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1.
Chinese Journal of Biotechnology ; (12): 4124-4133, 2021.
Article in Chinese | WPRIM | ID: wpr-921493

ABSTRACT

The existence of cancer stem cells is regarded as the major cause for therapeutic resistance and relapse of a variety of cancer types including hepatocellular carcinoma (HCC). However, the tracing of such a subpopulation in vivo has been challenging. We have previously demonstrated that the isoform 5 of the voltage-gated calcium channel α2δ1 subunit, which can be recognized specifically by a monoclonal antibody 1B50-1, is a bona fide surface marker for HCC stem cells. Here we developed a strategy for optical imaging of α2δ1-positive cells by using a fusion protein containing the single chain variable fragment (scFv) of Mab1B50-1 and the luciferase NanoLuc which was tagged with Flag in the C-terminal. The scFv of Mab1B50-1 was fused to the N-terminal of NanoLucFlag using overlap PCR, and the recombinant fragment, which was named as 1B50-1scFv-NanoLucFlag, was subsequently cloned into a eukaryotic expression vector. The resulting construct was transfected into FreeStyle 293F cells in suspension using PEI reagent. The expression of the fusion protein was identified as a protein with molecular weight about 50 kDa by Western blotting. After purification by ANTI-FLAG® M2 affinity chromatography, 1B50-1scFv-NanoLucFlag was demonstrated to bind to α2δ1 positive cells specifically with a Kd value of (18.62±1.84) nmol/L. Furthermore, a strong luciferase activity of 1B50-1scFv-NanoLucFlag was detected in α2δ1 positive cells following incubation with the fusion protein, indicating that the presence of α2δ1 could be quantified using this fusion protein. Hence, 1B50-1scFv-NanoLucFlag provides a potential tool for optical imaging of α2δ1 positive cancer stem cells both in vitro and in vivo.


Subject(s)
Carcinoma, Hepatocellular , Humans , Liver Neoplasms , Neoplastic Stem Cells , Recombinant Proteins/genetics , Single-Chain Antibodies/genetics
2.
Chinese Journal of Biotechnology ; (12): 2870-2877, 2021.
Article in Chinese | WPRIM | ID: wpr-887849

ABSTRACT

Asthma is a common respiratory disease that affects 300 million of people worldwide, posing a serious health risk and medical burden. Development of new anti-asthmatic drugs and alternative treatment regimens is therefore encouraged. Recent studies have shown that Epidermal Growth Factor Receptor (EGFR) is involved in asthma development. In order to construct nanoparticles targeting EGFR for asthma treatment, a single chain antibody fragment (scFv) against EGFR was genetically engineered and modified at the N-terminal end of the human ferritin H-chain (FTH1) to construct Anti EGFR scFv::FTH1/FTH1 nanoparticles. Transmission electron microscopy showed that the nanoparticles were self-assembled into hollow cage-like structures with the particle size of about 12 nm. Semi-quantitative analysis of the purified nanoparticles by SDS-PAGE revealed the mass ratio of FTH1 to Anti EGFR scFv::FTH1 was 7:3. In House Dust Mite (HDM) driven models, Anti EGFR scFv::FTH1/FTH1 nanoparticles efficiently attenuated several key features of asthma, including goblet cell hyperplasia, mucous metaplasia and subepithelial fibrosis, showing the potential of using ferritin based nanoparticle for asthma treatment.


Subject(s)
Asthma/drug therapy , Ferritins , Humans , Nanoparticles , Oxidoreductases , Single-Chain Antibodies/genetics
3.
São Paulo; s.n; s.n; 2018. 71 p. graf, tab.
Thesis in Portuguese | LILACS | ID: biblio-996644

ABSTRACT

Anticorpos são moléculas de grande interesse científico e farmacêutico, principalmente, devido a sua alta especificidade contra antígenos determinados. Atualmente, anticorpos monoclonais estão entre os medicamentos (biofármacos) mais vendidos do mundo. São utilizados para o tratamento das mais diversas doenças, como câncer, retinopatias, doenças inflamatórias e do sistema imune, entre outras. Nos últimos 30 anos, as tecnologias para a obtenção de anticorpos monoclonais evoluíram muito, desde a tecnologia do hibridoma, até os processos de humanização de anticorpos murinos. Entre os métodos mais utilizados para a produção de anticorpos humanos, destaca-se a tecnologia do Phage Display. Nesta técnica, os genes que codificam as regiões variáveis de imunoglobulinas são inseridos no genoma de um bacteriófago, resultando na produção de partículas virais híbridas que contém fragmentos de anticorpos em fusão com uma das proteínas do capsídeo viral. Neste trabalho, desenvolvemos novos vetores para a apresentação de fragmentos ScFv em fusão com duas proteínas das proteínas do capsídeo viral, a pIII e pVIII. Os oligonucleotídeos utilizados para amplificar os genes de imunoglobulinas foram redesenhados e para minimizar a perda do repertório durante a produção da biblioteca, avaliamos em bancos de dados enzimas de restrição que não apresentam sítios de restrição nas sequencias gênicas. Esses sítios de restrição foram utilizados para construir as regiões de clonagem do vetor Phagemid. Outra etapa crítica na produção de bibliotecas de anticorpos é a reação do PCR de overlap, que pode restringir a diversidade de anticorpos e resultar na produção de amplicons codificando anticorpos truncados. Por isso, nossos vetores foram desenhados para permitir a clonagem direta das regiões variáveis das imunoglobulinas humanas ou murinas, sem a necessidade do PCR de overlap. Nossa expectativa, é que estes novos reagentes serão mais efetivos para a produção de novas bibliotecas de anticorpos pelo sistema do Phage Display


