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Antibodies
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Antibody Constructs
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Antibody Constructs

A special issue of Antibodies (ISSN 2073-4468).

Deadline for manuscript submissions: closed (30 November 2014) | Viewed by 108432

Special Issue Editors

Dr. Satoshi Ohtake

E-Mail Website
Guest Editor
Pharmaceutical R&D, BioTherapeutics Pharmaceutical Sciences, Pfizer Inc., 700 Chesterfield Parkway West, Chesterfield, MO 63017, USA
Interests: formulation and manufacturing of biologics; novel biotherapeutic modalities; drying technologies; solid state stabilization; vaccines
Dr. Tsutomu Arakawa

E-Mail Website
Guest Editor
Alliance Protein Laboratories, 6042 Cornerstone Ct West, Suite A1, San Diego, CA 92121, USA
Interests: modulation of aqueous protein solution by solvents
Special Issues, Collections and Topics in MDPI journals

Special Issue Information

Dear Colleagues,

Antibody fragments are under development as a next generation antibody therapeutic that takes advantage of their smaller sizes in comparison to a full antibody. They have unique structural characteristics, and hence challenges, which may differ from those of full antibodies in regard to production, purification/refolding, and formulation. The current Special Issue welcomes contributions from experts in the field that address and review the current state-of-the art technology and developments in the field of antibody fragments.

Dr. Satoshi Ohtake
Dr. Tsutomu Arakawa
Guest Editors

Manuscript Submission Information

Manuscripts should be submitted online at www.mdpi.com by registering and logging in to this website. Once you are registered, click here to go to the submission form. Manuscripts can be submitted until the deadline. All submissions that pass pre-check are peer-reviewed. Accepted papers will be published continuously in the journal (as soon as accepted) and will be listed together on the special issue website. Research articles, review articles as well as short communications are invited. For planned papers, a title and short abstract (about 100 words) can be sent to the Editorial Office for announcement on this website.

Submitted manuscripts should not have been published previously, nor be under consideration for publication elsewhere (except conference proceedings papers). All manuscripts are thoroughly refereed through a single-blind peer-review process. A guide for authors and other relevant information for submission of manuscripts is available on the Instructions for Authors page. Antibodies is an international peer-reviewed open access quarterly journal published by MDPI.

Please visit the Instructions for Authors page before submitting a manuscript. The Article Processing Charge (APC) for publication in this open access journal is 1800 CHF (Swiss Francs). Submitted papers should be well formatted and use good English. Authors may use MDPI's English editing service prior to publication or during author revisions.

Keywords

  • antibody
  • antibody fragments
  • expression
  • purification
  • refolding
  • formulation

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Published Papers (7 papers)

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Research

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645 KiB  
Article
The Biotechnological Applications of Recombinant Single-Domain Antibodies are Optimized by the C-Terminal Fusion to the EPEA Sequence (C Tag)
by Selma Djender, Anne Beugnet, Aurelie Schneider and Ario De Marco
Antibodies 2014, 3(2), 182-191; https://doi.org/10.3390/antib3020182 - 2 Apr 2014
Cited by 21 | Viewed by 17725
Abstract
We designed a vector for the bacterial expression of recombinant antibodies fused to a double tag composed of 6xHis and the EPEA amino acid sequence. EPEA sequence (C tag) is tightly bound by a commercial antibody when expressed at the C-term end of [...] Read more.
We designed a vector for the bacterial expression of recombinant antibodies fused to a double tag composed of 6xHis and the EPEA amino acid sequence. EPEA sequence (C tag) is tightly bound by a commercial antibody when expressed at the C-term end of a polypeptide. The antigen is released in the presence of 2 M MgCl2. Consequently, constructs fused to the 6xHis-C tags can be purified by two successive and orthogonal affinity steps. Single-domain antibodies were produced either in the periplasmic or in the cytoplasmic space of E. coli. Surprisingly, the first affinity purification step performed using the EPEA-binding resin already yielded homogeneous proteins. The presence of the C tag did not interfere with the binding activity of the antibodies, as assessed by FACS and SPR analyses, and the C tag was extremely effective for immunoprecipitating HER2 receptor. Finally, the Alexa488-coupled anti-C tag allowed for simplification of FACS and IF analyses. These results show that a tag of minimal dimensions can be effectively used to improve the applicability of recombinant antibodies as reagents. In our hands, C tag was superior to His-tag in affinity purification and pull-down experiments, and practical in any other standard immune technique. Full article
(This article belongs to the Special Issue Antibody Constructs)
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Review

