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Antibodies
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Open Access journal
ISSN (Print) 2073-4468
Published by MDPI

[222 journals]

Antibodies, Vol. 8, Pages 39: Dynamic Views of the Fc Region of
Immunoglobulin G Provided by Experimental and Computational Observations
- Authors: Saeko Yanaka, Rina Yogo, Rintaro Inoue, Masaaki Sugiyama, Satoru G. Itoh, Hisashi Okumura, Yohei Miyanoiri, Hirokazu Yagi, Tadashi Satoh, Takumi Yamaguchi, Koichi Kato
  First page: 39
  Abstract: The Fc portion of immunoglobulin G (IgG) is a horseshoe-shaped homodimer, which interacts with various effector proteins, including Fcγ receptors (FcγRs). These interactions are critically dependent on the pair of N-glycans packed between the two CH2 domains. Fucosylation of these N-glycans negatively affects human IgG1-FcγRIIIa interaction. The IgG1-Fc crystal structures mostly exhibit asymmetric quaternary conformations with divergent orientations of CH2 with respect to CH3. We aimed to provide dynamic views of IgG1-Fc by performing long-timescale molecular dynamics (MD) simulations, which were experimentally validated by small-angle X-ray scattering and nuclear magnetic resonance spectroscopy. Our simulation results indicated that the dynamic conformational ensembles of Fc encompass most of the previously reported crystal structures determined in both free and complex forms, although the major Fc conformers in solution exhibited almost symmetric, stouter quaternary structures, unlike the crystal structures. Furthermore, the MD simulations suggested that the N-glycans restrict the motional freedom of CH2 and endow quaternary-structure plasticity through multiple intramolecular interaction networks. Moreover, the fucosylation of these N-glycans restricts the conformational freedom of the proximal tyrosine residue of functional importance, thereby precluding its interaction with FcγRIIIa. The dynamic views of Fc will provide opportunities to control the IgG interactions for developing therapeutic antibodies.
  Citation: Antibodies
  PubDate: 2019-07-01
  DOI: 10.3390/antib8030039
  Issue No: Vol. 8, No. 3 (2019)
Antibodies, Vol. 8, Pages 40: Integrated Clarification and Purification of
Monoclonal Antibodies by Membrane Based Separation of Aqueous Two-Phase
Systems
- Authors: Thomas Kruse, Axel Schmidt, Markus Kampmann, Jochen Strube
  First page: 40
  Abstract: Therapeutic monoclonal antibodies (mAb) are used for the treatment of numerous serious diseases, which have led to an increasing demand over the last decades. Increased cell density and mAb titer of the cultivation broth lead to great challenges for the subsequent clarification and capture operations in the downstream process. As an alternative approach to the conventional downstream process, a selective mAb extraction via an aqueous two-phase system (ATPS) directly from the cultivation broth of a mAb producing industrial relevant chinese hamster ovary (CHO) cell line was investigated. An efficient purification of the mAb was accomplished by the ATPS composition. The phase separation was realized by a newly developed membrane based phase separator. Moreover, a complete cell removal was integrated into this process by the used membrane. A selectivity between both phases was achieved by membrane modification. Yields up to 93% in the light phase and removal of process related impurities were obtained after aqueous two-phase extraction (ATPE). Phase separation performance as well as contact angles on the membrane were characterized for different ATPS. ATPE directly from the cultivation broth in combination with the new membrane based phase separation led to a mAb yield of 78% with a simultaneous reduction of deoxyribonucleic acid (DNA) and host cell protein (HCP) load.
  Citation: Antibodies
  PubDate: 2019-07-02
  DOI: 10.3390/antib8030040
  Issue No: Vol. 8, No. 3 (2019)
Antibodies, Vol. 8, Pages 41: Bispecific T-Cell Redirection versus
Chimeric Antigen Receptor (CAR)-T Cells as Approaches to Kill Cancer Cells
- Authors: William R. Strohl, Michael Naso
  First page: 41
  Abstract: The concepts for T-cell redirecting bispecific antibodies (TRBAs) and chimeric antigen receptor (CAR)-T cells are both at least 30 years old but both platforms are just now coming into age. Two TRBAs and two CAR-T cell products have been approved by major regulatory agencies within the last ten years for the treatment of hematological cancers and an additional 53 TRBAs and 246 CAR cell constructs are in clinical trials today. Two major groups of TRBAs include small, short-half-life bispecific antibodies that include bispecific T-cell engagers (BiTE®s) which require continuous dosing and larger, mostly IgG-like bispecific antibodies with extended pharmacokinetics that can be dosed infrequently. Most CAR-T cells today are autologous, although significant strides are being made to develop off-the-shelf, allogeneic CAR-based products. CAR-Ts form a cytolytic synapse with target cells that is very different from the classical immune synapse both physically and mechanistically, whereas the TRBA-induced synapse is similar to the classic immune synapse. Both TRBAs and CAR-T cells are highly efficacious in clinical trials but both also present safety concerns, particularly with cytokine release syndrome and neurotoxicity. New formats and dosing paradigms for TRBAs and CAR-T cells are being developed in efforts to maximize efficacy and minimize toxicity, as well as to optimize use with both solid and hematologic tumors, both of which present significant challenges such as target heterogeneity and the immunosuppressive tumor microenvironment.
  Citation: Antibodies
  PubDate: 2019-07-03
  DOI: 10.3390/antib8030041
  Issue No: Vol. 8, No. 3 (2019)
Antibodies, Vol. 8, Pages 42: Selection and Characterization of Monoclonal
Antibodies Targeting Middle East Respiratory Syndrome Coronavirus through
a Human Synthetic Fab Phage Display Library Panning
- Authors: Kim, Lee, Park, Park, Lim, So, Woo, Ko, Lee, Lim, Ko, Park, Choi, Song, Lee, Kim, Kim
  First page: 42
  Abstract: Since its first report in the Middle East in 2012, the Middle East respiratory syndrome-coronavirus (MERS-CoV) has become a global concern due to the high morbidity and mortality of individuals infected with the virus. Although the majority of MERS-CoV cases have been reported in Saudi Arabia, the overall risk in areas outside the Middle East remains significant as inside Saudi Arabia. Additional pandemics of MERS-CoV are expected, and thus novel tools and reagents for therapy and diagnosis are urgently needed. Here, we used phage display to develop novel monoclonal antibodies (mAbs) that target MERS-CoV. A human Fab phage display library was panned against the S2 subunit of the MERS-CoV spike protein (MERS-S2P), yielding three unique Fabs (S2A3, S2A6, and S2D5). The Fabs had moderate apparent affinities (Half maximal effective concentration (EC50 = 123–421 nM) for MERS-S2P, showed no cross-reactivity to spike proteins from other CoVs, and were non-aggregating and thermostable (Tm = 61.5–80.4 °C). Reformatting the Fabs into IgGs (Immunoglobulin Gs) greatly increased their apparent affinities (KD = 0.17–1.2 nM), presumably due to the effects of avidity. These apparent affinities were notably higher than that of a previously reported anti-MERS-CoV S2 reference mAb (KD = 8.7 nM). Furthermore, two of the three mAbs (S2A3 and S2D5) bound only MERS-CoV (Erasmus Medical Center (EMC)) and not other CoVs, reflecting their high binding specificity. However, the mAbs lacked MERS-CoV neutralizing activity. Given their high affinity, specificity, and desirable stabilities, we anticipate that these anti-MERS-CoV mAbs would be suitable reagents for developing antibody-based diagnostics in laboratory or hospital settings for point-of-care testing.
  Citation: Antibodies
  PubDate: 2019-07-31
  DOI: 10.3390/antib8030042
  Issue No: Vol. 8, No. 3 (2019)
Antibodies, Vol. 8, Pages 43: Design and Production of Bispecific
Antibodies
- Authors: Qiong Wang, Yiqun Chen, Jaeyoung Park, Xiao Liu, Yifeng Hu, Tiexin Wang, Kevin McFarland, Michael J. Betenbaugh
  First page: 43
  Abstract: With the current biotherapeutic market dominated by antibody molecules, bispecific antibodies represent a key component of the next-generation of antibody therapy. Bispecific antibodies can target two different antigens at the same time, such as simultaneously binding tumor cell receptors and recruiting cytotoxic immune cells. Structural diversity has been fast-growing in the bispecific antibody field, creating a plethora of novel bispecific antibody scaffolds, which provide great functional variety. Two common formats of bispecific antibodies on the market are the single-chain variable fragment (scFv)-based (no Fc fragment) antibody and the full-length IgG-like asymmetric antibody. Unlike the conventional monoclonal antibodies, great production challenges with respect to the quantity, quality, and stability of bispecific antibodies have hampered their wider clinical application and acceptance. In this review, we focus on these two major bispecific types and describe recent advances in the design, production, and quality of these molecules, which will enable this important class of biologics to reach their therapeutic potential.
