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ABSTRACT: The generation of a high productivity cell line is a critical step in the production of a therapeutic protein. Many innovative engineering strategies have been devised in order to maximize the expression rate of production cells for increased process efficiency. Less effort has focused on improvements to the cell line generation process, which is typically long and laborious when using mammalian cells. Based on unexpected findings when generating stable CHO cell lines expressing human IL-17F, we studied the benefit of expressing this protein during the establishment of production cell lines. We demonstrate that IL-17F expression enhances the rate of selection and overall number of selected cell lines as well as their transgene expression levels. We also show that this benefit is observed with different parental CHO cell lines and selection systems. Furthermore, IL-17F expression improves the efficiency of cell line subcloning processes. IL-17F can therefore be exploited in a standard manufacturing process to obtain higher productivity clones in a reduced time frame. Biotechnol. Bioeng. 2013; 110: 1153-1163. © 2012 Wiley Periodicals, Inc.
Biotechnology and Bioengineering 10/2012; · 3.95 Impact Factor
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ABSTRACT: The goal of rheumatoid arthritis (RA) treatment is to achieve clinical remission in order to limit structural damage and physical disability. To this end, recent emphasis has been placed on aggressive treatment early in the course of disease with drugs such as anti-tumor necrosis factor (anti-TNF) agents. As T cells are also thought to play an important role in the initiation of RA, we hypothesized that targeting both TNF and T cells would result in better outcomes. The aim of this study was to examine the efficacy of combined therapy with anti-CD3 and anti-TNF in experimental RA.
Two anti-mouse antibodies were developed as surrogate reagents for anti-TNF and anti-CD3 therapies. Collagen-induced arthritis (CIA) was induced in DBA/1 mice, and antibodies were injected intraperitoneally, either alone on in combination, at predetermined subtherapeutic doses. The frequency and number of pathogenic and regulatory CD4+ T cell subsets in the draining lymph nodes were determined in order to investigate the mechanisms of action.
Strikingly, the combination of the two antibodies demonstrated a potent synergy in established CIA, with long-term inhibition of disease progression and protection from joint destruction. The results did not demonstrate any enhancement of CD25+FoxP3+ regulatory T cells, but a profound depletion of pathogenic T cells from the draining lymph nodes was associated with reduced numbers of T cells in the joints.
A short course of combination therapy with anti-CD3 and anti-TNF efficiently depletes pathogenic T cells from the draining lymph nodes, reducing the numbers of T cells in the joints and affording long-lasting inhibition of established CIA.
Arthritis & Rheumatism 04/2012; 64(10):3189-98. · 7.87 Impact Factor
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ABSTRACT: Antibody repertoires are characterized by diversity as they vary not only amongst individuals and post antigen exposure but also differ significantly between vertebrate species. Such plasticity can be exploited to generate human antibody libraries featuring hallmarks of these diverse repertoires. In this study, the focus was to capture CDRH3 sequences, as this region generally accounts for most of the interaction energy with antigen. Sequences from human as well as non-human sources were successfully integrated into human antibody libraries. Next generation sequencing of these libraries proved that the CDRH3 lengths and amino acid composition corresponded to the species of origin. Specific CDRH3 sequences, biased towards the recognition of a model antigen either by immunizing mice or by selecting with phage display, were then integrated into another set of libraries. From these antigen biased libraries, highly potent antibodies were more frequently isolated, indicating that the characteristics of an immune repertoire is transferrable via CDRH3 sequences into a human antibody library. Taken together, these data demonstrate that the properties of naturally or experimentally biased repertoires can be effectively harnessed for the generation of targeted human antibody libraries, substantially increasing the probability of isolating antibodies suitable for therapeutic and diagnostic applications.
PLoS ONE 01/2012; 7(8):e43471. · 4.09 Impact Factor
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ABSTRACT: The MHC class-I related receptor or neonatal Fc receptor (FcRn) protects IgG and albumin from degradation by rescuing them in endothelial cells in a pH dependent fashion and consequently increases their respective half-lives. Monoclonal antibody-based therapies are of increasing interest and characterizing the interaction with FcRn is important for the development of an antibody candidate. In order to facilitate the production of soluble FcRn suitable for interaction studies, we generated semi-stable pools co-expressing FcRn α-chain, β2-microglobulin, biotin ligase and EGFP using a dual promoter, multi-cistronic vector. Human and mouse FcRn were purified in the mg/L range of culture medium and a single purification step was sufficient to reach a high level of purity. The receptors were characterized by ELISA, flow cytometry and surface plasmon resonance and shown to be functional. The single site biotinylation facilitated the directional immobilization of FcRn on the sensor chip and significantly increased the response level of the surface compared to amine coupling used in previous studies. Using this system, the affinity constants of seven IgGs, from various species and isotypes, were determined for human and mouse FcRn, including two hamster isotypes. These results confirm the higher selectivity of the human receptor and the promiscuous binding of mFcRn to IgGs from different species.
