Dissertations / Theses on the topic 'Antibody'
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Krawczyk, Konrad. "Computational antibody design." Thesis, University of Oxford, 2013. https://ora.ox.ac.uk/objects/uuid:530a7e81-8525-4cb6-a54f-76827b565ff9.
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Antibodies are a class of proteins vital in mediating immune responses in vertebrates. Their binding site is highly malleable, allowing them to bind virtually any antigen. The versatility of antibody binding sites has received much attention from the pharmaceutical industry, marking them out as the most important category of biopharmaceuticals. The development of antibodies which bind to a specific antigen has thus far been achieved by costly and time-consuming experimental screening campaigns. However, in recent years computational approaches to antibody design have started to emerge, which offer an alternative. Computational antibody design techniques focus on determination of the binding site on the antibody, antibody-modelling, antibody-antigen docking and prediction of the binding site on the antigen. Here, we explore aspects of computational antibody design with the aim of gaining a better understanding of antibody-antigen interactions and improving existing artificial antibody design tools. We start by demonstrating our structural antibody database which has become a primary resource for antibody structural information. This is followed by a detailed analysis of the antibodyantigen interactions. The information gathered from this analysis allowed us to create an antibody contact site prediction tool, Antibody i-Patch. This tool was then employed to develop a local antibody-antigen docking pipeline, which used knowledge of the binding site of the antigen. We then tackled the global antibody-antigen docking problem by developing EpiPred, antigen binding site predictor which was employed in our global antibody-antigen docking pipeline.
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Ohlin, Mats. "Human monoclonal antibody technology a tool to investigate human antibody repertoires /." Lund : Dept. of Immunotechnology, Lund University, 1992. http://catalog.hathitrust.org/api/volumes/oclc/39693827.html.
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Rostedt, Punga Anna. "MuSK Antibody(+) Versus AChR Antibody(+) Myasthenia Gravis : Clinical, Neurophysiological and Morphological Aspects." Doctoral thesis, Uppsala : Acta Universitatis Upsaliensis : Univ.-bibl [distributör], 2007. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-7408.
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Harasym, Troy O. "Antibody-targeted liposomal systems." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1997. http://www.collectionscanada.ca/obj/s4/f2/dsk3/ftp04/nq25066.pdf.
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Low, Nigel Murray. "Mimicking antibody affinity maturation." Thesis, University of Cambridge, 1996. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.364567.
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Marks, Cara. "Antibody structure and function." Thesis, University of Cambridge, 1992. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.260558.
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Nowak, Jaroslaw. "Understanding antibody binding sites." Thesis, University of Oxford, 2017. http://ora.ox.ac.uk/objects/uuid:5558a55e-bb47-4b29-a681-1e58771abd1d.
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Antibodies are soluble proteins produced by the adaptive immune system to bind and counteract invading pathogens. The binding properties of a typical human antibody are determined by the structure of its variable domain, composed of two chains â heavy and light and by the conformation of six loops located on the surface of the variable domain, known as Complementarity Determining Regions (CDRs). In the first chapter, we describe our analysis of the conformational space occupied by five out of six antibody CDRs (L1, L2, L3, H1 and H2) and the development of a novel, length-independent method for grouping these CDRs into structural clusters (canonical forms). We show that using our method we can increase coverage and precision of assigning CDR sequences into clusters. In the next chapter, we describe a method for ranking structural decoys of the CDR-H3 loop. We show that by computationally perturbing CDR-H3 decoys we can improve the performance of existing ranking methods. In the same chapter, we discuss the development of a method for high-throughput assignment of heavy-light chain orientation. The power of the method was demonstrated by assigning orientation to billions of potential Fv sequences. The third Chapter describes the analysis of a large dataset of CDR sequences with the aim of identifying sequence patterns responsible for the loops' structure. Using a neural network methodology, we found several groups of CDR sequences which might be indicative of previously-unseen conformations. In the final results Chapter, we describe how we used the structural knowledge developed throughout the rest of the thesis to create a novel pipeline for computational antibody design. We show that the binders developed using our methodology had similar features to available antibody therapeutics and low predicted propensity to cause an immunogenic response. These results demonstrate the potential for using computational methods for designing high affinity therapeutics with human properties.
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Kelly, Ryan L. (Ryan Lewis). "Determinants of antibody specificity." Thesis, Massachusetts Institute of Technology, 2017. http://hdl.handle.net/1721.1/112506.
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Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biological Engineering, 2017.Cataloged from PDF version of thesis.
Includes bibliographical references (pages 134-146).
High throughput screening methods such as yeast surface display (YSD) are frequently able to isolate high affinity antibodies against clinical targets; however, the success of these candidates depends on selecting for both on-target binding and desirable biophysical characteristics. Development liabilities, including antibody aggregation and nonspecificity, can lead to problems during production and poor pharmacokinetics (PK). The exact structural and sequence determinants causing this poor developability are unknown, which leads to inexact methods to correct otherwise promising clinical candidates. In this thesis we outline the development of high throughput methods to interrogate developability of candidate antibodies on the surface of yeast and apply these methods to both determine the causes of nonspecificity and create new libraries with improved biophysical properties. We first analyzed methods for early stage assessment of monoclonal antibodies, finding a polyspecificity reagent (PSR) binding assay on the surface of yeast which can accurately predict antibody clearance rates in mice. While robust, this assay relies on production of a poorly defined mixture of protein components, and thus, we next looked at potential alternatives to a multicomponent PSR reagent. We found that chaperone proteins may work as well-defined, easily producible reagents with similar broad predictive power to predict downstream antibody behavior. Next, we applied these assays to assess core determinants of nonspecificity. We first analyzed a case study of two antibodies with identical target antigens but vastly different performance on preclinical assessments of biophysical characteristics. Through this matched case, we found differences in clearance rates can be driven wholly by variable-region mediated effects independent of neonatal Fc receptor (FcRn) binding. Focused on the antibody variable region, we next utilized our nonspecificity assay as a sorting tool to look at a naive repertoire library. We found significant nonspecificity in the VH6 class of antibodies, driven by a poorly behaved complementarity determining region (CDR) H2 sequence. Subsequently, we applied a similar sorting technique to two synthetic library designs to identify a set of motifs that can drive nonspecificity. These included motifs containing tryptophan, valine, glycine and arginine located in CDR H3. We then applied these discoveries to the design of a new, semi-synthetic single chain variable fragment (scFv) library and demonstrated its ability to isolate high affinity, highly specific candidate clones against a panel of antigens. Finally, we explored the use of an alternate yeast display system capable of easily switching between scFv-Fc display and secretion, which may aid in the rapid development and testing of candidate antibodies. Taken in whole, the work in this thesis aids the clinical development of antibodies. We have presented both methods to assess nonspecificity at an early stage in the development process as well as a set of motifs to be eliminated in future library designs. With these combined findings, we hope to increase the utilization of in vitro screening methods such as yeast display for the isolation of clinical candidate antibodies with favorable biophysical characteristics.
by Ryan L. Kelly.
