attana - sensor technologies

Antibodies/Proteins

  • mAbs, DOI: 10.1080/19420862.2017.1319023 Inhibition of HER3 activation and tumor growth with a human antibody binding to a conserved epitope formed by domain III and IVLisa C. Schmitt, Alexander Rau, Oliver Seifert, Jonas Honer, Meike Hutt, Simone Schmid, Jonas Zantow, Michael Hust, Stefan Dübel, Monilola A. Olayioye & Roland E. Kontermann
    Abstract

    Human epidermal growth factor receptor 3 (HER3, also known as ErbB3) has emerged as relevant target for antibody-mediated tumor therapy. Here, we describe a novel human antibody, IgG 3-43, recognizing a unique epitope formed by domain III and parts of domain IV of the extracellular region of HER3, conserved between HER3 and mouse ErbB3. An affinity of 11 nM was determined for the monovalent interaction. In the IgG format, the antibody bound recombinant bivalent HER3 with subnanomolar affinity (KD = 220 pM) and HER3-expressing tumor cells with EC50 values in the low picomolar range (27-83 pM). The antibody competed with binding of heregulin to HER3-expressing cells, efficiently inhibited phosphorylation of HER3 as well as downstream signaling, and induced receptor internalization and degradation. Furthermore, IgG 3-43 inhibited heregulin-dependent proliferation of several HER3-positive cancer cell lines and heregulin-independent colony formation of HER2-overexpressing tumor cell lines. Importantly, inhibition of tumor growth and prolonged survival was demonstrated in a FaDu xenograft tumor model in SCID mice. These findings demonstrate that by binding to the membrane-proximal domains III and IV involved in ligand binding and receptor dimerization, IgG 3-43 efficiently inhibits activation of HER3, thereby blocking tumor cell growth both in vitro and in vivo.

    © 2017 Taylor & Francis Group. All rights reserved.

  • Nature: Sci. Rep. 7, 43006; doi: 10.1038/srep43006 (2017) Cellular glycosylation affects Herceptin binding and sensitivity of breast cancer cells to doxorubicin and growth factorsDiluka Peiris, Alexander F. Spector, Hannah Lomax-Browne, TayebehAzimi, Bala Ramesh, Marilena Loizidou, Hazel Welch and Miriam V. Dwek
    Abstract

    Alterations in protein glycosylation are a key feature of oncogenesis and have been shown to affect cancer cell behaviour perturbing cell adhesion, favouring cell migration and metastasis. This study investigated the effect of N-linked glycosylation on the binding of Herceptin to HER2 protein in breast cancer and on the sensitivity of cancer cells to the chemotherapeutic agent doxorubicin (DXR) and growth factors (EGF and IGF-1). The interaction between Herceptin and recombinant HER2 protein and cancer cell surfaces (on-rate/off-rate) was assessed using a quartz crystal microbalance biosensor revealing an increase in the accessibility of HER2 to Herceptin following deglycosylation of cell membrane proteins (deglycosylated cells Bmax: 6.83Hz; glycosylated cells Bmax: 7.35Hz). The sensitivity of cells to DXR and to growth factors was evaluated using an MTT assay. Maintenance of SKBR-3 cells in tunicamycin (an inhibitor of N-linked glycosylation) resulted in an increase in sensitivity to DXR (0.1μM DXR P<0.001) and a decrease in sensitivity to IGF-1 alone and to IGF-1 supplemented with EGF (P<0.001). This report illustrates the importance of N-linked glycosylation in modulating the response of cancer cells to chemotherapeutic and biological treatments and highlights the potential of glycosylation inhibitors as future combination treatments for breast cancer.

    © The Authors 2017 under license from Springer Nature

  • Sensing and Bio-Sensing Research 9 (2016) 23–30 Real-time and label free determination of ligand binding-kinetics to primary cancer tissue specimens; a novel tool for the assessment of biomarker targetingThomas Mandel Clausen, Marina Ayres Pereira, Htoo Zarni Oo, Mafalda Resende, Tobias Gustavson, Yang Mao, Nobuo Sugiura, Janet Liew, Ladan Fazli, Thor G. Theander, Mads Daugaard, Ali Salanti
    Abstract

    In clinical oncology, diagnosis and evaluation of optimal treatment strategies are mostly based on histopathological examination combined with immunohistochemical (IHC) expression analysis of cancer-associated antigens in formalin fixed paraffin-embedded (FFPE) tissue biopsies. However, informative IHC analysis depends on both the specificity and affinity of the binding reagent, which are inherently difficult to quantify in situ. Here we describe a label-free method that allows for the direct and real-time assessment of molecular binding kinetics in situ on FFPE tissue specimens using quartz crystal microbalance (QCM) enabled biosensor technology. We analysed the interaction between the rVAR2 protein and its placental-like chondroitin sulfate (pl-CS) receptor in primary human placenta tissue and in breast and prostate tumour specimens in situ. rVAR2 interacted with FFPE human placenta and cancer tissue with an affinity in the nanomolar range, and showed no detectable interaction with pl-CS negative normal tissue. We further validated the method by including analysis with the androgen receptor N-20 antibody (anti-AR). As the KD value produced by this method is independent of the number of epitopes available, this readout offers a quantitative and unbiased readout for in situ binding-avidity and amount of binding epitopes. In summary, this method adds a new and important dimension to classical IHC-based molecular pathology by adding information about the binding characteristics in biologically relevant conditions. This can potentially be used to select optimal biologics for diagnostic and for therapeutic applications as well as guide the development of novel high affinity binding drugs.

    © 2016 The Authors. Published by Elsevier B.V.

  • mAbs. 2015 Oct 29:0. [Epub ahead of print] Pharmacokinetic properties of IgG and various Fc fusion proteins in mice.Unverdorben F, Richter F, Hutt M, Seifert O, Malinge P, Fischer N and Kontermann RE
    Abstract

    Fusion to an IgG Fc region is an established strategy to extend the half-life of therapeutic proteins. Most Fc fusion proteins, however, do not achieve the long half-life of IgGs. Based on findings that scFv-Fc fusion proteins exhibit a shorter half-life than the corresponding IgG molecules, we performed a comparative study of different antibody-derived Fc fusion proteins. We could confirm that fusion of single-chain Fv (scFv) and single-chain diabody (scDb) molecules to an Fc region yields in fusion proteins with substantially extended half-lives compared with the single-chain versions. However, even fusion proteins with a size similar to that of IgG, e.g., scDb-Fc, did not have a half-life as long as an IgG molecule. Binding to the neonatal Fc receptor (FcRn) under acidic and neutral conditions was similar for IgG and all Fc fusion proteins. However, we observed differences between IgG and the Fc fusion proteins for dissociation of FcRn-bound proteins induced by shifting from acidic to neutral pH, reflecting the physiological release mechanism, further supporting a contribution of the kinetics of pH-dependent release from FcRn to the pharmacokinetic properties of IgG and Fc fusion proteins.

