RSS[68Ga-labeled peptides for clinical trials - Production according to the German Drug Act: The Göttingen experience.]
Source
Priv.-Doz. Dr. rer. nat. Birgit Meller, Abteilung für Nuklearmedizin, Universitätsmedizin Göttingen, Robert-Koch-Str. 40, 37075 Göttingen, Deutschland, Tel. 05 51/39 49 84, Fax 05 51/39 85 26, E-Mail: birgit.meller@medizin.uni-halle.de.
Abstract
The AMG implies far-reaching implications for the synthesis of new radiopharmaceuticals for clinical trials. Aim, methods: As a part of the DFG-funded Clinical Research Group (KFO 179) a project designated "Immuno-PET for assessment of early response to radiochemotherapy of advanced rectal cancer" was initiated. This trial is focused on a trivalent bispecific humanized monoclonal antibody, and a 68Ga-labeled peptide. Following the new regulatory framework we established a GMP-compliant cleanroom laboratory and applied for a manufacturing permission. Results: During the project constructural, personnel and organizational conditions for a successful application were established, including a quality management system. A GMP-conform cleanroom laboratory class C was constructed, equipped with a two-chamber lock. The actual manufacturing is performed in a closed system with subsequent sterile filtration. Themanufacturing processes have been automatised and validated as well as the necessary quality controls. Themanufacturing permission was granted after an official inspection. Conclusions: The new German Drug Act is considered as a break in the production practice of nuclear medicine. The early involvement and communication with the authorities avoids time-consuming and costly planning errors. It is much to be hoped that the new legal situation in Germany will not cause serious impairments in the realization of clinical trials in German nuclear medicine.
Clinical manufacturing of recombinant human interleukin 15. I. Production cell line development and protein expression in E. coli with stop codon optimization.
Source
Biopharmaceutical Development Program, SAIC-Frederick Inc., National Cancer Institute at Frederick (NCI-Frederick), Frederick, MD 21702.
Abstract
Interleukin 15 (IL-15) has shown remarkable biological properties of promoting NK- and T-cell activation and proliferation, as well as enhancing antitumor immunity of CD8(+) T cells in preclinical models. Here, we report the development of an E. coli cell line to express recombinant human Interleukin-15 (rhIL-15) for clinical manufacturing. Human IL-15 cDNA sequence was inserted into a pET28b plasmid and expressed in several E. coli BL21 strains. Through product quality comparisons among several E. coli strains, including E. coli BL21(DE3), BL21(DE3)pLysS, BLR(DE3)pLysS, and BL21-AI, E. coli BL21-AI was selected for clinical manufacturing. Expression optimization was carried out at shake flask and 20-L fermenter scales, and the product was expressed as inclusion bodies that were solubilized, refolded, and purified to yield active rhIL-15. Stop codons of the expression construct were further investigated after 15-20% of the purified rhIL-15 showed an extraneous peak corresponding to an extra tryptophan residue based on peptide mapping and mass spectrometry analysis. It was determined that the presence of an extra tryptophan was due to a stop codon wobble effect, which could be eliminated by replacing TGA (opal) stop codon with TAA (ochre). As a novel strategy, a simple method of demonstrating lack of tRNA suppressors in the production host cells was developed to validate the cells in this study. The E. coli BL21-AI cells containing the rhIL-15 coding sequence with a triplet stop codon TAATAATGA were banked for further clinical manufacturing. © 2011 American Institute of Chemical Engineers Biotechnol. Prog.,, 2011.
Copyright © 2011 American Institute of Chemical Engineers (AIChE).
Drug delivery applications for superporous hydrogels.
Source
Nova Southeastern University, College of Pharmacy, Department of Pharmaceutical Sciences , 3200 South University Dr, Fort Lauderdale , FL 33328, USA ; omidian@nova.edu.