Journal of immunological methods 09/2011; 375(1-2):20-9. · 2.35 Impact Factor
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Masayuki Fukata,
Limin Shang,
Rebeca Santaolalla,
John Sotolongo,
Cristhine Pastorini,
Cecilia España,
Ryan Ungaro,
Noam Harpaz,
Harry S Cooper,
Greg Elson, Marie Kosco-Vilbois,
Julia Zaias,
Maria T Perez,
Lloyd Mayer,
Arunan S Vamadevan,
Sergio A Lira,
Maria T Abreu
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ABSTRACT: Chronic intestinal inflammation culminates in cancer and a link to Toll-like receptor-4 (TLR4) has been suggested by our observation that TLR4 deficiency prevents colitis-associated neoplasia. In the current study we address the effect of the aberrant activation of epithelial TLR4 on induction of colitis and colitis-associated tumor development. We take a translational approach to address the consequences of increased TLR signaling in the intestinal mucosa.
Mice transgenic for a constitutively active TLR4 under the intestine-specific villin promoter (villin-TLR4 mice) were treated with dextran sodium sulfate (DSS) for acute colitis and azoxymethane (AOM)-DSS TLR4 expression was analyzed by immunohistochemistry in colonic tissue from patients with ulcerative colitis (UC) and UC-associated cancer. The effect of an antagonist TLR4 antibody (Ab) was tested in prevention of colitis-associated neoplasia in the AOM-DSS model.
Villin-TLR4 mice were highly susceptible to both acute colitis and colitis-associated neoplasia. Villin-TLR4 mice had increased epithelial expression of COX-2 and mucosal PGE₂ production at baseline. Increased severity of colitis in villin-TLR4 mice was characterized by enhanced expression of inflammatory mediators and increased neutrophilic infiltration. In human UC samples, TLR4 expression was upregulated in almost all colitis-associated cancer and progressively increased with grade of dysplasia. As a proof of principle, a TLR4/MD-2 antagonist antibody inhibited colitis-associated neoplasia in the mouse model.
Our results show that regulation of TLRs can affect the outcome of both acute colitis and its consequences, cancer. Targeting TLR4 and other TLRs may ultimately play a role in prevention or treatment of colitis-associated cancer.
Inflammatory Bowel Diseases 07/2011; 17(7):1464-73. · 4.86 Impact Factor
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Fabrizio Montecucco,
Vincent Braunersreuther,
Sébastien Lenglet,
Benedicte M A Delattre,
Graziano Pelli,
Vanessa Buatois,
Florence Guilhot,
Katia Galan,
Nicolas Vuilleumier,
Walter Ferlin,
Nicolas Fischer,
Jean-Paul Vallée, Marie Kosco-Vilbois,
François Mach
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ABSTRACT: The chemokine CCL5 plays a critical role as neutrophil and macrophage activator do in atherosclerosis and myocardial infarction. Thus, we investigated whether the treatment with a neutralizing monoclonal antibody (mAb) to mouse CCL5 would provide therapeutic benefit when provoking a coronary-associated ischaemic event.
C57Bl/6 mice were submitted to left coronary artery permanent ligature. Then, various parameters were monitored for up to 21 days. At5 min and 3 days after coronary occlusion, mice received one intravenous injection of the rat anti-mouse CCL5 mAb or isotype IgG control. Infarct size was assessed histologically and by measuring serum cardiac troponin I levels. Kinetics of CCL5 tissue expression, leucocyte infiltration, matrix metalloproteinase (MMP) levels, and collagen deposition were histologically assessed. Serum chemokine levels were measured by enzyme-linked immunosorbent assay. Cardiac function and dimensions were assessed by magnetic resonance imaging (MRI). Chronic ischaemia increased both circulating and intracardiac levels of CCL5. At 24 h, treatment with the anti-CCL5 mAb resulted in a smaller infarct size and reduced circulating levels of chemokines. This effect was associated with reduction of neutrophil and macrophage infiltration within the infarcted myocardium. After 3 days of chronic ischaemia, anti-CCL5 mAb treatment reduced cardiac MMP-9. At 7 days, collagen content was significantly lower. At 21 days, neutralizing CCL5 improved mouse survival, cardiac myocyte size, and cardiac function.
