Special Issue "Antibody Constructs"

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

Deadline for manuscript submissions: closed (30 November 2014)

Special Issue Editors

Guest Editor
Dr. Satoshi Ohtake

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

Alliance Protein Laboratories, 6042 Cornerstone Ct West, Suite A1, San Diego, CA 92121, USA
Website | E-Mail
Interests: modulation of aqueous protein solution by solvents

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

Submission

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. Papers will be published continuously (as soon as accepted) and will be listed together on the special issue website. Research articles, review articles as well as 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 refereed through a 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 300 CHF (Swiss Francs). English correction and/or formatting fees of 250 CHF (Swiss Francs) will be charged in certain cases for those articles accepted for publication that require extensive additional formatting and/or English corrections.

Keywords

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

Published Papers (7 papers)

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Research

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Open AccessArticle The Biotechnological Applications of Recombinant Single-Domain Antibodies are Optimized by the C-Terminal Fusion to the EPEA Sequence (C Tag)
Antibodies 2014, 3(2), 182-191; doi:10.3390/antib3020182
Received: 11 February 2014 / Revised: 18 March 2014 / Accepted: 20 March 2014 / Published: 2 April 2014
Cited by 3 | PDF Full-text (645 KB) | HTML Full-text | XML Full-text
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)

Review

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Open AccessReview Antibody Fragments and Their Purification by Protein L Affinity Chromatography
Antibodies 2015, 4(3), 259-277; doi:10.3390/antib4030259
Received: 29 June 2015 / Revised: 5 August 2015 / Accepted: 27 August 2015 / Published: 11 September 2015
Cited by 3 | PDF Full-text (1073 KB) | HTML Full-text | XML Full-text | Supplementary Files
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)
Figures

Open AccessReview Cell-Free Synthesis Meets Antibody Production: A Review
Antibodies 2015, 4(1), 12-33; doi:10.3390/antib4010012
Received: 2 December 2014 / Accepted: 13 January 2015 / Published: 26 January 2015
Cited by 4 | PDF Full-text (1525 KB) | HTML Full-text | XML Full-text
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)
Open AccessReview Antibody Fragments Defining Biologically Relevant Conformations of Target Proteins
Antibodies 2014, 3(4), 289-302; doi:10.3390/antib3040289
Received: 10 November 2014 / Revised: 2 December 2014 / Accepted: 8 December 2014 / Published: 11 December 2014
PDF Full-text (830 KB) | HTML Full-text | XML Full-text
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
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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)
Open AccessReview Efficient Expression of Antibody Fragments with the Brevibacillus Expression System
Antibodies 2014, 3(2), 242-252; doi:10.3390/antib3020242
Received: 13 March 2014 / Revised: 2 May 2014 / Accepted: 12 May 2014 / Published: 26 May 2014
Cited by 1 | PDF Full-text (550 KB) | HTML Full-text | XML Full-text
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
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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)
Open AccessReview Refolding Technologies for Antibody Fragments
Antibodies 2014, 3(2), 232-241; doi:10.3390/antib3020232
Received: 20 January 2014 / Revised: 26 April 2014 / Accepted: 13 May 2014 / Published: 23 May 2014
PDF Full-text (343 KB) | HTML Full-text | XML Full-text
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)
Open AccessReview Production of Single-Chain Variable-Fragments against Carbohydrate Antigens
Antibodies 2014, 3(1), 155-168; doi:10.3390/antib3010155
Received: 28 January 2014 / Revised: 18 February 2014 / Accepted: 24 February 2014 / Published: 12 March 2014
Cited by 1 | PDF Full-text (302 KB) | HTML Full-text | XML Full-text
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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