Abstract
Introduction: Considerable advances have been made to hydrogels with the development of faster swelling superporous hydrogels (SPHs). These new-generation hydrogels have large numbers of interconnected pores, giving them the capacity to absorb large amounts of water at an accelerated rate. This gives SPHs the ability to be used in a variety of novel drug delivery applications, such as gastric retention and peroral intestinal delivery of proteins and peptides. Areas covered: This review focuses on the applications of SPHs for drug transport and targeted drug therapies, as well as the characteristics and historical advancements made to SPH synthesis as it pertains to drug delivery. Manufacturingconsiderations and challenges that must be overcome are also discussed, such as scale-up, biocompatibility and safety. Expert opinion: Modern SPHs have high swelling and high mechanical strength making them suitable for many diverse pharmaceutical and biomedical applications. However, demonstrative preclinical animal studies still need to be confirmed in human trials, to further address safety issues and confirm therapeutic success when using SPHs as platforms for drug delivery. The focus of forthcoming applications of SPHs is likely to be in the area of oral site-specific delivery and regenerative medicine.
Simultaneous Generation of Multivirus-specific and Regulatory T Cells for Adoptive Immunotherapy.
Source
*Department of Molecular Immunology, Institute of Child Health ∥Department of Bone Marrow Transplantation, Great Ormond Street Hospital for Children NHS Trust, London, UK †Departments of Hematology ‡Clinical Pharmacy and Toxicology §Pediatrics, Leiden University Medical Centre, Leiden, The Netherlands.
Abstract
Previous studies have established that adoptive immunotherapy with donor-derived virus-specific T cells can prevent/treat viral complications post-stem cell transplant and regulatory T cells show promise as inhibitors of graft-versus-host disease. On the basis of flow cytometric analysis of upregulation of activation markers after stimulation with viral peptidepools, we have developed a rapid and clinically applicable protocol for the simultaneous selection of virus-specific T cells (after stimulation with peptide pools for the immunodominant antigens of cytomegalovirus, Epstein-Barr virus, and adenovirus) and regulatory T cells using CD25 immunomagnetic selection. Using tetramer staining, we detected enrichment of CD8 T cells recognizing peptide epitopes from cytomegalovirus and Epstein-Barr virus antigens after CD25 selection in 6 of 7 donors. Enzyme-linked immunospot assays demonstrated the simultaneous presence of bivirus-specific or trivirus-specific cells in all evaluated donors, with a median 29-fold (6 to 168), 40-fold (1 to 247), and 16-fold (1 to 219) enrichment of cells secreting interferon-γ in response to cytomegalovirus pp65, adenovirus hexon, and Epstein-Barr virus lymphoblastoid cells compared with unmanipulated peripheral blood mononuclear cells from the same donors. Furthermore, the CD25-enriched cells lost alloreactivity in H-thymidine proliferation assays and showed highly effective (median, 98%) suppression of alloreactivity in all evaluated donors. In summary, we have developed a rapid, simple Good Manufacturing Practice compliant methodology for the simultaneous selection of T cells with multiple viral specificities and regulatory T cells. Adoptive transfer of T cells generated using this strategy may enable restoration of cellular immunity to viruses after allogeneic stem cell transplant with a low risk of graft-versus-host disease. Owing to the speed and simplicity of this methodology, this approach may significantly broaden the applicability of adoptive immunotherapy.
Bioceramics: from bone regeneration to cancer nanomedicine.
Source
Departamento de Química Inorgánica y Bioinorgánica, Facultad de Farmacia, Universidad Complutense de Madrid, Plaza Ramón y Cajal s/n, 28040 Madrid, Spain, and Networking Research Center on Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN), Spain. vallet@farm.ucm.es.
Abstract
Research on biomaterials has been growing in the last few years due to the clinical needs in organs and tissues replacement and regeneration. In addition, cancer nanomedicine has recently appeared as an effective means to combine nanotechnology developments towards a clinical application. Ceramic materials are suitable candidates to be used in the manufacturing of bone-like scaffolds. Bioceramic materials may also be designed to deliver biologically active substances aimed at repairing, maintaining, restoring or improving the function of organs and tissues in the organism. Several materials such as calcium phosphates, glasses and glass ceramics able to load and subsequently release in a controlled fashion drugs, hormones, growth factors, peptides or nucleic acids have been developed. In particular, to prevent post surgical infections bioceramics may be surface modified and loaded with certain antibiotics, thus preventing the formation of bacterial biofilms. Remarkably, mesoporous bioactive glasses have shown excellent characteristics as drug carrying bone regeneration materials. These bioceramics are not only osteoconductive and osteoproductive, but also osteoinductive, and have therefore been proposed as ideal components for the fabrication of scaffolds for bone tissue engineering. A recent promising development of bioceramic materials is related to the design of magnetic mediators against tumors. Magnetic composites are suitable thermoseeds for cancer treatment by hyperthermia. Moreover, magnetic nanomaterials offer a wide range of possibilities for diagnosis and therapy. These nanoparticles may be conjugated with therapeutic agents and heat the surrounding tissue under the action of alternating magnetic fields, enabling hyperthermia of cancer as an effective adjunct to chemotherapy regimens.
Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
Plasma-derived clotting factor VIII: heterogeneity evaluation in the quest for potential inhibitory-antibody stimulating factors.
Source
Department of Environmental Sciences, Tuscia University, Viterbo, Italy.
Abstract
In this study, we investigated the heterogeneity and the purity grade of three commercially available plasma-derived clotting factor VIII (FVIII) concentrates, which highly differ with regard to purification strategies, relative concentrations of stabilizers (von Willebrand factor, with or without albumin) and virus inactivation strategies (solvent/detergent and/or heat/pasteurization treatments). Western blot analyses were used to evaluate product-specific variations from Emoclot(®) , Alphanate(®) and Haemate(®) both in the presence and absence of reducing agents (dithiotreithol). All the plasma-derived concentrates showed a strong heterogeneity, as they all included a significant amount of truncated forms of the full-length (FL) clotting FVIII protein. The intact protein accounted for the 38% of the total FVIII proteins in Haemate(®) and 29 and 23% in Alphanate(®) and Emoclot(®) , respectively. Lower intact FVIII amounts in Emoclot might be mainly due to the low von Willebrand factor dosage and the absence of albumin. Upon addition of thrombin, both the FL and truncated forms of the FVIII protein were almost completely digested. Indeed, after thrombin activation, we could still observe a mixture of B-domain truncated forms of the FL protein along with biologically active digested-A1 forms. Batch-to-batch variation was tested with no evident changes appearing among different batches. Despite the variables in manufacturing processes, inter-product comparisons yielded similar results for all the plasma-derived FVIII considered in this study. However, we could individuate in Emoclot a band that was not digested by thrombin, which we could characterize as the 200 kDa FVIII heavy chain. This investigation prompts new concerns about the strong heterogeneity observed upon thrombin digestion of plasma-derived FVIII, which might contribute to the development of inhibitory antibodies at an early stage of therapy, and to which extent these untoward phenomena could be avoided through direct intervention on routine manufacturing processes.
Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
Structure of peptides on metal oxide surfaces probed by NMR.
Source
Materials and Manufacturing Directorate, Nanostructured and Biological Materials Branch, Air Force Research Laboratories, Wright-Patterson AFB, Ohio 45433, United States. peter.mirau@wpafb.af.mil
Abstract
Peptides that bind inorganic surfaces and template the formation of nanometer-sized inorganic particles are of great interest for the self- or directed assembly of nanomaterials for sensors and diagnostic applications. These surface-recognizing peptides can be identified from combinatorial phage-display peptide libraries, but little experimental information is available for understanding the relationship between the peptide sequence, structure at the nanoparticle surface, and function. We have developed NMR methods to determine the structures of peptides bound to inorganic nanoparticles and report on the structure of three peptides bound to silica and titania surfaces. Samples were prepared under conditions leading to rapid peptide exchange at the surface such that solution-based nuclear Overhauser experiments can be used to determine the three-dimensional structure of the bound peptide. The binding motif is defined by a compact "C"-shaped structure for the first six amino acids in the 12-mer. The orientation of the peptide on the nanoparticle surface was determined by magnetization transfer from the nanoparticle surface to the nearby peptideprotons. These methods can be applied to a wide variety of abiotic interfaces to provide an insight into the relationship between the primary sequence of peptides and their functionality at the interface.
Quantitation of amyloid beta peptides Aβ(1-38), Aβ(1-40), and Aβ(1-42) in human cerebrospinal fluid by ultra-performance liquid chromatography-tandem mass spectrometry.
Source
Pfizer Global Research and Development, Groton Laboratories, Groton, CT 06340, USA.
