1.Advances in the study of site-specific antibody-drug conjugates.
Yu SUN ; Rong HUANG ; Bai-wang SUN
Acta Pharmaceutica Sinica 2015;50(10):1225-1231
Antibody drug conjugates (ADCs) are an emerging class of targeted therapeutics with the potential to improve therapeutic index over the traditional chemotherapy. However, it is difficult to control the site and stoichiometry of conjugation in mAb, typically resulting in heterogeneous mixtures of ADCs that are difficult to optimize. New methods for site-specific drug attachment allow development of more homogeneous conjugates and control of the site of drug attachment. In this article, the new literature on development of ADCs and site-specific ADCs is reviewed. In addition, we summarized the various strategies in production of site-specific ADCs.
Antibodies, Monoclonal
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chemistry
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Antibody Specificity
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Binding Sites, Antibody
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Immunoconjugates
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chemistry
2.Progress in shark single-domain antibody.
Chinese Journal of Biotechnology 2020;36(6):1069-1082
Monoclonal antibody (mAb) is an important biological macromolecule and widely used in immune detection, in vitro diagnostics, and drug discovery. However, the inherent properties of mAb restrict its further development, such as high molecular weight and complex structure. Therefore, there is an urgent need to develop alternatives for mAb. Various types of miniaturized antibodies have been developed, among which the variable domain of immunoglobulin new antigen receptor (VNAR) is very attractive. The shark single-domain antibody, also known as shark VNAR, is an antigen-binding domain obtained by genetic engineering technology based on the immunoglobulin new antigen receptor (IgNAR) that naturally exists in selachimorpha. It has a molecular weight of 12 kDa, which is the smallest antigen-binding domain found in the known vertebrates at present. Compared with mAb, the shark VNAR exhibits various superiorities, such as low molecular weight, high affinity, tolerance to the harsh environment, good water solubility, strong tissue penetration, and recognition of the hidden epitopes. It has attracted wide attention in the fields of immunochemical reagents and drug discovery. In this review, various aspects of shark VNAR are elaborated, including the structural and functional characteristics, generating and humanization techniques, affinity maturation strategies, application fields, advantages and disadvantages, and prospects.
Animals
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Antibodies, Monoclonal
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immunology
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Antibodies, Monoclonal, Humanized
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immunology
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Antigens
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Epitopes
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metabolism
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Protein Domains
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immunology
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Receptors, Antigen
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chemistry
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immunology
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Sharks
3.Construction and functional analysis of a bispecific antibody that targets TNF-α and ED-B.
Lu-Jun LI ; Yan-Qun YANG ; Xue-Ping HU ; Mian XIE ; Meng-Yuan LIU
Acta Pharmaceutica Sinica 2014;49(12):1665-1673
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.
Animals
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Antibodies, Bispecific
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chemistry
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Antibodies, Monoclonal
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chemistry
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Arthritis, Experimental
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Escherichia coli
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Fibronectins
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chemistry
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Half-Life
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Humans
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Mice
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Single-Chain Antibodies
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chemistry
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Tumor Necrosis Factor-alpha
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chemistry
4.Preparation of immunomagnetic nanoparticles for tumor targeting therapy with herceptin.
Gui-ping LI ; Kai HUANG ; Hui ZHANG ; Feng LIU ; Xu-jian CHEN
Journal of Southern Medical University 2010;30(11):2539-2542
OBJECTIVETo prepare immunomagnetic nanoparticles (IMNs) for HER2/neu-targeted radioimmunotherapy with herceptin, a humanized anti-p185-HER-2/neu monoclonal antibody targeting the extracellular domain of HER-2/neu receptor.
METHODSThe magnetic nanoparticles were synthesized by partial reductive precipitation method and the surface of the particles was chemically modified using silane coupling agent. Herceptin and histidine were covalently linked to the amine group upon the silica-coated magnetic nanoparticles modified by N-[3-(trimethoxysilyl) propyl]-ethylenediamine using glutaraldehyde method to prepare the IMNs. The nanoparticles were evaluated by diffraction (XRD), scanning electron microscope (SEM) and X- ray energy spectrometry (EDS), and the immunoreactivity of IMN was determined.