  • Sensors and Actuators B: Chemical Volume 224, 1 March 2016, Pages 814–822 Oriented and reversible immobilization of His-tagged proteins on two- and three-dimensional surfaces for study of protein–protein interactions by a QCM biosensorXueming Li, Siyu Song, Yuxin Pei, Hai Dong, Teodor Aastrup and Zhichao Pei
    Abstract

    A two-dimensional (2D) and a three-dimensional (3D) His-tag capture surfaces were fabricated for oriented and reversible immobilization of His-tagged proteins on quartz crystal microbalance (QCM) biosensor surfaces, which can be used for label-free and real-time detection of the interactions between His-tagged protein and its interacting protein (analyte). His-tagged proteins immobilized on the 2D His-tag capture surface maintained a higher binding activity than those immobilized on a 2D carboxyl surface via amine coupling. The 3D His-tag capture surface has about twice the amount of immobilization capacity as the 2D His-tag capture surface, which enables a higher sensitivity for detection. His-tag capture surface can be optionally regenerated to remove the His-tagged protein as well as the analyte for the next cycle of His-tagged protein immobilization, or to only selectively remove the analyte, leaving the His-tagged protein on the surface for the next cycle of analyte binding. Furthermore, the kinetic and affinity studies of the interactions between the His-tagged protein and its interacting protein were performed. This study provides an efficient way to study protein–protein interactions by oriented and reversible immobilization of His-tagged proteins on QCM biosensor surfaces.

    Copyright © 2017 Elsevier B.V. or its licensors or contributors.

  • PLOS ONE, DOI:10.1371, journal.pone.0139838, October 2, 2015 A Fab-Selective Immunoglobulin-Binding Domain from Streptococcal Protein G with Improved Half-Life Extension PropertiesFelix Unverdorben, Meike Hutt, Oliver Seifert and Roland E. Kontermann
    Abstract

    Half-life extension strategies have gained increasing interest to improve the pharmacokinetic and pharmacodynamic properties of protein therapeutics. Recently, we established an immunoglobulin-binding domain (IgBD) from streptococcal protein G (SpGC3) as module for half-life extension. SpGC3 is capable of binding to the Fc region as well as the CH1 domain of Fab arms under neutral and acidic conditions. Using site-directed mutagenesis, we generated a Fab-selective mutant (SpGC3Fab) to avoid possible interference with the FcRn-mediated recycling process and improved its affinity for mouse and human IgG by site-directed mutagenesis and phage display selections. In mice, this affinity-improved mutant (SpGC3FabRR) conferred prolonged plasma half-lives compared with SpGC3Fab when fused to small recombinant antibody fragments, such as singlechain Fv (scFv) and bispecific single-chain diabody (scDb). Hence, the SpGC3FabRR
    domain seems to be a suitable fusion partner for the half-life extension of small recombinant therapeutics. The half-life extension properties of SpGC3 can be retained by restricting binding to the Fab fragment of serum immunoglobulins and can be improved by increasing binding activity. The modified SpGC3 module should be suitable to extend the half-life of therapeutic proteins and, thus to improve therapeutic activity.

    ©2015 Unverdorben et al

  • mAbs, Volume 7, 2015 - Issue 1 Preclinical and early clinical development of GNbAC1, a humanized IgG4 monoclonal antibody targeting endogenous retroviral MSRV-Env proteinFrançois Curtin, Hervé Perron, Arno Kromminga, Hervé Porchet and Alois B Lang
    Abstract

    Monoclonal antibodies (mAbs) play an increasing important role in the therapeutic armamentarium against multiple sclerosis (MS), an inflammatory and degenerative disorder of the central nervous system. Most of the mAbs currently developed for MS are immunomodulators blocking the inflammatory immune process. In contrast with mAbs targeting immune function, GNbAC1, a humanized IgG4 mAb, targets the multiple sclerosis associated retrovirus envelope (MSRV-Env) protein, an upstream factor in the pathophysiology of MS. MSRV-Env protein is of endogenous retroviral origin, expressed in MS brain lesions, and it is pro-inflammatory and toxic to the remyelination process, by preventing the differentiation of oligodendrocyte precursor cells. We present the preclinical and early clinical development results of GNbAC1. The specificity of GNbAC1 for its endogenous retroviral target is described. Efficacy of different mAb versions of GNbAC1 were assessed in MSRV-Env induced experimental allergic encephalitis (EAE), an animal model of MS. Because the target MSRV-Env is not expressed in animals, no relevant animal model exists for a proper in vivo toxicological program. An off-target 2-week toxicity study in mice was thus performed, and it showed an absence of safety risk. Additional in vitro analyses showed an absence of complement or antibody-dependent cytotoxicity as well as a low level of cross-reactivity to human tissues. The first-in-man clinical study in 33 healthy subjects and a long-term clinical study in 10 MS patients showed that GNbAC1 is well tolerated in humans without induction of immunogenicity and that it induces a pharmacodynamic response on MSRV biomarkers. These initial results suggest that the mAb GNbAC1 could be a safe long-term treatment for patients with MS with a unique therapeutic mechanism of action.

    © 2017 The Author(s). Published with license by Taylor & Francis Group, LLC

  • Nucleic Acids Research, 2014-10-01 doi: 10.1093/nar/gku882 Highly potent dUTPase inhibition by a bacterial repressor protein reveals a novel mechanism for gene expression controlJudit E. Szabó, Veronika Németh, Veronika Papp-Kádár, Kinga Nyíri, Ibolya Leveles, Ábris Á. Bendes, Imre Zagyva, Gergely Róna, Hajnalka L. Pálinkás, Balázs Besztercei, Olivér Ozohanics, Károly Vékey, Károly Liliom, Judit Tóth and Béata G. Vértessy
    Abstract

    Transfer of phage-related pathogenicity islands of Staphylococcus aureus (SaPI-s) was recently reported to be activated by helper phage dUTPases.
    This is a novel function for dUTPases otherwise involved in preservation of genomic integrity by sanitizing the dNTP pool. Here we investigated the molecular mechanism of the dUTPase-induced gene expression control using direct techniques. The expression of SaPI transfer initiating proteins is repressed by proteins called Stl. We found that 11 helper phage dUTPase eliminates SaPIbov1 Stl binding to its cognate DNA by binding tightly to Stl protein. We also show that dUTPase enzymatic activity is strongly inhibited in the dUTPase:Stl complex and that the dUTPase:dUTP complex is inaccessible to the Stl repressor. Our results disprove the previously proposed G-protein-like mechanism of SaPI transfer activation. We propose that the transfer only occurs if dUTP is cleared from the nucleotide pool, a condition promoting genomic stability of the virulence elements.
    
    © The Authors 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

  • mAbs 6:2, 533–546; March/April 2014 3D modeling and characterization of the human CD115 monoclonal antibody H27K15 epitope and design of a chimeric CD115 targetBenoît Grellier, Fabrice Le Pogam, Marc Vitorino, Jean-Philippe Starck, Michel Geist, Vanessa Duong, Hélène Haegel, Thierry Menguy, Jean-Yves Bonnefoy, Jean-Baptiste Marchand, and Philippe Ancian
    Abstract

    The humanized monoclonal antibody H27K15 specifically targets human CD115, a type III tyrosine kinase receptor involved in multiple cancers and inflammatory diseases. Binding of H27K15 to hCD115 expressing cells inhibits the functional effect of colony-stimulating factor-1 (CSF-1), in a non-competitive manner. Both homology modeling and docking programs were used here to model the human CD115 extracellular domains, the H27K15 variable region and their interaction. The resulting predicted H27K15 epitope includes mainly the D1 domain in the N-terminal extracellular region of CD115 and some residues of the D2 domain. Sequence alignment with the non-binding murine CD115, enzyme-linked immunosorbent assay, nuclear magnetic resonance spectroscopy and affinity measurements by quartz crystal microbalance revealed critical residues of this epitope that are essential for H27K15 binding. A combination of computational simulations and biochemical experiments led to the design of a chimeric CD115 carrying the human epitope of H27K15 in a murine CD115 backbone that is able to bind both H27K15 as well as the murine ligands CSF-1 and IL-34. These results provide new possibilities to minutely study the functional effects of H27K15 in a transgenic mouse that would express this chimeric molecule.

