única/imunologia , Anticorpos de Cadeia Única/metabolismo
3.
Food Chem Toxicol ; 135: 110973, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31738983

RESUMO

The construction, expression and functional analysis of codon-optimized single-chain variable fragment (coscFv) against clenbuterol (CBL) prepared from the Escherichia coli system is described. First, the ionic concentration for coscFv expression was optimized through single-factor experiments. Then, the extraction conditions of inclusion bodies were optimized, and coscFv was affinity-purified. Finally, the functional analysis of coscFv was elucidated by indirect competitive enzyme-linked immunosorbent assay (icELISA) and molecular docking. After optimizing the ionic concentration, the yield of coscFv increased from 21.69% to 23.26%. The molecular weight of coscFv was determined to be approximately 27 kDa according to the SDS-PAGE and Western blot assay. The percentage of coscFv was as high as 43.9% after the inclusion bodies were extracted, washed, and dissolved. Functional analysis indicated that the coscFv recognized CBL, and the 50% inhibition average concentration of CBL (IC50) was 4.22 ± 0.01 (n = 3) ng/mL. The binding site between coscFv and CBL consisted of Asp33H, Met34H, Ser50H, Arg52H, Tyr57H, Leu59H, Asp99H, and Tyr93L. Our study confirms that coscFv can bind with CBL through the key amino acid residues and can be used to sensitively detect CBL.


Assuntos
Agonistas Adrenérgicos beta/imunologia , Clembuterol/imunologia , Anticorpos de Cadeia Única/imunologia , Agonistas Adrenérgicos beta/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Clembuterol/metabolismo , Clonagem Molecular/métodos , Ensaio de Imunoadsorção Enzimática , Escherichia coli/genética , Simulação de Acoplamento Molecular , Ligação Proteica , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Anticorpos de Cadeia Única/genética , Anticorpos de Cadeia Única/metabolismo
4.
PLoS One ; 14(12): e0226245, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31887144

RESUMO

Antibody therapies for Alzheimer's Disease (AD) hold promise but have been limited by the inability of these proteins to migrate efficiently across the blood brain barrier (BBB). Central nervous system (CNS) gene transfer by vectors like adeno-associated virus (AAV) overcome this barrier by allowing the bodies' own cells to produce the therapeutic protein, but previous studies using this method to target amyloid-ß have shown success only with truncated single chain antibodies (Abs) lacking an Fc domain. The Fc region mediates effector function and enhances antigen clearance from the brain by neonatal Fc receptor (FcRn)-mediated reverse transcytosis and is therefore desirable to include for such treatments. Here, we show that single chain Abs fused to an Fc domain retaining FcRn binding, but lacking Fc gamma receptor (FcγR) binding, termed a silent scFv-IgG, can be expressed and released into the CNS following gene transfer with AAV. While expression of canonical IgG in the brain led to signs of neurotoxicity, this modified Ab was efficiently secreted from neuronal cells and retained target specificity. Steady state levels in the brain exceeded peak levels obtained by intravenous injection of IgG. AAV-mediated expression of this scFv-IgG reduced cortical and hippocampal plaque load in a transgenic mouse model of progressive ß-amyloid plaque accumulation. These findings suggest that CNS gene delivery of a silent anti-Aß scFv-IgG was well-tolerated, durably expressed and functional in a relevant disease model, demonstrating the potential of this modality for the treatment of Alzheimer's disease.


Assuntos
Doença de Alzheimer/terapia , Sistema Nervoso Central/metabolismo , Vetores Genéticos/administração & dosagem , Fragmentos Fc das Imunoglobulinas/genética , Anticorpos de Cadeia Única/genética , Doença de Alzheimer/genética , Animais , Barreira Hematoencefálica , Linhagem Celular , Dependovirus/genética , Modelos Animais de Doenças , Progressão da Doença , Terapia Genética , Antígenos de Histocompatibilidade Classe I/metabolismo , Humanos , Fragmentos Fc das Imunoglobulinas/química , Fragmentos Fc das Imunoglobulinas/metabolismo , Camundongos , Camundongos Transgênicos , Domínios Proteicos , Receptores Fc/metabolismo , Receptores de IgG/metabolismo , Anticorpos de Cadeia Única/química , Anticorpos de Cadeia Única/metabolismo
5.
Life Sci ; 239: 117052, 2019 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-31733318