Antibodies are molecules of great scientific and pharmaceutical interest, mainly because of their high specificity against certain antigens. Currently, monoclonal antibodies are among the best selling drugs (biopharmaceuticals) in the world. They are used for the treatment of the most diverse disorders, such as cancer, retinopathies, inflammatory and immune system diseases, among others. In the past 30 years, technologies for obtaining monoclonal antibodies has greatly evolved from hybridoma technology to the humanization processes of murine antibodies. Among the methods used for the production of human antibodies, the technology of Phage Display stands out. In this technique, the genes encoding the immunoglobulin variable regions are inserted into the genome of a bacteriophage, resulting in the production of hybrid virus particles which contain fragments of antibodies in fusion with one of the viral capsid proteins. In this work, we developed new vectors for the presentation of ScFv fragments in fusion with two proteins of viral capsid proteins, pIII and pVIII. The oligonucleotides used to amplify the immunoglobulin genes were redesigned and to minimize repertory loss during library production, we evaluated restriction enzymes in databases that lack restriction sites in the gene sequences. These restriction sites were used to construct the cloning regions of the Phagemid vector. Another critical step in the production of antibody libraries is the overlap PCR reaction, which may restrict the diversity of antibodies and result in the production of amplicons encoding truncated antibodies. Therefore, our vectors were designed to allow the direct cloning of human or murine Immunoglobulins variable regions without the need for overlap PCR. Our expectation is that these new reagents will be more effective for the production of new antibody libraries by the Phage Display system


Subject(s)
Pharmaceutical Preparations , Cell Surface Display Techniques/instrumentation , Antibodies, Monoclonal/analysis , Immunoglobulins/classification , Single-Chain Antibodies
4.
Chinese Journal of Biotechnology ; (12): 2016-2024, 2018.
Article in Chinese | WPRIM | ID: wpr-771407

ABSTRACT

To screen the specific anti-human intercellular adhesion molecule-1 (ICAM-1) single chain fragment variable (scFv) using phage display library technology and to identify its biological activity. P1 peptide was used as antigen, and the phage antibodies against human ICAM-1 antigen were panned by four binding-eluting-amplifying cycles using Tomlinson I+J phage display library. After four rounds of selective enrichment screening, the positive clones were determined by PCR, enzyme linked immunosorbent assay (ELISA)-based antigenic cross reaction and Dot blotting. Then the binding specificity and biological activity of purified scFv were identified by Western blotting, competitive ELISA and cell adhesion inhibition assay respectively. Furthermore, four positive clones were first panned through P1 peptide coated-ELISA assay, and then J-A1 was obtained and identified by PCR, ELISA-based antigenic cross reaction and Dot blotting, which could show a specific binding between P1 peptide and human ICAM-1 protein antigen. Subsequently, the purified scFv showed a satisfactory specificity and anti-adhesive activity in competitive ELISA and the cell adhesion inhibition assay. The specific anti-human ICAM-1 scFv was prepared successfully from Tomlinson I+J phage display library, which pave the way for further application of anti-human ICAM-1 scFv for inflammation diseases therapeutics.


Subject(s)
Antibodies , Enzyme-Linked Immunosorbent Assay , Humans , Immunoglobulin Variable Region , Intercellular Adhesion Molecule-1 , Allergy and Immunology , Peptide Library , Single-Chain Antibodies
5.
Electron. j. biotechnol ; 29: 57-62, sept. 2017. ilus, graf, tab
Article in English | LILACS | ID: biblio-1017244

ABSTRACT

Background: Hydatid disease is a serious parasitic disease threatening public health. Because of its rarity in non-endemic coastal areas, determining the nature and origin of a chronic, enlarged liver cystic mass is challenging in these regions. Under these circumstances, physicians need a confirmatory diagnostic tool beyond immunological and radiological examinations. This study investigated a novel human single-chain fragment variable (scFv) antibody for the confirmative diagnosis of 18 atypical hydatid disease cases in non-endemic coastal areas. Results: A scFv antibody against cystic echinococcosis was produced by genetic engineering and then applied to the immunohistochemical diagnosis of 18 cases of cystic echinococcosis presented in non-endemic coastal areas. The diagnosis of these cases by ultrasound and serum-based examinations was inconclusive. The 750 bp scFv antibody gene was expressed in COS-7 cells, and the antibody localized in the cytoplasm. The scFv antibody can detect the germinal layer and protoscolices of actively growing cysts but not of the degenerating protoscolices and has a diagnostic efficiency higher than that of single serum or ultrasound testing (P b 0.05). The combined use of scFv antibodies with serology and ultrasound diagnostics results in a diagnostic efficiency comparable to that of surgery. The scFv antibody can be used as a confirmatory test for the diagnosis of hydatid disease in non-endemic areas, providing a beneficial supplementary diagnostic method that complements traditional immune testing and ultrasonic radiology and thus helping physicians to effectively differentiate hydatid disease.