  Citation: Antibodies
  PubDate: 2019-08-02
  DOI: 10.3390/antib8030043
  Issue No: Vol. 8, No. 3 (2019)
Antibodies, Vol. 8, Pages 44: Phage Display Libraries for Antibody
Therapeutic Discovery and Development
- Authors: Juan C. Almagro, Martha Pedraza-Escalona, Hugo Iván Arrieta, Sonia Mayra Pérez-Tapia
  First page: 44
  Abstract: Phage display technology has played a key role in the remarkable progress of discovering and optimizing antibodies for diverse applications, particularly antibody-based drugs. This technology was initially developed by George Smith in the mid-1980s and applied by John McCafferty and Gregory Winter to antibody engineering at the beginning of 1990s. Here, we compare nine phage display antibody libraries published in the last decade, which represent the state of the art in the discovery and development of therapeutic antibodies using phage display. We first discuss the quality of the libraries and the diverse types of antibody repertoires used as substrates to build the libraries, i.e., naïve, synthetic, and semisynthetic. Second, we review the performance of the libraries in terms of the number of positive clones per panning, hit rate, affinity, and developability of the selected antibodies. Finally, we highlight current opportunities and challenges pertaining to phage display platforms and related display technologies.
  Citation: Antibodies
  PubDate: 2019-08-23
  DOI: 10.3390/antib8030044
  Issue No: Vol. 8, No. 3 (2019)
Antibodies, Vol. 8, Pages 45: Back-To-Germline (B2G) Procedure for
Antibody Devolution
- Authors: Schrade, Bujotzek, Spick, Wagner, Goerl, Wezler, Georges, Kontermann, Brinkmann
  First page: 45
  Abstract: Bispecific antibodies (bsAbs) with avidity-enhanced specificity can be used to address target cells with increased specificity, ideally binding efficiently to cells that express two cognate antigens, yet not to cells that express only one of those. Building blocks required to generate such bsAbs are binders that recognize the two antigens with high specificity yet with various (including very low monovalent) affinities. The herein described ‘back-to-germline’ (B2G) procedure defines such derivatives. It converts parent antibodies with high specificity to derivatives that retain specificity but modulate affinity. The approach defines mutations to be introduced into antibody complementarity-determining regions (CDRs) regions without requiring structures of antibody-antigen complexes. Instead, it reverses the B-cell maturation process that increases affinities, with preference on CDR residues with high antigen contact probability. Placing germline residues at those positions generates VH and VL domains and Fv-combinations thereof that retain specificities but are ‘de-matured‘ to different degrees. De-maturation influences on-rates and off-rates, and can produce entities with extremely low affinity for which binding can only be detected in bivalent formats. A comparison with alanine replacement in CDRs (so far, the most frequently applied technology) indicates that B2G may be more reliable/predictable without introduction of stickiness or poly-reactivity. The applicability for generating sets of affinity-modulated monospecific variants is exemplarily shown for antibodies that bind CD138, Her2/neu, and EGFR.
  Citation: Antibodies
  PubDate: 2019-08-26
  DOI: 10.3390/antib8030045
  Issue No: Vol. 8, No. 3 (2019)
Antibodies, Vol. 8, Pages 26: VHH-Photosensitizer Conjugates for Targeted
Photodynamic Therapy of Met-Overexpressing Tumor Cells
- Authors: Raimond Heukers, Vida Mashayekhi, Mercedes Ramirez-Escudero, Hans de Haard, Theo C. Verrips, Paul. M.P. van Bergen en Henegouwen, Sabrina Oliveira
  First page: 26
  Abstract: Photodynamic therapy (PDT) is an approach that kills (cancer) cells by the local production of toxic reactive oxygen species upon the local illumination of a photosensitizer (PS). The specificity of PDT has been further enhanced by the development of a new water-soluble PS and by the specific delivery of PS via conjugation to tumor-targeting antibodies. To improve tissue penetration and shorten photosensitivity, we have recently introduced nanobodies, also known as VHH (variable domains from the heavy chain of llama heavy chain antibodies), for targeted PDT of cancer cells overexpressing the epidermal growth factor receptor (EGFR). Overexpression and activation of another cancer-related receptor, the hepatocyte growth factor receptor (HGFR, c-Met or Met) is also involved in the progression and metastasis of a large variety of malignancies. In this study we evaluate whether anti-Met VHHs conjugated to PS can also serve as a biopharmaceutical for targeted PDT. VHHs targeting the SEMA (semaphorin-like) subdomain of Met were provided with a C-terminal tag that allowed both straightforward purification from yeast supernatant and directional conjugation to the PS IRDye700DX using maleimide chemistry. The generated anti-Met VHH-PS showed nanomolar binding affinity and, upon illumination, specifically killed MKN45 cells with nanomolar potency. This study shows that Met can also serve as a membrane target for targeted PDT.
  Citation: Antibodies
  PubDate: 2019-04-04
  DOI: 10.3390/antib8020026
  Issue No: Vol. 8, No. 2 (2019)
Antibodies, Vol. 8, Pages 27: Single-Domain Antibodies as Therapeutic and
Imaging Agents for the Treatment of CNS Diseases
- Authors: Kasandra Bélanger, Umar Iqbal, Jamshid Tanha, Roger MacKenzie, Maria Moreno, Danica Stanimirovic
  First page: 27
  Abstract: Antibodies have become one of the most successful therapeutics for a number of oncology and inflammatory diseases. So far, central nervous system (CNS) indications have missed out on the antibody revolution, while they remain ‘hidden’ behind several hard to breach barriers. Among the various antibody modalities, single-domain antibodies (sdAbs) may hold the ‘key’ to unlocking the access of antibody therapies to CNS diseases. The unique structural features of sdAbs make them the smallest monomeric antibody fragments suitable for molecular targeting. These features are of particular importance when developing antibodies as modular building blocks for engineering CNS-targeting therapeutics and imaging agents. In this review, we first introduce the characteristic properties of sdAbs compared to traditional antibodies. We then present recent advances in the development of sdAbs as potential therapeutics across brain barriers, including their use for the delivery of biologics across the blood–brain and blood–cerebrospinal fluid (CSF) barriers, treatment of neurodegenerative diseases and molecular imaging of brain targets.
  Citation: Antibodies
  PubDate: 2019-04-05
  DOI: 10.3390/antib8020027
  Issue No: Vol. 8, No. 2 (2019)
Antibodies, Vol. 8, Pages 28: David vs. Goliath: The Structure, Function,
and Clinical Prospects of Antibody Fragments
- Authors: Adam Bates, Christine A. Power
  First page: 28
  Abstract: Since the licensing of the first monoclonal antibody therapy in 1986, monoclonal antibodies have become the largest class of biopharmaceuticals with over 80 antibodies currently approved for a variety of disease indications. The development of smaller, antigen binding antibody fragments, derived from conventional antibodies or produced recombinantly, has been growing at a fast pace. Antibody fragments can be used on their own or linked to other molecules to generate numerous possibilities for bispecific, multi-specific, multimeric, or multifunctional molecules, and to achieve a variety of biological effects. They offer several advantages over full-length monoclonal antibodies, particularly a lower cost of goods, and because of their small size they can penetrate tissues, access challenging epitopes, and have potentially reduced immunogenicity. In this review, we will discuss the structure, production, and mechanism of action of EMA/FDA-approved fragments and of those in clinical and pre-clinical development. We will also discuss current topics of interest surrounding the potential use of antibody fragments for intracellular targeting and blood–brain barrier (BBB) penetration.
  Citation: Antibodies
  PubDate: 2019-04-09
  DOI: 10.3390/antib8020028
  Issue No: Vol. 8, No. 2 (2019)
Antibodies, Vol. 8, Pages 29: IMGT® and 30 Years of Immunoinformatics
Insight in Antibody V and C Domain Structure and Function
- Authors: Marie-Paule Lefranc, Gérard Lefranc
  First page: 29
  Abstract: At the 10th Human Genome Mapping (HGM10) Workshop, in New Haven, for the first time, immunoglobulin (IG) or antibody and T cell receptor (TR) variable (V), diversity (D), joining (J), and constant (C) genes were officially recognized as ‘genes’, as were the conventional genes. Under these HGM auspices, IMGT®, the international ImMunoGeneTics information system®, was created in June 1989 at Montpellier (University of Montpellier and CNRS). The creation of IMGT® marked the birth of immunoinformatics, a new science, at the interface between immunogenetics and bioinformatics. The accuracy and the consistency between genes and alleles, sequences, and three-dimensional (3D) structures are based on the IMGT Scientific chart rules generated from the IMGT-ONTOLOGY axioms and concepts: IMGT standardized keywords (IDENTIFICATION), IMGT gene and allele nomenclature (CLASSIFICATION), IMGT standardized labels (DESCRIPTION), IMGT unique numbering and IMGT Collier de Perles (NUMEROTATION). These concepts provide IMGT® immunoinformatics insights for antibody V and C domain structure and function, used for the standardized description in IMGT® web resources, databases and tools, immune repertoires analysis, single cell and/or high-throughput sequencing (HTS, NGS), antibody humanization, and antibody engineering in relation with effector properties.