Abstract
Critical events in Alzheimer's disease (AD) involve an imbalance between the production and clearance of amyloid beta (Aβ) peptides from the brain. Current methods for Aβ quantitation rely heavily on immuno-based techniques. However, these assays require highly specific antibodies and reagents that are time-consuming and expensive to develop. Immuno-based assays are also characterized by poor dynamic ranges, cross-reactivity, matrix interferences, and dilution linearity problems. In particular, noncommercial immunoassays are especially subject to high intra- and interassay variability because they are not subject to more stringent manufacturing controls. Combinations of these factors make immunoassays more labor-intensive and often challenging to validate in support of clinical studies. Here we describe a mixed-mode solid-phase extraction method and an ultra-performance liquid chromatography tandem mass spectrometry (SPE UPLC-MS/MS) assay for the simultaneous quantitation of Aβ(1-38), Aβ(1-40), and Aβ(1-42) from human cerebrospinal fluid (CSF). Negative ion versus positive ion species were compared using their corresponding multiple reaction monitoring (MRM) transitions, and negative ions were approximately 1.6-fold greater in intensity but lacked selectivity in matrix. The positive ion MRM assay was more than sufficient to quantify endogenous Aβ peptides. Aβ standards were prepared in artificial CSF containing 5% rat plasma, and quality control samples were prepared in three pooled CSF sources. Extraction efficiency was greater than 80% for all three peptides, and the coefficient of variation during analysis was less than 15% for all species. Mean basal levels of Aβ species from three CSF pools were 1.64, 2.17, and 1.26 ng/ml for Aβ(1-38); 3.24, 3.63, and 2.55 ng/ml for Aβ(1-40); and 0.50, 0.63, and 0.46 ng/ml for Aβ(1-42).
Copyright © 2011 Elsevier Inc. All rights reserved.
Low Molecular Weight Opioid Peptide Esters Could be Developed as a New Class of Analgesics.
Source
Durham Veterans Affairs Medical Center and Duke University School of Medicine, Durham, NC, USA.
Abstract
Low molecular weight opioid peptide esters (OPE) could become a class of analgesics with different side effect profiles than current opiates. OPE may have sufficient plasma stability to cross the blood brain barrier (BBB), undergo ester hydrolysis and produce analgesia. OPE of dipeptides, tyr-pro and tyr-gly conjugated to ethanol have a structure similar to the anesthestic agent, etomidate. Based upon the analgesic activity of dipeptide opioids, Lipinski's criteria, and permeability of select GABA esters to cross the BBB, opioid peptides (OP) conjugated to ethanol, cholesterol or 3-glucose are lead recommendations. Preliminary animal data suggests that tyr-pro-ethyl ester crosses the BBB and unexpectedly produces hyperalgesia. Currently, there are no approved OP analgesics available for clinical use. Clinical trials of good manufacturing practice OP administered to patients suffering from chronic pain with indwelling intrathecal pumps could resolve the issue that OP may be superior to opiates and may redirect research.
Preferential binding of peptides to graphene edges and planes.
Source
Materials and Manufacturing Directorate, Air Force Research Laboratory, Wright-Patterson Air Force Base, Ohio 45433, USA.
Abstract
Peptides identified from combinatorial peptide libraries have been shown to bind to a variety of abiotic surfaces. Biotic-abiotic interactions can be exploited to create hybrid materials with interesting electronic, optical, or catalytic properties. Here we show that peptides identified from a combinatorial phage display peptide library assemble preferentially to the edge or planar surface of graphene and can affect the electronic properties of graphene. Molecular dynamics simulations and experiments provide insight into the mechanism of peptide binding to the graphene edge.
Characterisation of Lactobacillus helveticus strains producing antihypertensivepeptides by RAPD and inverse-PCR of IS elements.
Source
Sezione di Microbiologia Alimentare, Ambientale ed Industriale, Dipartimento di Scienze Alimentari, Agro-Ingegneristiche, Fisiche, Economico-Agrarie e del Territorio (SAIFET), Università Politecnica delle Marche, via Brecce Bianche, Ancona, Italy. l.aquilanti@univpm.it
Abstract
Lactobacillus helveticus is used for the manufacture of cheeses and milk-based products. Although it is not considered a probiotic microorganism, some strains demonstrated beneficial effects through the production of antihypertensivepeptides from the hydrolysis of casein during milk fermentation. Strain-specificity of bioactive peptide production by L. helveticus makes the availability of reliable typing methods essential for both legal and good manufacturing processes. Accordingly, RAPD and inverse-PCR of five insertion sequence elements were comparatively evaluated for the molecular characterisation of four L. helveticus dairy cultures producing antihypertensive peptides and fourteen reference strains. Calculation of discriminatory indices and cluster analysis of the DNA fingerprints confirmed the suitability of both approaches for acceptable strain differentiation. Although RAPD was more discriminating, for a few test strains a neat discrimination was only achieved through multiplex inverse-PCR, thus suggesting the suitability of a combined analytical approach for a finer strain discrimination.
