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Antibodies, Volume 2, Issue 2 (June 2013), Pages 168-391

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Research

Jump to: Review

Open AccessArticle Isolation of Panels of Llama Single-Domain Antibody Fragments Binding All Nine Neuraminidase Subtypes of Influenza A Virus
Antibodies 2013, 2(2), 168-192; doi:10.3390/antib2020168
Received: 16 February 2013 / Revised: 3 March 2013 / Accepted: 25 March 2013 / Published: 10 April 2013
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Abstract
Avian influenza A virus comprises sixteen hemagglutinin (HA) and nine neuraminidase (NA) subtypes (N1–N9). To isolate llama single-domain antibody fragments (VHHs) against all N subtypes, four llamas were immunized with mixtures of influenza viruses. Selections using influenza virus yielded predominantly VHHs binding to
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Avian influenza A virus comprises sixteen hemagglutinin (HA) and nine neuraminidase (NA) subtypes (N1–N9). To isolate llama single-domain antibody fragments (VHHs) against all N subtypes, four llamas were immunized with mixtures of influenza viruses. Selections using influenza virus yielded predominantly VHHs binding to the highly immunogenic HA and nucleoprotein. However, selection using enzymatically active recombinant NA (rNA) protein enabled us to isolate NA binding VHHs. Some isolated VHHs cross-reacted to other N subtypes. These were subsequently used for the capture of N subtypes that could not be produced as recombinant protein (rN6) or were enzymatically inactive (rN1, rN5) in phage display selection, yielding novel VHHs. In total we isolated 188 NA binding VHHs, 64 of which were expressed in yeast. Most VHHs specifically recognize a single N subtype, but some VHHs cross-react with other N-subtypes. At least one VHH bound to all N subtypes, except N4, identifying a conserved antigenic site. Thus, this work (1) describes methods for isolating NA binding VHHs, (2) illustrates the suitability of llama immunization with multiple antigens for retrieving many binders against different antigens and (3) describes 64 novel NA binding VHHs, including a broadly reactive VHH, which can be used in various assays for influenza virus subtyping, detection or serology. Full article
(This article belongs to the Special Issue Single-Domain Antibody)
Open AccessArticle Characterization of a Phospho-Specific Antibody to the Fcε Receptor γ Chain, Reveals Differences in the Regulation of Syk and Akt Phosphorylation
Antibodies 2013, 2(2), 321-337; doi:10.3390/antib2020321
Received: 10 April 2013 / Revised: 20 April 2013 / Accepted: 3 May 2013 / Published: 13 May 2013
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Abstract
We previously demonstrated that the Fc receptor γ-chain Y58(C-terminal tyrosine) is highly susceptible to dephosphorylation; a mechanism that controls the extent of Syk activation and the downstream signaling in mast cells. Here, we explored the importance of the γ-chain Y47
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We previously demonstrated that the Fc receptor γ-chain Y58(C-terminal tyrosine) is highly susceptible to dephosphorylation; a mechanism that controls the extent of Syk activation and the downstream signaling in mast cells. Here, we explored the importance of the γ-chain Y47 (N-terminal tyrosine) in mast cell signaling. We generated a highly sensitive and versatile phospho-specific antibody that recognized the phosphorylated Y47 in various species. Using this antibody, we found that mutation of the FcεRIβ Y219 to phenylalanine caused a loss in the phosphorylation of the γ-chain Y47, consistent with the previously described role of Y219 in Lyn association with FcεRIβ and subsequent FcεRIγ phosphorylation. These conditions also diminished the tyrosine phosphorylation of Syk and LAT1 but, surprisingly, not the phosphorylation of Akt at T308. Mutation of Y47 or Y58 of the γ-chain also caused a marked inhibition of Syk and LAT1 phosphorylation, but only the latter mutant showed a reduction in Akt phosphorylation. These findings show that the full phosphorylation of Syk and LAT1 requires the FcεRIβ Y219 and both Y47 and Y58 of the γ-chain. However, T308 phosphorylation of Akt is largely independent of FcεRIγ Y47 phosphorylation and of the Lyn-binding site (Y219) on the FcεRIβ. Full article
(This article belongs to the Special Issue Cytokine Growth Factor Antibodies in Immunotherapy)
Open AccessArticle Immunotherapy of B-Cell Lymphoma with an Engineered Bispecific Antibody Targeting CD19 and CD5
Antibodies 2013, 2(2), 338-352; doi:10.3390/antib2020338
Received: 26 March 2013 / Revised: 22 April 2013 / Accepted: 22 April 2013 / Published: 14 May 2013
Cited by 1 | PDF Full-text (588 KB) | HTML Full-text | XML Full-text
Abstract
Using genetic engineering a humanized Fab fragment with specificity for CD19 was fused to a disulfide-stabilized single-chain antibody (dsFv) recognizing CD5. This format should show reduced immunogenicity and improved tissue penetration. The specificity of bsAb FabCD19xdsFvCD5 binding to target cells was verified by