Treatment with anti-CCL5 mAb significantly reduced both infarct size and post-infarction heart failure in a mouse model of chronic cardiac ischaemia. Cardioprotective effects were associated with the reduction of leucocyte recruitment within infarcted hearts.
European Heart Journal 05/2011; 33(15):1964-74. · 10.48 Impact Factor
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Giovanni Magistrelli,
Pauline Malinge,
Rami Lissilaa,
Séverine Fagète,
Florence Guilhot,
Valéry Moine,
Vanessa Buatois,
Yves Delneste,
Stephan Kellenberger,
Franck Gueneau,
Ulla Ravn, Marie Kosco-Vilbois,
Nicolas Fischer
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ABSTRACT: Many research projects in life sciences require purified biologically active recombinant protein. In addition, different formats of a given protein may be needed at different steps of experimental studies. Thus, the number of protein variants to be expressed and purified in short periods of time can expand very quickly. We have therefore developed a rapid and flexible expression system based on described episomal vector replication to generate semi-stable cell pools that secrete recombinant proteins. We cultured these pools in serum-containing medium to avoid time-consuming adaptation of cells to serum-free conditions, maintain cell viability and reuse the cultures for multiple rounds of protein production. As such, an efficient single step affinity process to purify recombinant proteins from serum-containing medium was optimized. Furthermore, a series of multi-cistronic vectors were designed to enable simultaneous expression of proteins and their biotinylation in vivo as well as fast selection of protein-expressing cell pools. Combining these improved procedures and innovative steps, exemplified with seven cytokines and cytokine receptors, we were able to produce biologically active recombinant endotoxin free protein at the milligram scale in 4-6weeks from molecular cloning to protein purification.
Protein Expression and Purification 08/2010; 72(2):209-16. · 1.59 Impact Factor
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European Journal of Immunology 10/2009; 39(9):2310-2312. · 5.10 Impact Factor
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Ryan Ungaro,
Masayuki Fukata,
David Hsu,
Yasmin Hernandez,
Keith Breglio,
Anli Chen,
Ruliang Xu,
John Sotolongo,
Cecillia Espana,
Julia Zaias,
Greg Elson,
Lloyd Mayer, Marie Kosco-Vilbois,
Maria T Abreu
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ABSTRACT: Dysregulated innate immune responses to commensal bacteria contribute to the development of inflammatory bowel disease (IBD). TLR4 is overexpressed in the intestinal mucosa of IBD patients and may contribute to uncontrolled inflammation. However, TLR4 is also an important mediator of intestinal repair. The aim of this study is to examine the effect of a TLR4 antagonist on inflammation and intestinal repair in two murine models of IBD. Colitis was induced in C57BL/6J mice with dextran sodium sulfate (DSS) or by transferring CD45Rb(hi) T cells into RAG1-/- mice. An antibody (Ab) against the TLR4/MD-2 complex or isotype control Ab was administered intraperitoneally during DSS treatment, recovery from DSS colitis, or induction of colitis in RAG1-/- mice. Colitis severity was assessed by disease activity index (DAI) and histology. The effect of the Ab on the inflammatory infiltrate was determined by cell isolation and immunohistochemistry. Mucosal expression of inflammatory mediators was analyzed by real-time PCR and ELISA. Blocking TLR4 at the beginning of DSS administration delayed the development of colitis with significantly lower DAI scores. Anti-TLR4 Ab treatment decreased macrophage and dendritic cell infiltrate and reduced mucosal expression of CCL2, CCL20, TNF-alpha, and IL-6. Anti-TLR4 Ab treatment during recovery from DSS colitis resulted in defective mucosal healing with lower expression of COX-2, PGE(2), and amphiregulin. In contrast, TLR4 blockade had minimal efficacy in ameliorating inflammation in the adoptive transfer model of chronic colitis. Our findings suggest that anti-TLR4 therapy may decrease inflammation in IBD but may also interfere with colonic mucosal healing.