Ph. D.
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Bidgood, Susanna Ruth. "Antibody mediated intracellular immunity." Thesis, University of Cambridge, 2013. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.648288.
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Ozeren, Muserref. "Catalysis by polyclonal antibody." Thesis, University of Bath, 1997. https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.246006.
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Cecilia, Bill. "Improving anti-drug antibody assay performance in Gyrolab for therapeutic recombinant antibody Infliximab." Thesis, Linköpings universitet, Institutionen för klinisk och experimentell medicin, 2014. http://urn.kb.se/resolve?urn=urn:nbn:se:liu:diva-111527.
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Monoclonal antibodies can be used as targeting therapies for several diseases. One major concern when using these therapies is anti-drug antibodies which may hamper the drugs efficiency. Gyrolab is an automated platform which can be used to develop bridging immunoassays where the anti-drug antibodies affinity towards the monoclonal antibody is utilized. Anti-drug antibody immunoassay development on Gyrolab is limited mainly by three factors which may inappropriately affect signal intensity levels. In this project different variants of bridging immunoassays based on drug Fab fragments have been developed for monoclonal antibody Infliximab, with the purpose to illustrate the effects of these three factors. Findings indicate that an assay based completely on drug Fab fragments is more sensitive compared to an assay based on intact drug since less affected by unspecific interactions between drug reagents and complex formations. Surprisingly findings also indicate that an assay based completely on drug Fab fragments is affected by human anti-hinge antibodies which decrease assay sensitivity. The most optimal assay variant is based on the combination between intact capture drug and Fab fragment as detection. This variant is insensitive to false positive reactions caused by Rheumatoid factor and human anti-hinge antibodies, less prone to form unspecific interactions between drug reagents and complex formations in the presence of anti-drug antibodies. The optimal assay variant also demonstrates best drug tolerance in combination with acid dissociation.
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Björling, Erik. "Databases for antibody-based proteomics." Doctoral thesis, KTH, Skolan för bioteknologi (BIO), 2008. http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-9658.
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Humans are believed to have ~20,500 protein-coding genes andmuch effort has over the last years been put into the characterizationand localization of the encoded proteins in order to understand theirfunctions. One such effort is the Human Proteome Resource (HPR)project, started in Sweden 2003 with the aim to generate specificantibodies to each human protein and to use those antibodies toanalyze the human proteome by screening human tissues and cells.The work reported in this thesis deals with structuring of data fromantibody-based proteomics assays, with focus on the importance ofaggregating and presenting data in a way that is easy to apprehend.The goals were to model and build databases for collecting, searchingand analyzing data coming out of the large-scale HPR project and tomake all collected data publicly available. A public website, theHuman Protein Atlas, was developed giving all end-users in thescientific community access to the HPR database with proteinexpression data. In 2008, the Human Protein Atlas was released in its4th version containing more than 6000 antibodies, covering more than25% of the human proteins. All the collected protein expression datais searchable on the public website. End-users can query for proteinsthat show high expression in one tissue and no expression in anotherand possibly find tissue specific biomarkers. Queries can also beconstructed to find proteins with different expression levels in normalvs. cancer tissues. The proteins found by such a query could identifypotential biomarkers for cancer that could be used as diagnosticmarkers and maybe even be involved in cancer therapy in the future.Validation of antibodies is important in order to get reliable resultsfrom different assays. It has been noted that some antibodies arereliable in certain assays but not in others and therefore anotherpublicly available database, the Antibodypedia, has been createdwhere any antibody producer can submit their binders together withthe validation data in order for end users to purchase the bestantibody for their protein target and their intended assay.QC 20100708
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Saini, Surinder Singh. "Molecular immunogenetics of bovine antibody." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1999. http://www.collectionscanada.ca/obj/s4/f2/dsk1/tape8/PQDD_0005/NQ40388.pdf.
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Qundos, Ulrika. "Antibody based plasma protein profiling." Doctoral thesis, KTH, Proteomik och nanobioteknologi, 2013. http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-126270.
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This thesis is about protein profiling in serum and plasma using antibody suspension bead arrays for the analysis of biobanked samples and in the context of prostate cancer biomarker discovery. The influence of sample preparation methods on antibody based protein profiles were investigated (Papers I-III) and a prostate cancer candidate biomarker identified and verified (Papers III-V). Furthermore, a perspective on the research area affinity proteomics and its’ employment in biomarker discovery, for improved understanding and potentially improved disease diagnosis, is provided. Paper I presents the results of a comparative plasma and serum protein profiling study, with a targeted biomarker discovery approach in the context of metabolic syndrome. The study yielded a higher number of significant findings and a low experimental variability in blood samples prepared as plasma. Paper II investigated the effects from post-centrifugation delays at different temperatures prior sample storage of serum and plasma samples. Minor effects were found on the detected levels of more than 300 predicted or known plasma proteins. In Paper III, the detectability of proteins in plasma was explored by exposing samples to different pre-analytical heat treatments, prior target capture. Heat induced epitope retrieval was observed for approximately half of the targeted proteins, and resulted in the discovery of different candidate markers for prostate cancer. Several antibodies towards the prostate cancer candidate biomarker CNDP1 were generated, epitope mapped and evaluated in a bead based sandwich immunoassay, as presented in Papers IV and V. Furthermore, the developed sandwich immunoassay targeting multiple distinct CNDP1 epitopes in more than 1000 samples, confirmed the association of CNDP1 levels to aggres- sive prostate cancer and more specifically to prostate cancer patients with regional lymph node metastasis (Paper V). As an outcome of the present investigations and in parallel to studies within the Biobank profiling research group, valuable lessons from study design and multiplex antibody analysis of plasma within biomarker discovery to experimental, technical and biological verifications have been collected.QC 20130821
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Irani, Sarosh R. "Antibody targets in autoimmune encephalitis." Thesis, University of Oxford, 2010. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.533850.
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Graff, Christilyn Paula. "Antibody engineering for tumor immunotherapy." Thesis, Massachusetts Institute of Technology, 2002. http://hdl.handle.net/1721.1/29279.
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Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemical Engineering, 2002.Vita.
Includes bibliographical references (leaves 130-140).