    © 2014 Landes Bioscience

  • Anal Bioanal Chem 4 April 2014, DOI 10.1007/s00216-014-7821-9 Partial-filling affinity capillary electrophoresis and quartz crystal microbalance with adsorption energy distribution calculations in the study of biomolecular interactions with apolipoprotein E as interaction partnerLipponen, Tähkä, Samuelsson,Jauhiainen, Metso, Cilpa-Karhu, Fornstedt, Kostiainen, Riekkola
    Abstract

    Adsorption energy distribution (AED) calculations were successfully applied to partial-filling affinity capillary electrophoresis (PF-ACE) to facilitate more detailed studies of biomolecular interactions. PF-ACE with AED calculations was employed to study the interactions between two isoforms of apolipoprotein E (apoE) and dermatan sulfate (DS), and a quartz crystal microbalance (QCM) was used in combination with AED calculations to examine the interactions of the 15-amino-acid peptide fragment of apoE with DS. The heterogeneity of the interactions was elucidated. Microscale thermophoresis was used to validate the results. The interactions studied are of interest because, in vivo, apolipoprotein E localizes on DS-containing regions in the extracellular matrix of human vascular subendothelium. Two-site binding was demonstrated for the isoform apoE3 and DS, but only one-site binding for apoE2–DS. Comparable affinity constants were obtained for the apoE2–DS, apoE3–D3, and 15-amino-acid peptide of apoE–DS using the three techniques. The results show that combining AED calculations with modern biosensing techniques can open up another dimension in studies on the heterogeneity and affinity constants of biological molecules.

    Copyright © Springer-Verlag Berlin Heidelberg 2014

  • Biosensors 2014, 4(2), 137-149 A Phage Display Screening Derived Peptide with Affinity for the Adeninyl MoietyElmlund, Söderberg, Suriyanarayanan, Nicholls
    Abstract

    Phage display screening of a surface-immobilized adenine derivative led to the identification of a heptameric peptide with selectivity for adenine as demonstrated through quartz crystal microbalance (QCM) studies. The peptide demonstrated a concentration dependent affinity for an adeninyl moiety decorated surface (KD of 968 ± 53.3 μM), which highlights the power of piezoelectric sensing in the study of weak interactions.

    © 2014 Elmlund et al.

  • Analytical Biochemistry 443 (2013) 139–147 Three complementary techniques for the clarification of temperature effect on low-density lipoprotein–chondroitin-6-sulfate interactionCilpa-Karhu, Lipponen, Samuelsson, Öörni, Fornstedt, Riekkola
    Abstract

    A rigorous processing of adsorption data from quartz crystal microbalance technology was successfully combined with the data obtained by partial filling affinity capillary electrophoresis and molecular dynamics for the clarification of the temperature effect on the interaction of a major glycosaminoglycan chain chondroitin-6-sulfate (C6S) of proteoglycans with low-density lipoprotein (LDL) and with a peptide fragment of apolipoprotein B-100 (residues 3359–3377 of LDL, PPBS). Two experimental techniques and computational atomistic methods demonstrated a nonlinear pattern of the affinity of C6S at temperatures above 38.0 C to both LDL and PPBS. The temperature affects the interaction of C6S with LDL and PPBS by influencing the structural behavior of glycosaminoglycan C6S and/or that of LDL.

    © 2013 Elsevier Inc. All rights reserved.

  • mAbs 5:5, 736–747; September/October 2013 A unique anti-CD115 monoclonal antibody which inhibits osteolysis and skews human monocyte differentiation from M2-polarized macrophages toward dendritic cellsHélène Haegel, Christine Thioudellet, Rémy Hallet, Michel Geist, Thierry Menguy, Fabrice Le Pogam, Jean-Baptiste Marchand, Myew-Ling Toh, Vanessa Duong, Alexandre Calcei, Nathalie Settelen, Xavier Preville, Marie Hennequi, Benoit Grellier, Philippe Ancian, Jukka Rissanen, Pascal Clayette, Christine Guillen, Ronald Rooke and Jean-Yves Bonnefoy
    Abstract

    Cancer progression has been associated with the presence of tumor-associated M2-macrophages (M2-TAMs) able to inhibit anti-tumor immune responses. It is also often associated with metastasis-induced bone destruction mediated by osteoclasts. Both cell types are controlled by the CD115 (CSF-1R)/colony-stimulating factor-1 (CSF-1, M-CSF) pathway, making CD115 a promising target for cancer therapy. Anti-human CD115 monoclonal antibodies (mAbs) that inhibit the receptor function have been generated in a number of laboratories. These mAbs compete with CSF-1 binding to CD115, dramatically affecting monocyte survival and preventing osteoclast and macrophage differentiation, but they also block CD115/CSF-1 internalization and degradation, which could lead to potent rebound CSF-1 effects in patients after mAb treatment has ended. We thus generated and selected a non-ligand competitive anti-CD115 mAb that exerts only partial inhibitory effects on CD115 signaling without blocking the internalization or the degradation of the CD115/CSF-1 complex. This mAb, H27K15, affects monocyte survival only minimally, but downregulates osteoclast differentiation and activity. Importantly, it inhibits monocyte differentiation to CD163+CD64+ M2-polarized suppressor macrophages, skewing their
    differentiation toward CD14−CD1a+ dendritic cells (DCs). In line with this observation, H27K15 also drastically inhibits monocyte chemotactic protein-1 secretion and reduces interleukin-6 production; these two molecules are known to be involved in M2-macrophage recruitment. Thus, the non-depleting mAb H27K15 is a promising anti-tumor candidate, able to inhibit osteoclast differentiation, likely decreasing metastasis-induced osteolysis, and able to prevent M2 polarization of TAMs while inducing DCs, hence contributing to the creation of more efficient anti-tumor immune responses.

    ©2013 Landes Bioscience

  • PLoS ONE 8(8): e72156 August 19, 2013 Antagonistic TNF Receptor One-Specific Antibody (ATROSAB): Receptor Binding and In Vitro BioactivityRichter, Liebig, Guenzi, Herrmann, Scheurich, Pfizenmaier, Kontermann
    Abstract

    The epitope of ATROSAB resides in the N-terminal region of TNFR1 covering parts of CRD1 and CRD2. By site-directed mutagenesis, we identified Arg68 and His69 of TNFR1 as important residues for ATROSAB binding. ATROSAB inhibited binding of 125I-labeled TNF to HT1080 in the subnanomolar range. Furthermore, ATROSAB inhibited release of IL-6 and IL-8 from HeLa and HT1080 cells, respectively, induced by TNF or lymphotoxin alpha (LTα). Different from an agonistic antibody (Htr-9), which binds to a region close to the ATROSAB epitope but elicits strong TNFR1 activation, ATROSAB showed a negligible induction of IL-6 and IL-8 production over a broad concentration range. We further verified that ATROSAB, comprising mutations within the Fc region known to abrogate complement fixation and antibody-mediated cellular effector functions, indeed lacks binding activity for C1q, FcγRI (CD64), FcγRIIB (CD32b), and FcγRIII (CD16) disabling ADCC and CDC.

    © 2013 Richter et al.

