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Antibodies, Volume 6, Issue 4 (December 2017)

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Open AccessArticle Characterization of Monoclonal Antibody–Protein Antigen Complexes Using Small-Angle Scattering and Molecular Modeling
Antibodies 2017, 6(4), 25; https://doi.org/10.3390/antib6040025
Received: 16 November 2017 / Revised: 7 December 2017 / Accepted: 8 December 2017 / Published: 15 December 2017
Cited by 1 | Viewed by 1549 | PDF Full-text (35215 KB) | HTML Full-text | XML Full-text
Abstract
The determination of monoclonal antibody interactions with protein antigens in solution can lead to important insights guiding physical characterization and molecular engineering of therapeutic targets. We used small-angle scattering (SAS) combined with size-exclusion multi-angle light scattering high-performance liquid chromatography to obtain monodisperse samples
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The determination of monoclonal antibody interactions with protein antigens in solution can lead to important insights guiding physical characterization and molecular engineering of therapeutic targets. We used small-angle scattering (SAS) combined with size-exclusion multi-angle light scattering high-performance liquid chromatography to obtain monodisperse samples with defined stoichiometry to study an anti-streptavidin monoclonal antibody interacting with tetrameric streptavidin. Ensembles of structures with both monodentate and bidentate antibody–antigen complexes were generated using molecular docking protocols and molecular simulations. By comparing theoretical SAS profiles to the experimental data it was determined that the primary component(s) were compact monodentate and/or bidentate complexes. SAS profiles of extended monodentate complexes were not consistent with the experimental data. These results highlight the capability for determining the shape of monoclonal antibody–antigen complexes in solution using SAS data and physics-based molecular modeling. Full article
(This article belongs to the Special Issue Monoclonal Antibodies) Printed Edition available
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Open AccessArticle Process Analytical Approach towards Quality Controlled Process Automation for the Downstream of Protein Mixtures by Inline Concentration Measurements Based on Ultraviolet/Visible Light (UV/VIS) Spectral Analysis
Antibodies 2017, 6(4), 24; https://doi.org/10.3390/antib6040024
Received: 31 October 2017 / Revised: 29 November 2017 / Accepted: 6 December 2017 / Published: 12 December 2017
Cited by 3 | Viewed by 1762 | PDF Full-text (3618 KB) | HTML Full-text | XML Full-text
Abstract
Downstream of pharmaceutical proteins, such as monoclonal antibodies, is mainly done by chromatography, where concentration determination of coeluting components presents a major problem. Inline concentration measurements (ICM) by Ultraviolet/Visible light (UV/VIS)-spectral data analysis provide a label-free and noninvasive approach to significantly speed up
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Downstream of pharmaceutical proteins, such as monoclonal antibodies, is mainly done by chromatography, where concentration determination of coeluting components presents a major problem. Inline concentration measurements (ICM) by Ultraviolet/Visible light (UV/VIS)-spectral data analysis provide a label-free and noninvasive approach to significantly speed up the analysis and process time. Here, two different approaches are presented. For a test mixture of three proteins, a fast and easily calibrated method based on the non-negative least-squares algorithm is shown, which reduces the calibration effort compared to a partial least-squares approach. The accuracy of ICM for analytical separations of three proteins on an ion exchange column is over 99%, compared to less than 85% for classical peak area evaluation. The power of the partial least squares algorithm (PLS) is shown by measuring the concentrations of Immunoglobulin G (IgG) monomer and dimer under a worst-case scenario of completely overlapping peaks. Here, the faster SIMPLS algorithm is used in comparison to the nonlinear iterative partial least squares (NIPALS) algorithm. Both approaches provide concentrations as well as purities in real-time, enabling live-pooling decisions based on product quality. This is one important step towards advanced process automation of chromatographic processes. Analysis time is less than 100 ms and only one program is used for all the necessary communications and calculations. Full article