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Using genetic engineering a humanized Fab fragment with specificity for CD19 was fused to a disulfide-stabilized single-chain antibody (dsFv) recognizing CD5. This format should show reduced immunogenicity and improved tissue penetration. The specificity of bsAb FabCD19xdsFvCD5 binding to target cells was verified by flow cytometry on B and T lymphoma cell lines. Binding affinities of both arms were compared with the bivalent parental antibodies against CD19 and CD5 by binding competition assay. Redirected lysis of B lymphoma cells by preactivated PBMC from healthy donors was demonstrated in a chromium-release assay. A clear dose-response relationship could be established in the range from 1 ng/mL to 10 mg/mL bsAb. To evaluate the in vivo efficacy of bsAb FabCD19xdsFvCD5, NOD/SCID mice were intravenously injected with luciferase transfected Raji lymphoma cells together with pre-activated PBMC. Mice received five injections of therapeutic bsAb or control antibodies. While in the control groups all mice died within 40 to 50 days, 40% of bsAb treated animals survived longer than 60 days. Full article
(This article belongs to the Special Issue Bispecific Antibodies for Dual Targeting Strategies)

Review

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Open AccessReview In Vivo Applications of Single Chain Fv (Variable Domain) (scFv) Fragments
Antibodies 2013, 2(2), 193-208; doi:10.3390/antib2020193
Received: 4 February 2013 / Revised: 25 March 2013 / Accepted: 29 March 2013 / Published: 11 April 2013
Cited by 17 | PDF Full-text (397 KB) | HTML Full-text | XML Full-text
Abstract
Single chain variable domain (Fv) fragments (scFv) are powerful tools in research and clinical settings, owing to better pharmacokinetic properties compared to the parent monoclonal antibodies and the relative ease of producing them in large quantities, at low cost. Though they offer
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Single chain variable domain (Fv) fragments (scFv) are powerful tools in research and clinical settings, owing to better pharmacokinetic properties compared to the parent monoclonal antibodies and the relative ease of producing them in large quantities, at low cost. Though they offer several advantages, they suffer from lower binding affinity and rapid clearance from circulation, which limits their therapeutic potential. However, these fragments can be genetically modified to enhance desirable properties, such as multivalency, high target retention and slower blood clearance, and as such, a variety of scFv formats have been generated. ScFvs can be administered by systemic injection for diagnostic and therapeutic purposes. They can be expressed in vivo through viral vectors in instances where large infection rates and sustenance of high levels of the antibody is required. ScFvs have found applications as tools for in vivo loss-of-function studies and inactivation of specific protein domains, diagnostic imaging, tumor therapy and treatment for neurodegenerative and infectious diseases. This review will focus on their in vivo applications. Full article
(This article belongs to the Special Issue Single-Domain Antibody)
Open AccessReview Diving through Membranes: Molecular Cunning to Enforce the Endosomal Escape of Antibody-Targeted Anti-Tumor Toxins
Antibodies 2013, 2(2), 209-235; doi:10.3390/antib2020209
Received: 4 March 2013 / Revised: 27 March 2013 / Accepted: 5 April 2013 / Published: 17 April 2013
Cited by 7 | PDF Full-text (809 KB) | HTML Full-text | XML Full-text
Abstract
Membranes are vital barriers by which cells control the flux of molecules and energy between their exterior and interior and also between their various intracellular compartments. While numerous transport systems exist for ions and small molecules, the cytosolic uptake of larger biological molecules
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Membranes are vital barriers by which cells control the flux of molecules and energy between their exterior and interior and also between their various intracellular compartments. While numerous transport systems exist for ions and small molecules, the cytosolic uptake of larger biological molecules and in particular antibody-targeted drugs, is a big challenge. Inducing leakage of the plasma membrane is unfavorable since the target cell specificity mediated by the antibody would likely be lost in this case. After binding and internalization, the antibody drug conjugates reach the endosomes. Thus, enforcing the endosomal escape of anti-tumor toxins without affecting the integrity of other cellular membranes is of paramount importance. Different strategies have been developed in the last decades to overcome endosomal accumulation and subsequent lysosomal degradation of targeted protein-based drugs. In this review we summarize the various efforts made to establish efficient techniques to disrupt the endosomal membrane barrier including the use of molecular ferries such as cell penetrating peptides or viral membrane fusion proteins, endosomal leakage inducing molecules such as saponins or monensin and physicochemical methods as represented by photochemical internalization. Full article
(This article belongs to the Special Issue Recombinant Immunotoxins)