  • Protein Engineering, Design & Selection, 8 August, 2013 An anti-TNFR1 scFv-HSA fusion protein as selective antagonist of TNF actionVerena Berger, Fabian Richter, Kirstin Zettlitz, Felix Unverdorben, Peter Scheurich, Andreas Herrmann, Klaus Pfizenmaier and Roland E. Kontermann
    Abstract

    IZI-06.1 is a humanized anti-TNFR1 single-chain fragment variable (scFv) that selectively inhibits binding of tumor necrosis factor (TNF) and lymphotoxin alpha to tumor necrosis factor receptor 1 (TNFR1) but not TNFR2. Recently, IZI-06.1 was converted into a fully human IgG1 antibody (ATROSAB) for the treatment of inflammatory diseases. Here, we compare the bivalent ATROSAB with a monovalent scFv-human serum albumin (HSA) fusion protein lacking any antibody-associated effector functions and possessing approximately only half the molecular mass of an IgG, which should facilitate accumulation in inflamed tissues. Furthermore, the half-life of the scFv should be strongly extended while maintaining monovalent binding, avoiding a possible signal transduction by receptor cross-linking in the absence of TNF. The scFv-HSA fusion protein was produced by stably transfected Chinese hamster ovary cells and purified by affinity chromatography. The fusion protein bound specifically to TNFR1 in enzyme-linked immunosorbent assay and TNFR1-transfected mouse embryonic fibroblasts. Affinity determined by quartz crystal microbalance was reduced compared with ATROSAB, which resulted also in a reduced inhibitory activity. Compared with the scFv fragment, the halflife of the fusion protein was significantly increased, although not reaching the long half-life of ATROSAB. In summary, the scFv-HSA may provide an alternative to the full-length IgG1 with the ability to selectively inhibit TNFR1 and exploiting the pharmacokinetic properties of albumin.

    Protein Engineering, Design & Selection vol. 26 no. 10 pp. 581–587, 2013

    © The Author 2013. Published by Oxford University Press

  • J Virol. 2013 Jul;87(13):7747-53 Protection by immunoglobulin dual-affinity retargeting antibodies against dengue virusBrien, Sukupolvi-Petty, Williams, Lam, Schmid, Johnson, Harris, Diamond
    Abstract

    Dengue viruses are the most common arthropod-transmitted viral infection, with an estimated 390 million human infections annually and ∼3.6 billion people at risk. Currently, there are no approved vaccines or therapeutics available to control the global dengue virus disease burden. In this study, we demonstrate the binding, neutralizing activity, and therapeutic capacity of a novel bispecific dual-affinity retargeting molecule (DART) that limits infection of all four serotypes of dengue virus.

    Copyright © 2013, American Society for Microbiology. All Rights Reserved.

  • Analytical Biochemistry 421 (2012) 351–361 Domain-based assays of individual antibody concentrations in an oligoclonal combination targeting a single proteinQ. Meng, M. Li, M.A. Silberg, F. Conrad, J. Bettencourt, R. To, C. Huang, J. Ma, K. Meyer, R. Shimizu, L. Cao, M.T. Tomic, J.D. Marks
    Abstract

    Quantitation of individual monoclonal antibodies (mAbs) within a combined antibody drug product is required for preclinical and clinical drug development, including pharmacokinetic (PK), toxicology, stability, and biochemical characterization studies of such drugs. We have developed an antitoxin, XOMA 3AB, consisting of three recombinant mAbs that potently neutralize the known subtypes of type A botulinum neurotoxin (BoNT/A). The three mAbs bind nonoverlapping BoNT/A epitopes with high affinity. XOMA 3AB is being developed as a treatment for botulism resulting from BoNT/A. To develop antibody-specific assays, we cloned, expressed, and purified BoNT/A domains from Escherichia coli. Each mAb bound only to its specific domain with affinity comparable to the binding to holotoxin. mAb-specific domains were used to develop an enzyme-linked immunosorbent assay (ELISA) for characterization of the integrity and binding activity of the three mAbs in the drug product. An electrochemiluminescence bridging assay that is robust to interference from components in serum was also developed, and we demonstrate that it can be used for PK assays. This type of antigen engineering to generate mAb-specific domains is a general method allowing quantitation and characterization of individual mAbs in a mAb cocktail that binds the same protein and is superior to anti-idiotype approaches.

    © 2011 Elsevier Inc.

  • Protein Eng Des Sel. 2012 Feb;25(2):81-8. Half-life extension of a single-chain diabody by fusion to domain B of staphylococcal protein AUnverdorben, Färber-Schwarz, Richter, Hutt, Kontermann
    Abstract

    Binding of a therapeutic protein to a long-circulating plasma protein can result in a strongly extended half-life. Among these plasma proteins, albumin and immunoglobulins are of special interest because of their exceptionally long half-life, which is to a great extent determined by recycling through the neonatal Fc receptor (FcRn). Many strategies have been established employing reversible binding to albumin, e.g. using an albumin-binding domain from streptococcal protein G. We show here that the half-life of a recombinant antibody molecule can also be prolonged by fusion to a single immunoglobulin-binding domain (IgBD) from staphylococcal protein A. This domain (domain B, SpA(B)) is composed of 56 amino acid residues and was fused to the C-terminus of a bispecific single-chain diabody (scDb). The scDb-SpA(B) fusion protein was produced in HEK293 cells and retained its antigen-binding activity as shown by enzyme-linked immunosorbent assay and flow cytometry. Furthermore, the fusion protein was capable of binding to human and mouse IgG in a pH-dependent manner. In mice, the terminal half-life of the fusion protein was improved from ∼1-2 h of the unmodified scDb to 11.8 h. Although the fusion protein did not reach the long half-life seen for IgG, our results established the applicability of a single bacterial IgBD for half-life extension purposes.

    © Unverdorben et al. 2012. Published by Oxford University Press. All rights reserved.

  • Vaccine, 2012 Jan 11;30(3):572-9 The effect of adjuvants on the immune response induced by a DBL4ɛ-ID4 VAR2CSA based Plasmodium falciparum vaccine against placental malariaPinto VV, Salanti A, Joergensen LM, Dahlbäck M, Resende M, Ditlev SB, Agger EM, Arnot DE, Theander TG, Nielsen MA.
    Abstract

    A vaccine protecting women against placental malaria could be based on the sub-domains of the VAR2CSA antigen, since antibodies against the DBL4ɛ-ID4 subunit of the VAR2CSA protein can inhibit parasite binding to the placental ligand chondroitin sulphate A (CSA). Here we tested the ability ofDBL4ɛ-ID4 to induce binding-inhibitory antibodies when formulated with adjuvants approved for human use. We have characterized the immune response of DBL4ɛ-ID4 in combination with Freund's complete and incomplete adjuvant and with three adjuvants currently being used in clinical trials: Montanide(®) ISA 720, Alhydrogel(®) and CAF01. Antibodies induced against DBL4ɛ-ID4 in combination with these adjuvants inhibited parasite binding to CSA from 82% to 99%. Although, different epitope recognition patterns were obtained for the different formulations, all adjuvant combinations induced strong Th1 and Th2 type responses, resulting in IgG with similar binding strength, with to the DBL4ɛ-ID4 antigen. These results demonstrate that the DBL4ɛ-ID4 antigen is highly immunogenic and that binding inhibitory antibodies are induced when formulated with any of the tested adjuvants.

    Copyright © 2011 Elsevier Ltd. All rights reserved.

