Making cell-permeable antibodies (Transbody) through fusion of protein transduction domains (PTD) with single chain variable fragment (scFv) antibodies: Potential advantages over antibodies expressed within the intracellular environment (Intrabody)

2005 ◽  
Vol 64 (6) ◽  
pp. 1105-1108 ◽  
Author(s):  
Boon Chin Heng ◽  
Tong Cao
Keyword(s):  
Protein Transduction ◽  
Single Chain Variable Fragment ◽  
Single Chain ◽  
Protein Transduction Domains ◽  
Intracellular Environment

scholarly journals Antibody Mediated Transduction of Therapeutic Proteins into Living Cells

2005 ◽  
Vol 5 ◽  
pp. 782-788 ◽  
Author(s):  
James E. Hansen ◽  
Richard H. Weisbart ◽  
Robert N. Nishimura
Keyword(s):  
Therapeutic Proteins ◽  
Disease Process ◽  
Antibody Fragment ◽  
Living Cells ◽  
Cell Penetrating Peptides ◽  
Biologically Active ◽  
Protein Transduction ◽  
Protein Therapy ◽  
Single Chain ◽  
Protein Transduction Domains

Protein therapy refers to the direct delivery of therapeutic proteins to cells and tissues with the goal of ameliorating or modifying a disease process. Current techniques for delivering proteins across cell membranes include taking advantage of receptor-mediated endocytosis or using protein transduction domains that penetrate directly into cells. The most commonly used protein transduction domains are small cell-penetrating peptides derived from such proteins as the HIV-1 Tat protein. A novel protein transduction domain developed as the single chain fragment (Fv) of a murine anti-DNA autoantibody, mAb 3E10, has recently been developed and used to deliver biologically active proteins to living cellsin vitro. This review will provide a brief overview of the development of the Fv fragment and provide a summary of recent studies using Fv to deliver therapeutic peptides and proteins (such as a C-terminal p53 peptide, C-terminal p53 antibody fragment, full-length p53, and micro-dystrophin) to cells.

Procaryotic Expression of Single-Chain Variable-Fragment (scFv) Antibodies: Secretion in L-Form Cells of Proteus mirabilis Leads to Active Product and Overcomes the Limitations of Periplasmic Expression in Escherichia coli

1998 ◽  
Vol 64 (12) ◽  
pp. 4862-4869 ◽  
Author(s):  
Jörg F. Rippmann ◽  
Michaela Klein ◽  
Christian Hoischen ◽  
Bodo Brocks ◽  
Wolfgang J. Rettig ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Proteus Mirabilis ◽  
Binding Activity ◽  
Host Cells ◽  
Heterologous Proteins ◽  
Single Chain Variable Fragment ◽  
Single Chain ◽  
E Coli ◽  
Active Product ◽  
Periplasmic Expression

ABSTRACT Recently it has been demonstrated that L-form cells ofProteus mirabilis (L VI), which lack a periplasmic compartment, can be efficiently used in the production and secretion of heterologous proteins. In search of novel expression systems for recombinant antibodies, we compared levels of single-chain variable-fragment (scFv) production in Escherichia coliJM109 and P. mirabilis L VI, which express four distinct scFvs of potential clinical interest that show differences in levels of expression and in their tendencies to form aggregates upon periplasmic expression. Production of all analyzed scFvs in E. coli was limited by the severe toxic effect of the heterologous product as indicated by inhibition of culture growth and the formation of insoluble aggregates in the periplasmic space, limiting the yield of active product. In contrast, the L-form cells exhibited nearly unlimited growth under the tested production conditions for all scFvs examined. Moreover, expression experiments with P. mirabilis L VI led to scFv concentrations in the range of 40 to 200 mg per liter of culture medium (corresponding to volume yields 33- to 160-fold higher than those with E. coli JM109), depending on the expressed antibody. In a translocation inhibition experiment the secretion of the scFv constructs was shown to be an active transport coupled to the signal cleavage. We suppose that this direct release of the newly synthesized product into a large volume of the growth medium favors folding into the native active structure. The limited aggregation of scFv observed in the P. mirabilis L VI supernatant (occurring in a first-order-kinetics manner) was found to be due to intrinsic features of the scFv and not related to the expression process of the host cells. The P. mirabilis L VI supernatant was found to be advantageous for scFv purification. A two-step chromatography procedure led to homogeneous scFv with high antigen binding activity as revealed from binding experiments with eukaryotic cells.

scholarly journals Development of a bispecific immune engager using a recombinant malaria protein

2021 ◽  
Vol 12 (4) ◽  
Author(s):  
Mie A. Nordmaj ◽  
Morgan E. Roberts ◽  
Emilie S. Sachse ◽  
Robert Dagil ◽  
Anne Poder Andersen ◽  
...  
Keyword(s):  
Cancer Cells ◽  
Immune Evasion ◽  
Blood Cells ◽  
Xenograft Model ◽  
Cancer Targeting ◽  
Single Chain Variable Fragment ◽  
Single Chain ◽  
Conjugation System ◽  
Immune Mediated