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1073 KiB  
Review
Antibody Fragments and Their Purification by Protein L Affinity Chromatography
by Gustav Rodrigo, Mats Gruvegård and James M. Van Alstine
Antibodies 2015, 4(3), 259-277; https://doi.org/10.3390/antib4030259 - 11 Sep 2015
Cited by 74 | Viewed by 33986
Abstract
Antibodies and related proteins comprise one of the largest and fastest-growing classes of protein pharmaceuticals. A majority of such molecules are monoclonal antibodies; however, many new entities are antibody fragments. Due to their structural, physiological, and pharmacological properties, antibody fragments offer new biopharmaceutical [...] Read more.
Antibodies and related proteins comprise one of the largest and fastest-growing classes of protein pharmaceuticals. A majority of such molecules are monoclonal antibodies; however, many new entities are antibody fragments. Due to their structural, physiological, and pharmacological properties, antibody fragments offer new biopharmaceutical opportunities. In the case of recombinant full-length antibodies with suitable Fc regions, two or three column purification processes centered around Protein A affinity chromatography have proven to be fast, efficient, robust, cost-effective, and scalable. Most antibody fragments lack Fc and suitable affinity for Protein A. Adapting proven antibody purification processes to antibody fragments demands different affinity chromatography. Such technology must offer the unit operation advantages noted above, and be suitable for most of the many different types of antibody fragments. Protein L affinity chromatography appears to fulfill these criteria—suggesting its consideration as a key unit operation in antibody fragment processing. Full article
(This article belongs to the Special Issue Antibody Constructs)
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1525 KiB  
Review
Cell-Free Synthesis Meets Antibody Production: A Review
by Marlitt Stech and Stefan Kubick
Antibodies 2015, 4(1), 12-33; https://doi.org/10.3390/antib4010012 - 26 Jan 2015
Cited by 51 | Viewed by 21847
Abstract
Engineered antibodies are key players in therapy, diagnostics and research. In addition to full size immunoglobulin gamma (IgG) molecules, smaller formats of recombinant antibodies, such as single-chain variable fragments (scFv) and antigen binding fragments (Fab), have emerged as promising alternatives since they possess [...] Read more.
Engineered antibodies are key players in therapy, diagnostics and research. In addition to full size immunoglobulin gamma (IgG) molecules, smaller formats of recombinant antibodies, such as single-chain variable fragments (scFv) and antigen binding fragments (Fab), have emerged as promising alternatives since they possess different advantageous properties. Cell-based production technologies of antibodies and antibody fragments are well-established, allowing researchers to design and manufacture highly specific molecular recognition tools. However, as these technologies are accompanied by the drawbacks of being rather time-consuming and cost-intensive, efficient and powerful cell-free protein synthesis systems have been developed over the last decade as alternatives. So far, prokaryotic cell-free systems have been the focus of interest. Recently, eukaryotic in vitro translation systems have enriched the antibody production pipeline, as these systems are able to mimic the natural pathway of antibody synthesis in eukaryotic cells. This review aims to overview and summarize the advances made in the production of antibodies and antibody fragments in cell-free systems. Full article
(This article belongs to the Special Issue Antibody Constructs)
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830 KiB  
Review
Antibody Fragments Defining Biologically Relevant Conformations of Target Proteins
by Alastair D. G. Lawson
Antibodies 2014, 3(4), 289-302; https://doi.org/10.3390/antib3040289 - 11 Dec 2014
Cited by 2 | Viewed by 7630
Abstract
Antibody fragments have long been used as chaperones in crystallography, but have more recently been applied to the definition of biologically relevant conformations among the dynamic ensemble of target protein conformational sampling. This review charts the progress being made in understanding function in [...] Read more.
Antibody fragments have long been used as chaperones in crystallography, but have more recently been applied to the definition of biologically relevant conformations among the dynamic ensemble of target protein conformational sampling. This review charts the progress being made in understanding function in the context of structure using this approach, and highlights new opportunities for drug discovery. Full article
(This article belongs to the Special Issue Antibody Constructs)
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550 KiB  
Review
Efficient Expression of Antibody Fragments with the Brevibacillus Expression System
by Hiroshi Hanagata, Makoto Mizukami and Akira Miyauchi
Antibodies 2014, 3(2), 242-252; https://doi.org/10.3390/antib3020242 - 26 May 2014
Cited by 14 | Viewed by 9317
Abstract