  • Journal of Biological Chemistry, doi: 10.1074/jbc.M111.311522, December 6, 2011 Plasma half-life extension of small recombinant antibodies by fusion to immunoglobulin-binding domains (IgBD)Meike Hutt, Aline Färber-Schwarz, Felix Unverdorben, Fabian Richter & Roland E. Kontermann
    Abstract

    Many therapeutic proteins possessing a small size are rapidly cleared from circulation. Half-life extension strategies have therefore become increasingly important to improve the pharmacokinetic and pharmacodynamic properties of protein therapeutics. Here, we performed a comparative analysis of the half-life extension properties of various bacterial immunoglobulin-binding domains (IgBD) derived from Staphylococcus protein A (SpA), Streptococcus protein G (SpG) and Finegoldia (formerly Peptostreptococcus) protein L (PpL). These domains, composed of 50-60 amino acid residues, were fused to the C-terminus of a single-chain Fv (scFv) and a bispecific singlechain diabody (scDb), respectively. All fusion proteins were produced in mammalian cells and retained their antigen-binding properties. The half-lives of the antibody molecules were prolonged to varying extent for the different IgBDs. The strongest effects in mice were observed for domain C3 of SpG (SpGC3), followed by domains B and D of SpA, suggesting that SpGC3 is particularly useful to extend the plasma half-life of small proteins.

    © 2011 The American Society for Biochemistry and Molecular Biology, Inc.

  • PLoS ONE 6(9): e24558 2011, September 13, 2011 In Vitro Evolution of Allergy Vaccine Candidates, with Maintained Structure, but Reduced B Cell and T Cell Activation CapacityOla B. Nilsson, Justus Adedoyin, Claudio Rhyner, Theresa Neimert-Andersson, Jeanette Grundström, Kurt D. Berndt, Reto Crameri, Hans Grönlund
    Abstract

    Allergy and asthma to cat (Felis domesticus) affects about 10% of the population in affluent countries. Immediate allergic symptoms are primarily mediated via IgE antibodies binding to B cell epitopes, whereas late phase inflammatory reactions are mediated via activated T cell recognition of allergen-specific T cell epitopes. Allergen-specific immunotherapy relieves symptoms and is the only treatment inducing a long-lasting protection by induction of protective immune responses. The aim of this study was to produce an allergy vaccine designed with the combined features of attenuated T cell activation, reduced anaphylactic properties, retained molecular integrity and induction of efficient IgE blocking IgG antibodies for safer and efficacious treatment of patients with allergy and asthma to cat. The template gene coding for rFel d 1 was used to introduce random mutations, which was subsequently expressed in large phage libraries. Despite accumulated mutations by up to 7 rounds of iterative error-prone PCR and biopanning, surface topology and structure was essentially maintained using IgE-antibodies from cat allergic patients for phage enrichment. Four candidates were isolated, displaying similar or lower IgE binding, reduced anaphylactic activity as measured by their capacity to induce basophil degranulation and, importantly, a significantly lower T cell reactivity in lymphoproliferative assays compared to the original rFel d 1. In addition, all mutants showed ability to induce blocking antibodies in immunized mice.The approach presented here provides a straightforward procedure to generate a novel type of allergy vaccines for safer and efficacious treatment of allergic patients.

    © 2011 Nilsson et al.

  • J Immunol 2011; 186:959-968 Neonatal FcR Overexpression Boosts Humoral Immune Response in Transgenic MiceCervenak, Bender, Schneider, Magna, Valer Carstea, Liliom, Erdei, Bosze and Kacskovics
    Abstract

    The neonatal FcR (FcRn) regulates IgG and albumin homeostasis, mediates maternal IgG transport, takes active part in phagocytosis, and delivers Ag for presentation.We have previously shown that overexpression of FcRn in transgenic (Tg) mice extends the half-life of mouse IgG by reducing its clearance. In this paper, we demonstrate that immunization of these mice with OVA and trinitrophenyl-conjugated human IgG results in a 3- to 10-fold increase of Ag-specific IgM and IgG in serum. The IgM increase was unexpected because FcRn does not bind IgM. Our results showed that the affinity of the Ag-specific IgG was at least as good in Tg mice as in the wild-type (wt) controls, implying appropriate affinity maturation in both groups. Influenza vaccination produced a 2-fold increase in the amount of virus-specific Ab in Tg animals, which proved twice as efficient in a hemagglutination inhibition assay as was the case in wt controls. After immunization, Tg mice displayed significantly larger spleens containing a higher number of Ag-specific B cells and plasma cells, as well as many more granulocytes and dendritic cells, analyzed by ELISPOT and flow cytometric studies. The neutrophils from these Tg mice expressed the Tg FcRn and phagocytosed IgG immune complexes more efficiently than did those from wt mice. These results show that FcRn overexpression not only extends the IgG half-life but also enhances the expansion of Ag-specific B cells and plasma cells. Although both effects increase the level of Ag-specific IgG, the increase in immune response and IgG production seems to be more prominent compared with the reduced IgG clearance.

    Copyright © 2011 by The American Association of Immunologists

  • Innovations in Pharmaceutical Technology 2011, June; 34-40 Improving the Developability of BiopharmaceuticalsZurdo J, Michael R, Stallwood Y, Hedman K and Aastrup T
    Abstract

    Biopharmaceutical development is marred by multiple risks. High levels of attrition during preclinical and clinical development are posing a significant challenge, and pushing drug development costs to levels that are no longer sustainable. Diverse strategies for failing early and cheaply are currently being explored, with an emphasis on translational medicine, predictive technologies, scale-down models and further up-front product characterisation. One of these approaches involves the incorporation of a developability risk assessment – focusing on manufacturability and safety – in the early phases of development, in order to help with the selection and design of products with the right quality attributes. One such attribute is aggregation, which constitutes a significant hurdle for biopharmaceutical development and a potential risk of increased immunogenicity. In this article, we discuss how protein-engineering approaches based on predictive computational methods, together with early analytics, can be utilised to select candidates with enhanced developability. We also provide examples of new orthogonal strategies to assess aggregation and stability at an early stage.

    © 2011 Samedan Ltd

  • Appl Environ Microbiol. 2011 Sep;77(17):6165-71 Magnetosome expression of functional camelid antibody fragments (nanobodies) in Magnetospirillum gryphiswaldensePollithy, Romer, Lang, Müller, Helma, Leonhardt, Rothbauer, Schüler
    Abstract

    Numerous applications of conventional and biogenic magnetic nanoparticles (MNPs), such as in diagnostics, immunomagnetic separations, and magnetic cell labeling, require the immobilization of antibodies. This is usually accomplished by chemical conjugation, which, however, has several disadvantages, such as poor efficiency and the need for coupling chemistry. Here, we describe a novel strategy to display a functional camelid antibody fragment (nanobody) from an alpaca (Lama pacos) on the surface of bacterial biogenic magnetic nanoparticles (magnetosomes). Magnetosome-specific expression of a red fluorescent protein (RFP)-binding nanobody (RBP) in vivo was accomplished by genetic fusion of RBP to the magnetosome protein MamC in the magnetite-synthesizing bacterium Magnetospirillum gryphiswaldense. We demonstrate that isolated magnetosomes expressing MamC-RBP efficiently recognize and bind their antigen in vitro and can be used for immunoprecipitation of RFP-tagged proteins and their interaction partners from cell extracts. In addition, we show that coexpression of monomeric RFP (mRFP or its variant mCherry) and MamC-RBP results in intracellular recognition and magnetosome recruitment of RFP within living bacteria. The intracellular expression of a functional nanobody targeted to a specific bacterial compartment opens new possibilities for in vivo synthesis of MNP-immobilized nanobodies. Moreover, intracellular nanotraps can be generated to manipulate bacterial structures in live cells.