  Citation: Antibodies
  PubDate: 2019-04-11
  DOI: 10.3390/antib8020029
  Issue No: Vol. 8, No. 2 (2019)
Antibodies, Vol. 8, Pages 30: The Ligands for Human IgG and Their Effector
Functions
- Authors: Steven W. de Taeye, Theo Rispens, Gestur Vidarsson
  First page: 30
  Abstract: Activation of the humoral immune system is initiated when antibodies recognize an antigen and trigger effector functions through the interaction with Fc engaging molecules. The most abundant immunoglobulin isotype in serum is Immunoglobulin G (IgG), which is involved in many humoral immune responses, strongly interacting with effector molecules. The IgG subclass, allotype, and glycosylation pattern, among other factors, determine the interaction strength of the IgG-Fc domain with these Fc engaging molecules, and thereby the potential strength of their effector potential. The molecules responsible for the effector phase include the classical IgG-Fc receptors (FcγR), the neonatal Fc-receptor (FcRn), the Tripartite motif-containing protein 21 (TRIM21), the first component of the classical complement cascade (C1), and possibly, the Fc-receptor-like receptors (FcRL4/5). Here we provide an overview of the interactions of IgG with effector molecules and discuss how natural variation on the antibody and effector molecule side shapes the biological activities of antibodies. The increasing knowledge on the Fc-mediated effector functions of antibodies drives the development of better therapeutic antibodies for cancer immunotherapy or treatment of autoimmune diseases.
  Citation: Antibodies
  PubDate: 2019-04-25
  DOI: 10.3390/antib8020030
  Issue No: Vol. 8, No. 2 (2019)
Antibodies, Vol. 8, Pages 31: The Diagnostic and Clinical Utility of
Autoantibodies in Systemic Vasculitis
- Authors: Elena Csernok
  First page: 31
  Abstract: Considerable progress has been made in understanding the role of autoantibodies in systemic vasculitides (SV), and consequently testing for anti-neutrophil cytoplasmic antibodies (ANCA), anti-glomerular basement membrane antibodies (anti-GBM), and anti-C1q antibodies is helpful and necessary in the diagnosis, prognosis, and monitoring of small-vessel vasculitis. ANCA-directed proteinase 3 (PR3-) or myeloperoxidase (MPO-) are sensitive and specific serologic markers for ANCA-associated vasculitides (AAV), anti-GBM antibodies are highly specific for the patients with anti-GBM antibody disease (formerly Goodpasture’s syndrome), and autoantibodies to C1q are characteristic of hypocomlementemic urticarial vasculitis syndrome (HUVS; anti-C1q vasculitis). The results of a current EUVAS study have led to changes in the established strategy for the ANCA testing in small-vessel vasculitis. The revised 2017 international consensus recommendations for ANCA detection support the primary use PR3- and MPO-ANCA immunoassays without the categorical need for additional indirect immunofluorescence (IIF). Interestingly, the presence of PR3- and MPO-ANCA have led to the differentiation of distinct disease phenotype of AAV: PR3-ANCA-associated vasculitis (PR3-AAV), MPO-ANCA-associated vasculitis (MPO-AAV), and ANCA-negative vasculitis. Further studies on the role of these autoantibodies are required to better categorize and manage appropriately the patients with small-vessel vasculitis and to develop more targeted therapy.
  Citation: Antibodies
  PubDate: 2019-05-01
  DOI: 10.3390/antib8020031
  Issue No: Vol. 8, No. 2 (2019)
Antibodies, Vol. 8, Pages 32: Targeting the MHC Ligandome by Use of
TCR-Like Antibodies
- Authors: Lene Støkken Høydahl, Rahel Frick, Inger Sandlie, Geir Åge Løset
  First page: 32
  Abstract: Monoclonal antibodies (mAbs) are valuable as research reagents, in diagnosis and in therapy. Their high specificity, the ease in production, favorable biophysical properties and the opportunity to engineer different properties make mAbs a versatile class of biologics. mAbs targeting peptide–major histocompatibility molecule (pMHC) complexes are often referred to as “TCR-like” mAbs, as pMHC complexes are generally recognized by T-cell receptors (TCRs). Presentation of self- and non-self-derived peptide fragments on MHC molecules and subsequent activation of T cells dictate immune responses in health and disease. This includes responses to infectious agents or cancer but also aberrant responses against harmless self-peptides in autoimmune diseases. The ability of TCR-like mAbs to target specific peptides presented on MHC allows for their use to study peptide presentation or for diagnosis and therapy. This extends the scope of conventional mAbs, which are generally limited to cell-surface or soluble antigens. Herein, we review the strategies used to generate TCR-like mAbs and provide a structural comparison with the analogous TCR in pMHC binding. We further discuss their applications as research tools and therapeutic reagents in preclinical models as well as challenges and limitations associated with their use.
  Citation: Antibodies
  PubDate: 2019-05-09
  DOI: 10.3390/antib8020032
  Issue No: Vol. 8, No. 2 (2019)
Antibodies, Vol. 8, Pages 33: Ion Channel Targeting with Antibodies and
Antibody Fragments for Cancer Diagnosis
- Authors: Claudia Duranti, Annarosa Arcangeli
  First page: 33
  Abstract: The antibody era has greatly impacted cancer management in recent decades. Indeed, antibodies are currently applied for both cancer diagnosis and therapy. For example, monoclonal antibodies are the main constituents of several in vitro diagnostics, which are applied at many levels of cancer diagnosis. Moreover, the great improvement provided by in vivo imaging, especially for early-stage cancer diagnosis, has traced the path for the development of a complete new class of antibodies, i.e., engineered antibody fragments. The latter embody the optimal characteristics (e.g., low renal retention, rapid clearance, and small size) which make them ideal for in vivo applications. Furthermore, the present review focuses on reviewing the main applications of antibodies and antibody fragments for solid cancer diagnosis, both in vitro and in vivo. Furthermore, we review the scientific evidence showing that ion channels represent an almost unexplored class of ideal targets for both in vitro and in vivo diagnostic purposes. In particular, we review the applications, in solid cancers, of monoclonal antibodies and engineered antibody fragments targeting the voltage-dependent ion channel Kv 11.1, also known as hERG1.
  Citation: Antibodies
  PubDate: 2019-05-24
  DOI: 10.3390/antib8020033
  Issue No: Vol. 8, No. 2 (2019)
Antibodies, Vol. 8, Pages 34: Investigational Monoclonal Antibodies in the
Treatment of Multiple Myeloma: A Systematic Review of Agents under
Clinical Development
- Authors: Ahmad Iftikhar, Hamza Hassan, Nimra Iftikhar, Adeela Mushtaq, Atif Sohail, Nathaniel Rosko, Rajshekhar Chakraborty, Faryal Razzaq, Sonia Sandeep, Jason Neil Valent, Abraham Sebastian Kanate, Faiz Anwer
  First page: 34
  Abstract: Background: Immunotherapy for multiple myeloma (MM) has been the focus in recent years due to its myeloma-specific immune responses. We reviewed the literature on non-Food and Drug Administration (FDA) approved monoclonal antibodies (mAbs) to highlight future perspectives. We searched PubMed, EMBASE, Web of Science, Cochrane Library and ClinicalTrials.gov to include phase I/II clinical trials. Data from 39 studies (1906 patients) were included. Of all the agents, Isatuximab (Isa, anti-CD38) and F50067 (anti-CXCR4) were the only mAbs to produce encouraging results as monotherapy with overall response rates (ORRs) of 66.7% and 32% respectively. Isa showed activity when used in combination with lenalidomide (Len) and dexamethasone (Dex), producing a clinical benefit rate (CBR) of 83%. Additionally, Isa used in combination with pomalidomide (Pom) and Dex resulted in a CBR of 73%. Indatuximab Ravtansine (anti-CD138 antibody-drug conjugate) produced an ORR of 78% and 79% when used in combination with Len-Dex and Pom-Dex, respectively. Conclusions: Combination therapy using mAbs such as indatuximab, pembrolizumab, lorvotuzumab, siltuximab or dacetuzumab with chemotherapy agents produced better outcomes as compared to monotherapies. Further clinical trials investigating mAbs targeting CD38 used in combination therapy are warranted.