AJP Gastrointestinal and Liver Physiology 05/2009; 296(6):G1167-79. · 3.43 Impact Factor
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ABSTRACT: Hereditary haemophagocytic lymphohistiocytosis (HLH) is a fatal inflammatory disease and treatments currently may lead to serious side effects. There is a pressing need for effective, less toxic treatments for this disease. Previous reports have suggested that interferon gamma (IFNgamma) has a role in the pathogenesis of HLH. Here, we report that blocking IFNgamma had a therapeutic effect in two different murine models of human hereditary HLH (perforin-deficient and Rab27a-deficient mice, both infected with lymphocytic choriomeningitis virus). Therapeutic administration of an anti-IFNgamma antibody induced recovery from haemophagocytosis in both genetic models, as evidenced by increased survival in perforin-deficient mice and correction of blood cytopenia, moderation of body temperature changes, decreased cytokinaemia, restoration of splenic architecture and reduced haemophagocytosis in the liver of both murine models. Involvement of the central nervous system in Rab27a-deficient mice was prevented by anti-IFNgamma therapy. Hepatic T-cell infiltrates and virus persisted, with no detectable harm during the time course of these studies. These data strongly suggest that neutralization of IFNgamma could be used in humans to safely alleviate the clinical manifestations of haemophagocytosis.
EMBO Molecular Medicine 05/2009; 1(2):112-24. · 10.33 Impact Factor
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ABSTRACT: Recently, two-photon imaging has allowed intravital tracking of lymphocyte migration and cellular interactions during germinal center (GC) reactions. The implications of two-photon measurements obtained by several investigators are currently the subject of controversy. With the help of two mathematical approaches, we reanalyze these data. It is shown that the measured lymphocyte migration frequency between the dark and the light zone is quantitatively explained by persistent random walk of lymphocytes. The cell motility data imply a fast intermixture of cells within the whole GC in approximately 3 h, and this does not allow for maintenance of dark and light zones. The model predicts that chemotaxis is active in GCs to maintain GC zoning and demonstrates that chemotaxis is consistent with two-photon lymphocyte motility data. However, the model also predicts that the chemokine sensitivity is quickly down-regulated. On the basis of these findings, we formulate a novel GC lymphocyte migration model and propose its verification by new two-photon experiments that combine the measurement of B cell migration with that of specific chemokine receptor expression levels. In addition, we discuss some statistical limitations for the interpretation of two-photon cell motility measurements in general.
Journal of Experimental Medicine 01/2009; 205(13):3019-29. · 13.85 Impact Factor
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ABSTRACT: Analyzing the dynamics of cellular immune responses, although performed for decades in immunologic research, has seen an enormous increase in the number of studies using this approach since the development of intravital 2-photon microscopy. Meanwhile, new insights into the dynamics of cellular immunity are being published on a daily basis. This review gives a short overview of the currently most widely used techniques, both on the microscopy side as well as on the experimental part. Difficulties and promises will be discussed. Finally, a personal selection of the most interesting findings of the first 6 years of intravital 2-photon microscopy for immunological questions will be given. The overall aim is to get the reader interested into this fascinating way of investigating the immune response by means of "dynamic histology". This already has and will continue to broaden our view on how immune cells work in real life.
Histochemie 11/2008; 130(6):1053-62. · 2.59 Impact Factor
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ABSTRACT: Mechanical ventilation (MV) and lipopolysaccharide (LPS) synergistically increase inflammation and lung injury. The goal of this study was to determine whether blockade of CD14 or Toll-like receptor 4 (TLR4) would reduce inflammation caused by LPS and MV. Rabbits were pretreated with anti-TLR4 or anti-CD14 monoclonal antibodies, followed by endobronchial LPS and MV. Blockade of TLR4 reduced the number of neutrophils and the amount of CXCL8 in bronchoalveolar lavage fluid. In contrast, blockade of CD14 did not significantly decrease the number of neutrophils or the amount of CXCL8. These data show that TLR4 blockade reduces pulmonary inflammation caused by the combination of LPS and Mechanical ventilation.
Experimental Lung Research 07/2008; 34(5):225-43. · 1.22 Impact Factor
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Bruno Daubeuf,
John Mathison,
Stephan Spiller,
Stephanie Hugues,
Suzanne Herren,
Walter Ferlin, Marie Kosco-Vilbois,
Hermann Wagner,
Carsten J Kirschning,
Richard Ulevitch,
Greg Elson
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ABSTRACT: Overactivation of the immune system upon acute bacterial infection leads to septic shock. Specific bacterial products potently stimulate immune cells via toll-like receptors (TLRs). Gram-negative bacteria induce a predominantly TLR4-driven signal through LPS release. To neutralize LPS signaling in experimental models of sepsis, we generated mAbs toward the TLR4/myeloid differentiation protein-2 (MD-2) complex. The binding properties of an array of selected rat mAbs differed in respect to their specificity for TLR4/MD-2 complex. The specificity of one such mAb, 5E3, to murine TLR4 was confirmed by its recognition of an epitope within the second quarter of the ectodomain. 5E3 inhibited LPS-dependent cell activation in vitro and prevented proinflammatory cytokine production in vivo following LPS challenge in a dose-dependent manner. Furthermore, 5E3 protected mice from lethal shock-like syndrome when applied using both preventative and therapeutic protocols. Most notably, in the colon ascendens stent peritonitis model of polymicrobial abdominal sepsis, administration of a single dose of 5E3 (50 mug) protected mice against mortality. These results demonstrate that neutralizing TLR4/MD-2 is highly efficacious in protecting against bacterial infection-induced toxemia and offers TLR4/MD-2 mAb treatment as a potential therapy for numerous clinical indications.