AbstractAs an immune evasion and survival strategy, the Plasmodium falciparum malaria parasite has evolved a protein named VAR2CSA. This protein mediates sequestration of infected red blood cells in the placenta through the interaction with a unique carbohydrate abundantly and exclusively present in the placenta. Cancer cells were found to share the same expression of this distinct carbohydrate, termed oncofetal chondroitin sulfate on their surface. In this study we have used a protein conjugation system to produce a bispecific immune engager, V-aCD3, based on recombinant VAR2CSA as the cancer targeting moiety and an anti-CD3 single-chain variable fragment linked to a single-chain Fc as the immune engager. Conjugation of these two proteins resulted in a single functional moiety that induced immune mediated killing of a broad range of cancer cells in vitro and facilitated tumor arrest in an orthotopic bladder cancer xenograft model.

scholarly journals Single Chain Variable Fragment Fused to Maltose Binding Protein: A Modular Nanocarrier Platform for the Targeted Delivery of Antitumorals

2021 ◽  
Author(s):  
Francisco J. Reche-Perez ◽  
Simona Plesselova ◽  
Eduardo De los Reyes-Berbel ◽  
Mariano Ortega-Muñoz ◽  
F. Javier Lopez-Jaramillo ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Targeted Delivery ◽  
Specific Binding ◽  
Protein A ◽  
Antibody Fragments ◽  
Single Chain Variable Fragment ◽  
Single Chain ◽  
Binding Properties ◽  
Single Chain Variable Fragments ◽  
Selective Delivery

The use of the specific binding properties of monoclonal antibody fragments such as single-chain variable fragments (ScFv) for the selective delivery of antitumor therapeutics for cancer cells is attractive due...

Single-chain variable fragment (scFv) targeting streptolysin O controls group A Streptococcus infection

2021 ◽  
Vol 566 ◽  
pp. 177-183
Author(s):  
Chihiro Aikawa ◽  
Kiyosumi Kawashima ◽  
Chihiro Fukuzaki ◽  
Makoto Nakakido ◽  
Kazunori Murase ◽  
...  
Keyword(s):  
Group A Streptococcus ◽  
Single Chain Variable Fragment ◽  
Single Chain ◽  
Streptolysin O ◽  
Group A

Isolation of single chain variable fragment (scFv) specific for Cry1C toxin from human single fold scFv libraries

Toxicon ◽  
2012 ◽  
Vol 60 (7) ◽  
pp. 1290-1297 ◽  
Author(s):  
Yun Wang ◽  
Xiao Zhang ◽  
Cunzheng Zhang ◽  
Yuan Liu ◽  
Xianjin Liu
Keyword(s):  
Single Chain Variable Fragment ◽  
Single Chain ◽  
Single Fold

scholarly journals Protein Transduction Domains Fused to Virus Receptors Improve Cellular Virus Uptake and Enhance Oncolysis by Tumor-Specific Replicating Vectors

2004 ◽  
Vol 78 (24) ◽  
pp. 13743-13754 ◽  
Author(s):  
Florian Kühnel ◽  
Bernd Schulte ◽  
Thomas Wirth ◽  
Norman Woller ◽  
Sonja Schäfers ◽  
...  
Keyword(s):  
Viral Vectors ◽  
Protein Transduction ◽  
Virus Binding ◽  
Adenoviral Infection ◽  
Protein Transduction Domains ◽  
Oncolytic Therapy ◽  
Uptake Rates ◽  
Immunodeficiency Virus ◽  
Transactivator Of Transcription ◽  
Adenovirus Receptor

ABSTRACT Expression of cellular receptors determines viral tropism and limits gene delivery by viral vectors. Protein transduction domains (PTDs) have been shown to deliver proteins, antisense oligonucleotides, liposomes, or plasmid DNA into cells. In our study, we investigated the role of several PTD motifs in adenoviral infection. When physiologically expressed, a PTD from human immunodeficiency virus transactivator of transcription (Tat) did not improve adenoviral infection. We therefore fused PTDs to the ectodomain of the coxsackievirus-adenovirus receptor (CARex) to attach PTDs to adenoviral fiber knobs. CARex-Tat and CARex-VP22 allowed efficient adenoviral infection in nonpermissive cells and significantly improved viral uptake rates in permissive cells. Dose-dependent competition of CARex-PTD-mediated infection using CARex and inhibition experiments with heparin showed that binding of CARex-PTD to both adenoviral fiber and cellular glycosaminoglycans is essential for the improvement of infection. CARex-PTD-treated adenoviruses retained their properties after density gradient ultracentrifugation, indicating stable binding of CARex-PTD to adenoviral particles. Consequently, the mechanism of CARex-PTD-mediated infection involves coating of the viral fiber knobs by CARex-PTD, rather than placement of CARex domains on cell surfaces. Expression of CARex-PTDs led to enhanced lysis of permissive and nonpermissive tumor cells by replicating adenoviruses, indicating that CARex-PTDs are valuable tools to improve the efficacy of oncolytic therapy. Together, our study shows that CARex-PTDs facilitate gene transfer in nonpermissive cells and improve viral uptake at reduced titers and infection times. The data suggest that PTDs fused to virus binding receptors may be a valuable tool to overcome natural tropism of vectors and could be of great interest for gene therapeutic approaches.

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