Characterization of continuous B‐cell epitopes in the N‐terminus of glutamate decarboxylase67 using monoclonal antibodies

Glutamate decarboxylase (GAD) is an autoantigen associated with the autoimmune disorders Type‐1 diabetes (T1D) and stiff‐person syndrome (SPS). The protein, being an essential enzyme involved in the production of the inhibitory neurotransmitter γ‐aminobutyric acid, exists in two isoforms, GAD67 and GAD65. Both isoforms may be targeted by autoantibodies in SPS and T1D patients, although SPS primarily is associated with the presence of GAD67 autoantibodies, whereas T1D mainly is associated with the presence of GAD65 autoantibodies. In this study, we describe antibody reactivity to overlapping GAD67 peptides covering the complete protein sequence by modified peptide enzyme‐linked immunosorbent assay in order to identify potential GAD67 epitopes using two monoclonal antibodies (mAbs). Both GAD67 mAbs showed reactivity to linear epitopes located at the N‐terminal end of GAD67. The epitopes of GAD mAb 1 and 2 were identified as the amino acid sequences NAGADPNTTN and TETDFSNLF, respectively, corresponding to amino acids 14–23 and 91–99. Fine mapping of the epitopes revealed that antibody reactivity was related to amino acid side‐chain functionality, rather than amino acid side‐chain specificity. Additionally, results suggested that non‐contact amino acids in the epitope structure were essential for antibody reactivity. The exact role of these amino acids remains to be determined, but they are thought to be involved in backbone hydrogen bonds or stabilization of the epitope structure. As only limited knowledge is available in relation to antigenic regions of GAD67, this study contributes to characterization of GAD67 epitopes and may be a first step in the development of peptide‐based therapeutics against SPS. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.     

Insect cells for antibody production: evaluation of an efficient alternative

In recent years there has been an increase in both availability and demand for therapeutic monoclonal antibodies. Currently, most of these antibodies are produced by stably transfected mammalian cells. In this study we evaluated the use of different baculoviral insect cell systems as an alternative for commonly used production schemes. We expressed the human anti-gp41 antibody 3D6 in Spodoptera frugiperda Sf9, Trichoplusia ni BTI-TN5B1-4 "High Five", and Spodoptera frugiperda SfSWT-1 "Mimic?" insect cells and compared product yield, specificity and glycosylation patterns with a 3D6 antibody expressed in Chinese hamster ovary cells. Using "High Five" cells we achieved amounts of secreted antibody comparable to those resulting from transient expression in mammalian cells. We determined the N-linked oligosaccharide structures present on asparagine-297 in IgG? heavy chains and tested the functionality in terms of antigen binding and the ability to elicit effector functions. Antibodies expressed in all insect cell lines displayed highly specific antigen binding. In general, the insect-produced antibodies carried, as the CHO-produced form, fucosylated N-glycans, including, in the case of "High Five" cells, high levels of core α1,3-fucose. This indicates that in all systems glycoengineering may be required in order to produce optimal glycoforms of this antibody.     

Humanization of Murine Monoclonal anti-hTNF Antibody: The F10 Story

Tumor necrosis factor (TNF) is a proinflammatory cytokine implicated in pathogenesis of multiple autoimmune and inflammatory diseases. Anti-TNF therapy has revolutionized the therapeutic paradigms of autoimmune diseases and became one of the most successful examples of the clinical use of monoclonal antibodies. Currently, anti-TNF therapy is used by millions of patients worldwide. At the moment, fully human anti-TNF antibody Adalimumab is the best-selling anti-cytokine drug in the world. Here, we present a story about a highly potent anti-TNF monoclonal antibody initially characterized more than 20 years ago and further developed into chimeric and humanized versions. We present comparative analysis of this antibody with Infliximab and Adalimumab.     

Higher order structures of Adalimumab,Infliximab and their complexes with TNFα revealed by electron microscopy