(This article belongs to the Special Issue Monoclonal Antibodies) Printed Edition available
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Open AccessArticle Modulation of BCR Signaling by the Induced Dimerization of Receptor-Associated SYK
Antibodies 2017, 6(4), 23; https://doi.org/10.3390/antib6040023
Received: 26 October 2017 / Revised: 27 November 2017 / Accepted: 2 December 2017 / Published: 7 December 2017
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Abstract
Clustering of the B cell antigen receptor (BCR) by polyvalent antigens is transmitted through the SYK tyrosine kinase to the activation of multiple intracellular pathways that determine the physiological consequences of receptor engagement. To explore factors that modulate the quantity and quality of
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Clustering of the B cell antigen receptor (BCR) by polyvalent antigens is transmitted through the SYK tyrosine kinase to the activation of multiple intracellular pathways that determine the physiological consequences of receptor engagement. To explore factors that modulate the quantity and quality of signals sent by the crosslinked BCR, we developed a novel chemical mediator of dimerization to induce clustering of receptor-associated SYK. To accomplish this, we fused SYK with E. coli dihydrofolate reductase (eDHFR), which binds the small molecule trimethoprim (TMP) with high affinity and selectivity and synthesized a dimer of TMP with a flexible linker. The TMP dimer is able to induce the aggregation of eDHFR-linked SYK in live cells. The induced dimerization of SYK bound to the BCR differentially regulates the activation of downstream transcription factors, promoting the activation of Nuclear Factor of Activated T cells (NFAT) without affecting the activation of NFκB. The dimerization of SYK enhances the duration but not the amplitude of calcium mobilization by enhancing the extent and duration of its interaction with the crosslinked BCR at the plasma membrane. Full article
(This article belongs to the Special Issue B Cell Antigen Receptor: Structure and Function)
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Open AccessArticle Generation and Performance of R132H Mutant IDH1 Rabbit Monoclonal Antibody
Antibodies 2017, 6(4), 22; https://doi.org/10.3390/antib6040022
Received: 31 October 2017 / Revised: 21 November 2017 / Accepted: 22 November 2017 / Published: 1 December 2017
Cited by 1 | Viewed by 1286 | PDF Full-text (3849 KB) | HTML Full-text | XML Full-text
Abstract
Isocitrate dehydrogenase 1 (IDH1) gene mutations have been observed in a majority of diffuse astrocytomas, oligodendrogliomas, and secondary glioblastomas, and the mutant IDH1 R132H is detectable in most of these lesions. By specifically targeting the R132H mutation through B-cell cloning, a novel rabbit
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Isocitrate dehydrogenase 1 (IDH1) gene mutations have been observed in a majority of diffuse astrocytomas, oligodendrogliomas, and secondary glioblastomas, and the mutant IDH1 R132H is detectable in most of these lesions. By specifically targeting the R132H mutation through B-cell cloning, a novel rabbit monoclonal antibody, MRQ-67, was produced that can recognize mutant IDH1 R132H and does not react with the wild type protein as demonstrated by Enzyme-linked immunosorbent assay (ELISA) and Western blotting. Through immunohistochemistry, the antibody is able to highlight neoplastic cells in glioma tissue specimens, and can be used as a tool in glioma subtyping. Immunohistochemistry (IHC) detection of IDH1 mutant protein may also be used to visualize single infiltrating tumor cells in surrounding brain tissue with an otherwise normal appearance. Full article
(This article belongs to the Special Issue Monoclonal Antibodies) Printed Edition available
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Open AccessArticle Integration of Aqueous Two-Phase Extraction as Cell Harvest and Capture Operation in the Manufacturing Process of Monoclonal Antibodies
Antibodies 2017, 6(4), 21; https://doi.org/10.3390/antib6040021
Received: 30 October 2017 / Revised: 17 November 2017 / Accepted: 20 November 2017 / Published: 1 December 2017
Cited by 4 | Viewed by 1991 | PDF Full-text (7487 KB) | HTML Full-text | XML Full-text