Antibodies have been used as cancer therapeutics for several decades. One area in which this therapy may be improved is the retention time of antibody in the tumor relative to normal tissue. In this Thesis, we have attempted to elucidate the mechanisms that are most influential to improving antibodies as cancer therapeutics. Carcinoembryonic antigen (CEA) has long been identified as a tumor-associated antigen. CEA is also quite stable, with a cell-surface shedding half-life of approximately 7 days. Directed evolution methodology has been utilized to design an antibody fragment with properties that would improve tumor retention. Specifically, antibody engineering methods were used to produce a humanized, extremely high affinity and stable single chain antibody fragment (scFv) against CEA. Several mutant scFv libraries were constructed and screened against soluble CEA with yeast surface display and fluorescent activated cell sorting (FACS). A series of antibodies were engineered that span three orders of magnitude in off-rate improvement. These antibody fragments show excellent stability at physiologically relevant temperatures. In addition, soluble protein expression levels were greatly improved. The final product has a dissociation half-life of approximately 7 days, currently the longest engineered half-life of an scFv against a tumor-associated antigen. Binding and diffusion in micrometastases was also modeled to gain an improved understanding of the quantitative interplay among the rate processes of diffusion, binding, degradation, and plasma clearance in tumor microspheroids.
(cont.) Modeling studies illuminated the importance of targeting stable tumor-associated antigens. The elimination rate of the antigen was of critical importance to the change in the therapeutic effect of antibodies with increasing affinity. The significance of this result in the context of previous experimental studies will be discussed. By affinity maturing an antibody with a dissociation half-life equal to the turnover half-life of the antigen, we have engineered an antibody with effectively irreversible binding to CEA. Differences in retention for the series of scFvs will thus be dominated by the off-rate of the antibody and not the half-life of CEA. With this in mind, the molecules designed in this study can be used to reconcile the issue of affinity's impact on efficacy in tumor therapy.
by Christilyn Paula Graff.
Ph.D.
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Yeung, Yik Andy. "Antibody engineering for cancer therapy." Thesis, Massachusetts Institute of Technology, 2005. http://hdl.handle.net/1721.1/32325.
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Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemical Engineering, 2005.Vita.
Includes bibliographical references (leaves 131-141).
Antibodies targeting various tumor-associated antigens have been developed successfully to treat cancer. In this Thesis, novel antibodies and antibody-conjugate against two tumor antigens, AF-20 antigen and human aspartyl (asparaginyl) [beta]- hydroxylase (HAAH), were developed. Previously, these two tumor antigens have been shown to be present on a variety of tumor cells, while they have minimal expression on normal tissues, rendering them excellent targets for antibody therapy. For the AF-20 work, the variable region (V) gene of a previously isolated mouse monoclonal antibody (mAb) AF-20 was cloned from hybridoma mRNA and used to construct an AF-20 single-chain Fv (scFv). The AF-20 scFv was shown to bind specifically to the same epitope as mAb AF-20 with a binding affinity of 4nM. The AF- 20 scFv was also internalized into tumor cells in a manner identical to that of the original mAb AF-20. The scFv was later employed for cellular internalization of virus-sized fluorescent quantum dots. In addition, to demonstrate the versatility of this antibody, an immunotoxin composed of AF-20 scFv fused to the highly cytotoxic recombinant toxin gelonin was constructed, and its in-vitro efficacy against three different tumor cell lines were evaluated. The IC50 of the AF-20 scFv-gelonin fusion was consistently one to two logs lower than the IC50 of free gelonin on FOCUS (liver), L3.6pl (pancreas) and PC3 (prostate) cells, further demonstrating the capability of the AF-20 scFv as a targeting module. Therefore, this AF-20 scFv is a potential internalization vector for toxins, enzymes, radionuclides and virus for targeted therapy of AF-20-antigen expressing tumor cells.
For the HAAH study, twelve novel human scFv against HAAH were isolated from a human non-immune scFv library displayed on the surface of yeast. Five of the twelve scFv were reformatted as human IgG 1. One of the reformatted IgG, 6-22, showed significant binding to recombinant HAAH protein in ELISA, tumor cell lines, and tumor tissues. 6-22 IgG was also shown to target the catalytic domain of HAAH, and its apparent dissociation constant was determined to be 1.OnM. 6-22 IgG alone does not exhibit significant cytotoxicity toward the tumor cells. However, 6-22 IgG internalizes into tumor cells and can therefore be employed to deliver cytotoxic moieties into tumor cells. A goat anti-human IgG-saporin conjugate was delivered into tumor cells by 6-22 IgG and hence elicited cytotoxicity toward the tumor cells in vitro. Meanwhile, the monovalent affinity of 6-22 scFv was too low for therapeutic or diagnostic application, so 6-22 scFv was affinity matured using directed evolution and yeast surface display. After two rounds of mutagenesis, a mutant, C4-18, with an affinity of 0.6nM was isolated. Overall, these human [gamma]-HAAH scFv and IgG can potentially be used in the diagnosis and therapeutic treatment of HAAH-expressing tumor cells.
by Yik Andy Yeung.
Ph.D.
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Chapman, Miles David. "Antibody specificity in neurological disease." Thesis, University College London (University of London), 2006. http://discovery.ucl.ac.uk/1445388/.
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The study of antigen-specific intrathecal oligoclonal bands is well established and a number of methods have been used to demonstrate that the relative affinity of the antibody produced in multiple sclerosis is low, and in encephalitis, high. A method colloquially known as Eastern blotting was developed whereby relative affinity of individual clones, rather than total antibody, could be studied and quantified by antigen immunoblotting and investigation of a digitised blot using image-manipulating software. This method was used to show that the pixel density of a band in CSF was significantly greater than the same band in serum in a patient with SSPE, and was thus of intrathecal origin. Eastern blotting was then used on a series of samples from a patient with herpes encephalitis to demonstrate that affinity maturation of the immune response had occurred intrathecally. The method was used qualitatively to investigate a proposal that Acinetobacter sp. infection could be the primary cause of multiple sclerosis: no evidence could be found to support the hypothesis. Another suggested cause of multiple sclerosis, Chlamydophila pneumoniae, was studied using a variety of methods including Western blotting. Again, there was no evidence to support the hypothesis. During the project, an unexpected effect of high-strength thiocyanate was revealed, and limited study of this suggested that thiocyanate had an effect on IgG, possibly related to the age of the sample.
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Hoehn, Kenneth. "Evolutionary models of antibody lineages." Thesis, University of Oxford, 2017. https://ora.ox.ac.uk/objects/uuid:ae1fcd96-d858-4f6d-8d99-46b678b2625d.
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Antibodies are proteins released into the blood and mucosa to identify and neutralize invading pathogens by binding to structures on their surface (antigens). Before being exported as antibodies, these vital components of the adaptive immune system are expressed, and refined, as membrane-bound B cell receptors (BCRs). BCRs are initially formed through somatic recombination of germline DNA, creating a large repertoire of unique sequences. After encountering antigen for the first time, BCRs undergo an evolutionary process of somatic mutation and clonal selection leading to improved antigen binding. Recently, next-generation sequencing has provided an unprecedented ability to characterize the genetic diversity of BCRs within individuals. Chapter 1 of this thesis overviews the work done elsewhere in the field until now. Chapter 2 uses summary statistics applied to high-throughput sequence data from a clinical trial to explore the genetic changes that occur in the repertoire during HIV infection. The results of these analyses motivated a more rigorous, model-based approach to understanding BCR diversity. Chapter 3 introduces a new phylogenetic substitution model that relaxes common model assumptions that are violated by somatic hypermutation. Chapter 4 expands this model to incorporate previously defined empirical models of somatic hypermutation, providing a more complete model of B cell maturation. Chapter 5 uses the models developed in Chapters 4 and 5 to explore dynamics of clonal selection during the maturation of three HIV broadly neutralizing antibody lineages. Finally, Chapter 6 shows how this framework may be scaled up to characterize data from entire BCR repertoires from a phylogenetic perspective.