Adalimumab and Infliximab are recombinant IgG1 monoclonal antibodies (mAbs) that bind and neutralize human tumor necrosis factor alpha (TNFα). TNFα forms a stable homotrimer with unique surface‐exposed sites for Adalimumab, Infliximab, and TNF receptor binding. Here, we report the structures of Adalimumab‐TNFα and Infliximab‐TNFα complexes modeled from negative stain EM and cryo‐EM images. EM images reveal complex structures consisting of 1:1, 1:2, 2:2, and 3:2 complexes of Adalimumab‐TNFα and Infliximab‐TNFα. The 2:2 complex structures of Adalimumab‐TNFα and Infliximab‐TNFα show diamond‐shaped profiles and the 2D class averages reveal distinct orientations of the Fab domains, indicating different binding modes by Adalimumab and Infliximab to TNFα. After separation by size exclusion chromatography and analysis by negative stain EM, the 3:2 complexes of Adalimumab‐TNFα or Infliximab‐TNFα complexes are more complicated but retain features recognized in the 2:2 complexes. Preliminary cryo‐EM analysis of 3:2 Adalimumab‐TNFα complex generated a low‐resolution density consistent with a TNFα trimer bound with three Fab domains from three individual antibody molecules, while each antibody molecule binds to two molecules of TNFα trimer. The Fc domains are not visible in the reconstruction. These results show the two mAbs form structurally distinct complexes with TNFα.     

Determination and validation of off-target activities of anti-CD44 variant 6 antibodies using protein biochips and tissue microarrays

We describe a novel procedure for determination and validation of off-target activities of anti-cluster designation antigen identity 44 (CD44) variant 6 recombinant antibodies by combining two complementary technology platforms; namely UNIchip AV-400, containing a printed serial dilution of CD44 variant 6 and approximately 400 different human proteins, and TISSOMICS, enabling human tissue microarray analysis in high-throughput mode. We have analyzed the performance of two human monoclonal recombinant antibodies directed against CD44 variant 6 protein, BMS 116 and BMS 125, in a blinded study. The antibodies exhibit a clear differentiation with regard to their binding profiles in the two systems. BMS 116 shows a low degree of specificity in the normal human Food and Drug Administration (FDA) tissue panel, which was confirmed by binding to more than 206 proteins on the protein biochip. In contrast, BMS 125 gives a highly selective membranous staining on selected human epithelial tissue components with no off-target activities observed on the protein biochip. Additionally, antibody BMS 125 shows a higher sensitivity to its antigen CD44 variant 6 than antibody BMS 116 as determined by the protein biochip.     

The variable C-terminus of 14-3-3 proteins mediates isoform-specific interaction with sucrose-phosphate synthase in the yeast two-hybrid system

Sucrose-6-phosphate synthase (SPS) is a target for 14-3-3 protein binding in plants. Because several isoforms of the 14-3-3 protein are expressed in plants, I investigated which isoforms have the ability to bind SPS. Two 14-3-3 isoforms (T14-3d and a novel isoform designated T14-3 g) were found to interact with SPS from tobacco (Nicotiana tabacum L.) in a two-hybrid screen. To further address the question of isoform specificity of 14-3-3s, four additional isoforms were tested for their ability to interact with SPS in the yeast two-hybrid system. The results clearly revealed large differences in affinity between individual 14-3-3 isoforms toward SPS. Deletion analysis suggested that these differences were mediated by the variable C-terminus of 14-3-3s. Site-directed mutagenesis of candidate 14-3-3 binding sites on SPS demonstrated that interaction could be independent of a phosphorylated serine residue within conserved binding motifs in the yeast system. These findings suggest that the large number of 14-3-3 isoforms present in plants reflects functional specificity.     

In vivo production of scFv-displaying biopolymer beads using a self-assembly-promoting fusion partner

Recombinant production and, in particular, immobilization of antibody fragments onto carrier materials are of high interest with regard to diagnostic and therapeutic applications. In this study, the recombinant production of scFv-displaying biopolymer beads intracellularly in Escherichia coli was investigated. An anti-beta-galactosidase scFv (single chain variable fragment of an antibody) was C-terminally tagged with the polymer-synthesizing enzyme PhaC from Cupriavidus necator by generating the respective hybrid gene. The functionality of the anti-beta-galactosidase scFv-PhaC fusion protein was assessed by producing the respective soluble fusion protein in an Escherichia coli AMEF mutant strain. AMEF (antibody-mediated enzyme formation) strains contain an inactive mutant beta-galactosidase, which can be activated by binding of an anti-beta-galactosidase antibody. In vivo activation of AMEF beta-galactosidase indicated that the scFv is functional with the C-terminal fusion partner PhaC. It was further demonstrated that polymer biosynthesis and bead formation were mediated by the scFv-PhaC fusion protein in the cytoplasm of recombinant E. coli when the polymer precursor was metabolically provided. This suggested that the C-terminal fusion partner PhaC acts as a functional insolubility partner, providing a natural cross-link to the bead and leading to in vivo immobilization of the scFv. Overproduction of the fusion protein at the polymer bead surface was confirmed by SDS-PAGE and MALDI-TOF/MS analysis of purified beads. Antigen binding functionality and specificity of the beads was assessed by analyzing the binding of beta-galactosidase to scFv-displaying beads and subsequently eluting the bound protein at pH 2.7. A strong enrichment of beta-galactosidase suggested the functional display of scFv at the bead surface as well as the applicability of these beads for antigen purification. Binding of beta-galactosidase to the scFv-displaying beads was quantitatively analyzed by enzyme-linked assays measuring beta-galactosidase activity. These indicated that the anti-beta-galactosidase scFv-displaying beads bound a maximum of 38 ng of beta-galactosidase per 1 microg of bead protein, showing an apparent equilibrium dissociation constant ( KD) of 12 x 10 (-7) M. This study clearly demonstrated that anti-beta-galactosidase scFv-displaying polymer beads can be produced in engineered E. coli in a one-step process by using PhaC as a self-assembly-promoting fusion partner.     