    Copyright © 2011, American Society for Microbiology. All Rights Reserved.

  • Analytical Chemistry, 2011, 83, 6040–6046 Three Different Approaches for the Clarification of the Interactions between Lipoproteins and Chondroitin-6-sulfateK. Lipponen, P.W. Stege, G. Cilpa, J. Samuelsson, T. Fornstedt and M.-L. Riekkola
    Abstract

    Two different experimental approaches were used for obtaining a comprehensive view and understanding of the interactions between apolipoprotein B-100 (ApoB-100) of low-density lipoprotein and apolipoprotein E (ApoE) of high-density lipoprotein and chondroitin-6-sulfate (C6S) of arterial proteoglycan. The techniques employed were partial filling affinity capillary electrophoresis (PF-ACE) and continuous flow quartz crystal microbalance (QCM). In addition, molecular dynamic (MD) simulations were used to provide a supportive visual insight into the interaction mechanism. A new tool for analysis of QCM-data was utilized, i.e., adsorption energy distribution calculations, which allowed a deeper understanding of the interactions, especially at different temperatures. The PF-ACE technique probed mainly the strong adsorption interactions whereas in the MD calculations short- and long-range interactions could be distinguished. Although there are differences in the techniques, a pretty good agreement was achieved between the three approaches for the interaction of 19 amino acid peptide of ApoB with C6S giving log affinity constants of 4.66 by QCM, 5.02 by PF-ACE, and 7.39 by MD, and for 15 amino acid peptide of ApoE with C6S 5.34 by QCM, 5.28 by PT-ACE, and 4.60 by MD at physiological temperature 37.0°C.

    © 2011 American Chemical Society

  • Journal of Biological Chemistry (JBC) 2011, May; vol 286:18, 15908-17 The Chondroitin Sulfate A-binding Site of the VAR2CSA Protein Involves Multiple N-terminal DomainsDahlbäck M, Jørgensen LM, Nielsen MA, Clausen TM, Ditlev SB, Resende M, Pinto VV, Arnot DE, Theander TG, Salanti A
    Abstract

    Malaria during pregnancy is a major health problem for African women. The disease is caused by Plasmodium falciparum malaria parasites, which accumulate in the placenta by adhering to chondroitin sulfate A (CSA). The interaction between infected erythrocytes and the placental receptor is mediated by a parasite expressed protein named VAR2CSA. A vaccine protecting pregnant women against placental malaria should induce antibodies inhibiting the interaction between VAR2CSA and CSA. Much effort has been put into defining the part of the 350 kDa VAR2CSA protein that is responsible for binding. It has been shown that full-length recombinant VAR2CSA binds specifically to CSA with high affinity, however to date no sub-fragment of VAR2CSA has been shown to interact with CSA with similar affinity or specificity. In this study, we used a biosensor technology to examine the binding properties of a panel of truncated VAR2CSA proteins. The experiments indicate that the core of the CSA-binding site is situated in three domains, DBL2X-CIDRPAM and a flanking domain, located in the N-terminal part of VAR2CSA. Furthermore, recombinant VAR2CSA subfragments containing this region elicit antibodies with high parasite adhesion blocking activity in animal immunization experiments.

    © 2011 by The American Society for Biochemistry and Molecular Biology, Inc.

  • Protein Engineering, Design & Selection (PEDS) 2010, Nov;23(11):827-34. Epub 2010 Sep 3. The effects of affinity and valency of an albumin-binding domain (ABD) on the half-life of a single-chain diabody-ABD fusion proteinHopp J, Hornig N, Zettlitz KA, Schwarz A, Fuss N, Müller D, Kontermann RE.
    Abstract

    Fusion of small recombinant antibody fragments to an albumin-binding domain (ABD) from streptococcal protein G strongly extends their plasma half-life. This ABD binds with nanomolar affinity to human (HSA) and mouse serum albumin (MSA). It was speculated that an increase in albumin-binding affinity should lead to a further increase in half-life. In the present study, we analyzed the effects of affinity and valency of the ABD on the pharmacokinetic properties of a bispecific single-chain diabody (scDb), applied previously to investigate various half-life extension strategies. The scDb is directed against carcinoembryonic antigen (CEA) and CD3 capable of mediating T cell retargeting to tumor cells. Two scDb derivatives with increased (scDb-ABD-H) and decreased (scDb-ABD-L) affinity as well as an scDb molecule fused to two ABD (scDb-ABD(2)) were generated and produced in mammalian cells. The altered binding of these constructs to HSA and MSA was confirmed by ELISA and quartz crystal microbalance measurements. All constructs bound efficiently to CEA and CD3-positive cells and were able to activate T cells in a target cell-dependent manner, although T cell activation was reduced in the presence of serum albumin. All three derivatives showed a strongly increased half-life in mice as compared with scDb. Compared with the wild-type scDb-ABD, the half-life of scDb-ABD-H exhibited a prolonged half-life and scDb-ABD-L a reduced half-life, while the half-life scDb-ABD(2) was almost identical to that of scDb-ABD. However, these changes were only moderate, indicating that the half-life-extending property of the ABD in mice is only weakly influenced by affinity for serum albumin or valency of albumin binding.

    © Copyright 2010 Oxford University Press

  • Journal of Biological Chemistry (JBC) 2010 Oct 15;285(42):32638-46. Epub 2010 Aug 2 WSS25 inhibits growth of xenografted hepatocellular cancer cells in nude mice by disrupting angiogenesis via blocking bone morphogenetic protein (BMP)/Smad/Id1 signalingQiu H, Yang B, Pei ZC, Zhang Z, Ding K.
    Abstract

    The highly expressed Id1 (inhibitor of DNA binding/differentiation) protein promotes angiogenesis in HCC and is a well established target for anti-angiogenesis therapeutic strategies. Heparan sulfate (HS) mimetics such as PI-88 can abrogate HS-protein interactions to inhibit angiogenesis. Id1 is the direct downstream effector of bone morphogenetic proteins (BMPs), which are angiogenic and HS-binding proteins. Thus, targeting BMPs by HS mimetics may inhibit angiogenesis via attenuating Id1 expression. We report here that a HS mimetic WSS25 potently inhibited the tube formation of HMEC-1 cells on Matrigel and their migration. Meanwhile, WSS25 (25 μg/ml) nearly completely blocked Id1 expression in the HMEC-1 cells as demonstrated by oligo-angiogenesis microarray analysis and further confirmed by RT-PCR and Western blotting. BMP/Smad/Id1 signaling also was blocked by WSS25 treatment in HMEC-1 cells. Importantly, Id1 knockdown in HMEC-1 cells caused the disruption of their tube formation on Matrigel. By employing quartz crystal microbalance analysis, we found that WSS25 strongly bound to BMP2. Moreover, WSS25 impaired BMP2-induced tube formation of HMEC-1 cells on Matrigel and angiogenesis in Matrigel transplanted into C57BL6 mice. Furthermore, WSS25 (100 mg/kg) abrogated the growth of HCC cells xenografted in male nude mice. Immunohistochemical analysis showed that both the expression of Id1 and the endothelial cell marker CD31 were lower in the WSS25-treated tumor tissue than in the control. Therefore, WSS25 is a potential drug candidate for HCC therapy as a tumor angiogenesis inhibitor.

    © 2010 by The American Society for Biochemistry and Molecular Biology, Inc.