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French, Alister Charles. "Covalent modification of antibody fragments." Thesis, University of Oxford, 2008. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.711604.
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Björling, Erik. "Databases for antibody-based proteomics /." Stockholm : Bioteknologi, Kungliga Tekniska högskolan, 2008. http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-9658.
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Voorhaar, Heather Marie. "Antibody architecture responding to bioterrorism /." Connect to this title online, 2009.
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PESSINO, GRETA. "Asparaginase-based antibody Drug Conjugates." Doctoral thesis, Università degli studi di Pavia, 2021. http://hdl.handle.net/11571/1435095.
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L-Asparaginase (ASNase, EC 3.5.1.1) is a key component of the established combined chemotherapy used for the treatment of pediatric acute lymphoblastic leukemia, having significantly increased the percentage of complete remissions in patients since its introduction in 1970s. The benefit of ASNase treatment is supported by extensive clinical data, while resistance to asparaginase is correlated with poor prognosis. ASNase is an amidohydrolase which shows a prevalent asparaginolytic and a secondary glutaminolytic activity; its therapeutic benefit comes from the depletion of asparagine from the blood stream, on which leukemic cells depends, given their absent or compromised capability to express asparagine synthetase (EC 6.3.5.4) under stress conditions. The ASNase molecules currently used in the clinics are derived from either E. coli (EcAII) or E. chrysanthemi, with the first line drug being PEG-Asparaginase (Oncaspar ®). However, most of the available ASNase products lack optimal pharmaceutical features, in particular because of ASNase high toxicity, due to its untargeted activity; high immunogenicity, due to its bacterial origin and large size; short blood serum half-life, and poor efficacy in specific sub-classes of patients (high risk). The aim of this work was to address these limitations, in order to improve EcAII efficacy, and in particular its high toxicity, on one side, by targeting the drug onto leukemic cells, and its high immunogenicity, on the other side, by miniaturizing the drug. It is expected that tackling these two points should also help to increase the drug efficacy in the treatment of high-risk patients. The adopted strategy consisted in the design of a radically new, anti-CD19 Asparaginase-based Antibody Drug Conjugate (ADC), which was conceived by our research group after the successful engineering of a single domain antibody (sdAb) with asparagynolitic activity, obtained through the rational transfer of E. coli type II asparaginase catalytic residues onto a camelid sdAb backbone (sdASNase) (PATENT# E0115946). The addition of a targeting domain nanobody to the catalytic sdASNase lead to the new concept of Targeted Catalytic Nanobodies (TCANs).In particular, the molecule designed in this work (TCAN3) was composed by a newly selected anti-CD19 nanobody (targeting domain) and by the catalytic sdASNase nanobody (catalytic domain). In order to produce such molecule, several steps were followed.
Firstly, the extracellular domain of CD19 was expressed as a C-terminal fusion with the human Fc fragment. For the selection of targeting nanobodies, the Phage Display technique was set up in house, but, due to obstacles encountered in both recombinant CD19 purification and classical Phage Display selection, the Yeast Two Hybrid system was chosen as an alternative strategy for the screening of anti-CD19 intracellular nanobodies. The collaboration with the group of Prof. Cattaneo (SNS, Pisa) resulted in the successful isolation of a nanobody, whose binding to CD19 was confirmed through ELISA tests. The TCAN3 was then assembled joining the selected anti-CD19 nanobody targeting domain to the available sdASNase nanobody through a linker. The TCAN3 was then expressed and purified, and its binding to CD19 was confirmed through ELISA tests. In the meantime, preliminary tests for co-localization studies of CD19 and lysosomes were set up.
In conclusion, in this work, TCAN3, a promising targeted asparaginase-based molecule which could help in leukemia therapy, and especially in high-risk forms, was designed and expressed. TCANs, focusing for the first time on the specificity of the metabolic traits of a given tumor and coupling the correct catalytic activity to the appropriate target specificity,might potentially represent a novel general approach to tackle any type of cancer which shows sensitivity to a specific metabolite deprivation.
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SARAGOZZA, SILVIA. "High-throughput antibody validation platforms." Doctoral thesis, Università del Piemonte Orientale, 2014. http://hdl.handle.net/11579/45955.
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Miret, Minard Joan. "Development of antibody-based antitumor therapies based on Trastuzumab: antibody-drug conjugates, immunocytokines and fragment conjugates." Doctoral thesis, Universitat Autònoma de Barcelona, 2019. http://hdl.handle.net/10803/669342.