Subject(s)
Humans , Male , Female , Middle Aged , Echinococcosis/diagnosis , Echinococcosis, Hepatic/diagnosis , Single-Chain Antibodies/chemistry , Immunoassay , Serologic Tests , Immunohistochemistry , COS Cells , Echinococcosis/diagnostic imaging , Echinococcosis, Hepatic/diagnostic imaging
6.
Electron. j. biotechnol ; 28: 14-19, July. 2017. ilus, graf
Article in English | LILACS | ID: biblio-1015726

ABSTRACT

Background: Gain-of-function of fibroblast growth factor receptor 3 (FGFR3) is involved in the pathogenesis of many tumors. More and more studies have focused on the potential usage of therapeutic single-chain Fv (ScFv) antibodies against FGFR3. RNA interference (RNAi) has been considered as a promising therapeutic method against cancer. A tool which can deliver small interference RNAs (siRNAs) into FGFR3 positive cancer cells is very promising for anti-tumor therapy. Results: In this study, a novel fusion protein R3P, which consists of FGFR3-ScFv and protamine, was generated in Escherichia coli by inclusion body expression strategy and Ni-NTA chromatography. Its yield reached 10 mg per liter of bacterial culture and its purity was shown to be higher than 95%. 1 µg of R3P could efficiently bind to about 2.5 pmol siRNAs and deliver siRNAs into FGFR3 positive RT112 and K562 cells. Annexin V staining results showed that R3P can deliver the amplified breast cancer 1 (AIB1) siRNAs to induce RT112 cell apoptosis. Conclusion: These results indicated that R3P was a promising carrier tool to deliver siRNAs into FGFR3 positive cancer cells and to exert anti-tumor effect.


Subject(s)
Urinary Bladder Neoplasms/metabolism , Recombinant Fusion Proteins/metabolism , Single-Chain Antibodies/metabolism , Recombinant Fusion Proteins/genetics , Protamines/metabolism , Inclusion Bodies , Cloning, Molecular , Apoptosis , RNA, Small Interfering , Escherichia coli/metabolism , Receptor, Fibroblast Growth Factor, Type 3 , Single-Chain Antibodies/isolation & purification , Single-Chain Antibodies/genetics , Flow Cytometry
7.
Electron. j. biotechnol ; 28: 52-57, July. 2017. ilus, graf, tab
Article in English | LILACS | ID: biblio-1015847

ABSTRACT

Background: Gardnerella vaginalis is a bacterial vaginosis (BV)-associated vaginal bacterium that produces the toxin vaginolysin (VLY). VLY is a pore-forming toxin that is suggested to be the main virulence factor of G. vaginalis. The high recurrence rate of BV and the emergence of antibiotic-resistant bacterial species demonstrate the need for the development of recombinant antibodies as novel therapeutic agents for disease treatment. Single-chain variable fragments (scFvs) generated against VLY exhibited reduced efficacy to neutralize VLY activity compared to the respective full-length antibodies. To improve the properties of scFvs, monospecific dimeric scFvs were generated by the genetic fusion of two anti-VLY scFv molecules connected by an alpha-helix-forming peptide linker. Results: N-terminal hexahistidine-tagged dimeric scFvs were constructed and produced in Escherichia coli and purified using metal chelate affinity chromatography. Inhibition of VLY-mediated human erythrocyte lysis by dimeric and monomeric scFvs was detected by in vitro hemolytic assay. The circulating half-life of purified scFvs in the blood plasma of mice was determined by ELISA. Dimeric anti-VLY scFvs showed higher neutralizing potency and extended circulating half-life than parental monomeric scFv. Conclusions: The protein obtained by the genetic fusion of two anti-VLY scFvs into a dimeric molecule exhibited improved properties in comparison with monomeric scFv. This new recombinant antibody might implement new possibilities for the prophylaxis and treatment of the diseases caused by the bacteria G. vaginalis.


Subject(s)
Animals , Mice , Bacterial Proteins/immunology , Bacterial Toxins/immunology , Antibodies, Neutralizing/metabolism , Single-Chain Antibodies/metabolism , Bacterial Proteins/toxicity , Bacterial Toxins/toxicity , Enzyme-Linked Immunosorbent Assay , Gardnerella vaginalis , Vaginosis, Bacterial , Dimerization , Virulence Factors , Gene Fusion , Antibodies, Neutralizing/immunology , Single-Chain Antibodies/immunology , Half-Life
8.
Braz. j. microbiol ; 48(3): 419-426, July-Sept. 2017. tab, graf
Article in English | LILACS | ID: biblio-889135

ABSTRACT

Abstract Antibodies and antibody fragments are nowadays among the most important biotechnological products, and Pichia pastoris is one of the most important vectors to produce them as well as other recombinant proteins. The conditions to effectively cultivate a P. pastoris strain previously genetically modified to produce the single-chain variable fragment anti low density lipoprotein (-) under the control of the alcohol oxidase promoter have been investigated in this study. In particular, it was evaluated if, and eventually how, the carbon source (glucose or glycerol) used in the preculture preceding cryopreservation in 20% glycerol influences both cell and antibody fragment productions either in flasks or in bioreactor. Although in flasks the volumetric productivity of the antibody fragment secreted by cells precultured, cryopreserved and reactivated in glycerol was 42.9% higher compared with cells precultured in glucose, the use of glycerol in bioreactor led to a remarkable shortening of the lag phase, thereby increasing it by no less than thrice compared to flasks. These results are quite promising in comparison with those reported in the literature for possible future industrial applications of this cultivation, taking into account that the overall process time was reduced by around 8 h.