  Citation: Antibodies
  PubDate: 2019-05-24
  DOI: 10.3390/antib8020034
  Issue No: Vol. 8, No. 2 (2019)
Antibodies, Vol. 8, Pages 35: EBNA1 IgM-Based Discrimination Between
Rheumatoid Arthritis Patients, Systemic Lupus Erythematosus Patients and
Healthy Controls
- Authors: Nicole Hartwig Trier, Anette Holck Draborg, Louise Sternbæk, Lone Troelsen, Janni Lisander Larsen, Søren Jacobsen, Gunnar Houen
  First page: 35
  Abstract: Epstein–Barr Virus (EBV) has been associated with development of rheumatic connective tissue diseases like rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE) in genetically susceptible individuals. Diagnosis of RA and SLE relies on clinical criteria in combination with the presence of characteristic autoantibodies. In addition, antibodies to several EBV antigens have been shown to be elevated in patients with these diseases compared to healthy controls (HC). Here, we elaborated improved enzyme-linked immunosorbent assays for antibodies (IgM, IgA, IgG) to the EBV proteins Epstein-Barr Virus nuclear antigen (EBNA)1 and early antigen diffuse (EAD) in order to determine their potential diagnostic role. We showed that especially EBNA1 IgM distinguished RA from SLE and HCs and also distinguished SLE from HCs. EBNA1 IgA was almost as effective in differentiating RA from SLE and HC, while EAD IgG and IgA were able to discern SLE patients from RA patients and HCs. Collectively, these findings illustrate the potential diagnostic use of antibodies to EBV proteins to diagnose RA and to differentiate SLE from RA.
  Citation: Antibodies
  PubDate: 2019-06-01
  DOI: 10.3390/antib8020035
  Issue No: Vol. 8, No. 2 (2019)
Antibodies, Vol. 8, Pages 36: Current Advancements in Addressing Key
Challenges of Therapeutic Antibody Design, Manufacture, and Formulation
- Authors: Vicki Sifniotis, Esteban Cruz, Barbaros Eroglu, Veysel Kayser
  First page: 36
  Abstract: Therapeutic antibody technology heavily dominates the biologics market and continues to present as a significant industrial interest in developing novel and improved antibody treatment strategies. Many noteworthy advancements in the last decades have propelled the success of antibody development; however, there are still opportunities for improvement. In considering such interest to develop antibody therapies, this review summarizes the array of challenges and considerations faced in the design, manufacture, and formulation of therapeutic antibodies, such as stability, bioavailability and immunological engagement. We discuss the advancement of technologies that address these challenges, highlighting key antibody engineered formats that have been adapted. Furthermore, we examine the implication of novel formulation technologies such as nanocarrier delivery systems for the potential to formulate for pulmonary delivery. Finally, we comprehensively discuss developments in computational approaches for the strategic design of antibodies with modulated functions.
  Citation: Antibodies
  PubDate: 2019-06-03
  DOI: 10.3390/antib8020036
  Issue No: Vol. 8, No. 2 (2019)
Antibodies, Vol. 8, Pages 37: Specificity of Anti-Citrullinated Protein
Antibodies in Rheumatoid Arthritis
- Authors: Nicole H. Trier, Bettina E. Holm, Paul R. Hansen, Ole Slot, Henning Locht, Gunnar Houen
  First page: 37
  Abstract: Rheumatoid arthritis (RA) is an autoimmune disease of unknown etiology. The majority of individuals with RA are positive for the disease-specific anti-citrullinated protein antibodies (ACPAs). These antibodies are primarily of cross-reactive nature, hence, the true autoantigen to ACPA remains unidentified. In this study, we analyzed the reactivity of RA sera to several post-translationally modified epitopes, in order to further characterize the specific nature of ACPAs by immunoassays. Substituting citrulline with other amino acids, e.g., D-citrulline, homo-citrulline and methyl-arginine illustrated that ACPAs are utmost specific for citrullinated targets. Collectively, these findings support that ACPAs and citrullinated targets are specific for RA, making citrulline-containing peptide targets the most effective assays for detection of ACPAs.
  Citation: Antibodies
  PubDate: 2019-06-07
  DOI: 10.3390/antib8020037
  Issue No: Vol. 8, No. 2 (2019)
Antibodies, Vol. 8, Pages 38: Super Potent Bispecific Llama VHH Antibodies
Neutralize HIV via a Combination of gp41 and gp120 Epitopes
- Authors: Nika M. Strokappe, Miriam Hock, Lucy Rutten, Laura E. Mccoy, Jaap W. Back, Christophe Caillat, Matthias Haffke, Robin A. Weiss, Winfried Weissenhorn, Theo Verrips
  First page: 38
  Abstract: Broad and potent neutralizing llama single domain antibodies (VHH) against HIV-1 targeting the CD4 binding site (CD4bs) have previously been isolated upon llama immunization. Here we describe the epitopes of three additional VHH groups selected from phage libraries. The 2E7 group binds to a new linear epitope in the first heptad repeat of gp41 that is only exposed in the fusion-intermediate conformation. The 1B5 group competes with co-receptor binding and the 1F10 group interacts with the crown of the gp120 V3 loop, occluded in native Env. We present biophysical and structural details on the 2E7 interaction with gp41. In order to further increase breadth and potency, we constructed bi-specific VHH. The combination of CD4bs VHH (J3/3E3) with 2E7 group VHH enhanced strain-specific neutralization with potencies up to 1400-fold higher than the mixture of the individual VHHs. Thus, these new bivalent VHH are potent new tools to develop therapeutic approaches or microbicide intervention.
  Citation: Antibodies
  PubDate: 2019-06-18
  DOI: 10.3390/antib8020038
  Issue No: Vol. 8, No. 2 (2019)
Antibodies, Vol. 8, Pages 3: Pharmacologic Considerations in the
Disposition of Antibodies and Antibody-Drug Conjugates in Preclinical
Models and in Patients
- Authors: Andrew T. Lucas, Ryan Robinson, Allison N. Schorzman, Joseph A. Piscitelli, Juan F. Razo, William C. Zamboni
  First page: 3
  Abstract: The rapid advancement in the development of therapeutic proteins, including monoclonal antibodies (mAbs) and antibody-drug conjugates (ADCs), has created a novel mechanism to selectively deliver highly potent cytotoxic agents in the treatment of cancer. These agents provide numerous benefits compared to traditional small molecule drugs, though their clinical use still requires optimization. The pharmacology of mAbs/ADCs is complex and because ADCs are comprised of multiple components, individual agent characteristics and patient variables can affect their disposition. To further improve the clinical use and rational development of these agents, it is imperative to comprehend the complex mechanisms employed by antibody-based agents in traversing numerous biological barriers and how agent/patient factors affect tumor delivery, toxicities, efficacy, and ultimately, biodistribution. This review provides an updated summary of factors known to affect the disposition of mAbs/ADCs in development and in clinical use, as well as how these factors should be considered in the selection and design of preclinical studies of ADC agents in development.
  Citation: Antibodies
  PubDate: 2019-01-01
  DOI: 10.3390/antib8010003
  Issue No: Vol. 8, No. 1 (2019)
Antibodies, Vol. 8, Pages 4: Acknowledgement to Reviewers of Antibodies in
2018
- Authors: Antibodies Editorial Office Antibodies Editorial Office
  First page: 4
  Abstract: Peer review is an essential part of the publication process, ensuring that Antibodies maintains high quality standards for its published papers. [...]
  Citation: Antibodies
  PubDate: 2019-01-01
  DOI: 10.3390/antib8010004
  Issue No: Vol. 8, No. 1 (2019)
Antibodies, Vol. 8, Pages 5: Current Approaches and Future Perspectives
for Nanobodies in Stroke Diagnostic and Therapy
- Authors: Larissa Jank, Carolina Pinto-Espinoza, Yinghui Duan, Friedrich Koch-Nolte, Tim Magnus, Björn Rissiek
  First page: 5
  Abstract: Antibody-based biologics are the corner stone of modern immunomodulatory therapy. Though highly effective in dampening systemic inflammatory processes, their large size and Fc-fragment mediated effects hamper crossing of the blood brain barrier (BBB). Nanobodies (Nbs) are single domain antibodies derived from llama or shark heavy-chain antibodies and represent a new generation of biologics. Due to their small size, they display excellent tissue penetration capacities and can be easily modified to adjust their vivo half-life for short-term diagnostic or long-term therapeutic purposes or to facilitate crossing of the BBB. Furthermore, owing to their characteristic binding mode, they are capable of antagonizing receptors involved in immune signaling and of neutralizing proinflammatory mediators, such as cytokines. These qualities combined make Nbs well-suited for down-modulating neuroinflammatory processes that occur in the context of brain ischemia. In this review, we summarize recent findings on Nbs in preclinical stroke models and how they can be used as diagnostic and therapeutic reagents. We further provide a perspective on the design of innovative Nb-based treatment protocols to complement and improve stroke therapy.