RESUMO

AIMS: A combination of biomarker and instrument technology diagnosis methods, especially antigen-targeted imaging methods, is required to increase the accuracy of the diagnosis of cancer. Currently, the targeting efficiency is limited by the conjugation methods used for the conjugation of antibodies and imaging materials. Here, a simple strategy for the conjugation of a probe and a single-chain fragment antibody (scFv) that does not change the characteristics of the antibody was shown. MAIN METHODS: An ScFv was conjugated with superparamagnetic iron oxide (SPIO) or indocyanine green (ICG) via a linker by utilizing the reaction between cysteine and maleimide. The characterization of the probe was performed by flow cytometry, confocal imaging, optical imaging and magnetic resonance imaging (MRI). KEY FINDINGS: After conjugation, the scFv retained high affinity, antigen specificity, and strong internalization ability. The application of the conjugated probe was also confirmed by optical imaging and MRI. SIGNIFICANCE: The proposed strategy provides a simple method for the production of high efficiency antigen-targeted imaging probes for tumor diagnosis.


Assuntos
Anticorpos Monoclonais/química , Imagem por Ressonância Magnética/métodos , Anticorpos de Cadeia Única/química , Anticorpos Monoclonais/metabolismo , Linhagem Celular Tumoral , Meios de Contraste , Compostos Férricos/química , Citometria de Fluxo , Humanos , Verde de Indocianina/química , Nanopartículas de Magnetita , Anticorpos de Cadeia Única/metabolismo
6.
Exp Parasitol ; 205: 107737, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31401060

RESUMO

Monoclonal antibodies have a wide range of applications in basic and applied research as well as in the medical and pharmaceutical industries. Phage display antibody libraries offer an alternative to hybridoma technology for the generation of monoclonal antibodies and can be applied to high-throughput screening and facilitate the generation of novel antibodies. Despite their utility in several fields of research there has been limited application of antibody libraries in the study of trematode parasites. Fasciola hepatica causes considerable loss to the agriculture sector and is also a human pathogen. The parasite's excretory/secretory material contains numerous molecules that facilitate its invasion and survival within the mammalian host, including cathepsin B and L proteases. F. hepatica cathepsin B2 is expressed during the initial weeks of infection and has suspected roles in immune evasion and as a digestive enzyme in the parasite's gut; it is considered a good target for vaccination or therapeutic inhibitors. In this study, we produced a single-chain variable fragment (scFv) phage display library from naïve mice. The library was used to identify several scFv that can bind to antigens from adult F. hepatica homogenate, and a scFv that can bind to F. hepatica cathepsin B2. The results highlight the potential applicability of such a library to facilitate the study of F. hepatica and other parasites. This is the first report of the application of a naïve phage display antibody library to the study of F. hepatica.


Assuntos
Anticorpos Anti-Helmínticos/metabolismo , Antígenos de Helmintos/imunologia , Fasciola hepatica/imunologia , Anticorpos de Cadeia Única/metabolismo , Animais , Anticorpos Monoclonais/metabolismo , Western Blotting , Catepsinas/imunologia , Técnicas de Visualização da Superfície Celular , Eletroforese em Gel de Ágar , Eletroforese em Gel de Poliacrilamida , Ensaio de Imunoadsorção Enzimática , Camundongos , Modelos Moleculares , Reação em Cadeia da Polimerase
7.
Cancer Sci ; 110(10): 3079-3088, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31432594

RESUMO

Chimeric antigen receptor-engineered T (CAR-T)-cell therapy holds significant promise for the treatment of hematological malignancies, especially for B-cell leukemia and lymphoma. However, its efficacy against non-hematological malignancies has been limited as a result of several biological problems characteristic of the tumor microenvironment of solid tumors. One of the main hurdles is the heterogeneous nature of tumor-associated antigens (TAA) expressed in solid tumors. Another hurdle is the inefficient activation and limited persistence of CAR-T cells, mainly as a result of T-cell exhaustion caused by immunosuppressive factors in the tumor microenvironment. In the present study, to address these problems, we engineered CAR-T cells to produce antagonistic anti-programmed cell death protein 1 (PD-1) single-chain variable fragment (scFv), by which PD-1-dependent inhibitory signals in CAR-T cells and adjacent tumor-specific non-CAR-T cells are attenuated. In mouse solid tumor models, PD-1 scFv-producing CAR-T cells induced potent therapeutic effects superior to those of conventional CAR-T cells, along with a significant reduction of apoptotic cell death not only in CAR-T cells themselves but also in TAA-specific T cells in the tumor tissue. In addition, the treatment with anti-PD-1 scFv-producing CAR-T cells resulted in an increased concentration of PD-1 scFv in tumor tissue but not in sera, suggesting an induction of less severe systemic immune-related adverse events. Hence, the present study developed anti-PD-1 scFv-producing CAR-T cell technology and explored its cellular mechanisms underlying potent antitumor efficacy.