Subject(s)
Pichia/metabolism , Industrial Microbiology/methods , Carbon/metabolism , Single-Chain Antibodies/biosynthesis , Antibodies/metabolism , Pichia/growth & development , Pichia/genetics , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Culture Media/metabolism , Culture Media/chemistry , Single-Chain Antibodies/genetics , Fermentation , Glycerol/metabolism , Lipoproteins, LDL/immunology , Antibodies/genetics
9.
Protein & Cell ; (12): 573-589, 2017.
Article in English | WPRIM | ID: wpr-756986

ABSTRACT

Chimeric antigen receptor (CAR) T cell therapy is a promising cancer treatment that has recently been undergoing rapid development. However, there are still some major challenges, including precise tumor targeting to avoid off-target or "on-target/off-tumor" toxicity, adequate T cell infiltration and migration to solid tumors and T cell proliferation and persistence across the physical and biochemical barriers of solid tumors. In this review, we focus on the primary challenges and strategies to design safe and effective CAR T cells, including using novel cutting-edge technologies for CAR and vector designs to increase both the safety and efficacy, further T cell modification to overcome the tumor-associated immune suppression, and using gene editing technologies to generate universal CAR T cells. All these efforts promote the development and evolution of CAR T cell therapy and move toward our ultimate goal-curing cancer with high safety, high efficacy, and low cost.


Subject(s)
Cell Movement , Allergy and Immunology , Cell Proliferation , Gene Expression , Genetic Vectors , Chemistry , Metabolism , Humans , Immunotherapy, Adoptive , Methods , Lymphocyte Activation , Lymphocytes, Tumor-Infiltrating , Cell Biology , Allergy and Immunology , Transplantation , Neoplasms , Genetics , Allergy and Immunology , Pathology , Therapeutics , Patient Safety , Receptors, Antigen, T-Cell , Chemistry , Genetics , Allergy and Immunology , Recombinant Fusion Proteins , Chemistry , Genetics , Allergy and Immunology , Signal Transduction , Single-Chain Antibodies , Chemistry , Genetics , T-Lymphocytes , Cell Biology , Allergy and Immunology , Transplantation , Treatment Outcome
10.
Article in English | WPRIM | ID: wpr-194442

ABSTRACT

Phage display technology provides a powerful tool to screen a library for a binding molecule via an enrichment process. It has been adopted as a critical technology in the development of therapeutic antibodies. However, a major drawback of phage display technology is that because the degree of the enrichment cannot be controlled during the bio-panning process, it frequently results in a limited number of clones. In this study, we applied next-generation sequencing (NGS) to screen clones from a library and determine whether a greater number of clones can be identified using NGS than using conventional methods. Three chicken immune single-chain variable fragment (scFv) libraries were subjected to bio-panning on prostate-specific antigen (PSA). Phagemid DNA prepared from the original libraries as well as from the Escherichia coli pool after each round of bio-panning was analyzed using NGS, and the heavy chain complementarity-determining region 3 (HCDR3) sequences of the scFv clones were determined. Subsequently, through two-step linker PCR and cloning, the entire scFv gene was retrieved and analyzed for its reactivity to PSA in a phage enzyme immunoassay. After four rounds of bio-panning, the conventional colony screening method was performed for comparison. The scFv clones retrieved from NGS analysis included all clones identified by the conventional colony screening method as well as many additional clones. The enrichment of the HCDR3 sequence throughout the bio-panning process was a positive predictive factor for the selection of PSA-reactive scFv clones.


Subject(s)
Antibodies , Bacteriophages , Chickens , Clone Cells , Cloning, Organism , Complementarity Determining Regions , DNA , Escherichia coli , Immunoenzyme Techniques , Mass Screening , Methods , Polymerase Chain Reaction , Prostate-Specific Antigen , Single-Chain Antibodies
11.
São Paulo; s.n; s.n; 2016. 273 p. ilus, tab, graf.
Thesis in Portuguese | LILACS | ID: biblio-881874

ABSTRACT

As doenças cardiovasculares são a principal causa de mortalidade no mundo. A aterosclerose é a base fisiopatológica dessas doenças, sendo definida como um processo crônico-inflamatório multifatorial, resultando da interação de diferentes células como linfócitos, macrófagos, células endoteliais e células musculares lisas na parede arterial. A lipoproteína de baixa densidade eletronegativa [LDL(-)], uma subfração modificada da LDL nativa, desempenha um papel-chave na aterosclerose, uma vez que as modificações sofridas por esta partícula são capazes de induzir o acúmulo de ésteres de colesterol em macrófagos e a subsequente formação de células espumosas. O sistema imunológico é crucial no processo aterogênico e estratégias terapêuticas direcionadas à imunoregulação deste processo têm sido utilizadas como novas alternativas tanto na prevenção do desenvolvimento quanto da progressão desta doença. Dentre essas estratégias, destaca-se o uso de fragmentos de anticorpos como o scFv (do inglês, single chain fragment variable), que podem ainda estar conjugados a nanopartículas com o intuito de aumentar sua eficiência de ação no organismo. Diante do papel da LDL(-) na aterosclerose, este projeto objetivou avaliar os efeitos in vitro e in vivo de um sistema nanoestruturado contendo fragmentos scFv anti-LDL(-) derivatizados na superfície de nanocápsulas sobre macrófagos murinos e humanos primários e em camundongos knockout para o gene do receptor da LDL (Ldlr-/-) no desenvolvimento e na progressão dessa doença. Demonstrou-se que o tratamento de macrófagos com a formulação scFv anti-LDL(-)-MCMN-Zn diminuiu de forma significativa a captação de LDL(-), assim como a expressão de IL-1ß (mRNA e proteína) e MCP-1 (mRNA). Foi demonstrada a internalização da nanoformulação pelos macrófagos via diferentes mecanismos de endocitose, demonstrando seu potencial uso como carreador de fármacos. In vivo, a nanoformulação diminuiu de forma significativa a área da lesão aterosclerótica em camundongos Ldlr-/- submetidos à avaliação pela técnica de tomografia por emissão de pósitrons (do inglês, PET), utilizando o radiotraçador 18F-FDG (18F-desoxiglicose), associada à tomografia computadorizada (CT) com agente de contraste iodado, além da análise morfométrica das lesões no arco aórtico. O conjunto dos resultados obtidos evidenciou a ação ateroprotetora da formulação scFv anti-LDL(-)-MCMN-Zn, reforçando seu potencial como estratégia terapêutica na aterosclerose