Abstract
Substantial improvements have been made to cell culturing processes (e.g., higher product titer) in recent years by raising cell densities and optimizing cultivation time. However, this has been accompanied by an increase in product-related impurities and therefore greater challenges in subsequent clarification and
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Substantial improvements have been made to cell culturing processes (e.g., higher product titer) in recent years by raising cell densities and optimizing cultivation time. However, this has been accompanied by an increase in product-related impurities and therefore greater challenges in subsequent clarification and capture operations. Considering the paradigm shift towards the design of continuously operating dedicated plants at smaller scales—with or without disposable technology—for treating smaller patient populations due to new indications or personalized medicine approaches, the rising need for new, innovative strategies for both clarification and capture technology becomes evident. Aqueous two-phase extraction (ATPE) is now considered to be a feasible unit operation, e.g., for the capture of monoclonal antibodies or recombinant proteins. However, most of the published work so far investigates the applicability of ATPE in antibody-manufacturing processes at the lab-scale and for the most part, only during the capture step. This work shows the integration of ATPE as a combined harvest and capture step into a downstream process. Additionally, a model is applied that allows early prediction of settler dimensions with high prediction accuracy. Finally, a reliable process development concept, which guides through the necessary steps, starting from the definition of the separation task to the final stages of integration and scale-up, is presented. Full article
(This article belongs to the Special Issue Monoclonal Antibodies) Printed Edition available
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Open AccessFeature PaperArticle Pyrrolobenzodiazepine Antibody-Drug Conjugates Designed for Stable Thiol Conjugation
Antibodies 2017, 6(4), 20; https://doi.org/10.3390/antib6040020
Received: 17 October 2017 / Revised: 20 November 2017 / Accepted: 21 November 2017 / Published: 28 November 2017
Cited by 1 | Viewed by 1985 | PDF Full-text (3382 KB) | HTML Full-text | XML Full-text | Supplementary Files
Abstract
Thiosuccinimide-linked antibody-drug conjugates (ADCs) are susceptible to drug loss over time due to a retro-Michael reaction, which can be prevented by selecting stable conjugation positions or hydrolysis of the thiosuccinimide. Here, we investigate pyrrolobenzodiazepine (PBD) ADC drug-linkers equipped with N-phenyl maleimide functionality
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Thiosuccinimide-linked antibody-drug conjugates (ADCs) are susceptible to drug loss over time due to a retro-Michael reaction, which can be prevented by selecting stable conjugation positions or hydrolysis of the thiosuccinimide. Here, we investigate pyrrolobenzodiazepine (PBD) ADC drug-linkers equipped with N-phenyl maleimide functionality for stable thiol conjugation via thiosuccinimide hydrolysis. Two PBD drug-linker formats (enzyme-cleavable and non-cleavable) were evaluated following site-specific conjugation to an engineered cysteine incorporated at position T289, which is known to be unstable for N-alkyl maleimide conjugates. N-phenyl maleimide PBDs conjugated to antibodies with similar efficiencies as N-alkyl maleimide PBDs and enhanced thiosuccinimide hydrolysis for N-phenyl maleimide PBDs was confirmed by mass spectrometry, capillary isoelectric focusing, and a SYPRO Orange dye binding assay. All of the PBD ADCs were highly potent in vitro regardless of maleimide- or linker-type, exhibiting low pM EC50 values. Thiol conjugation to N-phenyl maleimide PBD minimized the retro-Michael reaction in both rat and mouse serum. However, cleavage of the valine-alanine dipeptide in mouse serum for ADCs containing cleavable drug-linker led to drug loss regardless of maleimide type, which impacted ADC potency in tumor growth inhibition studies that were conducted in mouse models. Therapeutic improvement in mouse tumor models was realized for ADCs prepared with non-cleavable PBD drug-linkers that were conjugated through N-phenyl maleimide, where a stronger tumor growth inhibition (TGI) response was achieved when compared to the analogous N-alkyl maleimide drug-linker ADC. Altogether, our findings highlight the stability and efficacy benefits of N-phenyl maleimide functionality for ADCs that are produced with thiol-maleimide conjugation chemistry. Full article