Assuntos
Imunoterapia Adotiva/métodos , Neoplasias/terapia , Receptor de Morte Celular Programada 1/imunologia , Receptores de Antígenos Quiméricos/metabolismo , Anticorpos de Cadeia Única/metabolismo , Animais , Apoptose , Linhagem Celular Tumoral , Técnicas de Cocultura , Masculino , Camundongos , Neoplasias/imunologia , Linfócitos T/citologia , Linfócitos T/imunologia , Ensaios Antitumorais Modelo de Xenoenxerto
8.
Molecules ; 24(14)2019 Jul 18.
Artigo em Inglês | MEDLINE | ID: mdl-31323851

RESUMO

Single-chain Fv (scFv) antibodies are recombinant proteins in which the variable regions of the heavy chain (VH) and light chain (VL) are connected by a short flexible polypeptide linker. ScFvs have the advantages of easy genetic manipulation and low-cost production using Escherichia coli compared with monoclonal antibodies, and are thus expected to be utilized as next-generation medical antibodies. However, the practical use of scFvs has been limited due to low homogeneity caused by their aggregation propensity mediated by inter-chain VH-VL interactions. Because the interactions between the VH and VL domains of antibodies are generally weak, individual scFvs are assumed to be in equilibrium between a closed state and an open state, in which the VH and VL domains are assembled and disassembled, respectively. This dynamic feature of scFvs triggers the formation of dimer, trimer, and larger aggregates caused by the inter-chain VH-VL interactions. To overcome this problem, the N-terminus and C-terminus were herein connected by sortase A-mediated ligation to produce a cyclic scFv. Open-closed dynamics and aggregation were markedly suppressed in the cyclic scFv, as judged from dynamic light scattering and high-speed atomic force microscopy analyses. Surface plasmon resonance and differential scanning fluorometry analysis revealed that neither the affinity for antigen nor the thermal stability was disrupted by the scFv cyclization. Generality was confirmed by applying the present method to several scFv proteins. Based on these results, cyclic scFvs are expected to be widely utilized in industrial and therapeutic applications.


Assuntos
Cadeias Pesadas de Imunoglobulinas/química , Cadeias Leves de Imunoglobulina/química , Região Variável de Imunoglobulina/química , Anticorpos de Cadeia Única/química , Varredura Diferencial de Calorimetria , Ciclização , Difusão Dinâmica da Luz , Humanos , Cadeias Pesadas de Imunoglobulinas/metabolismo , Cadeias Leves de Imunoglobulina/metabolismo , Região Variável de Imunoglobulina/metabolismo , Microscopia de Força Atômica , Agregados Proteicos , Ligação Proteica , Anticorpos de Cadeia Única/metabolismo , Ressonância de Plasmônio de Superfície
9.
Nat Commun ; 10(1): 2947, 2019 07 03.
Artigo em Inglês | MEDLINE | ID: mdl-31270320

RESUMO

To expand the toolbox of imaging in living cells, we have engineered a single-chain variable fragment binding the linear HA epitope with high affinity and specificity in vivo. The resulting probe, called the HA frankenbody, can light up in multiple colors HA-tagged nuclear, cytoplasmic, membrane, and mitochondrial proteins in diverse cell types. The HA frankenbody also enables state-of-the-art single-molecule experiments in living cells, which we demonstrate by tracking single HA-tagged histones in U2OS cells and single mRNA translation dynamics in both U2OS cells and neurons. Together with the SunTag, we also track two mRNA species simultaneously to demonstrate comparative single-molecule studies of translation can now be done with genetically encoded tools alone. Finally, we use the HA frankenbody to precisely quantify the expression of HA-tagged proteins in developing zebrafish embryos. The versatility of the HA frankenbody makes it a powerful tool for imaging protein dynamics in vivo.