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La tesi està centrada en el desenvolupament de molècules terapèutiques basades en anticossos per al tractament de càncer de mama HER2 positiu. Les molècules generades consisteixen en anticossos conjugats a drogues (ADCs) i una immunocitoquina, totes aquestes molècules basades en l’anticòs monoclonal model Trastuzumab. Aquest treball comprèn el procés producció i caracterització de les molècules, partint del clonatge de les seqüències dels anticossos i anant fins l’anàlisi in vitro de la seva activitat antiproliferativa, i, per a un dels candidats desenvolupats, analitzant també el seu potencial terapèutic in vivo. Com a primer pas, l’anticòs de referència Trastuzumab ha estat produït mitjançant la línia cel·lular HEK293, s’ha caracteritzat la producció a escala matràs així com en sistemes d’un sol ús de 5 i 50 litres. També s’ha definit una seqüència d’operacions per a la recuperació i purificació del producte, amb la qual s’ha obtingut una puresa de l’ordre del 99%. Així mateix, s’han implementat eines per a la caracterització fisicoquímica del producte obtingut. Les estratègies de producció, purificació i caracterització desenvolupades per a l’anticòs Trastuzumab han estat aplicades a la resta de molècules derivades d’anticòs, produïdes posteriorment. En un primer intent per a desenvolupar molècules derivades de Trastuzumab amb una activitat terapèutica incrementada, l’anticòs Trastuzumab ha estat fusionat a la citoquina interferó-2, en forma d’immunocitoquina. Aquest constructe, però, no ha generat una activitat antiproliferativa més potent que la del Trastuzumab, ni tampoc ha resultat en una activació del sistema immunitari. Ambdues activitats han estat analitzades en assajos in vitro. L’increment de la potència antiproliferativa de l’anticòs Trastuzumab ha estat aconseguida a través de la seva conjugació a les drogues citotòxiques DM1 i vcMMAE, amb ràtios droga-anticòs (DAR) de 3.1 i 4, respectivament. Aquesta estratègia implica l’aplicació de dos dels principals mètodes de conjugació de referència, utilitzats en ADCs aprovats comercialment: conjugació a residus endògens de l’anticòs de lisina (DM1) i de cisteïna, després d’una reducció parcial (vcMMAE). També s’han aplicat estratègies de conjugació dirigida, les quals generen ADCs homogenis, que presenten un índex terapèutic més favorable. Tres estratègies diferents han estat implementades per la conjugació homogènia de l’anticòs Trastuzumab. En una primera estratègia, l’anticòs ha estat conjugat a la droga vcMMAE amb un DAR objectiu de 8, mitjançant una reducció completa de l’anticòs. DARs elevats, però, poden causar inestabilitat de la molècula i perjudicar-ne l’efecte in vivo. Així, s’ha realitzat la conjugació homogènia amb un DAR objectiu de 2, a través de la inserció d’una cisteïna a la seqüència del Trastuzumab, la qual ha estat conjugada al vcMMAE, amb un DAR resultant d’1.79. També s’ha desenvolupat una estratègia innovadora per tal d’obtenir un ADC homogeni amb un DAR de 2, basada en la conjugació del residu de cisteïna de cadenes lleugeres produïdes independentment, després unides a cadenes pesades també produïdes independentment: s’obté un ADC homogeni amb un DAR de 2. Les estratègies de conjugació heterogènies i homogènies implementades per a l’anticòs Trastuzumab sencer han estat també aplicades a fragments scFv, en aquest cas, han resultat en processos amb un baix rendiment de recuperació a causa de la poca estabilitat dels conjugats. Els diversos ADCs generats en aquest treball han estat analitzats en models in vitro 2D amb la línia cel·lular SKBR3, model de càncer de mama, amb els quals s’ha confirmat el seu potencial anticancerós. També s’han desenvolupat models 3D, els quals han estat més resistents que els models 2D. Finalment, l’efecte terapèutic de l’ADC homogeni format per la unió de les cadenes independents ha demostrat una forta activitat terapèutica en un model de ratolí in vivo, validant, així, el procés de producció d’ADCs desenvolupat.The thesis is focused on the development of antibody based therapeutic molecules for the treatment of HER2 positive breast cancer. The generated molecules consist in antibody-drug conjugates (ADCs) including whole antibody and antibody fragment conjugates, and an immunocytokine, all of these molecules being based on the model monoclonal antibody Trastuzumab. This work comprises the whole production and characterization process of the molecules, going from the cloning of the genetic sequences of the antibody molecules to the in vitro analysis of their antiproliferation activity, and, for one of the developed candidates, an in vivo assessment of its therapeutic potential was also performed. In a first step, the reference antibody Trastuzumab was produced using the mammalian cell line HEK293, its production being characterized in shake flasks as well as in 5L and 50L single use culture strategies. An operation sequence was defined for recovering and purifying the product, obtaining purity levels in the order of 99% and physicochemical characterization tools were implemented in order to assess the quality of the product. The production, purification and characterization strategies developed for Trastuzumab were applied to the other antibody-derived molecules later produced. In a first attempt to develop a Trastuzumab antibody-derived molecule with an enhanced therapeutic activity, Trastuzumab was fused to the cytokine interferon-2, forming an immunocytokine. This construct, however, did not result in an improved antiproliferative activity with respect to Trastuzumab, as assessed in an in vitro 2D proliferation assay. It did not result neither in an activation of the immune system, as analyzed in an in vitro lymphocyte proliferation assay. An increased potency of the antiproliferative activity of Trastuzumab was achieved by heterogeneously conjugating it to the cytotoxic drugs DM1 and vcMMAE, with drug-antibody ratios (DARs) of 3.1 and 4, respectively, applying two of the main reference conjugation strategies found in commercially approved ADCs: conjugation to endogenous lysine residues of the antibody (DM1), and conjugation to endogenous interchain cysteine residues after a partial reduction step (vcMMAE). After successfully implementing the conjugation strategies and DAR characterization tools, site-directed conjugation strategies (yielding homogeneous products that have therapeutic advantages in the form of a wider therapeutic window) were attempted. Three different strategies were implemented for the homogeneous conjugation of Trastuzumab. In a first simple and straightforward strategy, Trastuzumab was conjugated to vcMMAE with an aimed DAR of 8, by applying a complete reduction of the antibody. High DARs, however, can result in instability and impaired in vivo therapeutic efficacy of the ADC. Therefore, the homogeneous conjugation for a DAR of 2 was attempted by inserting a cysteine in the sequence of Trastuzumab and conjugating the vcMMAE drug to it, obtaining a DAR of 1.79. An innovative alternative strategy to obtain an homogeneous ADC with a DAR of 2 was also developed, consisting in the cysteine conjugation of an independently produced light chain, which is then assembled with independently produced heavy chains, forming an homogeneous ADC with a DAR of 2. The heterogeneous and homogeneous conjugation strategies implemented for whole Trastuzumab antibody were also applied to Trastuzumab-based scFv antibody fragments, resulting in conjugation processes with a low recovery yield due to precipitation issues. The different generated ADCs had their antiproliferation activity tested in 2D in vitro models using the breast cancer model SKBR3 cell line, confirming their antitumor potential. 3D culture models were also generated, showing a higher resistance to the developed drugs than the 2D display. Finally, the therapeutic effect of the homogeneous ADC formed by the chains assembly was tested on a developed mouse model in vivo, displaying a strong antitumor response, and therefore validating the whole developed ADC production process.
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Gozin, Yael. "Catalytic antibody 1E9: properties and selectivity /." Zürich : ETH, 2006. http://e-collection.ethbib.ethz.ch/show?type=diss&nr=16475.
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Forrer, Nicola. "Antibody purification with ion-exchange chromatography." Zürich : ETH, 2008. http://e-collection.ethbib.ethz.ch/show?type=diss&nr=17784.
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West, Natasha. "Nanocomposite immunosensor for anti-transglutaminase antibody." Thesis, University of the Western Cape, 2009. http://etd.uwc.ac.za/index.php?module=etd&action=viewtitle&id=gen8Srv25Nme4_6426_1298354109.
