Abstract B247: Integration of a modularized protein engineering technology and the RheoSwitch Therapeutic System® platform to develop high affinity trastuzumab single chain variable fragment-Fc proteins for gene therapy applications.

2013 ◽  
Author(s):  
Charles Reed ◽  
Vernon Dailey ◽  
Anissa Elayadi ◽  
Lindsay Williams ◽  
Dina Schneider ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Protein Engineering ◽  
Therapeutic System ◽  
Single Chain Variable Fragment ◽  
Single Chain ◽  
Engineering Technology ◽  
High Affinity

scholarly journals The intrabody targeting of hTERT attenuates the immortality of cancer cells

2010 ◽  
Vol 15 (1) ◽  
Author(s):  
Xiangying Zhu ◽  
Nan Yang ◽  
Jianguo Cai ◽  
Guimei Yang ◽  
Shenghua Liang ◽  
...  
Keyword(s):  
Cancer Cells ◽  
Malignant Tumors ◽  
Single Chain Variable Fragment ◽  
Single Chain ◽  
Human Telomerase Reverse Transcriptase ◽  
High Affinity ◽  
Promising Tool ◽  
Cell Immortalization ◽  
Synthesis Activity ◽  
Human Telomerase

AbstracthTERT (human telomerase reverse transcriptase) plays a key role in the process of cell immortalization. Overexpression of hTERT has been implicated in 85% of malignant tumors and offers a specific target for cancer therapy. In this paper, we describe an effective approach using a single-chain variable fragment (scFv) intrabody derived from monoclonal hybridoma directed against hTERT to attenuate the immortalization of human uterine cervix and hepatoma cells. The scFv we constructed had a high affinity to hTERT, and specifically neutralized over 70% of telomere synthesis activity, thereby inhibiting the viability and proliferation of the cancer cells. Our results indicate that this anti-hTERT intrabody is a promising tool to target hTERT and intervene in the immortalization process of cancer cells.

scholarly journals Screening for a Single-Chain Variable-Fragment Antibody That Can Effectively Neutralize the Cytotoxicity of the Vibrio parahaemolyticus Thermolabile Hemolysin

2012 ◽  
Vol 78 (14) ◽  
pp. 4967-4975 ◽  
Author(s):  
Rongzhi Wang ◽  
Sui Fang ◽  
Dinglong Wu ◽  
Junwei Lian ◽  
Jue Fan ◽  
...  
Keyword(s):  
Vibrio Parahaemolyticus ◽  
Animal Health ◽  
Halophilic Bacterium ◽  
Cell Toxicity ◽  
Pathogenic Factor ◽  
Single Chain Variable Fragment ◽  
Single Chain ◽  
Content Type ◽  
High Affinity ◽  
Food Borne

ABSTRACTVibrio parahaemolyticusis a halophilic bacterium that is widely distributed in water resources. The bacterium causes lethal food-borne diseases and poses a serious threat to human and animal health all over the world. The major pathogenic factor ofV. parahaemolyticusis thermolabile hemolysin (TLH), encoded by thetlhgene, but its toxicity mechanisms are unknown. A high-affinity antibody that can neutralize TLH activity effectively is not available. In this study, we successfully expressed and purified the TLH antigen and discovered a high-affinity antibody to TLH, named scFv-LA3, by phage display screening. Cytotoxicity analysis showed that scFv-LA3 has strong neutralization effects on TLH-induced cell toxicity.

scholarly journals Construction of a Single-Chain Variable-Fragment Antibody against the Superantigen Staphylococcal Enterotoxin B

2010 ◽  
Vol 76 (24) ◽  
pp. 8184-8191 ◽  
Author(s):  
Pawan Kumar Singh ◽  
Ranu Agrawal ◽  
Dev Vrat Kamboj ◽  
Garima Gupta ◽  
M. Boopathi ◽  
...  
Keyword(s):  
Phage Display ◽  
Staphylococcal Enterotoxin ◽  
Scfv Antibody ◽  
Affinity Constant ◽  
Enzyme Linked Immunosorbent Assay ◽  
Staphylococcal Enterotoxin B ◽  
Single Chain Variable Fragment ◽  
Single Chain ◽  
High Affinity ◽  
Enterotoxin B