  Citation: Antibodies
  PubDate: 2019-01-03
  DOI: 10.3390/antib8010005
  Issue No: Vol. 8, No. 1 (2019)
Antibodies, Vol. 8, Pages 6: Binding of Immunoglobulin G to Protoporphyrin
IX and Its Derivatives: Evidence the Fab Domain Recognizes the
Protoporphyrin Ring
- Authors: Koichi Orino
  First page: 6
  Abstract: Immunoglobulin G (IgG) is known to bind zinc via the Fc domain. In this study, biotinylated protoporphyrin IX (PPIX) was incubated with human IgG and then zinc-immobilized Sepharose beads (Zn-beads) were added to the mixture. After washing the beads, the binding of biotinylated PPIX with IgG trapped on Zn-beads was detected using alkaline phosphatase (ALP)-labeled avidin. Human IgG and its Fab domain coated on microtiter plate wells recognized biotin-labeled PPIX and its derivatives, Fe-PPIX and Zn-PPIX, whereas the Fc domain showed some extent of reaction only with Zn-PPIX. When rabbit anti-bovine transferrin (Tf) antibodies were incubated with biotinylated PPIX, the binding of anti-Tf antibodies with apo-Tf was indirectly detected using ALP-labeled avidin, suggesting that even if the antibody is modified with PPIX, the antibody-antigen reaction occurs. These results suggest that the IgG Fab domain recognizes PPIX and its derivatives, probably via the recognition of the PPIX ring. It is unlikely that binding between the Fab domain and PPIX affects the Fc domain-zinc interaction or antigen-antibody reaction.
  Citation: Antibodies
  PubDate: 2019-01-04
  DOI: 10.3390/antib8010006
  Issue No: Vol. 8, No. 1 (2019)
Antibodies, Vol. 8, Pages 7: A RAGE-Targeted Antibody-Drug Conjugate:
Surface Plasmon Resonance as a Platform for Accelerating Effective ADC
Design and Development
- Authors: Gareth D. Healey, Asa Frostell, Tim Fagge, Deyarina Gonzalez, R. Steven Conlan
  First page: 7
  Abstract: Antibodies, antibody-like molecules, and therapeutics incorporating antibodies as a targeting moiety, such as antibody-drug conjugates, offer significant potential for the development of highly efficacious drugs against a wide range of disorders. Despite some success, truly harnessing the superior targeting properties of these molecules requires a platform from which to effectively identify the best candidates for drug development. To streamline the development of antibody-drug conjugates targeting gynecological cancers within our laboratory, we incorporated surface plasmon resonance analysis (Biacore™ T200) into our development toolkit. Antibodies, selected based on positive ELISA screens as suitable for development as antibody-drug conjugates, were evaluated using surface plasmon resonance to determine a wide range of characteristics including specificity, kinetics/affinity, the effect of linker binding, the impact of the drug to antibody ratio, and the effect of endosomal pH on antibody-antigen binding. Analysis revealed important kinetics data and information regarding the effect of conjugation and endosomal pH on our antibody candidates that correlated with cell toxicity and antibody internalization data. As well as explaining observations from cell-based assays regarding antibody-drug conjugate efficacies, these data also provide important information regarding intelligent antibody selection and antibody-drug conjugate design. This study demonstrates the application of surface plasmon resonance technology as a platform, where detailed information can be obtained, supporting the requirements for rapid and high-throughput screening that will enable enhanced antibody-drug conjugate development.
  Citation: Antibodies
  PubDate: 2019-01-07
  DOI: 10.3390/antib8010007
  Issue No: Vol. 8, No. 1 (2019)
Antibodies, Vol. 8, Pages 8: Selection and Characterization of a Nanobody
Biosensor of GTP-Bound RHO Activities
- Authors: Laura Keller, Nicolas Bery, Claudine Tardy, Laetitia Ligat, Gilles Favre, Terence H. Rabbitts, Aurélien Olichon
  First page: 8
  Abstract: RHO (Ras HOmologous) GTPases are molecular switches that activate, in their state bound to Guanosine triphosphate (GTP), key signaling pathways, which involve actin cytoskeleton dynamics. Previously, we selected the nanobody RH12, from a synthetic phage display library, which binds the GTP-bound active conformation of RHOA (Ras Homologous family member A). However, when expressed as an intracellular antibody, its blocking effect on RHO signaling led to a loss of actin fibers, which in turn affected cell shape and cell survival. Here, in order to engineer an intracellular biosensor of RHOA-GTP activation, we screened the same phage nanobody library and identified another RHO-GTP selective intracellular nanobody, but with no apparent toxicity. The recombinant RH57 nanobody displays high affinity towards GTP-bound RHOA/B/C subgroup of small GTPases in vitro. Intracellular expression of the RH57 allowed selective co-precipitation with the GTP-bound state of the endogenous RHOA subfamily. When expressed as a fluorescent fusion protein, the chromobody GFP-RH57 was localized to the inner plasma membrane upon stimulation of the activation of endogenous RHO. Finally, the RH57 nanobody was used to establish a BRET-based biosensor (Bioluminescence Resonance Energy Transfer) of RHO activation. The dynamic range of the BRET signal could potentially offer new opportunities to develop cell-based screening of RHOA subfamily activation modulators.
  Citation: Antibodies
  PubDate: 2019-01-09
  DOI: 10.3390/antib8010008
  Issue No: Vol. 8, No. 1 (2019)
Antibodies, Vol. 8, Pages 9: Impact of Acetylated and Non-Acetylated
Fucose Analogues on IgG Glycosylation
- Authors: Martina Zimmermann, Janike Ehret, Harald Kolmar, Aline Zimmer
  First page: 9
  Abstract: The biological activity of therapeutic antibodies is highly influenced by their glycosylation profile. A valuable method for increasing the cytotoxic efficacy of antibodies, which are used, for example, in cancer treatment, is the reduction of core fucosylation, as this enhances the elimination of target cells through antibody-dependent cell-mediated cytotoxicity. Development of fucose analogues is currently the most promising strategy to reduce core fucosylation without cell line engineering. Since peracetylated sugars display enhanced cell permeability over the highly polar free hydroxy sugars, this work sought to compare the efficacy of peracetylated sugars to their unprotected forms. Two potent fucose analogues, 2-deoxy-2-fluorofucose and 5-alkynylfucose, and their acetylated forms were compared for their effects on fucosylation. 5-alkynylfucose proved to be more potent than 2-deoxy-2-fluorofucose at reducing core fucosylation but was associated with a significant higher incorporation of the alkynylated fucose analogue. Acetylation of the sugar yielded only slightly lower fucosylation levels suggesting that acetylation has a minor impact on cellular entry. Even though the efficacy of all tested components was confirmed, results presented in this study also show a significant incorporation of unnatural fucose analogues into the glycosylation pattern of the produced IgG, with unknown effect on safety and potency of the monoclonal antibody.
  Citation: Antibodies
  PubDate: 2019-01-10
  DOI: 10.3390/antib8010009
  Issue No: Vol. 8, No. 1 (2019)
Antibodies, Vol. 8, Pages 10: A Strategy to Optimize the Generation of
Stable Chromobody Cell Lines for Visualization and Quantification of
Endogenous Proteins in Living Cells
- Authors: Bettina-Maria Keller, Julia Maier, Melissa Weldle, Soeren Segan, Bjoern Traenkle, Ulrich Rothbauer
  First page: 10
  Abstract: Single-domain antibodies have emerged as highly versatile nanoprobes for advanced cellular imaging. For real-time visualization of endogenous antigens, fluorescently labelled nanobodies (chromobodies, CBs) are introduced as DNA-encoded expression constructs in living cells. Commonly, CB expression is driven from strong, constitutively active promoters. However, high expression levels are sometimes accompanied by misfolding and aggregation of those intracellular nanoprobes. Moreover, stable cell lines derived from random genomic insertion of CB-encoding transgenes bear the risk of disturbed cellular processes and inhomogeneous CB signal intensities due to gene positioning effects and epigenetic silencing. In this study we propose a strategy to generate optimized CB expressing cell lines. We demonstrate that expression as ubiquitin fusion increases the fraction of intracellularly functional CBs and identified the elongation factor 1α (EF1-α) promoter as highly suited for constitutive CB expression upon long-term cell line cultivation. Finally, we applied a CRISPR/Cas9-based gene editing approach for targeted insertion of CB expression constructs into the adeno-associated virus integration site 1 (AAVS1) safe harbour locus of human cells. Our results indicate that this combinatorial approach facilitates the generation of fully functional and stable CB cell lines for quantitative live-cell imaging of endogenous antigens.