Assuntos
Epitopos/metabolismo , Sondas Moleculares/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Imagem Individual de Molécula , Animais , Linhagem Celular Tumoral , Embrião não Mamífero/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Neurônios/metabolismo , Biossíntese de Proteínas , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas Recombinantes de Fusão/isolamento & purificação , Anticorpos de Cadeia Única/metabolismo , Coloração e Rotulagem , Peixe-Zebra/embriologia
10.
Nat Commun ; 10(1): 2681, 2019 06 18.
Artigo em Inglês | MEDLINE | ID: mdl-31213606

RESUMO

Although chimeric antigen receptor (CAR) T cell therapies have demonstrated considerable success in treating hematologic malignancies, they have simultaneously been plagued by a cytokine release syndrome (CRS) that can harm or even kill the cancer patient. We describe a CAR T cell strategy in which CAR T cell activation and cancer cell killing can be sensitively regulated by adjusting the dose of a low molecular weight adapter that must bridge between the CAR T cell and cancer cell to initiate tumor eradication. By controlling the concentration and dosing schedule of adapter administration, we document two methods that can rapidly terminate (<3 h) a pre-existing CRS-like toxicity and two unrelated methods that can pre-emptively prevent a CRS-like toxicity that would have otherwise occurred. Because all four methods concurrently enhance CAR T cell potency, we conclude that proper use of bispecific adapters could potentially avoid a life-threatening CRS while enhancing CAR T cell tumoricidal activity.


Assuntos
Doenças do Sistema Imunitário/prevenção & controle , Imunoterapia Adotiva/efeitos adversos , Neoplasias/terapia , Receptores de Antígenos Quiméricos/imunologia , Linfócitos T/imunologia , Animais , Engenharia Celular/métodos , Linhagem Celular Tumoral , Citocinas/imunologia , Fluoresceína/metabolismo , Receptores de Folato com Âncoras de GPI/metabolismo , Ácido Fólico/metabolismo , Humanos , Doenças do Sistema Imunitário/etiologia , Imunoterapia Adotiva/métodos , Ativação Linfocitária/imunologia , Camundongos , Neoplasias/imunologia , Receptores de Antígenos Quiméricos/metabolismo , Anticorpos de Cadeia Única/imunologia , Anticorpos de Cadeia Única/metabolismo , Síndrome , Linfócitos T/metabolismo , Linfócitos T/transplante , Ensaios Antitumorais Modelo de Xenoenxerto
11.
BMC Biotechnol ; 19(1): 38, 2019 06 25.
Artigo em Inglês | MEDLINE | ID: mdl-31238939

RESUMO

BACKGROUND: Anti-Tumor Necrosis Factor-alpha therapy has become clinically important for treating inflammatory bowel disease. However, the use of conventional immunotherapy requires a systemic exposure of patients and collateral side effects. Lactic acid bacteria have been shown to be effective as mucosal delivering system for cytokine and single domain antibodies, and it is amenable to clinical purposes. Therefore, lactic acid bacteria may function as vehicles for delivery of therapeutic antibodies molecules to the gastrointestinal tract restricting the pharmacological effect towards the gut. Here, we use the mucosal delivery of Lactococcus lactis carrying an anti-TNFα scFv expression plasmid on a DSS-induced colitis model in mice. RESULTS: Experimental colitis was induced with DSS administered in drinking water. L. lactis carrying the scFv expression vector was introduced by gavage. After four days of treatment, animals showed a significant improvement in histological score and disease activity index compared to those of untreated animals. Moreover, treated mice display IL-6, IL17A, IL1ß, IL10 and FOXP3 mRNA levels similar to health control mice. Therefore, morphological and molecular markers suggest amelioration of the experimentally induced colitis. CONCLUSION: These results provide evidence for the use of this alternative system for delivering therapeutic biopharmaceuticals in loco for treating inflammatory bowel disease, paving the way for a novel low-cost and site-specific biotechnological route for the treatment of inflammatory disorders.