Digestive enzymes of the crustaceans Munida and their application in cheesemanufacturing: a review.
Source
Department of Biology, Defence and Agro-Forestal Biotechnology, Center of Bioproteomics, University of Basilicata, 85100 Potenza, Italy. rocco.rossano@unibas.it
Abstract
Crustaceans Munida (fam. Galatheideae, ord. Decapodi) were fished in the Southern Adriatic Sea and their proteolytic activities were characterized and tested for potential application in cheese manufacturing. Enzymes extracted from whole crustaceans, mainly serine proteases, showed high caseinolytic and moderate clotting activities. Analysis by 2D zymography of the digestive enzymes extracted from Munida hepatopancreas, showed the presence of several isotrypsin- and isochymotrypsin-like enzymes in the range of 20-34 kDa and 4.1-5.8 pI. Moreover, specific enzymatic assays showed the presence of aminopeptidases and carboxypeptidases A and B. Overall, optimum activity was achieved at pH 7.5 and 40-45 °C. Caseinolytic activity, determined both spectrophotometrically and by SDS gel electrophoresis, indicated higher activity on β-casein than on α-casein. Miniature cheddar-type cheeses and Pecorino-type cheeses were manufactured by adding starter, rennet and Munida extracts to milk. Reverse-phase HPLC and MALDI-ToF mass spectrometry showed a more complex pattern of proteolytic products in cheeses made using Munida instead of chymosin. Munida extracts were found to degrade the chymosin-derived β-casein fragment f193-209, one of the peptides associated with bitterness in cheese. In conclusion, Munida digestive enzymes represent a promising tool for development of new cheese products and shorten cheese ripening when used either alone or in addition to calf rennet.
Modification of a novel angiogenic peptide, AG30, for the development of novel therapeutic agents.
Source
Division of Gene Therapy Science, Osaka University Graduate School of Medicine Division of Vascular Medicine and Epigenetics, Osaka University United Graduate School of Child Development, 2-1 Yamada-oka, Suita, Osaka 565-0871 Anges-MG, Inc. 7-7-15 Saito Bio-Incubator, Ibaraki, Osaka 567-0085 Skin Regeneration, PIAS Collaborative Research, The Center for Advanced Science and Innovation, Osaka University Peptide Institute, Inc. 7-7-15 Saito Asagi, Ibaraki, Osaka 567-0085 Research Department, NISSEI BILIS Co. Ltd., Koka, Shiga, 528-0052 Department of Clinical Gene Therapy, Osaka University Graduate School of Medicine Division of Infection Control and Prevention, Graduate School of Medicine, 2-2 Yamada-oka, Suita, Osaka 565-0871.
Abstract
We previously identified a novel angiogenic peptide, AG30, with antibacterial effects that could serve as a foundation molecule for the design of wound healing drugs. Toward clinical application, in this study we have developed a modified version of the AG30 peptide characterized by improved antibacterial and angiogenic action, thus establishing a lead compound for a feasibility study. Since AG30 has an α-helix structure with a number of hydrophobic and cationic amino acids, we designed a modified AG30 peptide by replacing several of the amino acids. The replacement of cationic amino acids (yielding a new molecule, AG30/5C), but not hydrophobic amino acids, increased both the angiogenic and the antimicrobial properties of the peptide. AG30/5C was also effective against methicillin-resistant Staphylococcus aureus (MRSA) and antibiotic-resistant Pseudomonas aeruginosa. In a diabetic mouse wound-healing model, the topical application of AG30/5C accelerated wound healing with increased angiogenesis and attenuated MRSA infection. To facilitate the eventual clinical investigation/application of these compounds, we developed a large-scale procedure for the synthesis of AG30/5C that employed the conventional solution method and met Good Manufacturing Practice guidelines. In the evaluation of stability of this peptide in saline solution, RP-HPLC analysis revealed that AG30/5C was fairly stable under 5°C for 12 months. Therefore, we propose the use of AG30/5C as a wound healing drug with antibacterial and angiogenic actions.