  Citation: Antibodies
  PubDate: 2019-01-10
  DOI: 10.3390/antib8010010
  Issue No: Vol. 8, No. 1 (2019)
Antibodies, Vol. 8, Pages 11: Multiplex LC-MS/MS Assays for Clinical
Bioanalysis of MEDI4276, an Antibody-Drug Conjugate of Tubulysin Analogue
Attached via Cleavable Linker to a Biparatopic Humanized Antibody against
HER-2
- Authors: Morse Faria, Marlking Peay, Brandon Lam, Eric Ma, Moucun Yuan, Michael Waldron, William R. Mylott, Meina Liang, Anton I. Rosenbaum
  First page: 11
  Abstract: Bioanalysis of complex biotherapeutics, such as antibody-drug conjugates (ADCs), is challenging and requires multiple assays to describe their pharmacokinetic (PK) profiles. To enable exposure-safety and exposure-efficacy analyses, as well as to understand the metabolism of ADC therapeutics, three bioanalytical methods are typically employed: Total Antibody, Antibody Conjugated Toxin or Total ADC and Unconjugated Toxin. MEDI4276 is an ADC comprised of biparatopic humanized antibody attached via a protease-cleavable peptide-based maleimidocaproyl linker to a tubulysin toxin (AZ13599185) with an approximate average drug-antibody ratio of 4. The conjugated payload of MEDI4276 can undergo ester hydrolysis to produce the conjugated payload AZ13687308, leading to the formation of MEDI1498 (de-acetylated MEDI4276). In this report, we describe the development, validation and application of three novel multiplex bioanalytical methods. The first ligand-binding liquid chromatography coupled with tandem mass spectrometry (LBA-LC-MS/MS) method was developed and validated for simultaneous measurement of total antibody and total ADC (antibody-conjugated AZ13599185) from MEDI4276. The second LBA-LC-MS/MS assay quantified total ADC (antibody-conjugated AZ13687308) from MEDI1498. The third multiplex LC-MS/MS assay was used for simultaneous quantification of unconjugated AZ13599185 and AZ13687308. Additional stability experiments confirmed that quantification of the released warhead in the presence of high concentrations of MEDI4276 was acceptable. Subsequently, the assays were employed in support of a first-in-human clinical trial (NCT02576548).
  Citation: Antibodies
  PubDate: 2019-01-11
  DOI: 10.3390/antib8010011
  Issue No: Vol. 8, No. 1 (2019)
Antibodies, Vol. 8, Pages 12: Targeted Nanobody-Based Molecular Tracers
for Nuclear Imaging and Image-Guided Surgery
- Authors: Pieterjan Debie, Nick Devoogdt, Sophie Hernot
  First page: 12
  Abstract: Molecular imaging is paving the way towards noninvasive detection, staging, and treatment follow-up of diseases such as cancer and inflammation-related conditions. Monoclonal antibodies have long been one of the staples of molecular imaging tracer design, although their long blood circulation and high nonspecific background limits their applicability. Nanobodies, unique antibody-binding fragments derived from camelid heavy-chain antibodies, have excellent properties for molecular imaging as they are able to specifically find their target early after injection, with little to no nonspecific background. Nanobody-based tracers using either nuclear or fluorescent labels have been heavily investigated preclinically and are currently making their way into the clinic. In this review, we will discuss different important factors in nanobody-tracer design, as well as the current state of the art regarding their application for nuclear and fluorescent imaging purposes. Furthermore, we will discuss how nanobodies can also be exploited for molecular therapy applications such as targeted radionuclide therapy and photodynamic therapy.
  Citation: Antibodies
  PubDate: 2019-01-11
  DOI: 10.3390/antib8010012
  Issue No: Vol. 8, No. 1 (2019)
Antibodies, Vol. 8, Pages 13: Nanobody Engineering: Toward Next Generation
Immunotherapies and Immunoimaging of Cancer
- Authors: Timothée Chanier, Patrick Chames
  First page: 13
  Abstract: In the last decade, cancer immunotherapies have produced impressive therapeutic results. However, the potency of immunotherapy is tightly linked to immune cell infiltration within the tumor and varies from patient to patient. Thus, it is becoming increasingly important to monitor and modulate the tumor immune infiltrate for an efficient diagnosis and therapy. Various bispecific approaches are being developed to favor immune cell infiltration through specific tumor targeting. The discovery of antibodies devoid of light chains in camelids has spurred the development of single domain antibodies (also called VHH or nanobody), allowing for an increased diversity of multispecific and/or multivalent formats of relatively small sizes endowed with high tissue penetration. The small size of nanobodies is also an asset leading to high contrasts for non-invasive imaging. The approval of the first therapeutic nanobody directed against the von Willebrand factor for the treatment of acquired thrombotic thrombocypenic purpura (Caplacizumab, Ablynx), is expected to bolster the rise of these innovative molecules. In this review, we discuss the latest advances in the development of nanobodies and nanobody-derived molecules for use in cancer immunotherapy and immunoimaging.
  Citation: Antibodies
  PubDate: 2019-01-21
  DOI: 10.3390/antib8010013
  Issue No: Vol. 8, No. 1 (2019)
Antibodies, Vol. 8, Pages 14: Cross-Reactive and Lineage-Specific Single
Domain Antibodies against Influenza B Hemagglutinin
- Authors: Walter Ramage, Tiziano Gaiotto, Christina Ball, Paul Risley, George W. Carnell, Nigel Temperton, Chung Y. Cheung, Othmar G. Engelhardt, Simon E. Hufton
  First page: 14
  Abstract: Influenza B virus (IBV) circulates in the human population and causes considerable disease burden worldwide, each year. Current IBV vaccines can struggle to mount an effective cross-reactive immune response, as strains become mismatched, due to constant antigenic changes. Additional strategies which use monoclonal antibodies, with broad reactivity, are of considerable interest, both, as diagnostics and as immunotherapeutics. Alternatives to conventional monoclonal antibodies, such as single domain antibodies (NanobodiesTM) with well-documented advantages for applications in infectious disease, have been emerging. In this study we have isolated single domain antibodies (sdAbs), specific to IBV, using alpacas immunised with recombinant hemagglutinin (HA) from two representative viruses, B/Florida/04/2006 (B/Yamagata lineage) and B/Brisbane/60/2008 (B/Victoria lineage). Using phage display, we have isolated a panel of single domain antibodies (sdAbs), with both cross-reactive and lineage-specific binding. Several sdAbs recognise whole virus antigens, corresponding to influenza B strains included in vaccines spanning over 20 years, and were capable of neutralising IBV pseudotypes corresponding to prototype strains from both lineages. Lineage-specific sdAbs recognised the head domain, whereas, sdAbs identified as cross-reactive could be classified as either head binding or stem binding. Using yeast display, we were able to correlate lineage specificity with naturally occurring sequence divergence, at residue 122 in the highly variable 120 loop of the HA1 domain. The single domain antibodies described, might have applications in IBV diagnostics, vaccine potency testing and as immunotherapeutics.
  Citation: Antibodies
  PubDate: 2019-02-10
  DOI: 10.3390/antib8010014
  Issue No: Vol. 8, No. 1 (2019)
Antibodies, Vol. 8, Pages 15: Canine CD117-Specific Antibodies with
Diverse Binding Properties Isolated from a Phage Display Library Using
Cell-Based Biopanning
- Authors: Mohamed A. Alfaleh, Neetika Arora, Michael Yeh, Christopher J. de Bakker, Christopher B. Howard, Philip Macpherson, Rachel E. Allavena, Xiaoli Chen, Linda Harkness, Stephen M. Mahler, Martina L. Jones
  First page: 15
  Abstract: CD117 (c-Kit) is a tyrosine kinase receptor that is overexpressed in multiple dog tumors. There is 100% homology between the juxtamembrane domain of human and canine CD117, and many cancer-causing mutations occur in this region in both species. Thus, CD117 is an important target for cancer treatment in dogs and for comparative oncology studies. Currently, there is no monoclonal antibody (mAb) specifically designed to target the exposed region of canine CD117, although there exist some with species cross-reactivity. We panned a naïve phage display library to isolate antibodies against recombinant CD117 on whole cells. Several mAbs were isolated and were shown to bind recombinant canine CD117 at low- to sub-nanomolar affinity. Additionally, binding to native canine CD117 was confirmed by immunohistochemistry and by flow cytometry. Competitive binding assays also identified mAbs that competed with the CD117 receptor-specific ligand, the stem cell factor (SCF). These results show the ability of our cell-based biopanning strategy to isolate a panel of antibodies that have varied characteristics when used in different binding assays. These in vitro/ex vivo assessments suggest that some of the isolated mAbs might be promising candidates for targeting overexpressed CD117 in canine cancers for different useful applications.