Analysis of GAD65 autoantibodies in Stiff-Person syndrome patients

Autoantibodies to the 65-kDa isoform of glutamate decarboxylase GAD65 (GAD65Ab) are strong candidates for a pathological role in Stiff-Person syndrome (SPS). We have analyzed the binding specificity of the GAD65Ab in serum and cerebrospinal fluid (CSF) of 12 patients with SPS by competitive displacement studies with GAD65-specific rFab-derived from a number of human and mouse mAbs specific for different determinants on the Ag. We demonstrate considerable differences in the epitope specificity when comparing paired serum and CSF samples, suggesting local stimulation of B cells in the CSF compartment of these patients. Moreover, these autoantibodies strongly inhibit the enzymatic activity of GAD65, thus blocking the formation of the neurotransmitter gamma-aminobutyric acid. The capacity of the sera to inhibit the enzymatic activity of GAD65 correlated with their binding to a conformational C-terminal Ab epitope. Investigation of the inhibitory mechanism revealed that the inhibition could not be overcome by high concentrations of glutamate or the cofactor pyridoxal phosphate, suggesting a noncompetitive inhibitory mechanism. Finally, we identified a linear epitope on amino acids residues 4-22 of GAD65 that was recognized solely by autoantibodies from patients with SPS but not by serum from type 1 diabetes patients. A mAb (N-GAD65 mAb) recognizing this N-terminal epitope was successfully humanized to enhance its potential therapeutic value by reducing its overall immunogenicity.     

Active bovine selenophosphate synthetase 2, not having selenocysteine

During the course of studying selenocysteine (Sec) synthesis mechanisms in mammals, we prepared selenophosphate synthetase (SPS) from bovine liver by 4-step chromatography. In the last step of chromatography of hydroxyapatite, we found a protein band of molecular mass 33 kDa on SDS-PAGE, consistent with the pattern of SPS activity that was indirectly manifested by [⁷⁵Se]Sec production activity; however, we could not detect significant Se content in this active fraction. We also found a clear band of 33 kDa by Western blotting with antibody against a common peptide (387-401) in SPS2. We detected selenophosphate as the product of this active enzyme in the reaction mixture, composed of ATP, [⁷⁵Se]H₂Se and SPS. Chemically synthesized selenophosphate plays a role in Sec synthesis, not the addition of this enzyme. These results support that the product of SPS2 is selenophosphate itself. During this investigation, the probable sequence of bovine SPS2 not having Sec was reported in the blast information and the molecular mass was near with the protein in this report. Thus, bovine active SPS2 of molecular mass 33 kDa does not contain Sec. K. Furumiya and K. Kanaya contributed equally to this work.     

Fluorobodies combine GFP fluorescence with the binding characteristics of antibodies

The difficulty of deriving binding ligands to targets identified by genomic sequencing has led to a bottleneck in genomic research. By inserting diverse antibody binding loops into four of the exposed loops at one end of green fluorescent protein (GFP), we have mimicked the natural antibody binding footprint to create robust binding ligands that combine the advantages of antibodies (high affinity and specificity) with those of GFP (intrinsic fluorescence, high stability, expression and solubility). These 'fluorobodies' have been used effectively in enzyme-linked immunosorbent assays (ELISAs), flow cytometry, immuno-fluorescence, arrays and gel shift assays, and show affinities as high as antibodies. Furthermore, the intrinsic fluorescence of fluorobodies correlates with binding activity, allowing the rapid determination of functionality, concentration and affinity. These properties render them especially suitable for the high-throughput genomic scale selections required in proteomics, as well as in diagnostics, target validation and drug development.     