© 2011 The Authors Journal of Cellular and Molecular Medicine © 2011 Foundation for Cellular and Molecular Medicine/Blackwell Publishing Ltd.
Examination of a synthetic benzophenone membrane-targeted antibiotic.
Source
Department of Pharmaceutical Sciences, Eugene Applebaum College of Pharmacy and Health Sciences, Wayne State University, Detroit, MI 48201, USA.
Abstract
The enormous success of antibiotics is seriously threatened by the development of resistance to most of the drugs available on the market. Thus, novel antibiotics are needed that are less prone to bacterial resistance and are directed toward novel biological targets. Antimicrobial peptides (AMPs) have attracted considerable attention due to their unique mode of action and broad spectrum activity. However, these agents suffer from liability to proteases and the high cost ofmanufacturing has impeded their development. Previously, we have reported on a novel class of benzophenone-based antibiotics and early studies suggested that these agents might target the bacterial membrane. In this study, we present our work on the mechanism of action of these novel membrane targeted antibiotics. These compounds have good affinities to polyanionic components of the cell wall such as lipoteichoic acid (LTA) and lipopolysaccharide (LPS). We found that these agents release potassium ions from treated bacteria; thus, resulting in disruption of the bacterial membrane potential. Benzophenone-based membrane targeted antibiotics (BPMTAs) cause membrane disruption in synthetic lipid vesicles that mimic Gram-positive or Gram-negative bacteria. The compounds display no hemolytic activity up to a concentration that is 100 times the MIC values and they are capable of curing mice of a lethal MRSA infection. Repeated attempts to develop a mutant resistant to these agents has failed. Taken together, BPMTAs represent a promising new class of membrane-targeted antibacterial agents.
Epidermal growth factor: layered silicate nanocomposites for tissue regeneration.
Source
Materials and Manufacturing Directorate, Air Force Research Laboratory, Wright-Patterson AFB, OH 45433, United States.
Abstract
Wound healing is a complex, multistep process that can be summarized into three stages, namely, hemostasis and inflammation, proliferation, and finally, tissue remodeling. Battlefield wound healing demands rapid hemostasis using clotting or cauterizing agents to immediately limit blood loss, but this occurs at the expense of proper tissue repair beyond hemostasis. Layered silicate clays such as kaolin and montmorillonite (MMT) have been previously shown to induce blood clotting due to their ability to form charged interactions with clotting factors. The charge characteristics of sodium MMT (Na-MMT) also enable functionalization with active biomolecules. Herein we functionalized Na-MMT with epidermal growth factor (EGF) via ion exchange reaction to create a nanocomposite (MMT-EGF) with approximately 0.004 EGF molecules per Na(+) exchange site and conduct biochemical analyses of keratinocytes after treatment with MMT-EGF. Our results demonstrate that EGF immobilized on MMT retains the ability to activate the epidermal growth factor receptor (EGRF), causing phosphorylation of the AKT and MEK1 pathways, as well as upregulation of its downstream target gene expression involved in cell growth and migration. This study also shows that like EGF, MMT-EGF treatment can stimulate cell migration in vitro, which is dependent on ERK1/2 phosphorylation.
Immunological aspects of scorpion toxins: current status and perspectives.
Source
Laboratoire des Venins et Toxines, Institut Pasteur de Tunis, Tunisia. balkiss.bouhaouala@pasteur.rns.tn
Abstract
Significant progress has been made in immunological studies of scorpion toxins and several formats of antibodies directed against scorpion toxins have been reported. Some of these are commonly used in a specific treatment against envenoming; others are primarily used for immuno-biochemical characterizations. The preparation protocol of the antibody or its fragments can be substantially different from one laboratory to another, which complicates a direct comparison of the potency of the antivenom. The use of immune sera, the total immunoglobulin fraction or Fab and Fab'2 fragments as the therapeutic agent is widespread. A number of monoclonal antibodies have also been reported and used for engineering of Fv, ScFv or Fab fragments. Recently, a novel antibody format - known as nanobodies - derived from HCAbs of camelids and selected after phage display shows great potential to provide a more efficient therapy against scorpion envenoming. Subsequent bispecific derivatives have been designed and their pharmacokinetics have been studied. Distinct advantages and disadvantages have been attributed to these equine, murine or camelid antibodies and their derived fragments. Some fragments are easily amenable into next generation therapeutics after proper manufacturing and provide an ensured availability of the product to the medical community. Through examples, we will show how the comparison of the serotherapeutic effectiveness is compromised due to the absence of standardization, on the preparation of immunogens, production processes and / or nature of the products. We will report on recent advances in the field.