RESULTSThe average diameter of the decanoic acid-coated Fe(3)O(4) nanoparticle was about 20 nm with a magnetic saturation of 65 emu/g. The surface amino group was 0.5 µmol/mg after modification with the amid functional group, and the mean size of Herceptin-loaded IMNs was about 60 nm. The IMN retained good immunoreactivity of Herceptin.
CONCLUSIONThe IMNs exhibit good properties for potential application in tumor targeting therapy using Herceptin against HER-2/nue proto-oncogene.
Antibodies, Monoclonal, Humanized ; chemistry ; Drug Carriers ; chemical synthesis ; Magnetics ; Nanoparticles ; chemistry ; Trastuzumab
5.Colloidal gold immunochromatographic test strip for virus detection: a review.
Xuxu DONG ; Wei SUN ; Pan CAO ; Xiaodan LIU
Chinese Journal of Biotechnology 2022;38(9):3243-3254
Colloidal gold immunochromatographic strip is a fast, sensitive and accurate solid-phase labeling detection technology, which has the advantages of low price, easy operation, rapid detection and high specificity, with the potential to qualitatively detect the relevant viruses in a short time with desired sensitivity and accuracy. It effectively addresses the disadvantages of long detection time, equipment inconvenience and professionalism requirement of the traditional detection methods used in the medical, veterinary, animal, plant virus detection, pesticide residue detection and other areas. Presently, the technology has been applied in the detection of bacterial diseases, viral diseases and prevention of extensive spread of infectious diseases, and has sufficient room for further development. This review summarizes the application of colloidal gold immunochromatography strip for biological virus detection, followed by prospecting future perspectives.
Animals
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Antibodies, Monoclonal/chemistry*
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Chromatography, Affinity
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Gold Colloid/chemistry*
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Pesticide Residues
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Sensitivity and Specificity
6.Antibody-drug conjugates: recent advances in conjugation and linker chemistries.
Kyoji TSUCHIKAMA ; Zhiqiang AN
Protein & Cell 2018;9(1):33-46
The antibody-drug conjugate (ADC), a humanized or human monoclonal antibody conjugated with highly cytotoxic small molecules (payloads) through chemical linkers, is a novel therapeutic format and has great potential to make a paradigm shift in cancer chemotherapy. This new antibody-based molecular platform enables selective delivery of a potent cytotoxic payload to target cancer cells, resulting in improved efficacy, reduced systemic toxicity, and preferable pharmacokinetics (PK)/pharmacodynamics (PD) and biodistribution compared to traditional chemotherapy. Boosted by the successes of FDA-approved Adcetris and Kadcyla, this drug class has been rapidly growing along with about 60 ADCs currently in clinical trials. In this article, we briefly review molecular aspects of each component (the antibody, payload, and linker) of ADCs, and then mainly discuss traditional and new technologies of the conjugation and linker chemistries for successful construction of clinically effective ADCs. Current efforts in the conjugation and linker chemistries will provide greater insights into molecular design and strategies for clinically effective ADCs from medicinal chemistry and pharmacology standpoints. The development of site-specific conjugation methodologies for constructing homogeneous ADCs is an especially promising path to improving ADC design, which will open the way for novel cancer therapeutics.
Amino Acids
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metabolism
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Animals
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Antibodies, Monoclonal
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chemistry
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metabolism
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Antigens
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metabolism
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Genetic Engineering
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Humans
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Immunoconjugates
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chemistry
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metabolism
7.Preparation and properties of SiO2 tubes immobilized antibody for HCAg detection.
Li XIE ; Yueping GUAN ; Ying GE ; Hongbo SHI
Chinese Journal of Biotechnology 2010;26(4):545-549
In order to optimize the fabrication of SiO2 tubes immobilized with antibody for hepatitis C virus antigen (HCAg) detection, we formed the activated amino on the surface of SiO2 tubes by using the activation of aminosilane. Then we immobilized the hepatitis C virus (HCV) monoclonal antibody on the surface of SiO2 tubes by using glutaraldehyde as a chemical cross-linker, followed by detecting HCAg. Sequence tests showed that when the SiO2 tubes were treated in 10% (V/V) aminosilane solution and 3% (V/V) glutaraldehyde solution for 3 hours and 2 hours, respectively, the HCV monoclonal antibody had high immobilization efficiency and low nonspecificity, and the HCAg was detected to 1 ng/mL. This experiment can provide principle and experimental data for establishment of HCAg magnetic immunoassay system.