Cardiovascular diseases are the leading cause of mortality worldwide. Atherosclerosis is the pathophysiological basis of these diseases, defined as a chronic inflammatory multifactorial process, resulting from the interaction of several cells such as lymphocytes macrophages, endothelial cells and smooth muscle cells within the arterial wall. The electronegative low-density lipoprotein [LDL(-)], a modified subfraction of native LDL, plays a key role in atherosclerosis, since its modifications are capable of inducing the accumulation of cholesteryl esters in macrophages and the subsequent foam cells formation. The immune system is crucial in atherogenic process and therapeutic strategies directed to the immunoregulation of this process have been used as a new alternative in the prevention of the development as well as the progression of this disease. Among these strategies, it is the use of antibody fragments such as scFv (single chain fragment variable), which may be also conjugated to nanoparticles in order to increase their efficiency in the body. Given the role of LDL(-) in atherosclerosis, the aim of this project was to evaluate the in vitro and in vivo effects of a nanostructured system containing scFv anti-LDL(-) fragments derivatized on the surface of nanocapsules on murine and human primary macrophages and in the development and progression of the disease in LDL receptor knockout mice (Ldlr-/-). It was demonstrated that the treatment of macrophages with scFv anti-LDL(-)-MCMN-Zn formulation significantly decreases the uptake of LDL(-) and the expression IL-1ß (mRNA and protein) and MCP-1 (mRNA). Moreover, the internalization of the nanoformulation by macrophages through different endocytosis mechanisms was shown, demonstrating its potential use as a nanocarrier. In vivo, the nanoformulation decreased the area of atherosclerotic lesions in Ldlr-/- mice evaluated by positron emission tomography with 18F-FDG associated with computed tomography with iodinated contrast agent (PET/CT), besides the lesion morphometric analysis at the aortic arch Thus, these data provide evidence of the atheroprotection action of the ateroprotection action of the scFv anti-LDL(-)-MCMN-Zn formulation, suggesting its promising use as a therapeutic strategy for atherosclerosis


Subject(s)
Animals , Male , Arteriosclerosis/pathology , Nanocapsules/statistics & numerical data , Single-Chain Antibodies/analysis , Flow Cytometry/methods , Low Density Lipoprotein Receptor-Related Protein-1/analysis , Microscopy, Confocal/instrumentation , Positron Emission Tomography Computed Tomography/methods
12.
Electron. j. biotechnol ; 18(4): 302-306, July 2015. ilus
Article in English | LILACS | ID: lil-757868

ABSTRACT

Background Overexpression or mutated activation of Fibroblast growth factor receptor 3 (FGFR3) is involved in the pathogenesis of many tumors. More and more studies focus on the potential usage of therapeutic antibodies against FGFR3. Results In this study, a novel single-chain Fv (ScFv) against FGFR3 was prepared and characterized. To achieve the soluble expression, ScFv was fused with Sumo (Small ubiquitin-related modifier) by polymerase chain reaction (PCR), and cloned into pET-20b. The recombinant bacteria were induced by 0.5 mM Isopropyl-ß-d-thiogalactopyranoside (IPTG) for 16 h at 20°C, and the supernatant liquid of Sumo-ScFv was harvested and purified by Ni-NTA chromatography. After being cleaved by the Sumo protease, the recombinant ScFv was released from the fusion protein, and further purified by Ni-NTA chromatography. The purity of ScFv was shown to be higher than 95% and their yield reached 4 mg per liter of bacterial culture. In vitro data showed that ScFv can significantly attenuate FGF9-induced phosphorylation of FGFR3. Conclusion We provide a novel method to produce soluble expression and bioactive functions of ScFv in Escherichia coli.