  • Malaria Journal 2010, 9:100 The kinetics of antibody binding to Plasmodium falciparum VAR2CSA PfEMP1 antigen and modeling of PfEMP1 antigen packing on the membrane knobsLars M Jørgensen, Ali Salanti, Tina Dobrilovic, Lea Barfod, Tue Hassenkam, Thor G Theander, Lars Hviid and David E Arnot
    Abstract

    Infected humans make protective antibody responses to the PfEMP1 adhesion antigens exported by Plasmodium falciparum parasites to the erythrocyte membrane, but little is known about the kinetics of this antibody-receptor binding reaction or how the topology of PfEMP1 on the parasitized erythrocyte membrane influences antibody association with, and dissociation from, its antigenic target.

    © 2010 Jørgensen et al; licensee BioMed Central Ltd.

  • Molecular Biotechnology (2010) 46:265–278 Humanization of a Mouse Monoclonal Antibody Directed Against a Cell Surface-Exposed Epitope of Membrane-Associated Heat Shock Protein 70 (Hsp70)Kirstin A. Zettlitz, Julia Seitter, Dafne Müller, Roland E. Kontermann
    Abstract

    The translocation of heat shock protein 70 (mHsp70) into the plasma membrane has been found to be associated with various cancers including breast cancer, head-and-neck cancer, and acute myeloid leukemia. Parts of the C-terminal substrate-binding domain (SBD) of mHsp70 are accessible to binding by monoclonal antibodies (mAb). One of these mAbs, cmHsp70.1, has been extensively studied and showed promising results as diagnostic and therapeutic antibody. Here, we describe cloning and humanization of cmHsp70.1 by complementarity determining region grafting resulting in an antibody (humex) possessing a similar affinity (3 nM) as the parental antibody and an improved production and thermal stability. Epitope mapping confirmed that the parental, chimeric, and humanized antibodies recognize the same region including amino acids 473–504 of the SBD. Hence, this humanized antibody provides a basis for further development of an anti-mHsp70 antibody therapy.

    © Springer Science+Business Media, LLC 2010

  • THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 286, NO. 30, pp. 26616 –26627 Heparin Impairs Angiogenesis through Inhibition of MicroRNA-10bXiaokun Shen, Jianping Fang, Xiaofen Lv, Zhicao Pei, Ying Wang, Songshan Jiang, and Kan Ding
    Abstract

    Heparin, which has been used as an anticoagulant drug for decades, inhibits angiogenesis, whereas thrombin promotes tumor-associated angiogenesis. However, the mechanisms underlying the regulation of angiogenesis by heparin and thrombin are not well understood. Here, we show that microRNA-10b (miR-10b) is down-regulated by heparin and up-regulated by thrombin in human microvascular endothelial cells (HMEC-1). Overexpression of miR-10b induces HMEC-1 cell migration, tube formation, and angiogenesis, and downregulates homeobox D10 (HoxD10) expression via direct binding of miR-10b to the putative 3 UTR of HoxD10. In addition, HMEC-1 cell migration and tube formation are induced by HoxD10 knockdown, whereas angiogenesis is arrested when HoxD10 expression is increased after anti-miR-10b or heparin treatments. Furthermore, expression of miR-10b and its transcription factor Twist are up-regulated by thrombin, whereas
    HoxD10 expression is impaired by thrombin. Using quartz crystal microbalance analysis, we show that heparin binds to thrombin, thereby inhibiting thrombin-induced expression of Twist
    and miR-10b. However, the expression of miR-10b is not attenuated by heparin any more after thrombin expression is silenced by its siRNA. Interestingly, we find that heparin attenuates miR-10b expression and induces HoxD10 expression in vivo to inhibit angiogenesis and impair the growth of MDA-MB-231 tumor xenografts. These results provide insight into the molecular
    mechanism by which heparin and thrombin regulate angiogenesis.

    © 2011 by The American Society for Biochemistry and Molecular Biology, Inc

  • Nature Structural & Molecular Biology vol. 17, no. 1, January 2010, (13 December, 2009) Modulation of protein properties in living cells using nanobodiesAxel Kirchhofer, Jonas Helma, Katrin Schmidthals, Carina Frauer, Sheng Cui, Annette Karcher, Mireille Pellis, Serge Muyldermans, Corella S. Casas-Delucchi, M. Cristina Cardoso, Heinrich Leonhardt, Karl-Peter P. Hopfner and Ulrich Rothbauer
    Abstract

    Protein conformation is critically linked to function and often controlled by interactions with regulatory factors. Here we report the selection of camelid-derived single-domain antibodies (nanobodies) that modulate the conformation and spectral properties of the green fluorescent protein (GFP). One nanobody could reversibly reduce GFP fluorescence by a factor of 5, whereas its displacement by a second nanobody caused an increase by a factor of 10. Structural analysis of GFP-nanobody complexes revealed that the two nanobodies induce subtle opposing changes in the chromophore environment, leading to altered absorption properties. Unlike conventional antibodies, the small, stable nanobodies are functional in living cells. Nanobody-induced changes were detected by ratio imaging and used to monitor protein expression and subcellular localization as well as translocation events such as the tamoxifen-induced nuclear localization of estrogen receptor. This work demonstrates that protein conformations can be manipulated and studied with nanobodies in living cells.

    © 2010 Nature America, Inc.

  • Innovations in Pharmaceutical Technology - Issue 29(2009) (Online) Crude Sample Analysis Made EasyAlexander Kovacs
    Abstract

    A new system enables biomolecular interaction analyses to be performed on impure samples – thereby saving on time, labour and costs. Knowing the kinetic properties of biomolecules is increasingly important in drug research. Previously, it has been necessary to obtain purified molecules for such studies as impure samples pose challenges to non-specific binding and microfluidics. The ability to analyse impure samples would thus provide the benefits of saving time, labour and cost. At Attana, we have developed a system that makes this possible. In this article, we review the system and describe how it can be used in the screening of antibodies and determining their offrates in serum containing hybridoma supernatants.

    IPTonline © 2009 The Pharmaceutical Technology Journal

  • The Journal of Biological Chemistry (2009) 284, 25612-25619 Biodistribution of a Bispecific Single-chain Diabody and Its Half-life Extended DerivativesRoland Stork, Emmanuelle Campigna, Bruno Robert, Dafne Müller and Roland E. Kontermann
    Abstract

    Small recombinant antibody molecules such as bispecific single-chain diabodies (scDb) possessing a molecular mass of approximately 55 kDa are rapidly cleared from circulation. We have recently extended the plasma half-life of scDb applying various strategies including PEGylation, N-glycosylation and fusion to an albumin-binding domain (ABD) from streptococcal protein G. Here, we further analyzed the influence of these modifications on the biodistribution of a scDb directed against carcinoembryonic antigen (CEA) and CD3 capable of retargeting T cells to CEA-expressing tumor cells. We show that a prolonged circulation time results in an increased accumulation in CEA+ tumors, which was most pronounced for scDb-ABD and PEGylated scDb. Interestingly, tumor accumulation of the scDb-ABD fusion protein was approximately 2-fold higher compared with PEGylated scDb, although both molecules exhibit similar plasma half-lives and similar affinities for CEA. Comparing half-lives in neonatal Fc receptor (FcRn) wild-type and FcRn heavy chain knock-out mice the contribution of the FcRn to the long plasma half-life of scDb-ABD was confirmed. The half-life of scDb-ABD was approximately 2-fold lower in the knock-out mice, while no differences were observed for PEGylated scDb. Binding of the scDb derivatives to target and effector cells was not or only marginally affected by the modifications, although, compared with scDb, a reduced cytotoxic activity was observed for scDb-ABD, which was further reduced in the presence of albumin. In summary, these findings demonstrate that the extended half-life of a bispecific scDb translates into improved accumulation in antigen-positive tumors but that modifications might also affect scDb-mediated cytotoxicity.