  Citation: Antibodies
  PubDate: 2019-02-12
  DOI: 10.3390/antib8010015
  Issue No: Vol. 8, No. 1 (2019)
Antibodies, Vol. 8, Pages 16: Using Nanobodies to Study Protein Function
in Developing Organisms
- Authors: Gustavo Aguilar, Shinya Matsuda, M. Alessandra Vigano, Markus Affolter
  First page: 16
  Abstract: Polyclonal and monoclonal antibodies have been invaluable tools to study proteins over the past decades. While indispensable for most biological studies including developmental biology, antibodies have been used mostly in fixed tissues or as binding reagents in the extracellular milieu. For functional studies and for clinical applications, antibodies have been functionalized by covalently fusing them to heterologous partners (i.e., chemicals, proteins or other moieties). Such functionalized antibodies have been less widely used in developmental biology studies. In the past few years, the discovery and application of small functional binding fragments derived from single-chain antibodies, so-called nanobodies, has resulted in novel approaches to study proteins during the development of multicellular animals in vivo. Expression of functionalized nanobody fusions from integrated transgenes allows manipulating proteins of interest in the extracellular and the intracellular milieu in a tissue- and time-dependent manner in an unprecedented manner. Here, we describe how nanobodies have been used in the field of developmental biology and look into the future to imagine how else nanobody-based reagents could be further developed to study the proteome in living organisms.
  Citation: Antibodies
  PubDate: 2019-02-12
  DOI: 10.3390/antib8010016
  Issue No: Vol. 8, No. 1 (2019)
Antibodies, Vol. 8, Pages 17: Preferential Identification of Agonistic
OX40 Antibodies by Using Cell Lysate to Pan Natively Paired, Humanized
Mouse-Derived Yeast Surface Display Libraries
- Authors: Angélica V. Medina-Cucurella, Rena A. Mizrahi, Michael A. Asensio, Robert C. Edgar, Jackson Leong, Renee Leong, Yoong Wearn Lim, Ayla Nelson, Ariel R. Niedecken, Jan Fredrik Simons, Matthew J. Spindler, Kacy Stadtmiller, Nicholas Wayham, Adam S. Adler, David S. Johnson
  First page: 17
  Abstract: To discover therapeutically relevant antibody candidates, many groups use mouse immunization followed by hybridoma generation or B cell screening. One modern approach is to screen B cells by generating natively paired single chain variable fragment (scFv) display libraries in yeast. Such methods typically rely on soluble antigens for scFv library screening. However, many therapeutically relevant cell-surface targets are difficult to express in a soluble protein format, complicating discovery. In this study, we developed methods to screen humanized mouse-derived yeast scFv libraries using recombinant OX40 protein in cell lysate. We used deep sequencing to compare screening with cell lysate to screening with soluble OX40 protein, in the context of mouse immunizations using either soluble OX40 or OX40-expressing cells and OX40-encoding DNA vector. We found that all tested methods produce a unique diversity of scFv binders. However, when we reformatted forty-one of these scFv as full-length monoclonal antibodies (mAbs), we observed that mAbs identified using soluble antigen immunization with cell lysate sorting always bound cell surface OX40, whereas other methods had significant false positive rates. Antibodies identified using soluble antigen immunization and cell lysate sorting were also significantly more likely to activate OX40 in a cellular assay. Our data suggest that sorting with OX40 protein in cell lysate is more likely than other methods to retain the epitopes required for antibody-mediated OX40 agonism.
  Citation: Antibodies
  PubDate: 2019-02-19
  DOI: 10.3390/antib8010017
  Issue No: Vol. 8, No. 1 (2019)
Antibodies, Vol. 8, Pages 18: Macro- and Micro-Heterogeneity of Natural
and Recombinant IgG Antibodies
- Authors: Alain Beck, Hongcheng Liu
  First page: 18
  Abstract: Recombinant monoclonal antibodies (mAbs) intended for therapeutic usage are required to be thoroughly characterized, which has promoted an extensive effort towards the understanding of the structures and heterogeneity of this major class of molecules. Batch consistency and comparability are highly relevant to the successful pharmaceutical development of mAbs and related products. Small structural modifications that contribute to molecule variants (or proteoforms) differing in size, charge or hydrophobicity have been identified. These modifications may impact (or not) the stability, pharmacokinetics, and efficacy of mAbs. The presence of the same type of modifications as found in endogenous immunoglobulin G (IgG) can substantially lower the safety risks of mAbs. The knowledge of modifications is also critical to the ranking of critical quality attributes (CQAs) of the drug and define the Quality Target Product Profile (QTPP). This review provides a summary of the current understanding of post-translational and physico-chemical modifications identified in recombinant mAbs and endogenous IgGs at physiological conditions.
  Citation: Antibodies
  PubDate: 2019-02-19
  DOI: 10.3390/antib8010018
  Issue No: Vol. 8, No. 1 (2019)
Antibodies, Vol. 8, Pages 19: IgE Antibodies: From Structure to Function
and Clinical Translation
- Authors: Brian J. Sutton, Anna M. Davies, Heather J. Bax, Sophia N. Karagiannis
  First page: 19
  Abstract: Immunoglobulin E (IgE) antibodies are well known for their role in mediating allergic reactions, and their powerful effector functions activated through binding to Fc receptors FcεRI and FcεRII/CD23. Structural studies of IgE-Fc alone, and when bound to these receptors, surprisingly revealed not only an acutely bent Fc conformation, but also subtle allosteric communication between the two distant receptor-binding sites. The ability of IgE-Fc to undergo more extreme conformational changes emerged from structures of complexes with anti-IgE antibodies, including omalizumab, in clinical use for allergic disease; flexibility is clearly critical for IgE function, but may also be exploited by allosteric interference to inhibit IgE activity for therapeutic benefit. In contrast, the power of IgE may be harnessed to target cancer. Efforts to improve the effector functions of therapeutic antibodies for cancer have almost exclusively focussed on IgG1 and IgG4 subclasses, but IgE offers an extremely high affinity for FcεRI receptors on immune effector cells known to infiltrate solid tumours. Furthermore, while tumour-resident inhibitory Fc receptors can modulate the effector functions of IgG antibodies, no inhibitory IgE Fc receptors are known to exist. The development of tumour antigen-specific IgE antibodies may therefore provide an improved immune functional profile and enhanced anti-cancer efficacy. We describe proof-of-concept studies of IgE immunotherapies against solid tumours, including a range of in vitro and in vivo evaluations of efficacy and mechanisms of action, as well as ex vivo and in vivo safety studies. The first anti-cancer IgE antibody, MOv18, the clinical translation of which we discuss herein, has now reached clinical testing, offering great potential to direct this novel therapeutic modality against many other tumour-specific antigens. This review highlights how our understanding of IgE structure and function underpins these exciting clinical developments.
  Citation: Antibodies
  PubDate: 2019-02-22
  DOI: 10.3390/antib8010019
  Issue No: Vol. 8, No. 1 (2019)
Antibodies, Vol. 8, Pages 20: Evaluation of the NovaLisa™ Leishmania
Infantum IgG ELISA in A Reference Diagnostic Laboratory in A Non-Endemic
Country
- Authors: Christen Rune Stensvold, Amalie Vang Høst, Salem Belkessa, Henrik Vedel Nielsen
  First page: 20
  Abstract: Anti-Leishmania antibodies may be detectable in patients with leishmaniasis. Here, we compared a commercial enzyme-linked immunosorbent assay (ELISA) for the detection of anti-Leishmania antibodies, with an immunofluorescence antibody test (IFAT) that is no longer commercially available. Eighty-six serum samples from 73 patients were tested. The results obtained by the NovaLisa™ Leishmania infantum IgG ELISA, interpreted according to the instructions of the manufacturer, but with a modified cut-off for borderline positive values, were compared with the IFAT results that were already available. Moreover, Leishmania Western blot IgG results were available for 43 of the samples. The overall concordance of ELISA and IFAT was 67%. The ELISA and IFAT tests scored as 24% and 15% of the samples being positive, respectively, while 13% and 33% scored as borderline-positive, respectively. Using a Western blot (WB) as the reference, the sensitivities and specificities for the positive plus borderline-positive samples combined was 95.5% (95% confidence interval (CI), 77.2–99.9%) and 81.0% (95% CI, 58.1–94.6%) for ELISA, and 95.5% (95% CI, 77.2–99.9%) and 42.9% (95% CI, 21.8–66.0%) for IFAT, respectively. Overall, the ELISA proved to be a cost-effective alternative to the IFAT, due to its higher accuracy and specificity, and with a consequently lower number of confirmatory WB tests being required. Lastly, we also present data on the associations between seroconversion and the type of leishmaniasis.