Subject(s)
Receptor, Fibroblast Growth Factor, Type 3/metabolism , Single-Chain Antibodies/isolation & purification , Single-Chain Antibodies/metabolism , Solubility , Mass Spectrometry , Recombinant Proteins , Blotting, Western , Escherichia coli
13.
Chinese Journal of Biotechnology ; (12): 722-733, 2015.
Article in Chinese | WPRIM | ID: wpr-240605

ABSTRACT

To enhance the specificity of anti-TNF-α single chain Fv antibody (TNF-scFv) to inflamed site, we constructed a bispecific antibody BsDb that targets TNF-α and ED-B-containing fibronectin (B-FN) by covalently linking TNF-scFv and the anti-ED-B scFv L19 at the gene level via a flexible peptide linker deriving from human serum albumin. BsDb was successfully secreted from Pichia pastoris as functional protein, identified by immunoblotting, and purified to homogeneity with affinity chromatography. BsDb retained the immunoreactivity of its original antibodies TNF-scFv and L19, and showed a marked gain in antigen-binding affinity and in TNF-α-neutralizing ability, when compared to TNF-scFv and L19 that were produced in Escherichia coli. In the adjuvant-induced arthritis (AIA) mice model, BsDb showed selective accumulation and retention in the inflamed paws but rapid clearance from blood, resulting in high arthritic paw to blood ratios. These data indicate that BsDb is endowed with high specificity to inflamed site and low toxicity to normal tissues and holds great potential for in vivo application for the targeted therapy of RA and other chronic inflammatory diseases.


Subject(s)
Animals , Antibodies, Bispecific , Allergy and Immunology , Antibodies, Neutralizing , Allergy and Immunology , Escherichia coli , Fibronectins , Chemistry , Allergy and Immunology , Humans , Mice , Single-Chain Antibodies , Allergy and Immunology , Tumor Necrosis Factor-alpha , Allergy and Immunology
14.
Journal of Experimental Hematology ; (6): 1272-1276, 2015.
Article in Chinese | WPRIM | ID: wpr-274052

ABSTRACT

<p><b>OBJECTIVE</b>To clone the variable region genes of human anti-IL1RAP (IL-1 receptor accessory protein) monoclonal antibodies (McAb) and to construct IL1RAP chimeric antigen receptors (CARs).</p><p><b>METHODS</b>The VH and VL DNA of IL1RAP single chain antibodies were amplified by RACE and overlap extension PCR from total RNA extracted from 3H6E10 and 10D8A7 hybridoma and ligated into specific IL1RAP single-chain variable fragments (scFv). CD8α transmembrane domain, CD137 intracellular domain, TCR ζ chain, human CD8α signal peptide and scFv-anti-IL1RAP were cloned into plasmid LV-lac. Recombinant lentiviruses were generated by co-transfection of recombinant plasmid LV-lac, pMD2. G, and psPAX2 helper vectors into 293FT packing cells.</p><p><b>RESULTS</b>The VH and VL genes of 2 human anti-IL1RAP McAb were acquired. The 3H6E10 VH and VL genes consisted of 402 bp and 393 bp encoding 134 and 131 aminoacid residues, respectively; 10D8A7 VH and VL genes consisted of 423 bp and 381 bp encoding 141 and 127 amine acid residues, respectively. Recombinant expression vertors LV-3H6E10 scFv-ICD and LV-10D8A7 scFv-ICD (ICD: CD8α transmembrane domain-CD137 intracellular domain-TCR ζ chain) were constructed. The target fragments were demonstrated by sequencing analysis. Recombinant plasmids were transfected into 293FT cells and lentiviral particles were acquired.</p><p><b>CONCLUSION</b>Human anti-IL1RAP recombinant receptors are constructed successfully and lay a good foundation for the construction of IL1RAP-CAR killer T cell vaccine.</p>


Subject(s)
Antibodies, Monoclonal , Genetics , Cloning, Molecular , Genetic Vectors , Humans , Hybridomas , Immunoglobulin Variable Region , Genetics , Interleukin-1 Receptor Accessory Protein , Allergy and Immunology , Plasmids , Polymerase Chain Reaction , Receptors, Antigen , Genetics , Single-Chain Antibodies
15.
Braz. j. med. biol. res ; 47(7): 540-547, 07/2014. tab, graf
Article in English | LILACS | ID: lil-712968

ABSTRACT

Intercellular adhesion molecule-1 (ICAM-1) is an important factor in the progression of inflammatory responses in vivo. To develop a new anti-inflammatory drug to block the biological activity of ICAM-1, we produced a monoclonal antibody (Ka=4.19×10−8 M) against human ICAM-1. The anti-ICAM-1 single-chain variable antibody fragment (scFv) was expressed at a high level as inclusion bodies in Escherichia coli. We refolded the scFv (Ka=2.35×10−7 M) by ion-exchange chromatography, dialysis, and dilution. The results showed that column chromatography refolding by high-performance Q Sepharose had remarkable advantages over conventional dilution and dialysis methods. Furthermore, the anti-ICAM-1 scFv yield of about 60 mg/L was higher with this method. The purity of the final product was greater than 90%, as shown by denaturing gel electrophoresis. Enzyme-linked immunosorbent assay, cell culture, and animal experiments were used to assess the immunological properties and biological activities of the renatured scFv.


Subject(s)
Animals , Female , Humans , Male , Mice , Gene Expression/physiology , Immunoglobulin Fragments/biosynthesis , Intercellular Adhesion Molecule-1/immunology , Protein Refolding , Protein Renaturation , Single-Chain Antibodies/biosynthesis , Antigen-Antibody Complex , Anti-Inflammatory Agents/pharmacology , Antibodies, Monoclonal/biosynthesis , Cell Adhesion , Chromatography , Dialysis , Enzyme-Linked Immunosorbent Assay , Ear Auricle/drug effects , Escherichia coli/genetics , Genetic Vectors , Immunoglobulin Fragments/pharmacology , Inclusion Bodies/metabolism , Intercellular Adhesion Molecule-1/drug effects , Leukocytes, Mononuclear/metabolism , Plasmids , Protein Engineering/methods , Single-Chain Antibodies/pharmacology , Xylenes/pharmacology
16.
Article in English | WPRIM | ID: wpr-819674