Assuntos
Colite/terapia , Citocinas/metabolismo , Vetores Genéticos/administração & dosagem , Lactococcus lactis/imunologia , Administração Oral , Animais , Anticorpos/genética , Anticorpos/imunologia , Anticorpos/metabolismo , Colite/induzido quimicamente , Colite/imunologia , Citocinas/genética , Citocinas/imunologia , Sulfato de Dextrana , Modelos Animais de Doenças , Vetores Genéticos/genética , Vetores Genéticos/metabolismo , Células HEK293 , Humanos , Lactococcus lactis/genética , Lactococcus lactis/metabolismo , Camundongos Endogâmicos C57BL , Anticorpos de Cadeia Única/genética , Anticorpos de Cadeia Única/imunologia , Anticorpos de Cadeia Única/metabolismo , Fator de Necrose Tumoral alfa/imunologia
12.
Nat Commun ; 10(1): 2301, 2019 05 24.
Artigo em Inglês | MEDLINE | ID: mdl-31127102

RESUMO

Genomic DNA in eukaryotes is organized into chromatin through association with core histones to form nucleosomes, each distinguished by their DNA sequences and histone variants. Here, we used a single-chain antibody fragment (scFv) derived from the anti-nucleosome antibody mAb PL2-6 to stabilize human CENP-A nucleosome containing a native α-satellite DNA and solved its structure by the cryo-electron microscopy (cryo-EM) to 2.6 Å resolution. In comparison, the corresponding cryo-EM structure of the free CENP-A nucleosome could only reach 3.4 Å resolution. We find that scFv binds to a conserved acidic patch on the histone H2A-H2B dimer without perturbing the nucleosome structure. Our results provide an atomic resolution cryo-EM structure of a nucleosome and insight into the structure and function of the CENP-A nucleosome. The scFv approach is applicable to the structural determination of other native-like nucleosomes with distinct DNA sequences.


Assuntos
Proteína Centromérica A/ultraestrutura , DNA Satélite/ultraestrutura , Nucleossomos/ultraestrutura , Proteína Centromérica A/imunologia , Proteína Centromérica A/metabolismo , Microscopia Crioeletrônica , DNA Satélite/metabolismo , Histonas/metabolismo , Histonas/ultraestrutura , Modelos Moleculares , Nucleossomos/metabolismo , Anticorpos de Cadeia Única/imunologia , Anticorpos de Cadeia Única/metabolismo , Anticorpos de Cadeia Única/ultraestrutura
13.
Bioengineered ; 10(1): 162-171, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31088324

RESUMO

Tonsillitis is the inflammation of the tonsils due to infection, many patients ultimately have to undergo tonsillectomy. In order to improve the accuracy of diagnosis and even create a new treatment for tonsillitis, we constructed a prokaryotic expression single-chain antibody fragment library against Streptococcus pneumoniae with immunoglobulin heavy chain variable region (VH), κ light chain (Vκ), and λ light chain (Vλ) genes by using human tonsil tissue. Plasmid DNA sequencing showed that single-chain antibodies were complete and constructed correctly. The binding activity of recombinant clones was detected by enzyme-linked immunosorbent assay (ELISA), results showed that the binding activity and specificity of anti-S. pneumoniae single-chain fragment variable (scfv) is proved to be successful. The single-chain antibody may be an attractive strategy for tonsillitis etiologic diagnosis and therapy.


Assuntos
Tonsila Palatina/citologia , Anticorpos de Cadeia Única/imunologia , Anticorpos de Cadeia Única/metabolismo , Streptococcus pneumoniae/imunologia , Streptococcus pneumoniae/metabolismo , Ensaio de Imunoadsorção Enzimática , Humanos , Plasmídeos/genética
14.
Mol Biol Rep ; 46(3): 3257-3264, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31073913

RESUMO

The aim of this work was to study the effect of the carbon source (glycerol, sucrose, glucose or a sucrose/glucose mixture) on the production of the anti LDL (-) single-chain variable fragment (scFv) by the recombinant Pichia pastoris SMD 1168 strain as well as on the cell size. The use of glucose as a carbon source in the growth phase led to a remarkable increase in cell size compared with glycerol, while the smallest cells were obtained with sucrose likely due to the occurrence of an energetic stress. The scFv concentration seemed to be related to cell number rather than to cell concentration, which in its turn showed no significant dependence on the carbon source. Yeast cells grown on sucrose had a mean diameter (0.736 ± 0.097 µm) about 35% shorter than those grown on glucose and allowed for the highest final concentration of the scFv antibody fragment (93.7 ± 0.2 mg/L). These results demonstrate that sucrose is the best carbon source for the expression of such an antibody fragment by the recombinant P. pastoris strain, which may be very useful for the diagnostic analysis of the so-called "bad cholesterol".