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Coeliac disease (CD) is a gluten intolerance condition that results in the flattening of the villi, which line the bowel. It is the most common cause of malabsorption of food nutrients. This inability to absorb sufficient levels of nutrients causes many of the common symptoms experienced by CD patients. Some of the symptoms, which lead to an increase in mortality rate, include chronic diarrhea, fatigue, iron-deficient anemia and osteoporosis. People with CD have higher than normal levels of certain antibodies in their blood. Thus, the concentration of anti-transglutaminase antibody (anti-tTG) in human sera is an important analytical marker for the diagnosis of CD. An immunosensor is a type of biosensor that has an antigen or antibody fragment as its biological recognition component. The specificity of the molecular recognition of antigens by antibodies to form a stable complex is the basis of immunosensor technology. In this work, overoxidized polypyrrole (OvoxPpy) was electrosynthesized as a noval sensor platform on a glassy carbon electrode (GCE). The OvoxPpy was then doped with gold-nanoparticles (GNP) by electrodeposition using cyclic voltammetry to form GNP|OvoxPpy||GCE electrode system. Morphology and size of the GNP|OvoxPpy||GCE nanocomposite were determined using scanning electron microscopy. The electrochemical immunosensor for anti-tTG antibodies was prepared by immobilizing transglutaminase antigen (tTG-antigen) onto the GNP|OvoxPpy||GCE by drop coating and allowed to incubate for 2 hrs. The electrochemical characterization of the nanocomposite platform and immunosensor were studied by voltammetry and electrochemical impedance spectroscopy (EIS)...
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Sabatte, Gwenola. "Development of a SERRS antibody assay." Thesis, University of Strathclyde, 2007. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.486539.
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Surf.'1ce enhanced resonance Raman scattering (SERRS) is an extremely sensitive detection technique, comparable to or, in some circumstances, better than fluorescence. Thanks to the molecularly specific vibrational signals, SERRS enables the identification of a specific label in situ in the presence of other materials or other labels. SERRS is produced when an analyte containing a chromophore is bound onto a.suitably roughened metal surface. The signals are recorded using Raman spectroscopy and the excitation frequency must match or be close to both the plasmon resonance frequency of the metal and an electronic transition of the chromophore. This thesis reports the development of an antibody assay using SERRS detection using colloidal silver particles as the substrate. An appropriate SERRS labelling chemistry was developed, by synthesising new SERRS labels using dyes, a polymer dye and SERRS active beads. They were conjugated to an antibody. SERRS of the labelled antibody is intense and gives specitic peaks which identify the label. Under the conditions used, antibodies were detected with a better sensitivity by SERRS detection (2.79 x 10-13 mol.dm-1 than by fluorescence (3.46 x 10-10 mol.dm·3 ). A sandwich assay was developed for two targets, brain natriuretic peptide and holotranscobalamin: The first target was used to assess the potential of several bioassay formats. The second target was added later to determine the potential for a multiple target assay. Optimisation of a complete SERRS assay using magnetic microparticles was carried out. Quantitative results were obtained with anyone run but there was variability between runs. A final investigation focused on trying to understand the source of the variability.
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Carlsson, Fredrik. "Antibody Feedback Regulation and T Cells." Doctoral thesis, Uppsala : Acta Universitatis Upsaliensis : Univ.-bibl. [distributör], 2007. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-7631.
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Falk, Ronny. "Systems enabling antibody-mediated proteomics research." Doctoral thesis, Stockholm, 2006. http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-4025.
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Hultberg, Anna. "Lactobacilli expressing antibody fragments against pathogens /." Stockholm, 2006. http://diss.kib.ki.se/2006/91-7140-862-2/.
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Karlnoski, Rachel Anne. "Optimization of anti-Abeta antibody therapy." [Tampa, Fla.] : University of South Florida, 2007. http://purl.fcla.edu/usf/dc/et/SFE0002145.
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Hilyard, Katherine L. "Protein engineering of antibody combining sites." Thesis, University of Oxford, 1991. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.291278.
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Khalili, Hanieh. "Disulfide-bridging PEGylation of antibody fragments." Thesis, University College London (University of London), 2012. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.553694.
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Monoclonal antibodies are routinely used in the clinic. There is also a small number of antibody fragments (e.g. Fabs) that are clinically used. In many applications where antibody binding is required to antagonise a receptor or simply to bind a ligand, there is no need for the Fe properties that are associated with effector functions. Unfortunately, without the Fe region, monovalent antibody fragments rapidly clear from the blood circulation. PEGylation is the most successful and clinically used approach to date for increasing the circulation half-life of therapeutic proteins. Disulfide bridging PEGylation is an appropriate method to site-specifically conjugate a molecule of PEG at the interchain disulfide of a Fab. Since a PEGylated Fab will be monovalent, it is not possible to exploit the cooperative binding or avidity that is associated with the bivalency of a full IgG. An objective of this PhD project was firstly to understand if a monovalent PEGylated Fab can bind effectively to its antigen. A bis-alkylation PEG reagent with a functional group at each terminus of the PEG could then be used to conjugate two Fabs, one at each end of the PEG molecule, to generate either a homo Fab-PEG-Fab or a hetero Fab-PEG-Fab* conjugate. An important objective of this PhD was to determine the structure-property correlations of a small family of PEGylated-Fabs. It was hypothesised that Fab-PEG- Fab conjugates would display comparable binding properties to the full parent IgG. Three Fabs were PEGylated in this project. Fabbeva and Fabtrast were obtained by papain digestion of bevacizumab and trastuzumab respectively. Ranibizumab is a clinically used Fab and therefore did not require digestion of a full antibody. Both Fabbeva and Fabrani bind to VEGF and Fabtrast binds to HER-2. After treament with DTT to open the interchain disulfide, each Fab underwent reaction with a Fab reagent capable of thiol specific, bis-alkylation. PEGylation was accomplished at near quantitative conversion with 1-2 equivalents with reproducible result. A single step ion-exchange purification process was used to obtain purified mono PEG-Fabs. The PEG-Fab conjugates were stable during a 3 months stability study at 4 °c with no de- PEGylation. A PEG2x2o-Fab' beva construct was also generated by conjugation of two molecules of the PEG reagent to intrachain disulfide bonds of the Fab beva- BIAcore and ELISA studies confirmed that compared with the unPEGylated Fabs, the PEGylated Fabbeva, Fabrani and Fabtrast displayed a 2 fold decrease in binding affinity I for their respective ligands. This decrease in binding affinity was much less than had been reported in the literature. PEG-Fabbeva conjugates comprising 20, 30 and 40 kDa PEG all displayed similar binding affinities. The binding affinity of the PEG2x20- Fab beva was decreased compared with mono PEG2o-Fabbeva as a result of a change in the dissociation rate constant. The homodimer Fab-PEG-Fab constructs comprising 6, 10 and 20 kDa PEG and Fabbeva, Fabrani and Fabtrast maintained their binding affinities compared with the parent IgGs. BIAcore kinetic studies showed there was greater binding affinity and slower dissociation rate for the Fabbeva-PEG-Fabbeva than the native Fabbeva. While similar binding affinity to bevacizumab was observed for the Fabbeva-PEG-Fabbeva, the dissociation rates of the the Fabbeva-PEG-Fabbeva were slower than for bevacizumab. It was also found that using a longer PEG in the Fabbeva-PEG-Fabbeva resulted in slower dissociation. The heterodimer Fabbeva-PEG2o-Fabtrast* that was produced maintained binding to VEGF and HER-2. An in vitro angiogenesis assay suggested that the Fabbeva-PEG20-Fabbeva and Fabrani-PEG20-Fabrani inhibit angiogenesis more effectively than bevacizumab. Using PEG as a linking molecule to conjugate two Fabs would appear to be a valid way to provide bivalency to the molecule, resulting in at least the functional activity expected of a full IgG. Since the PEG is a flexible coil, it may be the case that the two conjugated Fabs in the Fab-PEG-Fab homodimer are brought towards the VEGF in a manner that is more efficient than for a native IgG, resulting in a stronger binding interaction and hence enhanced functional activity. These results for Fab- PEG-Fab homodimer are encouraging and together with the results for the Fab-PEG- Fab* bring the potential to aid development of bivalent and bispecific protein-based medicines.