Directed Evolution of a Virus Exclusively Utilizing Human Epidermal Growth Factor Receptor as the Entry Receptor

Rational design and directed evolution are powerful tools to generate and improve protein function; however, their uses are mostly limited to enzyme and antibody engineering. Here we describe a directed-evolution strategy, named the tandem selection and enrichment system (TSES), and its use in generating virus with exclusive specificity for a particular cellular receptor. In TSES, evolving viruses are sequentially and iteratively transferred between two different host cells, one for selection of receptor specificity and the other for enrichment of the fittest virus. By combining rational design and TSES, we generated human epidermal growth factor receptor (EGFR)-specific virus 1 (ESV1). ESV1 has the backbone of Sindbis virus (SINV) and displays an EGF domain engrafted onto structural protein E2 after residue Pro192, together with eight amino acid changes stabilizing the E2-EGF chimera. ESV1 uses EGFR to initiate infection and has lost the capacity to interact with all known SINV receptors. A 12.2-Å cryoelectron microscopic (cryoEM) reconstruction of ESV1 reveals that the E2-EGF fusion adopts a fixed conformation, with EGF sitting at the top of the E2 spike; The EGFR binding interface faces outward, and the EGF domain completely masks SINV receptor binding. The cryoEM structure of ESV1 explains the desirable properties of ESV1 and provides insights for its further modification. TSES expands the scope of directed evolution and can be easily extended to other targeting molecules and viral systems.     

Binding characteristics to mosquito-larval midgut proteins of the cloned domain II-III fragment from the Bacillus thuringiensis Cry4Ba toxin

Receptor binding plays an important role in determining host specificity of the Bacillus thuringiensis Cry delta-endotoxins. Mutations in domains II and III have suggested the participation of certain residues in receptor recognition and insect specificity. In the present study, we expressed the cloned domain II-III fragment of Cry4Ba and examined its binding characteristics to mosquito-larval midgut proteins. The 43-kDa Cry4Ba-domain II-III protein over-expressed in Escherichia coli as inclusion bodies was only soluble when carbonate buffer, pH 10.0 was supplemented with 4 M urea. After renaturation via stepwise dialysis and subsequent purification, the refolded domain II-III protein, which specifically reacts with anti Cry4Ba-domain III monoclonal antibody, predominantly exists as a beta-sheet structure determined by circular dichroism spectroscopy. In vitro binding analysis to both histological midgut tissue sections and brush border membrane proteins prepared from susceptible Aedes aegypti mosquito-larvae revealed that the isolated Cry4Ba-domain II-III protein showed binding functionality comparable to the 65-kDa full-length active toxin. Altogether, the data present the 43-kDa Cry4Ba fragment comprising domains II and III that was produced in isolation was able to retain its receptor-binding characteristics to the target larval midgut proteins.     

Monomeric IgA can be produced in planta as efficient as IgG,yet receives different N‐glycans

The unique features of IgA, such as the ability to recruit neutrophils and suppress the inflammatory responses mediated by IgG and IgE, make it a promising antibody isotype for several therapeutic applications. However, in contrast to IgG, reports on plant production of IgA are scarce. We produced IgA1κ and IgG1κ versions of three therapeutic antibodies directed against pro‐inflammatory cytokines in Nicotiana benthamiana: Infliximab and Adalimumab, directed against TNF‐α, and Ustekinumab, directed against the interleukin‐12p40 subunit. We evaluated antibody yield, quality and N‐glycosylation. All six antibodies had comparable levels of expression between 3.5 and 9% of total soluble protein content and were shown to have neutralizing activity in a cell‐based assay. However, IgA1κ‐based Adalimumab and Ustekinumab were poorly secreted compared to their IgG counterparts. Infliximab was poorly secreted regardless of isotype backbone. This corresponded with the observation that both IgA1κ‐ and IgG1κ‐based Infliximab were enriched in oligomannose‐type N‐glycan structures. For IgG1κ‐based Ustekinumab and Adalimumab, the major N‐glycan type was the typical plant complex N‐glycan, biantennary with terminal N‐acetylglucosamine, β1,2‐xylose and core α1,3‐fucose. In contrast, the major N‐glycan on the IgA‐based antibodies was xylosylated, but lacked core α1,3‐fucose and one terminal N‐acetylglucosamine. This type of N‐glycan occurs usually in marginal percentages in plants and was never shown to be the main fraction of a plant‐produced recombinant protein. Our data demonstrate that the antibody isotype may have a profound influence on the type of N‐glycan an antibody receives.     