The Journal of Immunology 12/2007; 179(9):6107-14. · 5.79 Impact Factor
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ABSTRACT: The mammalian Toll-like receptor (TLR) family has evolved to sense pathogens in the environment and protect the host against infection. TLR4 recognizes lipopolysaccharide (LPS) from Gram-negative bacteria and induces a signaling cascade that, when exaggerated, has been associated with severe sepsis. We have generated a TLR4-specific monoclonal antibody, 15C1, which neutralizes LPS-induced TLR4 activation in a dose-dependent manner. 15C1 potently blocks the effects of LPS on a panel of primary cells and cell lines in vitro. The binding of 15C1 was mapped to an epitope in the second portion of the extracellular region of TLR4, which has been shown previously to be functionally important in the recognition of LPS. Furthermore, we demonstrate a novel mechanism of inhibition, as the effects of 15C1 are partially Fc-dependent, involving the regulatory Fcgamma receptor IIA (CD32A). In addition to introducing 15C1 as a potent clinical candidate for use in the treatment of LPS-mediated indications, our work demonstrates a newly discovered pathway whose manipulation is pivotal in achieving optimal neutralizing benefit.
Journal of Biological Chemistry 12/2007; 282(48):34817-27. · 4.77 Impact Factor
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ABSTRACT: Atherosclerosis is a chronic inflammatory disease of the large arteries that is the primary cause of heart disease and stroke. Anti-CD3-specific antibodies suppress immune responses by antigenic modulation of the CD3 antibody/T-cell receptor complex. Their unique capacity to restore self-tolerance in a mouse model of diabetes and, importantly, in patients with recent-onset type 1 diabetes involves transforming growth factor-beta-dependent mechanisms via expansion and/or activation of regulatory T cells. We hypothesized that treatment with anti-CD3-specific antibodies might inhibit atherosclerosis development and progression in mice.
Low-density lipoprotein receptor-deficient mice were fed a high-cholesterol diet for 13 or 24 weeks. Anti-CD3 antibody was administered on 5 consecutive days beginning 1 week before or 13 weeks after the high-cholesterol diet was initiated, respectively. Control mice were injected in parallel with phosphate-buffered saline. Anti-CD3 antibody therapy reduced plaque development when administered before a high-cholesterol diet and markedly decreased lesion progression in mice with already established atherosclerosis. We found increased production of the antiinflammatory cytokine transforming growth factor-beta in concanavalin A-stimulated lymph node cells and enhanced expression of the regulatory T-cell marker Foxp3 in spleens of anti-CD3 antibody-treated mice. A higher percentage of apoptotic cells within the plaques of anti-CD3 antibody-treated mice was also observed.
Altered disease progression, combined with the emergence of this particular cytokine pattern, indicates that short-term treatment with an anti-CD3 antibody induces a regulatory T-cell phenotype that restores self-tolerance in a mouse model of atherosclerosis.
Circulation 11/2006; 114(18):1977-84. · 14.74 Impact Factor
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ABSTRACT: Chemokines are a class of low molecular weight proteins that are involved in leukocytes trafficking. Due to their involvement in recruiting immune cells to sites of inflammation, chemokines, and chemokine receptors have become an attractive class of therapeutic targets. However, when expressed in Escherichia coli chemokines are poorly soluble and accumulate in inclusion bodies. Several purification methods have been described but involve time-consuming refolding, buffer exchange, and purification steps that complicate expression of these proteins. Here, we describe a simple and reliable method to express chemokines as fusions to the protein NusA. The fusion proteins were largely found in the soluble fraction and could be readily purified in a single step. Proteolytic cleavage was used to obtain soluble recombinant chemokines that were found to be very active in a novel in vitro chemotaxis assays. This method could be applied to several alpha and beta human chemokines, suggesting that it is generally applicable to this class of proteins.
Biochemical and Biophysical Research Communications 09/2005; 334(2):370-5. · 2.48 Impact Factor