An improved Fc function assay utilizing CMV antigen-coated red blood cells generated with synthetic function-spacer-lipid constructs.
Source
Research and Development Department, CSL Biotherapies, Broadmeadows, Vic., Australia Biotechnology Research Institute, AUT University, Auckland, New Zealand.
Abstract
Background and Objectives The Fc function assessment of immunoglobulin products is performed to confirm that the biological activity of immunoglobulin products is not affected by manufacturing and storage conditions. The European Pharmacopoeia (EP) test is cumbersome and time-consuming especially with respect to the derivatization of red blood cells (RBC) with rubella antigen via tanninization. We investigated the KODE biosurface engineering technology for inserting specific antigens into red blood cell membranes to create kodecytes and evaluated their suitability for use in the Fc function assay. Materials and Methods Human RBC were derivatized with function-spacer-lipid (FSL) constructs comprising of a peptide epitope of a cytomegalovirus (CMV) surface protein, conjugated to a spacer and lipid tail. These kodecytes were used in an Fc function assay based on the monitoring of complement-mediated haemolysis of immunoglobulin-coated red cells. Optimization of FSL construct, immunoglobulin and complement concentration was undertaken. Fc values obtained for various immunoglobulin batches were compared with those from the EP-based method. Carbohydrate-bearing FSLs Galili (Galα1-3Galβ1-4GlcNAcβ), Galα1-4GlcNAcβ and GalNAcα1-3Galβ were also evaluated. Results Fc function indices determined with CMV peptide construct-coated red cells were comparable to those obtained with the EP-based method with respect to specificity and precision. Carbohydrate-bearing FSLs revealed Fc indices lower than expected. Conclusion The use of CMV kodecytes was shown to be a convenient means of generating red cells for the determination of Fc function of immunoglobulin products and offers the possibility of significantly reducing the time required to perform this assay.
© 2011 The Author(s). Vox Sanguinis © 2011 International Society of Blood Transfusion.
Associations of polychlorinated biphenyl exposure and endogenous hormones with diabetes in post-menopausal women previously employed at a capacitormanufacturing plant.
Source
University of Illinois School of Public Health, 1603 West Taylor St., Chicago, IL 60612, USA. vwpersky@uic.edu
Abstract
There is an increasing body of literature showing associations of organochlorine exposure with risk of diabetes and insulin resistance. Some studies suggest that associations differ by gender and that diabetes risk, in turn, may be affected by endogenous steroid hormones. This report examines the relationships of serum PCBs and endogenous hormones with history of diabetes in a cohort of persons previously employed at a capacitor manufacturing plant. A total of 118 women were post-menopausal with complete data, of whom 93 were not using steroid hormones in 1996, at the time of examination, which included a survey of exposure and medical history, height, weight and collection of blood and urine for measurements of lipids, liver function, hematologic markers and endogenous hormones. This analysis examines relationships of serum polychlorinated biphenyls (PCBs), work exposure and endogenous hormones with self-reported history of diabetes after control for potential confounders. All PCB exposure groups were significantly related to history of diabetes, but not to insulin resistance as measured by the homeostatic model assessment of insulin resistance (HOMA-IR) in non-diabetics. Diabetes was also independently and inversely associated with follicle stimulating hormone (FSH), dehydroepiandrosterone sulfate (DHEAS) and triiodothyronine (T3) uptake. HOMA-IR was positively associated with body mass index (BMI) and C-reactive protein (CRP) and inversely associated with sex hormone binding globulin (SHBG) and T3 uptake after control for PCB exposure. Possible biologic mechanisms are discussed. This study confirms previous reports relating PCB exposure to diabetes and suggests possible hormonal pathways deserving further exploration.