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Goh, Y. S. "Antibody-mediated protection against Salmonella infections." Thesis, University of Cambridge, 2011. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.599460.
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In this project, I examined how antibody targeting different bacterial proteins and how the different antibody subclasses affect the interaction of opsonised bacteria with their host cells, using Salmonella strains expressing a foreign CD52 mimotope either in the flagellin protein, FliC, or in the outer membrane protein, OmpA, that can be recognized by a panel of chimaeric antibodies. Immunofluorescence studies examining the percentage of infected cells and the bacterial load per infected cell, and experiments examining intracellular bacterial viability revealed that the greatest enhancement of phagocytosis and the following antibacterial activity was observed with IgG3 opsonisation, followed by IgG1, IgG4, and IgG2, IgG3 was also more efficient in mediating phagolysosome fusion, followed by IgG1, IgG4, and IgG2, indicated by a higher percentage of bacterial co-localisation with TROv and cathepsin D. In addition, similar studies on cells differentially stimulated to induce different Fcγ receptor profiles have shown that cells expressing higher levels of FcγRI, FcγRIIA and FcγRIII are more efficient in the above mentioned phagocyte functions. Further investigations, using anti-FcγR antibodies to block host Fcγ receptors, revealed that IgG1-, IgG3- and IgG4-mediated phagocyte functions have a higher dependency on FcγRI over FcγRIIA, while IgG2-mediated phagocyte functions have a higher dependency on FcγRIIA over FcγRI. Taken together, the findings suggest that FliC and OmpA could be promising bacterial targets for vaccine development, and the distribution of the different antibody subclasses in the antibody profile might be important in antibody-mediated protection.
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Coles, A. "Monoclonal antibody therapy of multiple sclerosis." Thesis, University of Cambridge, 1998. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.597844.
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T cells mediate the inflammatory activity of multiple sclerosis, which is directed against an unknown autoantigen. A non-antigen specific treatment strategy was tested, drawing on the experimental demonstration of long term allograft acceptance following short term therapy with monoclonal antibodies against T cells. The treatment of 27 patients with multiple sclerosis using a single pulse of humanised anti-lymphocyte (CD52) antibody, Campath-1H, was investigated. With the first dose of monoclonal antibody, patients experienced a rehearsal of previous relapses that fully resolved and was associated with a transient rise in serum TNF-α and IFN-γ. This suggested that inflammatory mediators may impede axonal conduction at previously demyelinated sites. After five consecutive daily doses of Campath-1H, the circulating T lymphocyte count was suppressed for at least 18 months, without any serious infective complications. The in vitro mitogen induced proliferation, and IFN-γ secretion, of patients' peripheral blood mononuclear cells was reduced after treatment and there was also a signficant rise in the peripheral B cell count above pre-treatment levels. This deviation of immune responses away from the Th1 phenotype would, under the hypothesis that multiple sclerosis is a "Th1 disease", be expected to abrogate cerebal inflammation. Unexpectedly, one third of patients developed Graves' disease, an adverse effect not induced by any other therapies nor by Campath-1H treatment of other diseases. By analogy with experimental models of autoimmunity following prolonged lymphocyte depletion, Campath-1H may have selectively depleted T cell clones which suppress autoreactive T cells, although why Graves' disease specifically was induced remains unexplained. Radiological markers of cerebral inflammation were suppressed for at least 18 months in all patients, who experienced no new relapses. However half the patients continued to accumulate disability from deficits acquired prior to monoclonal antibody treatment. In these patients there was both a higher inflammatory load at baseline and progressive cerebral atrophy, which may represent axonal degeneration. It is concluded that inflammation is a necessary prerequisite for new lesion formation and that the secondary progressive phase of multiple sclerosis is initiated by demyelination but proceeds by non-inflammatory mechanisms.
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Holt, L. J. "Direct screening for antibody-antigen interactions." Thesis, University of Cambridge, 2001. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.604196.
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Array screening has been applied to the identification of antibodies from recombinant libraries, resulting in the identification of antibody-antigen interactions without the need for in vivo or in vitro antibody selection. In order to further increase the efficiency of array-based identification of molecular interactions, a novel two-dimensional arraying approach was invented and developed. Lines of biological sample from one repertoire were intersected with lines of sample from a second repertoire allowing interactions to be tested where the lines crossed. To test a given number of molecular pairs, the number of samples to be isolated, dispensed and stored and the time taken to create the array are greatly reduced when compared to one-dimensional arraying. The two-dimensional approach was applied to screening repertoires of antigens against repertoires of antibodies that bind to a variety of different proteins could be identified in a single experiment. In a second application of two-dimensional screening, large repertoires of antibodies were created on arrays by combining a few antibody heavy and light chains in all possible pairings. An array of 1.47 x 105 distinct antigen-binding sites was created by exhaustively combining 384 heavy chains and 384 light chains. It is likely that such repertoires, perhaps constructed from unselected antibody heavy and light chains, will provide a highly efficient method for the construction of antibody repertoires with which to analyse the human proteome.
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Ahmed, Sarah. "Antibody-enzyme conjugates for cancer therapy." Thesis, Imperial College London, 2008. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.500308.
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Advances in cancer therapy have led to the exploitation of the biological differences between cancer and normal cells. A popular approach is to attach a targeting moiety such as an antibody to a toxin or toxic enzyme in order to specifically locate and kill cancer cells only. However, many immunotoxins have fared poorly in their clinical trials, having shown to elicit immunogenic responses and toxicity in the patient. This problem is commonly attributed to the non-human sources of the toxins and enzymes being delivered. Targeting cytotoxic agents from human sources is therefore a prospective solution. Accordingly, this project aims to develop an immunoconjugate using mammalian or 'mammalian-like' enzymes for the treatment of epithelial cancers over-expressing the MUCl tumour antigen.
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Smith, Christopher Roy. "Quantum dots for antibody based sensors." Thesis, Imperial College London, 2010. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.528301.
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Hills, Anna E. "Control of monoclonal antibody N-glycosylation." Thesis, University of Kent, 2001. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.344101.
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Hamilton, Stephen. "Targeting melanoma using specific antibody fragments." Thesis, University College London (University of London), 2002. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.399235.