(This article belongs to the Special Issue Antibody Drug Conjugates)
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Open AccessFeature PaperReview Antagonist Anti-CD28 Therapeutics for the Treatment of Autoimmune Disorders
Antibodies 2017, 6(4), 19; https://doi.org/10.3390/antib6040019
Received: 26 October 2017 / Revised: 16 November 2017 / Accepted: 18 November 2017 / Published: 21 November 2017
Cited by 1 | Viewed by 1172 | PDF Full-text (480 KB) | HTML Full-text | XML Full-text
Abstract
The effector functions of T lymphocytes are responsible for most autoimmune disorders and act by directly damaging tissues or by indirectly promoting inflammation and antibody responses. Co-stimulatory and co-inhibitory T cell receptor molecules are the primary pharmacological targets that enable interference with immune-mediated
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The effector functions of T lymphocytes are responsible for most autoimmune disorders and act by directly damaging tissues or by indirectly promoting inflammation and antibody responses. Co-stimulatory and co-inhibitory T cell receptor molecules are the primary pharmacological targets that enable interference with immune-mediated diseases. Among these, selective CD28 antagonists have drawn special interest, since they tip the co-stimulation/co-inhibition balance towards efficiently inhibiting effector T cells while promoting suppression by pre-existing regulatory T-cells. After having demonstrated outstanding therapeutic efficacy in multiple models of autoimmunity, inflammation and transplantation, and safety in phase-I studies in humans, selective CD28 antagonists are currently in early clinical development for the treatment of systemic lupus erythematous and rheumatoid arthritis. Here, we review the available proof of concept studies for CD28 antagonists in autoimmunity, with a special focus on the mechanisms of action. Full article
(This article belongs to the Special Issue Therapeutic Antibodies)
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Open AccessReview Monoclonal Antibody: A New Treatment Strategy against Multiple Myeloma
Antibodies 2017, 6(4), 18; https://doi.org/10.3390/antib6040018
Received: 20 October 2017 / Revised: 9 November 2017 / Accepted: 10 November 2017 / Published: 14 November 2017
Cited by 2 | Viewed by 2390 | PDF Full-text (1485 KB) | HTML Full-text | XML Full-text
Abstract
2015 was a groundbreaking year for the multiple myeloma community partly due to the breakthrough approval of the first two monoclonal antibodies in the treatment for patients with relapsed and refractory disease. Despite early disappointments, monoclonal antibodies targeting CD38 (daratumumab) and signaling lymphocytic
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2015 was a groundbreaking year for the multiple myeloma community partly due to the breakthrough approval of the first two monoclonal antibodies in the treatment for patients with relapsed and refractory disease. Despite early disappointments, monoclonal antibodies targeting CD38 (daratumumab) and signaling lymphocytic activation molecule F7 (SLAMF7) (elotuzumab) have become available for patients with multiple myeloma in the same year. Specifically, phase 3 clinical trials of combination therapies incorporating daratumumab or elotuzumab indicate both efficacy and a very favorable toxicity profile. These therapeutic monoclonal antibodies for multiple myeloma can kill target cells via antibody-dependent cell-mediated cytotoxicity, complement-dependent cytotoxicity, and antibody-dependent phagocytosis, as well as by direct blockade of signaling cascades. In addition, their immunomodulatory effects may simultaneously inhibit the immunosuppressive bone marrow microenvironment and restore the key function of immune effector cells. In this review, we focus on monoclonal antibodies that have shown clinical efficacy or promising preclinical anti-multiple myeloma activities that warrant further clinical development. We summarize mechanisms that account for the in vitro and in vivo anti-myeloma effects of these monoclonal antibodies, as well as relevant preclinical and clinical results. Monoclonal antibody-based immunotherapies have already and will continue to transform the treatment landscape in multiple myeloma. Full article