ABSTRACT

OBJECTIVE@#To investigate the inhibitory effect of humanized anti-VEGFR-2 ScFv-As2O3-stealth nanoparticles conjugate on growth of human hepatocellular carcinoma both in vitro and in vivo, which may be a potential agents with sensitivity and targeting ability for human hepatocellular cancer.@*METHODS@#Humanized anti-VEGFR-2 ScFv-As2O3-stealth nanoparticles conjugate was previously constructed using ribosome display technology and antibody conjugate technology. In this combined in vitro and in vivo study, the inhibitory effects of anti-VEGFR-2 ScFv-As2O3-stealth nanoparticles conjugate on tumor growth, invasion, and metastasis was observed with human liver carcinoma cell line Bel7402 and normal cell L02 by MTT assay, Tanswell assay, Hochest33258 staining, and DNA ladder analysis. The anticancer activity and distribution of anti-VEGFR-2 ScFv-As2O3-stealth nanoparticles was then verified in a mouse model of Bel7402 xenografts.@*RESULTS@#Anti-VEGFR-2 ScFv-As2O3-stealth nanoparticles significantly inhibited the proliferation of Bel7402 in the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay while had almost no effects on L02 cells. And the apoptosis inducing effects were proved by Hochest33258 staining and DNA ladder analysis. Transwell assay found that the drug also inhibited the metastasis ability of tumor cells. Furthermore, anti-VEGFR-2 ScFv-As2O3-stealth nanoparticles significantly delayed the growth of Bel7402 xenografts after administration (92.9%), followed by As2O3-stealth nanoparticles, anti-VEGFR-2 ScFv, and As2O3 (61.4%, 58.8%, 20.5%, P<0.05). The concentration of As2O3 in anti-VEGFR-2 ScFv-As2O3-stealth nanoparticles group was more selectively.@*CONCLUSIONS@#Anti-VEGFR-2 ScFv-As2O3-stealth nanoparticles is a potent and selective anti-hepatocellular carcinoma agent which could inhibit the growth of liver cancer as a targeting agent both in vitro and in vivo and also significantly inhibit angiogenesis.


Subject(s)
Animals , Antineoplastic Agents , Chemistry , Pharmacokinetics , Pharmacology , Apoptosis , Arsenic Trioxide , Arsenicals , Chemistry , Pharmacokinetics , Pharmacology , Cell Line, Tumor , Cell Movement , Cell Proliferation , Drug Delivery Systems , Humans , Liver Neoplasms , Liver Neoplasms, Experimental , Mice , Microvessels , Nanoparticles , Chemistry , Metabolism , Neovascularization, Pathologic , Pathology , Oxides , Chemistry , Pharmacokinetics , Pharmacology , Single-Chain Antibodies , Chemistry , Metabolism , Vascular Endothelial Growth Factor Receptor-2 , Metabolism
17.
Chinese Journal of Hepatology ; (12): 843-848, 2014.
Article in Chinese | WPRIM | ID: wpr-337094

ABSTRACT

<p><b>OBJECTIVE</b>To construct a single-chain human anti-EGFR antibody (scFv) and truncated protamine (tP) fusion protein, ScFv/tP, carrying small interfering (si)RNA directed against the heat shock protein Hsp47, a collagen-binding glycoprotein, in order to evaluate the role Hsp47 in apoptosis of hepatic stellate cells.</p><p><b>METHODS</b>A single chain of the human variable fragment was obtained by phage display and fused with the tP gene and with or without (negative control) the Hsp47 siRNA sequences. Following expression and purification of the scFv/tP fusion protein and the scFv/tPHsp47 siRNA fusion protein, internalization capabilities were tested in isolated human hepatic stellate cells and the QSG-7701 human hepatocyte cells with visualization by immunofluorescent staining. The DNA binding ability of the fusion proteins were verified by gel shift assay.Following ScFv/tP-Hsp47 siRNA fusion protein transfection into the human hepatic stellate cells, the levels of Hsp47 mRNA and protein expression were tested by RT-PCR and Western blotting; in addition, effects of siRNA-mediated silencing of Hsp47 on cell proliferation and apoptosis were analyzed by the cell counting kit (CCK)-8, flow cytometry and Western blot detection of the apoptosis marker poly (ADP-ribose) polymerase (PARP).</p><p><b>RESULTS</b>Indirect immunofluorescence revealed that the ScFv/tP fusion proteins were internalized into human hepatic stellate cells but not into the QSG-7701 cells.The ScFv/tP-Hsp47 siRNA fusion protein caused reduced expression of Hsp47 mRNA and protein expression in the human hepatic stellate cells, as well as increased the cells' apoptosis remarkably.</p><p><b>CONCLUSION</b>The ScFv/tP fusion protein can be used as a transfection reagent to deliver Hsp47 siRNA into hepatic stellate cells and to mediate apoptosis via blockade of Hsp47 expression.</p>


Subject(s)
Apoptosis , Cell Proliferation , HSP47 Heat-Shock Proteins , Genetics , Hepatic Stellate Cells , Cell Biology , Humans , Protamines , Metabolism , RNA, Messenger , RNA, Small Interfering , ErbB Receptors , Allergy and Immunology , Single-Chain Antibodies , Transfection
18.
National Journal of Andrology ; (12): 1063-1067, 2014.
Article in Chinese | WPRIM | ID: wpr-319567