Assuntos
Carbono/metabolismo , LDL-Colesterol/imunologia , Pichia/metabolismo , Tamanho Celular , Fermentação , Expressão Gênica , Glicerol , Pichia/genética , Proteínas Recombinantes/genética , Anticorpos de Cadeia Única/metabolismo
15.
Mol Biol Rep ; 46(4): 4027-4037, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31073914

RESUMO

Mu-2-related death-inducing (MuD) gene is involved in apoptosis in tumor cells. Although we have previously produced mouse monoclonal antibodies (MAbs) that specifically recognize human MuD, the application scope of MuD MAbs was restricted due to their mouse origin. Therefore, we attempted the generation of single-chain variable fragment (scFv) against MuD. The heavy- and light-chain variable region genes from two MuD hybridomas were isolated by PCR and joined by DNA encoding a (Gly4Ser1)3 linker. These scFv fragments were cloned into a phagemid vector and expressed as E-tagged fusion proteins in Escherichia coli HB2151. The reactivity of selected Abs was evaluated using ELISA. Selected MuDscFv Abs specifically recognized human MuD, retaining ~ 50% potency of the parent MAbs. MuDscFv-M3H9 recognized the middle region of MuD, while MuDscFv-C22B3 recognized a broad region. Intracellular expression of MuDscFvs-C22B3 protected cells from TRAIL-induced apoptosis. These MuDscFv Abs may help in the study of intracellular signaling pathway centered on MuD and of drug use target and points.


Assuntos
Proteínas Reguladoras de Apoptose/imunologia , Clonagem Molecular/métodos , Anticorpos de Cadeia Única/biossíntese , Anticorpos de Cadeia Única/metabolismo , Sequência de Aminoácidos/genética , Animais , Anticorpos Monoclonais/imunologia , Especificidade de Anticorpos/imunologia , Proteínas Reguladoras de Apoptose/genética , Ensaio de Imunoadsorção Enzimática , Escherichia coli/genética , Expressão Gênica/genética , Engenharia Genética/métodos , Humanos , Hibridomas/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Proteínas Recombinantes/genética , Anticorpos de Cadeia Única/imunologia
16.
Talanta ; 201: 397-405, 2019 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-31122440

RESUMO

This article reports the identification, engineering and characterisation of recombinant single chain variable fragment (scFv) antibody against Zearalenone (ZEN), an oestrogenic mycotoxin, using phage display antibody technology. To increase the chance of obtaining clones that can bind to free toxin, the conjugated proteins of the target antigen, i.e. bovine serum albumin ZEN-BSA and ovalbumin ZEN-OVA, were switched during the biopanning. One phage-displayed scFv clone specific to free ZEN, designated yZEN2A8, could be isolated. The gene encoding the yZEN2A8 scFv was sub-cloned into the pET-21d (+) and pKP300 delta III vectors to generate the recombinant scFv and scFv-AP antibody formats, respectively. After ELISA optimisation by checkerboard titration, the sensitivities of the recombinant yZEN2A8 scFv antibody and scFv-AP fusion were improved approx. 2 and 60 folds, respectively. Competitive ELISA indicated that the median inhibition concentration (IC50) of recombinant yZEN2A8 scFv antibody and scFv-AP fusion after ELISA optimisation were 90 and 14 ng mL-1, with a limit of detection (LOD) of 20 and 2 ng mL-1, respectively. No cross-reactivity to other common mycotoxins was observed. Homology modelling illustrated specific binding of the recombinant antibody to ZEN and demonstrated the role of complementary determining regions (CDRs) of both the variable heavy and light chains in antibody-antigen interactions. Efficient application of scFv-AP for the detection of ZEN contamination in corns and wheat samples were investigated for the first time. The antibody in the form of scFv-AP can be used as a prototype for the development of a convenient reagent for the detection of ZEN contamination in various format, including biosensor-based.