ABSTRACT Staphylococcal food poisoning (SFP) is one of the most prevalent causes of food-borne illness throughout the world. SFP is caused by 21 different types of staphylococcal enterotoxins produced by Staphylococcus aureus. Among these, staphylococcal enterotoxin B (SEB) is the most potent toxin and is a listed biological warfare (BW) agent. Therefore, development of immunological reagents for detection of SEB is of the utmost importance. High-affinity and specific monoclonal antibodies are being used for detection of SEB, but hybridoma clones tend to lose their antibody-secreting ability over time. This problem can be overcome by the use of recombinant antibodies produced in a bacterial system. In the present investigation, genes from a hybridoma clone encoding monoclonal antibody against SEB were immortalized using antibody phage display technology. A murine phage display library containing single-chain variable-fragment (ScFv) antibody genes was constructed in a pCANTAB 5E phagemid vector. Phage particles displaying ScFv were rescued by reinfection of helper phage followed by four rounds of biopanning for selection of SEB binding ScFv antibody fragments by using phage enzyme-linked immunosorbent assay (ELISA). Soluble SEB-ScFv antibodies were characterized from one of the clones showing high affinity for SEB. The anti-SEB ScFv antibody was highly specific, and its affinity constant was 3.16 nM as determined by surface plasmon resonance (SPR). These results demonstrate that the recombinant antibody constructed by immortalizing the antibody genes from a hybridoma clone is useful for immunodetection of SEB.

scholarly journals A genetically encoded probe for imaging HA-tagged protein translation, localization, and dynamics in living cells and animals

2018 ◽  
Author(s):  
Ning Zhao ◽  
Kouta Kamijo ◽  
Philip D. Fox ◽  
Haruka Oda ◽  
Tatsuya Morisaki ◽  
...  
Keyword(s):  
Single Molecule ◽  
Mrna Translation ◽  
Protein Translation ◽  
Cell Types ◽  
Living Cells ◽  
Single Chain Variable Fragment ◽  
Single Chain ◽  
High Affinity ◽  
Single Molecule Studies

ABSTRACTTo expand the toolbox of imaging in living cells, we have engineered a new single chain variable fragment (scFv) that binds the classic linear HA epitope with high affinity and specificity in vivo. The resulting probe, which we call the HA frankenbody, is capable of lighting up in multiple colors HA-tagged nuclear, cytoplasmic, and membrane proteins in diverse living cell types. The HA frankenbody also enables state-of-the-art single-molecule experiments, which we demonstrate by tracking single mRNA translation dynamics in living U2OS cells and neurons. In combination with the SunTag, we 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 new tool for imaging protein dynamics in vivo.One-sentence summaryA genetically encodable intracellular single-chain variable fragment that selectively binds the HA epitope (YPYDVPDYA) with high affinity in living cells and organisms can be used to quantify HA-tagged protein translation, localization, and dynamics.

scholarly journals High-Affinity, Human Antibody-Like Antibody Fragment (Single-Chain Variable Fragment) Neutralizing the Lethal Factor (LF) of Bacillus anthracis by Inhibiting Protective Antigen-LF Complex Formation

2007 ◽  
Vol 51 (8) ◽  
pp. 2758-2764 ◽  
Author(s):  
Thibaut Pelat ◽  
Michael Hust ◽  
Emmanuelle Laffly ◽  
Florence Condemine ◽  
Chantal Bottex ◽  
...  
Keyword(s):  
Protective Antigen ◽  
Germ Line ◽  
Lethal Factor ◽  
Antibody Fragment ◽  
Human Antibody ◽  
Single Chain Variable Fragment ◽  
Single Chain ◽  
Vitro Assay ◽  
High Affinity

ABSTRACT The anthrax lethal toxin (LT) consists of two subunits, the protective antigen (PA) and the lethal factor (LF), and is essential for anthrax pathogenesis. Several recombinant antibodies directed against PA and intended for medical use have been obtained, but none against LF, despite the recommendations of anthrax experts. Here we describe an anti-LF single-chain variable fragment (scFv) that originated from an immunized macaque (Macaca fascicularis) and was obtained by phage display. Panning of the library of 1.8 × 108 clones allowed the isolation of 2LF, a high-affinity (equilibrium dissociation constant, 1.02 nM) scFv, which is highly neutralizing in the standardized in vitro assay (50% inhibitory concentration, 1.20 ± 0.06 nM) and in an in vivo assay. The scFv neutralizes anthrax LT by inhibiting the formation of the LF-PA complex. The genes encoding 2LF are very similar to those of human immunoglobulin germ line genes, sharing substantial (84.2%) identity with their most similar, germinally encoded counterparts; this feature favors medical applications. These results, and others formerly published, demonstrate that our approach can generate antibody fragments suitable for prophylaxis and therapeutics.

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