Tuning the specificity of a Two-in-One Fab against three angiogenic antigens by fully utilizing the information of deep mutational scanning

Monoclonal antibodies developed for therapeutic or diagnostic purposes need to demonstrate highly defined binding specificity profiles. Engineering of an antibody to enhance or reduce binding to related antigens is often needed to achieve the desired biologic activity without safety concern. Here, we describe a deep sequencing-aided engineering strategy to fine-tune the specificity of an angiopoietin-2 (Ang2)/vascular endothelial growth factor (VEGF) dual action Fab, 5A12.1 for the treatment of age-related macular degeneration. This antibody utilizes overlapping complementarity-determining region (CDR) sites for dual Ang2/VEGF interaction with K_D in the sub-nanomolar range. However, it also exhibits significant (K_D of 4 nM) binding to angiopoietin-1, which has high sequence identity with Ang2. We generated a large phage-displayed library of 5A12.1 Fab variants with all possible single mutations in the 6 CDRs. By tracking the change of prevalence of each mutation during various selection conditions, we identified 35 mutations predicted to decrease the affinity for Ang1 while maintaining the affinity for Ang2 and VEGF. We confirmed the specificity profiles for 25 of these single mutations as Fab protein. Structural analysis showed that some of the Fab mutations cluster near a potential Ang1/2 epitope residue that differs in the 2 proteins, while others are up to 15 Å away from the antigen-binding site and likely influence the binding interaction remotely. The approach presented here provides a robust and efficient method for specificity engineering that does not require prior knowledge of the antigen antibody interaction and can be broadly applied to antibody specificity engineering projects.     

A multifactorial screening strategy to identify anti-idiotypic reagents for bioanalytical support of antibody therapeutics

Antibodies are critical tools for protein bioanalysis; their quality and performance dictate the caliber and robustness of ligand binding assays. After immunization, polyclonal B cells generate a diverse antibody repertoire against constant and variable regions of the therapeutic antibody immunogen. Herein we describe a comprehensive and multifactorial screening strategy to eliminate undesirable constant region-specific antibodies and select for anti-idiotypic antibodies with specificity for the unique variable region. Application of this strategy is described for the therapeutic antibody Mab-A case study. Five different factors were evaluated to select a final antibody pair for the quantification of therapeutics in biological matrices: (i) matrix effect in preclinical and clinical matrices, (ii) assay sensitivity with lower limit of quantification goal of single-digit ng/ml (low pM) at a signal-to-background ratio greater than 5, (iii) epitope distinction or nonbridging antibody pair, (iv) competition with target and inhibitory capacity enabling measurement of free drug, and (v) neutralizing bioactivity using bioassay. The selected antibody pair demonstrated superior assay sensitivity with no or minimal matrix effect in common biological samples, recognized two distinct binding epitopes on the therapeutic antibody variable region, and featured inhibitory and neutralizing effects with respect to quantification of free drug levels.     

Production and characterization of scFvA33T1, an immunoRNase targeting colon cancer cells

Within the last 10?years, the use of different RNases as therapeutic agents for various diseases has been pursued. Furthermore, the advancements of recombinant technology have allowed the assembly of proteins with different functions. In this regard, immunoribonucleases (immunoRNases) stand out as some of the most promising therapeutic candidates given their enzymatic and non-mutagenic character. Accordingly, the work reported here describes fusing RNase T1, one of the most studied members of the microbial RNase family, to the single-chain variable fragment (scFv) of a monoclonal antibody that targets the glycoprotein A33 antigen (GPA33) from human colon cancer cells. A heterologous production system, which employs the yeast Pichia?pastoris, has been optimized to produce this immunoRNase (scFvA33T1) with yields of ～?5-10?mg·L(-1) . The purified protein appears to be correctly folded as it retains its antigen specificity and ribonucleolytic activity. Finally, it also shows specific binding to, internalization into and toxicity against GPA33-positive cell lines compared with the control, GPA33-negative cells. Overall, it can be concluded that scFvA33T1 is a promising therapeutic fusion protein with the additional advantage that presumably it can be produced and purified in large amounts using an easily scalable yeast-based system.     