Assuntos
Ração Animal/análise , Contaminação de Alimentos/análise , Proteínas Recombinantes de Fusão/imunologia , Anticorpos de Cadeia Única/imunologia , Zearalenona/análise , Fosfatase Alcalina/genética , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/imunologia , Técnicas de Visualização da Superfície Celular/métodos , Ensaio de Imunoadsorção Enzimática/métodos , Escherichia coli/genética , Humanos , Simulação de Acoplamento Molecular , Biblioteca de Peptídeos , Ligação Proteica , Coelhos , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Anticorpos de Cadeia Única/biossíntese , Anticorpos de Cadeia Única/genética , Anticorpos de Cadeia Única/metabolismo , Triticum/química , Zea mays/química , Zearalenona/imunologia , Zearalenona/metabolismo
17.
Appl Environ Microbiol ; 85(13)2019 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-31003980

RESUMO

Many recombinant proteins that are produced in Escherichia coli have to be targeted to the periplasm to be functional. N-terminal signal peptides can be used to direct recombinant proteins to the membrane-embedded Sec translocon, a multiprotein complex that translocates proteins across the membrane into the periplasm. We have recently shown that the cotranslational targeting of the single-chain variable antibody fragment BL1 saturates the capacity of the Sec translocon leading to impaired translocation of secretory proteins and protein misfolding/aggregation in the cytoplasm. In turn, protein production yields and biomass formation were low. Here, we study the consequences of targeting BL1 posttranslationally to the Sec translocon. Notably, the posttranslational targeting of BL1 does not saturate the Sec translocon capacity, and both biomass formation and protein production yields are increased. Analyzing the proteome of cells producing the posttranslationally targeted BL1 indicates that the decreased synthesis of endogenous secretory and membrane proteins prevents a saturation of the Sec translocon capacity. Furthermore, in these cells, highly abundant chaperones and proteases can clear misfolded/aggregated proteins from the cytoplasm, thereby improving the fitness of these cells. Thus, the posttranslational targeting of BL1 enables its efficient production in the periplasm due to a favorable adaptation of the E. coli proteome. We envisage that our observations can be used to engineer E. coli for the improved production of recombinant secretory proteins.IMPORTANCE The bacterium Escherichia coli is widely used to produce recombinant proteins. To fold properly, many recombinant proteins have to be targeted to the E. coli periplasm, but so far the impact of the targeting pathway of a recombinant protein to the periplasm has not been extensively investigated. Here, we show that the targeting pathway of a recombinant antibody fragment has a tremendous impact on cell physiology, ultimately affecting protein production yields in the periplasm and biomass formation. This indicates that studying the targeting and secretion of proteins into the periplasm could be used to design strategies to improve recombinant protein production yields.


Assuntos
Escherichia coli/metabolismo , Proteínas Recombinantes/metabolismo , Anticorpos de Cadeia Única/metabolismo , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Periplasma/metabolismo , Transporte Proteico , Proteínas Recombinantes/genética , Canais de Translocação SEC/genética , Canais de Translocação SEC/metabolismo , Anticorpos de Cadeia Única/genética
18.
Comput Biol Chem ; 80: 147-151, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30959270

RESUMO

A computational study of the interaction of cardiac troponin I (cTnI) with its specific antibody and of that antibody with skeletal troponin I (sTnI), the principal interferon of cTnI, is carried out. Computational and simulation tools such as FTSite, FTMap, FTDock and pyDock are used to determine the binding sites of these molecules and to study their interactions and molecular docking performance, allowing us to obtain relevant information related with the antigen-antibody interaction for each of the targets. In the context of the development of immunosensing platforms, this type of computational analysis allows performing a preliminary in-silico affinity study of the available bioreceptors for a better selection when moving to the experimental stage, with the subsequent saving in cost and time.