Antibodies, Immobilized
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immunology
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Antibodies, Monoclonal
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chemistry
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immunology
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Hepatitis C Antibodies
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chemistry
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immunology
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Hepatitis C Antigens
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analysis
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immunology
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Humans
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Silicon Dioxide
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chemistry
8.Building immune microsphere against tumor necrosis factor-alpha (TNF-alpha).
Qin WANG ; Xiongfei WU ; Junxia WANG ; Hong LIU ; Lian LI ; Xiyu JIN
Journal of Biomedical Engineering 2005;22(6):1219-1222
We have constructed the immune microsphere against tumor necrosis factor-alpha (TNF-alpha) prospectively, hoping to establish the experiment groundwork in more researches which could be used in specific elimination of the TNF-alpha by blood purification method for the future. The recombinant human tumor necrosis factor-alpha monoclonal antibody (rHTNF-alpha McAb) was wrapped on the polystyrene microsphere (PSM) carrier connecting poly-L-lysine (PLL) beforehand. They were earmarked by the fluorescein isothiocyanate (FITC) respectively. The packing conditions were examined using the inversted and fluorescence microscopes and the spectrophotometer. The results showed that the best conditions for wrapping were 20 degrees C, pH9.5 and 60 minutes. The PLL content was not changed in the washing fluid after coating, which indicated the wrapping was quite firm. At the same temperature and same coating time, the rHTNF-alpha McAb coated on the PLL was obviously substantial when the concentration of glutaraldehyde solution was 0.2%. The findings demonstrated that the built immune microsphers can be used as a novel adsorption material. This method is simple and economic, and it offers a new approach to the related studies.
Antibodies, Monoclonal
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immunology
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Fluorescein
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chemistry
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Humans
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Microspheres
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Polylysine
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chemistry
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Polystyrenes
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chemistry
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Recombinant Proteins
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immunology
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Tumor Necrosis Factor-alpha
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immunology
9.The study of in vitro refolding conditions for humanized anti-CTLA4-scFv expressed in E. coli.
Lingyu ZENG ; Qiang HUANG ; Lihong CHEN ; Lin WAN ; Xiaofeng LU
Journal of Biomedical Engineering 2005;22(3):588-592
The purpose of the study is to explore the in vitro refolding protocol for humanized anti-CTLA4-scFv expressed in E. coli. The inclusion bodies are denatured and then diluted or dialyzed into a refolding buffer. We analyzed several factors affecting the refolding yield, including refolding time, temperature, and redox environment. The refolded target proteins are analyzed by non-reducing SDS-PAGE, and the concentration of refolded proteins are examined by Bio-Rad Dc Protein Assay kit. The result shows that a high yield of the protein with natural conformation can be acquired in the condition of 0.15 mol x L(-1) sodium chloride, 50 mmol x L(-1) Tirs-HCl, pH 8.0 buffer containing 1 micromol x L(-1) reduced glutathione and 3 micromol x L(-1) oxidized glutathione. The refolding time is 48 to 54 h at 4 degrees C. 28 mg refolded proteins are produced from 3.9g E. coli.
Antibodies, Monoclonal, Humanized
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chemistry
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CTLA-4 Antigen
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chemistry
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Escherichia coli
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metabolism
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Humans
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Inclusion Bodies
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chemistry
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Protein Folding
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Single-Chain Antibodies
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chemistry
10.Strategies for antibody immobilization during the fabrication of antibody microarrays.
Journal of Biomedical Engineering 2006;23(4):907-910
As one of the proteomic technologies, antibody microarrays can provide a powerful tool for the high-throughput parallel analysis of thousands of proteins in a single experiment. Because antibodies are chemically and structurally complex; immobilizing antibody on solid phase surface of substrates has been a key step during the development of antibody microarrays. This review will focus on the current strategies for antibody immobilization and discuss the advantages and disadvantages of these methods.
Adsorption
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Antibodies, Monoclonal
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chemistry
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metabolism
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Protein Array Analysis
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methods
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Protein Binding