(This article belongs to the Special Issue Monoclonal Antibodies) Printed Edition available
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Open AccessReview Therapeutic and Diagnostic Antibodies to CD146: Thirty Years of Research on Its Potential for Detection and Treatment of Tumors
Antibodies 2017, 6(4), 17; https://doi.org/10.3390/antib6040017
Received: 2 October 2017 / Revised: 26 October 2017 / Accepted: 1 November 2017 / Published: 5 November 2017
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Abstract
CD146 (MCAM, MUC18, S-Endo1) is a transmembrane glycoprotein belonging to both CAM and mucin families. It exists as different splice variants and is cleaved from the membrane by metalloproteases to generate a soluble form. CD146 is expressed by numerous cancer cells as well
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CD146 (MCAM, MUC18, S-Endo1) is a transmembrane glycoprotein belonging to both CAM and mucin families. It exists as different splice variants and is cleaved from the membrane by metalloproteases to generate a soluble form. CD146 is expressed by numerous cancer cells as well as being one of the numerous proteins expressed by the vascular endothelium. It has also been identified on smooth muscle cells, pericytes, and some immune cells. This protein was initially described as an actor involved in tumor growth and metastatic dissemination processes. Some recent works highlighted the role of CD146 in angiogenesis. Interestingly, this knowledge allowed the development of therapeutic and diagnostic tools specifically targeting the different CD146 variants. The first anti-CD146 antibody designed to study the function of this molecule, MUC18, was described by the Pr. J.P. Jonhson in 1987. In this review, we will discuss the 30 following years of research focused on the detection, study, and blocking of this protein in physiological and pathological processes. Full article
(This article belongs to the Special Issue Cancer Antibodies)
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Open AccessReview Antibodies and Derivatives Targeting DR4 and DR5 for Cancer Therapy
Antibodies 2017, 6(4), 16; https://doi.org/10.3390/antib6040016
Received: 15 September 2017 / Revised: 16 October 2017 / Accepted: 19 October 2017 / Published: 25 October 2017
Cited by 4 | Viewed by 2340 | PDF Full-text (4410 KB) | HTML Full-text | XML Full-text
Abstract
Developing therapeutics that induce apoptosis in cancer cells has become an increasingly attractive approach for the past 30 years. The discovery of tumor necrosis factor (TNF) superfamily members and more specifically TNF-related apoptosis-inducing ligand (TRAIL), the only cytokine of the family capable of
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Developing therapeutics that induce apoptosis in cancer cells has become an increasingly attractive approach for the past 30 years. The discovery of tumor necrosis factor (TNF) superfamily members and more specifically TNF-related apoptosis-inducing ligand (TRAIL), the only cytokine of the family capable of eradicating selectively cancer cells, led to the development of numerous TRAIL derivatives targeting death receptor 4 (DR4) and death receptor 5 (DR5) for cancer therapy. With a few exceptions, preliminary attempts to use recombinant TRAIL, agonistic antibodies, or derivatives to target TRAIL agonist receptors in the clinic have been fairly disappointing. Nonetheless, a tremendous effort, worldwide, is being put into the development of novel strategic options to target TRAIL receptors. Antibodies and derivatives allow for the design of novel and efficient agonists. We summarize and discuss here the advantages and drawbacks of the soar of TRAIL therapeutics, from the first developments to the next generation of agonistic products, with a particular insight on new concepts. Full article
(This article belongs to the Special Issue Cancer Antibodies)
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Open AccessFeature PaperArticle Antibody Selection for Cancer Target Validation of FSH-Receptor in Immunohistochemical Settings
Antibodies 2017, 6(4), 15; https://doi.org/10.3390/antib6040015
Received: 15 September 2017 / Revised: 7 October 2017 / Accepted: 13 October 2017 / Published: 18 October 2017