ABSTRACT

<p><b>OBJECTIVE</b>To construct, express and purify human fusion proteins composed of a single-chain antibody fragment scFv that recognizes the prostate specific membrane antigen (PSMA) protein, Fdt, HA2 and tp, and to analyze the binding activity of the expressed fusion proteins.</p><p><b>METHODS</b>The fusion protein genes scFv, scFv-tp, and scFv-Fdt-HA2-tp were amplified by PCR, and the genes obtained were then cloned into the expression vector pET28 and expressed in E. coli BL21. The expressed products were identified by SDS-PAGE and Western blot and purified with Ni(2+)-NTA chelating agarose. The antigen-binding activity of the fusion proteins was determined by ELISA.</p><p><b>RESULTS</b>The human anti-PSMA fusion gene was successfully constructed and expressed in M15 as the inclusion body after induced with IPTG. All the target proteins expressed could bind the PSMA antigen.</p><p><b>CONCLUSION</b>Fusion proteins can specifically bind the PSMA antigen. This finding contributes to the study of the targeted delivery of siRNA.</p>


Subject(s)
Antigens, Surface , Allergy and Immunology , Cloning, Molecular , Electrophoresis, Polyacrylamide Gel , Escherichia coli , Genetics , Allergy and Immunology , Glutamate Carboxypeptidase II , Allergy and Immunology , Humans , Male , Polymerase Chain Reaction , RNA, Small Interfering , Allergy and Immunology , Recombinant Fusion Proteins , Genetics , Allergy and Immunology , Single-Chain Antibodies , Genetics , Allergy and Immunology
19.
Acta Pharmaceutica Sinica ; (12): 1665-1673, 2014.
Article in Chinese | WPRIM | ID: wpr-251838

ABSTRACT

In order to enhance the specificity of TNF-α monoclonal antibody to inflamed site, a bispecific antibody BsDb that targets TNF-α and the extra-domain B (ED-B) of fibronectin (FN) was constructed by covalently linking the anti-TNF-α single chain Fv antibody (TNF-scFv) and the anti-ED-B scFv L19 via a flexible peptide linker deriving from human serum albumin (HSA). ED-B is an antigen specifically expressed at the inflamed site. BsDb is expressed in E. coli, identified by immunoblot, and purified with affinity chromatography. This was followed by further examination of its bioactivities and pharmacokinetics. We demonstrated that BsDb retained the immunoreactivity of its original antibodies as it could simultaneously bind to TNF-α and ED-B and neutralize the biological action of TNF-α. In the collagen-induced arthritis mice model, BsDb selectively accumulate in the inflamed joint with a maximal uptake of (12.2 ± 1.50)% ID/g in a single inflamed paw and retain in the inflamed paw for at least 72 h. In contrast, BsDb showed a short serum half-life of (0.50 ± 0.05) h and a rapid clearance from normal tissues. The findings reported herein indicate that BsDb has good specificity to the inflamed site and low toxicity to normal tissues. BsDb is therefore likely to have greater clinical applications in the treatment of rheumatoid arthritis and other autoimmune diseases. This laid a stable basis for its preclinical study.


Subject(s)
Animals , Antibodies, Bispecific , Chemistry , Antibodies, Monoclonal , Chemistry , Arthritis, Experimental , Escherichia coli , Fibronectins , Chemistry , Half-Life , Humans , Mice , Single-Chain Antibodies , Chemistry , Tumor Necrosis Factor-alpha , Chemistry
20.
Article in Chinese | WPRIM | ID: wpr-251684

ABSTRACT

<p><b>OBJECTIVE</b>To construct human phage single-chain antibody (scFv) library against breast cancer, and to identify anti-HER2 specific antibodies from the human phage display scFv library to offer a stronger affinity sequence targeting HER2 for fusion protein targeting HER2 and CXCR4.</p><p><b>METHODS</b>Total RNA was extracted from the adjacent lymphatic tissue harvested from breast cancer patients. The variable regions of the whole antibody were amplified by using RT-PCR and were cloned into the vector pCANTAB-5E through a linker. The products were electroporated into competent E.coli TG1 cells. Recombinant phages specific for breast cancer cells were enriched in SKBR-3 after four rounds. The antigen-positive clones were selected by ELISA and immunohistochemistry.</p><p><b>RESULTS</b>The fragment of VH and VL were about 375 and 330 bp and were linked in vitro to form scFv of 750 bp that was resistant to the breast cancer HER2 single strand. A fusion phage display library that contained total of 2.48×10(8) pfu /ml was established. ELISA and immunohistochemical results confirmed that the antibody has a strong affinity with HER2 antigen in breast cancer tissue. Compared to human IgG antibody, a scFv phage library against human breast cancer was successfully constructed with high capacity. The scFv was highly specific to HER2 antigen and the sequencing results indicated that VL and VH genes were highly homologous with the variable region of human antibody.</p><p><b>CONCLUSION</b>This strategy may achieve new targeted antibody resistant to the breast cancer for clinical treatment and provide a carrier that uses HER2 as a target of the fusion protein for anti-tumor therapy.</p>


Subject(s)
Breast Neoplasms , Genetics , Allergy and Immunology , Female , Humans , Peptide Library , Receptor, ErbB-2 , Allergy and Immunology , Single-Chain Antibodies , Allergy and Immunology
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