Copyright © 2011 Elsevier Inc. All rights reserved.
Pulsatile culture of a poly(DL-lactic-co-glycolic acid) sandwiched cell/hydrogel construct fabricated using a step-by-step mold/extraction method.
Source
Key Laboratory for Advanced Materials Processing Technology, Ministry of Education & Center of Organ Manufacturing, Department of Mechanical Engineering, Tsinghua University Institute of Life Science & Medicine, Tsinghua University, Beijing, China. wangxiaohong@tsinghua.edu.cn
Abstract
To overcome the weak mechanical properties of cell/hydrogel composites, a poly(DL-lactic-co-glycolic acid) sandwiched adipose-derived stem cell (ADSC)/fibrin construct was fabricated using a step-by-step mold/extraction method to generate the middle smooth muscle layer of natural blood vessels. A pulse bioreactor with an adjustable 0-0.2 MPa pressure, 0-7% pulse amplitude, and 0-80 times/min pulse frequency was developed to mimic the liquid movement in the natural blood vessels. This new type of pulse bioreactor is sterilizable and dismantles easily. A comparative study was conducted with static and dynamic in vitro cultures. Exogenous growth factors, such as hepatocyte growth factor, platelet-derived growth factor BB, transforming growth factor β1, and basic fibroblast growth factor were used as additives in the culture medium for inducing the ADSCs into smooth muscle cells. The dynamic training, integrated with the growth factor, induced the transformation of ADSCs into smooth muscle-like cells with regular arrangement. This strategy shows promise of being widely used in tissue engineering and complex organ manufacturing.
© 2011, Copyright the Authors. Artificial Organs © 2011, International Center for Artificial Organs and Transplantation and Wiley Periodicals, Inc.
Comparative study of clinical grade human tolerogenic dendritic cells.
Source
Laboratory of Immunobiology for Research and Diagnosis, Blood and Tissue Bank, Dept. of Cell Biology, Physiology and Immunology, Universitat Autònoma de Barcelona, Institut Investigació Germans Trias i Pujol, Spain.
Abstract
BACKGROUND:
The use of tolerogenic DCs is a promising therapeutic strategy for transplantation and autoimmune disorders. Immunomodulatory DCs are primarily generated from monocytes (MDDCs) for in vitro experiments following protocols that fail to fulfil the strict regulatory rules of clinically applicable products. Here, we compared the efficacy of three different tolerance-inducing agents, dexamethasone, rapamycin and vitamin D3, on DC biology using GMP (GoodManufacturing Practice) or clinical grade reagents with the aim of defining their use for human cell therapy.
METHODS:
Tolerogenic MDDCs were generated by adding tolerogenic agents prior to the induction of maturation using TNF-α, IL-β and PGE2. We evaluated the effects of each agent on viability, efficiency of differentiation, phenotype, cytokine secretion and stability, the stimulatory capacity of tol-DCs and the T-cell profiles induced.
RESULTS:
Differences relevant to therapeutic applicability were observed with the cellular products that were obtained. VitD3-induced tol-DCs exhibited a slightly reduced viability and yield compared to Dexa-and Rapa-tol-DCs. Phenotypically, while Dexa-and VitD3-tol-DCs were similar to immature DCs, Rapa-tol-DCs were not distinguishable from mature DCs. In addition, only Dexa-and moderately VitD3-tol-DCs exhibited IL-10 production. Interestingly, in all cases, the cytokine secretion profiles of tol-DCs were not modified by a subsequent TLR stimulation with LPS, indicating that all products had stable phenotypes. Functionally, clearly reduced alloantigen T cell proliferation was induced by tol-DCs obtained using any of these agent. Also, total interferon-gamma (IFN-γ) secretion by T cells stimulated with allogeneic tol-DCs was reduced in all three cases, but only T cells co-cultured with Rapa-tol-DCs showed impaired intracellular IFN-γ production. In addition, Rapa-DCs promoted CD4+ CD127 low/negative CD25high and Foxp3+ T cells.
CONCLUSIONS:
Our results demonstrate contrasting influences of different clinical-grade pharmacological agents on human tol-DC generation. This should be taken into account for decisions on the use of a specific agent for the appropriate cellular therapy in the context of a particular disease.
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