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Open AccessReview Ricin and Ricin-Containing Immunotoxins: Insights into Intracellular Transport and Mechanism of action in Vitro
Antibodies 2013, 2(2), 236-269; doi:10.3390/antib2020236
Received: 27 December 2012 / Revised: 8 April 2013 / Accepted: 11 April 2013 / Published: 19 April 2013
Cited by 7 | PDF Full-text (337 KB) | HTML Full-text | XML Full-text
Abstract
Ricin is a type II ribosome inactivating protein (RIP) isolated from castor beans. Its high toxicity classifies it as a possible biological weapon. On the other hand, ricin linked to specific monoclonal antibodies or used in other conjugates has powerful medical applications. Ricin
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Ricin is a type II ribosome inactivating protein (RIP) isolated from castor beans. Its high toxicity classifies it as a possible biological weapon. On the other hand, ricin linked to specific monoclonal antibodies or used in other conjugates has powerful medical applications. Ricin consists of an A-chain (RTA) that damages ribosomes and inhibits protein synthesis, and a B-chain that plays a role in binding and cellular uptake. A number of recent studies have demonstrated that ricin-induced inhibition of protein synthesis is not the only mechanism responsible for cell death. It turns out that ricin is able to induce apoptosis in different cell lines and multiple organs in animals. However, the molecular link between protein synthesis inhibition and ricin-dependent triggering of apoptotic cell death is unclear. This review describes the intracellular transport of ricin and ricin-based immunotoxins and their mechanism of action in different non-malignant and cancer cell lines. Moreover, various ricin-containing immunotoxins, their composition, medical applications and side-effects will be described and discussed. Understanding the mechanism of action of ricin-based immunotoxins will facilitate construction of effectively acting immunotoxins that can be used in the clinic for cancer treatment. Full article
(This article belongs to the Special Issue Recombinant Immunotoxins)
Open AccessReview Antibody-Directed Phototherapy (ADP)
Antibodies 2013, 2(2), 270-305; doi:10.3390/antib2020270
Received: 25 February 2013 / Revised: 3 April 2013 / Accepted: 4 April 2013 / Published: 25 April 2013
Cited by 7 | PDF Full-text (2166 KB) | HTML Full-text | XML Full-text
Abstract
Photodynamic therapy (PDT) is a clinically-approved but rather under-exploited treatment modality for cancer and pre-cancerous superficial lesions. It utilises a cold laser or LED to activate a photochemical reaction between a light activated drug (photosensitiser-drug) and oxygen to generate cytotoxic oxygen species. These
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Photodynamic therapy (PDT) is a clinically-approved but rather under-exploited treatment modality for cancer and pre-cancerous superficial lesions. It utilises a cold laser or LED to activate a photochemical reaction between a light activated drug (photosensitiser-drug) and oxygen to generate cytotoxic oxygen species. These free radical species damage cellular components leading to cell death. Despite its benefits, the complexity, limited potency and side effects of PDT have led to poor general usage. However, the research area is very active with an increasing understanding of PDT-related cell biology, photophysics and significant progress in molecular targeting of disease. Monoclonal antibody therapy is maturing and the next wave of antibody therapies includes antibody-drug conjugates (ADCs), which promise to be more potent and curable. These developments could lift antibody-directed phototherapy (ADP) to success. ADP promises to increase specificity and potency and improve drug pharmacokinetics, thus delivering better PDT drugs whilst retaining its other benefits. Whole antibody conjugates with first generation ADP-drugs displayed problems with aggregation, poor pharmacokinetics and loss of immuno-reactivity. However, these early ADP-drugs still showed improved selectivity and potency. Improved PS-drug chemistry and a variety of conjugation strategies have led to improved ADP-drugs with retained antibody and PS-drug function. More recently, recombinant antibody fragments have been used to deliver ADP-drugs with superior drug loading, more favourable pharmacokinetics, enhanced potency and target cell selectivity. These improvements offer a promise of better quality PDT drugs. Full article
(This article belongs to the Special Issue Antibody-Drug Conjugates)
Open AccessReview Single Domain Antibody Fragments as Drug Surrogates Targeting Protein–Protein Interactions inside Cells
Antibodies 2013, 2(2), 306-320; doi:10.3390/antib2020306
Received: 3 April 2013 / Revised: 23 April 2013 / Accepted: 24 April 2013 / Published: 2 May 2013
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Abstract
Many human diseases are caused by mutant or abnormal protein functions that are largely confined to the inside of cells, rather than being displayed on the abnormal cell surface. Furthermore, many of the functional consequences of aberrant proteins, such as in cancer cells,