    © 2009 by American Society for Biochemistry and Molecular Biology

  • PNAS vol. 106, no. 12, 4623–4628 (Mar. 2009) Controlled release of functional proteins through designer self-assembling peptide nanofiber hydrogel scaffoldSotirios Koutsopoulos, Larry D. Unsworth, Yusuke Nagai, and Shuguang Zhang
    Abstract

    The release kinetics for a variety of proteins of a wide range of molecular mass, hydrodynamic radii, and isoelectric points through a nanofiber hydrogel scaffold consisting of designer self-assembling peptides were studied by using single-molecule fluorescence correlation spectroscopy (FCS). In contrast to classical diffusion experiments, the single-molecule approach allowed for the direct determination of diffusion coefficients for lysozyme, trypsin inhibitor, BSA, and IgG both inside the hydrogel and after being released into the solution. The results of the FCS analyses and the calculated pristine in-gel diffusion coefficients were compared with the values obtained from the Stokes–Einstein equation, Fickian diffusion models, and the literature. The release kinetics suggested that protein diffusion through nanofiber hydrogels depended primarily on the size of the protein. Protein diffusivities decreased, with increasing hydrogel nanofiber density providing a means of controlling the release kinetics. Secondary and tertiary structure analyses and biological assays of the released proteins showed that encapsulation and release did not affect the protein conformation and functionality. Our results show that this biocompatible and injectable designer self-assembling peptide hydrogel system may be useful as a carrier for therapeutic proteins for sustained release applications.

    © 2009 PNAS by the National Academy of Sciences

  • Tumor Biology 30:26–36 (Feb. 2009) Characterization of Monoclonal Antibodies Directed against Squamous Cell Carcinoma Antigens: Report of the Second TD-10 WorkshopK. Nustad, O. Nilsson, K. Majnesjö, A. Murakami, N. Sugino, D.J. Warren, and H. Kato
    Abstract

    Eight monoclonal antibodies directed against Squamous Cell Carcinoma Antigens (A1 and A2) were collected and evaluated by three working groups. Recombinant antigens, fusion proteins and native antigens from normal tissue were used to evaluate antibody specificity. Five antibodies reacted with both A1 and A2. Two of these antibodies (K123 and K131) showed related binding characteristics, whereas SCC140, K182 and SCC111 demonstrated unique epitope specificity and were not related to the reference antibodies included (F1H3, F2H7 and SCC107). SCC111 reacted particularly well with antigen on Western blot, indicating that the epitope was partly hidden when the antigen was in solution. Two antibodies (SCC103 and SCC109) reacted only with A2 and the fusion protein A1/A2, indicating that they recognized an A2 epitope in exon 8. The A2-specific antibodies are unique in their binding to A2 and are different from the reference antibodies included (SCC104 and K122). SCC103 is probably the best A2-specific antibody available. One antibody, K136, was A1-specific and is related to reference antibody K135. The new antibodies can be used to establish immunometric assays for specific measurement of A1, A2 or both A1 and A2 together.

    © 2009 S. Karger AG, Basel

  • Journal of Molecular Biology (2008) 384, 1143–1156 Generation, Affinity Maturation, and Characterization of a Human Anti-Human NKG2D Monoclonal Antibody with Dual Antagonistic and Agonistic ActivityKa Yin Kwong, Sivasubramanian Baskar, Hua Zhang, Crystal L. Mackall and Christoph Rader
    Abstract

    In humans,NKG2Dis an activating receptor on natural killer (NK) cells and a costimulatory receptor on certain Tcells and plays a central role in mediating immune responses in autoimmune diseases, infectious diseases, and cancer. Monoclonal antibodies that antagonize or agonize immune responses mediated by human NKG2D are considered to be of broad and potent therapeutic utility. Nonetheless, monoclonal antibodies to NKG2D that are suitable for clinical investigations have not been published yet. Here, we describe the generation, affinity maturation, and characterization of a fully human monoclonal antibody to human NKG2D. Using phage display technology based on a newly generated naïve human Fab library in phage display vector pC3C followed by a tandem chain shuffling process designed for minimal deviation from natural human antibody sequences,we selected a human Fab, designated KYK-2.0, with high specificity and affinity to human NKG2D. KYK-2.0 Fab blocked the binding of the natural human NKG2D ligands MICA, MICB, and ULBP2 as potently as a commercially available mouse anti-human NKG2D monoclonal antibody in immunoglobulin G (IgG) format. Conversion of KYK-2.0 Fab to IgG1 resulted in subnanomolar avidity for human NKG2D. KYK-2.0 IgG1 was found to selectively recognize defined subpopulations of human lymphocytes known to express NKG2D, that is, the majority of human CD8+, CD16+, and CD56+ cells as well as a small fraction of human CD4+ cells. In solution, KYK-2.0 IgG1 interfered with the cytolytic activity of ex vivo expanded human NK cells. By contrast, immobilized KYK-2.0 IgG1 was found to strongly induce human NK cell activation. The dual antagonistic and agonistic activity promises a wide range of therapeutic applications for KYK-2.0 IgG1 and its derivatives.

    © 2008 Published by Elsevier Inc.

  • Analytical Biochemistry 341 (2005) 89-93 Electro-immobilization of proinsulin C-peptide to a quartz crystal microbalance sensor chip for protein affinity purificationErmias Melles, Henrik Anderson, Daniel Wallinder, Jawed Shafqat, Tomas Bergman, Teodor Aastrup and Hans Jörnvall
    Abstract

    Proinsulin C-peptide was electro-immobilized to a quartz crystal microbalance sensor chip, localizing this low-pI peptide for covalent attachment to activated surface carboxyl groups. The resulting chip was used in a continuous flow biosensor to capture anti-C-peptide antibodies, which could subsequently be eluted in 5% formic acid between air bubbles for efficient recovery and mass spectrometric identification. The method is reproducible through repeated cycles, providing affinity purification of proteins under real-time monitoring of the binding and elution processes.

    © 2005 Elsevier Ltd. All rights reserved.

  • Protein Engineering, Design & Selection (2005) 18(11):537-546 Isolation of novel single-chain Cro proteins targeted for binding to the bcl-2 transcription initiation site by repertoire selection and subunit combinatoricsKristina Jonas, Erhard Van Der Vries, Mikael T.I.Nilsson and Mikael Widersten
    Abstract

    New designed DNA-binding proteins may be recruited to act as transcriptional regulators and could provide new therapeutic agents in the treatment of genetic disorders such as cancer. We have isolated tailored DNA-binding proteins elected for affinity to a region spanning the transcription initiation site of the human bcl-2 gene. The proteins were derived from a single-chain derivative of the lambda Cro protein (scCro), randomly mutated in its recognition helices to construct libraries of protein variants of distinct DNA binding properties. By phage display-afforded affinity selections combined with recombination of shuffled subunits, protein variants were isolated, which displayed high affinity for the target bcl-2 sequence, as determined by electrophoretic mobility shift and biosensor assays. The proteins analyzed were moderately sequence-specific but provide a starting point for further maturation of desired function.

    © 2005 Oxford Journals. All rights reserved.