Influence of glycosylation pattern on the molecular properties of monoclonal antibodies

Glycosylation is an important post-translational modification during protein production in eukaryotic cells, and it is essential for protein structure, stability, half-life, and biological functions. In this study, we produced various monoclonal antibody (mAb) glycoforms from Chinese hamster ovary (CHO) cells, including the natively glycosylated antibody, the enriched G0 form, the deglycosylated form, the afucosylated form, and the high mannose form, and we compared their intrinsic properties, side-by-side, through biophysical and biochemical approaches. Spectroscopic analysis indicates no measureable secondary or tertiary structural changes after in vitro or in vivo modification of the glycosylation pattern. Thermal unfolding experiments show that the high mannose and deglycosylated forms have reduced thermal stability of the CH2 domain compared with the other tested glycoforms. We also observed that the individual domain’s thermal stability could be pH dependent. Proteolysis analysis indicates that glycosylation plays an important role in stabilizing mAbs against proteases. The stability of antibody glycoforms at the storage condition (2–8 °C) and at accelerated conditions (30 and 40 °C) was evaluated, and the results indicate that glycosylation patterns do not substantially affect the storage stability of the antibody we studied.     

Monoclonal antibodies: technologies for early discovery and engineering

Antibodies are essential in modern life sciences biotechnology. Their architecture and diversity allow for high specificity and affinity to a wide array of biochemicals. Combining monoclonal antibody (mAb) technology with recombinant DNA and protein expression links antibody genotype with phenotype. Yet, the ability to select and screen for high affinity binders from recombinantly-displayed, combinatorial libraries unleashes the true power of mAbs and a flood of clinical applications. The identification of novel antibodies can be accomplished by a myriad of in vitro display technologies from the proven (e.g. phage) to the emerging (e.g. mammalian cell and cell-free) based on affinity binding as well as function. Lead candidates can be further engineered for increased affinity and half-life, reduced immunogenicity and/or enhanced manufacturing, and storage capabilities. This review begins with antibody biology and how the structure and genetic machinery relate to function, diversity, and in vivo affinity maturation and follows with the general requirements of (therapeutic) antibody discovery and engineering with an emphasis on in vitro display technologies. Throughout, we highlight where antibody biology inspires technology development and where high-throughput, “big data” and in silico strategies are playing an increasing role. Antibodies dominate the growing class of targeted therapeutics, alone or as bioconjugates. However, their versatility extends to research, diagnostics, and beyond.     

Influence of glycosylation pattern on the molecular properties of monoclonal antibodies

Glycosylation is an important post-translational modification during protein production in eukaryotic cells, and it is essential for protein structure, stability, half-life, and biological functions. In this study, we produced various monoclonal antibody (mAb) glycoforms from Chinese hamster ovary (CHO) cells, including the natively glycosylated antibody, the enriched G0 form, the deglycosylated form, the afucosylated form, and the high mannose form, and we compared their intrinsic properties, side-by-side, through biophysical and biochemical approaches. Spectroscopic analysis indicates no measureable secondary or tertiary structural changes after in vitro or in vivo modification of the glycosylation pattern. Thermal unfolding experiments show that the high mannose and deglycosylated forms have reduced thermal stability of the CH2 domain compared with the other tested glycoforms. We also observed that the individual domain’s thermal stability could be pH dependent. Proteolysis analysis indicates that glycosylation plays an important role in stabilizing mAbs against proteases. The stability of antibody glycoforms at the storage condition (2–8 °C) and at accelerated conditions (30 and 40 °C) was evaluated, and the results indicate that glycosylation patterns do not substantially affect the storage stability of the antibody we studied.     

An automated immunoassay for early specificity profiling of antibodies

Antibody-based therapeutics are of great value for the treatment of human diseases. In addition to functional activity, affinity or physico-chemical properties, antibody specificity is considered to be one of the most crucial attributes for safety and efficacy. Consequently, appropriate studies are required before entering clinical trials.

High content protein arrays are widely applied to assess antibody specificity, but this commercial solution can only be applied to final therapeutic antibody candidates because such arrays are expensive and their throughput is limited. A flexible, high-throughput and economical assay that allows specificity testing of IgG or Fab molecules during early discovery is described here. The 384-well microtiter plate assay contains a comprehensive panel of 32 test proteins and uses electrochemiluminescence as readout.

The Protein Panel Profiling (3P) was used to analyze marketed therapeutic antibodies that all showed highly specific binding profiles. Subsequently, 3P was applied to antibody candidates from early discovery and the results compared well with those obtained with a commercially available high content protein chip. Our results suggest that 3P can be applied as an additional filter for lead selection, allowing the identification of favorable antibody candidates in early discovery and thereby increasing the speed and possibility of success in drug development.