Cited by 2 | Viewed by 1291 | PDF Full-text (11348 KB) | HTML Full-text | XML Full-text | Correction | Supplementary Files
Abstract
Background: The follicle-stimulating hormone (FSH)-receptor (FSHR) has been reported to be an attractive target for antibody therapy in human cancer. However, divergent immunohistochemical (IHC) findings have been reported for FSHR expression in tumor tissues, which could be due to the specificity of the
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Background: The follicle-stimulating hormone (FSH)-receptor (FSHR) has been reported to be an attractive target for antibody therapy in human cancer. However, divergent immunohistochemical (IHC) findings have been reported for FSHR expression in tumor tissues, which could be due to the specificity of the antibodies used. Methods: Three frequently used antibodies (sc-7798, sc-13935, and FSHR323) were validated for their suitability in an immunohistochemical study for FSHR expression in different tissues. As quality control, two potential therapeutic anti-hFSHR Ylanthia® antibodies (Y010913, Y010916) were used. The specificity criteria for selection of antibodies were binding to native hFSHR of different sources, and no binding to non-related proteins. The ability of antibodies to stain the paraffin-embedded Flp-In Chinese hamster ovary (CHO)/FSHR cells was tested after application of different epitope retrieval methods. Results: From the five tested anti-hFSHR antibodies, only Y010913, Y010916, and FSHR323 showed specific binding to native, cell-presented hFSHR. Since Ylanthia® antibodies were selected to specifically recognize native FSHR, as required for a potential therapeutic antibody candidate, FSHR323 was the only antibody to detect the receptor in IHC/histochemical settings on transfected cells, and at markedly lower, physiological concentrations (ex., in Sertoli cells of human testes). The pattern of FSH323 staining noticed for ovarian, prostatic, and renal adenocarcinomas indicated that FSHR was expressed mainly in the peripheral tumor blood vessels. Conclusion: Of all published IHC antibodies tested, only antibody FSHR323 proved suitable for target validation of hFSHR in an IHC setting for cancer. Our studies could not confirm the previously reported FSHR overexpression in ovarian and prostate cancer cells. Instead, specific overexpression in peripheral tumor blood vessels could be confirmed after thorough validation of the antibodies used. Full article
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Open AccessArticle Epitope Sequences in Dengue Virus NS1 Protein Identified by Monoclonal Antibodies
Antibodies 2017, 6(4), 14; https://doi.org/10.3390/antib6040014
Received: 8 August 2017 / Revised: 22 September 2017 / Accepted: 22 September 2017 / Published: 15 October 2017
Cited by 1 | Viewed by 2274 | PDF Full-text (3356 KB) | HTML Full-text | XML Full-text | Supplementary Files
Abstract
Dengue nonstructural protein 1 (NS1) is a multi-functional glycoprotein with essential functions both in viral replication and modulation of host innate immune responses. NS1 has been established as a good surrogate marker for infection. In the present study, we generated four anti-NS1 monoclonal
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Dengue nonstructural protein 1 (NS1) is a multi-functional glycoprotein with essential functions both in viral replication and modulation of host innate immune responses. NS1 has been established as a good surrogate marker for infection. In the present study, we generated four anti-NS1 monoclonal antibodies against recombinant NS1 protein from dengue virus serotype 2 (DENV2), which were used to map three NS1 epitopes. The sequence 193AVHADMGYWIESALNDT209 was recognized by monoclonal antibodies 2H5 and 4H1BC, which also cross-reacted with Zika virus (ZIKV) protein. On the other hand, the sequence 25VHTWTEQYKFQPES38 was recognized by mAb 4F6 that did not cross react with ZIKV. Lastly, a previously unidentified DENV2 NS1-specific epitope, represented by the sequence 127ELHNQTFLIDGPETAEC143, is described in the present study after reaction with mAb 4H2, which also did not cross react with ZIKV. The selection and characterization of the epitope, specificity of anti-NS1 mAbs, may contribute to the development of diagnostic tools able to differentiate DENV and ZIKV infections. Full article
(This article belongs to the Special Issue Monoclonal Antibodies) Printed Edition available
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