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Many human diseases are caused by mutant or abnormal protein functions that are largely confined to the inside of cells, rather than being displayed on the abnormal cell surface. Furthermore, many of the functional consequences of aberrant proteins, such as in cancer cells, are due to protein–protein interactions (PPIs). Developing reagents that can specifically interfere with PPI is an important goal for both therapeutic use and as reagents to interrogate the functional importance of PPI. Antibody fragments can be used for inhibiting PPI. Our recent technology development has provided a set of simple protocols that allow development of single antibody variable (V) region domains that can function inside the reducing environment of the cell. The heavy chain variable region (VH) segments mainly used in this technology are based on a designer framework that folds inside cells without the need for the intra-chain disulphide bond and can be used as drug surrogates to determine on-target effects (target validation) and as templates for small molecule drug development. In this review, we discuss our work on single domains as intracellular antibodies and where this work might in the future. Full article
(This article belongs to the Special Issue Single-Domain Antibody)
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Open AccessReview The Development of Bispecific Hexavalent Antibodies as a Novel Class of DOCK-AND-LOCKTM (DNLTM) Complexes
Antibodies 2013, 2(2), 353-370; doi:10.3390/antib2020353
Received: 1 March 2013 / Revised: 9 May 2013 / Accepted: 13 May 2013 / Published: 23 May 2013
Cited by 3 | PDF Full-text (532 KB) | HTML Full-text | XML Full-text
Abstract
The DOCK-AND-LOCKTM (DNLTM) method provides a modular approach to develop multivalent, multifunctional complexes of defined structures, of which bispecific hexavalent antibodies (bsHexAbs) are prominent examples with potential applications in targeted therapy for malignant, autoimmune, and infectious diseases. Currently, bsHexAbs are
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The DOCK-AND-LOCKTM (DNLTM) method provides a modular approach to develop multivalent, multifunctional complexes of defined structures, of which bispecific hexavalent antibodies (bsHexAbs) are prominent examples with potential applications in targeted therapy for malignant, autoimmune, and infectious diseases. Currently, bsHexAbs are constructed by derivatizing a divalent IgG, at the carboxyl termini of either the heavy chain (the CH3-format) or the light chain (the Ck-format), to contain two stabilized dimers of Fab having a different specificity from the IgG. In this review, we briefly outline the features of the DNLTM method and describe key aspects of bsHexAbs examined with diverse preclinical studies, which include binding affinity to target cells, induction of signaling pathways, effector functions, serum stability, pharmacokinetics, and antitumor activity in human tumor xenograft models. Our findings favor the selection of the CK- over the CH3-format for further exploration of bsHexAbs in clinical trials. Full article
(This article belongs to the Special Issue Bispecific Antibodies for Dual Targeting Strategies)
Open AccessReview Role and Redirection of IgE against Cancer
Antibodies 2013, 2(2), 371-391; doi:10.3390/antib2020371
Received: 4 April 2013 / Revised: 13 May 2013 / Accepted: 14 May 2013 / Published: 28 May 2013
Cited by 1 | PDF Full-text (299 KB) | HTML Full-text | XML Full-text
Abstract
IgE is a highly elusive antibody class, yet a tremendously powerful elicitor of immune reactions. Despite huge efforts spent on the characterization and understanding of the IgE system many questions remain either unanswered or only marginally addressed. One above all relates to the
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IgE is a highly elusive antibody class, yet a tremendously powerful elicitor of immune reactions. Despite huge efforts spent on the characterization and understanding of the IgE system many questions remain either unanswered or only marginally addressed. One above all relates to the role of IgE. A common doubt is based on whether IgE mode of action should only be relegated to anti-parasite immunity and allergic manifestations. In search for a hidden role of IgE, reports from several laboratories are described herein in which a natural IgE link to cancer or the experimental redirection of IgE against cancer have been investigated. Epidemiological and investigational studies are trying to elucidate a possible direct intervention of endogenous IgE against cancer, raising thus far no definitive evidence. Conversely, experimental approaches implementing several strategies and engineered IgE formats built up a series of convincing results indicating that cancer might be tackled by the effector functions of this immunoglobulin class. Because of its peculiar immune features, IgE may present a superior anti-tumor performance as compared to IgG. However, extreme care should be taken on how IgE-based anti-tumor approaches should be devised. Overall, IgE appears as a promising resource, likely destined to enrich the anti-cancer arsenal. Full article
(This article belongs to the Special Issue Modes of Antibody Action for Cancer Therapy)

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