  Citation: Antibodies
  PubDate: 2019-02-27
  DOI: 10.3390/antib8010020
  Issue No: Vol. 8, No. 1 (2019)
Antibodies, Vol. 8, Pages 21: Immunogenicity of Innovative and Biosimilar
Monoclonal Antibodies
- Authors: Erik Doevendans, Huub Schellekens
  First page: 21
  Abstract: The development of hybridoma technology for producing monoclonal antibodies (mAbs) by Kohler and Milstein (1975) counts as one of the major medical breakthroughs, opening up endless possibilities for research, diagnosis and for treatment of a whole variety of diseases. Therapeutic mAbs were introduced three decades ago. The first generation of therapeutic mAbs of murine origin showed high immunogenicity, which limited efficacy and was associated with severe infusion reactions. Subsequently chimeric, humanized, and fully human antibodies were introduced as therapeutics, these mAbs were considerably less immunogenic. Unexpectedly humanized mAbs generally show similar immunogenicity as chimeric antibodies; based on sequence homology chimeric mAbs are sometimes more “human” than humanized mAbs. With the introduction of the regulatory concept of similar biological medicines (biosimilars) a key concern is the similarity in terms of immunogenicity of these biosimilars with their originators. This review focuses briefly on the mechanisms of induction of immunogenicity by biopharmaceuticals, mAbs in particular, in relation to the target of the immune system.
  Citation: Antibodies
  PubDate: 2019-03-05
  DOI: 10.3390/antib8010021
  Issue No: Vol. 8, No. 1 (2019)
Antibodies, Vol. 8, Pages 22: Kinetic Analysis and Epitope Mapping of
Monoclonal Antibodies to Salmonella Typhimurium Flagellin Using a Surface
Plasmon Resonance Biosensor
- Authors: Devendra Bhandari, Fur-Chi Chen, Shreya Hamal, Roger C. Bridgman
  First page: 22
  Abstract: Salmonella Typhimurium is one of the leading causes of foodborne diseases worldwide. Biosensors and immunoassays utilizing monoclonal antibodies are widely used for the detection and subtyping of S. Typhimurium. However, due to insufficient information on the nature of binding with S. Typhimurium flagellin, the selection of appropriate antibodies for assay development is a cumbersome task. Hence, we aimed to compare the binding kinetics of a panel of monoclonal antibodies and their relative binding sites to flagellin antigen using a surface plasmon resonance biosensor. Initially, the flagellin was captured on the sensor surface through an immobilized anti-flagellin antibody. The interactions of different concentrations of monoclonal antibodies to flagellin were determined, and binding curves were fitted using 1:1 bio-interaction model to calculate the kinetic parameters. For epitope mapping, pairwise comparisons were completed to determine the binding inhibition of each paired combination of monoclonal antibodies. It was found that these monoclonal antibodies differed significantly (p < 0.05) in association rate, dissociation rate, and equilibrium dissociation constants. Of the five monoclonal antibodies, only two interfered with the binding of each other. Four distinct epitopes located within a 23 kDa domain of flagellin were identified. Findings from this study provide crucial information needed for the further development and optimization of biosensors and other immunoassays for the detection and subtyping of Salmonella.
  Citation: Antibodies
  PubDate: 2019-03-06
  DOI: 10.3390/antib8010022
  Issue No: Vol. 8, No. 1 (2019)
Antibodies, Vol. 8, Pages 23: Peptides for Infectious Diseases: From Probe
Design to Diagnostic Microarrays
- Authors: Marina Cretich, Alessandro Gori, Ilda D’Annessa, Marcella Chiari, Giorgio Colombo
  First page: 23
  Abstract: Peptides and peptidomimetics have attracted revived interest regarding their applications in chemical biology over the last few years. Their chemical versatility, synthetic accessibility and the ease of storage and management compared to full proteins have made peptides particularly interesting in diagnostic applications, where they proved to efficiently recapitulate the molecular recognition properties of larger protein antigens, and were proven to be able to capture antibodies circulating in the plasma and serum of patients previously exposed to bacterial or viral infections. Here, we describe the development, integration and application of strategies for computational prediction and design, advanced chemical synthesis, and diagnostic deployment in multiplexed assays of peptide-based materials which are able to bind antibodies of diagnostic as well as therapeutic interest. By presenting successful applications of such an integrated strategy, we argue that they will have an ever-increasing role in both basic and clinical realms of research, where important advances can be expected in the next few years.
  Citation: Antibodies
  PubDate: 2019-03-12
  DOI: 10.3390/antib8010023
  Issue No: Vol. 8, No. 1 (2019)
Antibodies, Vol. 8, Pages 24: IgG Charge: Practical and Biological
Implications
- Authors: Danlin Yang, Rachel Kroe-Barrett, Sanjaya Singh, Thomas Laue
  First page: 24
  Abstract: Practically, IgG charge can contribute significantly to thermodynamic nonideality, and hence to solubility and viscosity. Biologically, IgG charge isomers exhibit differences in clearance and potency. It has been known since the 1930s that all immunoglobulins carry a weak negative charge in physiological solvents. However, there has been no systematic exploration of this fundamental property. Accurate charge measurements have been made using membrane confined electrophoresis in two solvents (pH 5.0 and pH 7.4) on a panel of twelve mAb IgGs, as well as their F(ab’)2 and Fc fragments. The following observations were made at pH 5.0: (1) the measured charge differs from the calculated charge by ~40 for the intact IgGs, and by ~20 for the Fcs; (2) the intact IgG charge depends on both Fv and Fc sequences, but does not equal the sum of the F(ab)’2 and Fc charge; (3) the Fc charge is consistent within a class. In phosphate buffered saline, pH 7.4: (1) the intact IgG charges ranged from 0 to −13; (2) the F(ab’)2 fragments are nearly neutral for IgG1s and IgG2s, and about −5 for some of the IgG4s; (3) all Fc fragments are weakly anionic, with IgG1 < IgG2 < IgG4; (4) the charge on the intact IgGs does not equal the sum of the F(ab’)2 and Fc charge. In no case is the calculated charge, based solely on H+ binding, remotely close to the measured charge. Some mAbs carried a charge in physiological salt that was outside the range observed for serum-purified human poly IgG. To best match physiological properties, a therapeutic mAb should have a measured charge that falls within the range observed for serum-derived human IgGs. A thermodynamically rigorous, concentration-dependent protein–protein interaction parameter is introduced. Based on readily measured properties, interaction curves may be generated to aid in the selection of proteins and solvent conditions. Example curves are provided.
  Citation: Antibodies
  PubDate: 2019-03-14
  DOI: 10.3390/antib8010024
  Issue No: Vol. 8, No. 1 (2019)
Antibodies, Vol. 8, Pages 25: Human Domain Antibodies to Conserved
Epitopes on HER2 Potently Inhibit Growth of HER2-Overexpressing Human
Breast Cancer Cells In Vitro
- Authors: Hongqian Wang, Yanping Wang, Zuoxiang Xiao, Wei Li, Dimiter S. Dimitrov, Weizao Chen
  First page: 25
  Abstract: The FDA approval of two anti-HER2 monoclonal antibodies, trastuzumab and pertuzumab, and an antibody-drug conjugate, trastuzumab emtansine, has transformed clinical practice for HER2-positive cancers. However, not all patients respond to therapy, and the majority of responders eventually develop resistance. In addition, cardiotoxicity is a major safety concern for their clinical use. Thus, there remains a need for the discovery and development of novel classes of HER2-targeted therapeutics with high efficacy and specificity. In this study, we report the identification and characterization of fully human single-domain antibodies (dAbs) targeting HER2 epitopes that are highly conserved among various species and non-overlapping with those of trastuzumab and pertuzumab. An Fc-fusion protein of the best binder demonstrated much higher inhibitory activity against HER2-amplified human breast cancer cell lines than trastuzumab and pertuzumab. Unlike the latter, however, it did not have an effect on gastric and ovarian cancer cell lines with HER2 overexpression. The dAb-Fc fusion protein showed poor pharmacokinetics in mice, thus limiting its potential for therapeutic use. It could be useful as an agent for the exploration of functionally important conserved structures on HER2 with implications for the design of novel therapeutics and elucidation of mechanisms of HER2-mediated tumorigenesis.
  Citation: Antibodies
  PubDate: 2019-03-18
  DOI: 10.3390/antib8010025
  Issue No: Vol. 8, No. 1 (2019)