Assuntos
Anticorpos de Cadeia Única/imunologia , Troponina I/imunologia , Animais , Sítios de Ligação , Galinhas , Simulação de Acoplamento Molecular , Ligação Proteica , Conformação Proteica , Anticorpos de Cadeia Única/metabolismo , Troponina I/classificação , Troponina I/metabolismo
19.
Theranostics ; 9(4): 974-985, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30867810

RESUMO

Epidermal growth factor receptor (EGFR) is a transmembrane cell surface receptor that is frequently overexpressed and/or mutated in many cancers. Therapies targeting EGFR have poor outcomes due to the lack of reliable diagnostic tests to monitor EGFR. Current in vitro EGFR diagnostic methods are invasive, requiring biopsies, which limits tumor sampling and availability. EGFR molecular imaging provides non-invasive whole-body images capable of detecting primary tumors and metastases, which can be used to diagnose and monitor response to therapy. Methods: We evaluated properties of two anti-EGFR fragments, 8708 and 8709, as molecular-targeted imaging probes. 8708 and 8709 are anti-EGFR antigen binding fragments (Fabs) that recognize domain I/II of EGFR, which is distinct from epitopes recognized by current anti-EGFR therapeutic antibodies. We used complementarity determining region sequences from 8708 and 8709 Fabs to generate an anti-EGFR IgG and (scFv)2 and scFv-Fc antibody fragments. We expressed, purified, and labeled the IgG and fragments with IRDye800CW and used them to image EGFR-positive and -negative xenografts in CD-1 nude mice. 8709 scFv-Fc was also tested for competitive binding with the therapeutic anti-EGFR antibody nimotuzumab and for quantifying ratios of EGFR and EGFRvIII deletion mutant. Results: IRDye800CW-labeled 8708 (scFv)2 and 8709 scFv-Fc imaging probes showed high levels of accumulation and good retention in EGFR-positive xenografts, with peak accumulation occurring at 24 and 48 hours post injection, respectively. IRDye680RD-labeled 8709 scFv-Fc did not compete with IRDye800CW-labeled nimotuzumab for EGFR binding as assayed by flow cytometry using an EGFR-positive cell line. IRDye680RD-labeled 8709 scFv-Fc and IRDye800CW-labeled nimotuzumab used in combination were able to determine the ratio of cells expressing EGFR and a deletion mutant EGFRvIII. Conclusion: IRDye800CW-labeled 8708 (scFv)2 and 8709 scFv-Fc had desirable binding affinities, clearance times, and tumor accumulation to be used for imaging in combination with current EGFR targeted therapies. This study highlights the potential for using 8708 (scFv)2 and 8709 scFv-Fc as EGFR diagnostic and therapy monitoring tools.


Assuntos
Receptores ErbB/análise , Corantes Fluorescentes/metabolismo , Xenoenxertos/diagnóstico por imagem , Fragmentos de Imunoglobulinas/metabolismo , Neoplasias/diagnóstico por imagem , Anticorpos de Cadeia Única/metabolismo , Animais , Camundongos Nus , Transplante de Neoplasias , Coloração e Rotulagem , Transplante Heterólogo
20.
MAbs ; 11(4): 632-638, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30898021

RESUMO

The complex molecular formats of recent therapeutic antibodies, including bispecific antibodies, antibody fragments, and other fusion proteins, makes the task of purifying the desired molecules in a limited number of purification steps more and more challenging. Manufacturing these complicated biologics can be substantially improved in the affinity capture stage if the simple bind-and-elute mode is accompanied by targeted removal of the impurities, such as mis-paired antibodies and oligomers or aggregates. Here, we report a method, based on the binding valency to Protein L resin, of separating proteins during the elution step by simply controlling the conductivity at low pH. We show that the method efficiently separated targeted antibodies from mis-paired and aggregated species. Notably, the number of Protein L binding sites can be built into the molecule by design to facilitate the purification. This method may be useful for purifying various antibody formats at laboratory and manufacturing scales.


Assuntos
Anticorpos Biespecíficos/isolamento & purificação , Anticorpos Monoclonais/isolamento & purificação , Proteínas de Bactérias/metabolismo , Cromatografia de Afinidade/métodos , Anticorpos de Cadeia Única/isolamento & purificação , Anticorpos Biespecíficos/metabolismo , Anticorpos Monoclonais/metabolismo , Complexo CD3/imunologia , Condutividade Elétrica , Antígeno HLA-A2/imunologia , Humanos , Concentração de Íons de Hidrogênio , Resinas de Troca Iônica , Ligação Proteica , Engenharia de Proteínas , Receptor ErbB-2/imunologia , Anticorpos de Cadeia Única/metabolismo
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