Antibodies, owing to their capability to bind specifically to a target molecule, have been and will continue to be applied in various areas, including research, diagnosis and therapy. In particular, antibody fragments, which are size-reduced antibodies comprising functional variable domains, are suited for [...] Read more.
Antibodies, owing to their capability to bind specifically to a target molecule, have been and will continue to be applied in various areas, including research, diagnosis and therapy. In particular, antibody fragments, which are size-reduced antibodies comprising functional variable domains, are suited for production in bacteria. They also are useful in applications requiring intracellular delivery and for further engineering toward molecules possessing multiple custom functions. An expression system based on Brevibacillus is characterized by high efficiency and simple genetic recombination for secretory production. The Brevibacillus expression system has been successfully utilized for the efficient production of antibody fragments, e.g., scFvs (single-chain antibody fragments) comprising heavy-chain and light-chain variable domains, linked by a spacer sequence. Expression in fusion with a Halobacterium-derived secretory protein was shown to confer enhanced productivity. In the case of Fabs, productivity as high as 100 mg/L was accomplished in a simple system, i.e., shake flask cultures. The Brevibacillus expression system offers several advantages, shared by other bacterial systems, such as E. coli, in particular, for the ease in genetic engineering and culture production. Full article
(This article belongs to the Special Issue Antibody Constructs)
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343 KiB  
Review
Refolding Technologies for Antibody Fragments
by Tsutomu Arakawa and Daisuke Ejima
Antibodies 2014, 3(2), 232-241; https://doi.org/10.3390/antib3020232 - 23 May 2014
Cited by 17 | Viewed by 8947
Abstract
Refolding is one of the production technologies for pharmaceutical grade antibody fragments. Detergents and denaturants are primarily used to solubilize the insoluble proteins. The solubilized and denatured proteins are refolded by reducing the concentration of the denaturants or detergents. Several refolding technologies have [...] Read more.
Refolding is one of the production technologies for pharmaceutical grade antibody fragments. Detergents and denaturants are primarily used to solubilize the insoluble proteins. The solubilized and denatured proteins are refolded by reducing the concentration of the denaturants or detergents. Several refolding technologies have been used for antibody fragments, comprising dilution, dialysis, solid phase solvent exchange and size exclusion chromatography, as reviewed here. Aggregation suppressor or folding-assisting agents, including arginine hydrochloride, ionic liquids and detergents or denaturants at low concentrations, are included in the refolding solvent to enhance refolding yield. Full article
(This article belongs to the Special Issue Antibody Constructs)
302 KiB  
Review
Production of Single-Chain Variable-Fragments against Carbohydrate Antigens
by Yoko Fujita-Yamaguchi
Antibodies 2014, 3(1), 155-168; https://doi.org/10.3390/antib3010155 - 12 Mar 2014
Cited by 5 | Viewed by 8078
Abstract
The production of human single-chain variable-fragments (scFvs) against carbohydrate antigens by phage display technology is seemingly a logical strategy towards the development of antibody therapeutics, since carbohydrates are self-antigens. Panning and screening of phages displaying human scFvs using a variety of neoglycolipids presenting [...] Read more.
The production of human single-chain variable-fragments (scFvs) against carbohydrate antigens by phage display technology is seemingly a logical strategy towards the development of antibody therapeutics, since carbohydrates are self-antigens. Panning and screening of phages displaying human scFvs using a variety of neoglycolipids presenting structurally-defined carbohydrates resulted in a number of candidate phage clones as judged by cautious evaluation of DNA sequences and specific binding to carbohydrate moieties of interest. ScFv proteins were expressed in prokaryotic or eukaryotic cells from the respective genes. The characterization of isolated scFvs gene products after establishing expression, production and purification of scFv protein in different expression systems demonstrated that the production of scFv-human IgG1 Fc conjugates were originally sufficient in the media of stably-transfected cells, but declined during early passages. Bacterial expression of soluble scFv proteins with binding activity suffered low yields, whereas overexpressed scFv proteins formed inclusion bodies, which required refolding. An insect cell expression system producing soluble and active scFv proteins was found to be cost- and time-effective. The best expression system and fine adjustments for the conditions to prepare active forms had to be determined for each scFv protein. The successful production of active scFv proteins seems to be dependent on their DNA and/or amino acid sequences. Full article
(This article belongs to the Special Issue Antibody Constructs)
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