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Bristow, Richard G. W. "Antibody recognition of HIV-1 glycoproteins." Thesis, Open University, 1992. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.315370.
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Pei, Xue Yuan. "Structural study of multivalent antibody fragments." Thesis, University of Cambridge, 1998. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.310912.
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Bojö, Peter. "Towards a carbon nanotube antibody sensor." Thesis, Massachusetts Institute of Technology, 2011. http://hdl.handle.net/1721.1/71463.
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Thesis (M. Eng.)--Massachusetts Institute of Technology, Dept. of Biological Engineering, February 2012.Cataloged from PDF version of thesis.
Includes bibliographical references (p. 46-51).
This work investigated single-walled carbon nanotube (SWNT)/polymer-protein A complexes for optically reporting antibody concentration via a change in near infrared fluorescent emission after antibody binding. SWNT have potential as biosensors because of extraordinary sensitivity, lack of photobleaching, and optical activity in a near-infrared window. A SWNT sensor could provide label-free measurements of antibody concentration in a continuous fashion, which may aid selection of production strains. Protein A itself, dextran, poly vinyl alcohol, DNA sequences, and chitosan were used as polymers for wrapping SWNT. Nonspecific binding to solution-phase constructs was found to be a major problem with these approaches. Chitosan hydrogels encapsulating SWNT also show nonspecific responses.
by Peter Bojö.
M.Eng.
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Simpson, Christina M. (Christina Margaret). "Cost modeling for monoclonal antibody manufacturing." Thesis, Massachusetts Institute of Technology, 2011. http://hdl.handle.net/1721.1/66050.
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Thesis (M.B.A.)--Massachusetts Institute of Technology, Sloan School of Management; and, (S.M.)--Massachusetts Institute of Technology, Dept. of Mechanical Engineering; in conjunction with the Leaders for Global Operations Program at MIT, 2011.Cataloged from PDF version of thesis.
Includes bibliographical references (p. 75-76).
The Novartis BioPharmOps division is responsible for manufacturing large molecule products, including monoclonal antibodies, for late stage clinical trials and commercial sales. The BioPharmOps site in Huningue, France is expanding their product line but is also trying to reduce costs; cost pressures are increasing as biotech products become a larger part of Novartis' pipeline. The site uses a standard cost method to calculate their product costs. However, when using standard costs it can be time-consuming to extrapolate and predict costs when inputs and assumptions (such as product mix or process parameters) are changed. This project describes development of a model that allows the factory to quickly and easily simulate new product mixes and process flows. This model provides the site with a different view of their costs that will help them understand their cost drivers more completely and thereby help enable strategic decision-making at the site. A model of this type can be used to provide unexpected insights but the data in it are not meant to stand alone. By using results from a cost model like this along with operational metrics like throughput time or changeover time, a site should be able to quickly predict the cost impact of process changes or changes in the production plan.
by Christina M. Simpson.
S.M.
M.B.A.
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Qian, Qi. "Intracellular delivery of rabbit monoclonal antibody." Scholarly Commons, 2007. https://scholarlycommons.pacific.edu/uop_etds/679.
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In the past decades, a series of small peptides, Protein Transduction Domain (PTD), were discovered to be able to facilitate the delivery of small proteins into living cells. With the specific feature, researchers have successfully delivered some functional proteins into living cells. To fully explore and understand the functions and structures of intracellular proteins, more powerful tools are under demand. Recently, an increasing number of rabbit monoclonal antibodies (RabMAbs) have been approved to able to recognize subtle distinctions between the changes of intracellular proteins status. They could be good tools for researchers with the ability to traverse through cell membrane into living cells.
In this dissertation, a novel delivery technology for RabMAbs was established. Transcriptional activator of transcription (TAT) peptide was utilized as a delivery carrier for RabMAbs. It was demonstrated that RabMAbs could be delivered into living cells by conjugating with TAT peptide. Different cell lines, including adherent and suspension cells, were tested for the delivery of RabMAbs. The delivery process was studied in terms of incubation concentration and time, and an optimal delivery condition was established.
To investigate the biological function of delivered RabMAbs inside cytoplasm, three RabMAbs against actin, procaspase-3 and NF-κB respectively were studied. Their binding activities after delivery were verified via sandwich-ELISA data. The immunofluorescent staining of the delivered RabMAb against actin showed it specifically bound to the actin filament in its native morphology. The quantitative analysis of the delivered RabMAb against procaspase-3 showed that approximately 60% of delivered antibody bound to the antigen proteins. The delivered RabMAb against NF-KB apparently blocked the nuclear translocation of NF-KB introduced by TNF-a. The success of delivering the three rabbit monoclonal antibodies with binding or inhibiting functions demonstrated the feasibility of delivering various RabMAbs into living cells by TAT peptide for studying the biological functions of intracellular proteins.
Furthermore, to overcome the efficiency and cost issues of the RabMAb delivery system, a universal delivery platform for RabMAbs was developed. This platform uses goat-anti-rabbit polyclonal antibody conjugated with TAT peptide as delivery vehicle. It was confirmed that the goat-anti-rabbit polyclonal antibody modified with TAT peptide was able to capture RabMAbs and deliver RabMAbs into living cells by the conjugated TAT peptide. The results provide a promising delivery platform for all RabMAbs.
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Watkins, Sarah Jane. "Adenoviral targeting with antibody fusion proteins." Thesis, University of Cambridge, 1998. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.624907.
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Okou, David. "Engineering of fluorescent antibody in bacteria." DigitalCommons@Robert W. Woodruff Library, Atlanta University Center, 2002. http://digitalcommons.auctr.edu/dissertations/3222.
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Towards the construction of protein-based biological sensors, chimeric proteins comprised of an antibody single chain antigen-binding protein (scFv) and the green fluorescent protein (GFP) were constructed. Although correct folding of the scFv domain typically requires the oxidizing conditions of extracellular compartments, such as the periplasmic space of E. coli, GFP is unable to mature under these conditions. Using DNA recombinant technology, fusion constructs were made in the cytoplasm under control of the araBAD promoter. Weak fluorescence of the GFP domain and antigen binding activity of the sFv domain were obtained in the cytoplasm of E. coli BL21, but improved expression and activities of both domains were obtained by using a trxB- mutant of E. coli, as well as by modifying physical and genetic conditions for expression of the fusion proteins. Assessment of the fluorescence and antigen binding activity of the fusion proteins indicates that GFP fluorescence can serve as an indicator of correct folding of fusion proteins.
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Fernandes, Telma Godinho Barroso Maciel. "Functional monolithic platforms for antibody purification." Doctoral thesis, Faculdade de Ciências e Tecnologia, 2014. http://hdl.handle.net/10362/11550.
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Dissertação para obtenção do Grau de Doutor em Química SustentávelFundação para a Ciência e Tecnologia - contracts PEst-C/EQB/LA0006/2011, MIT-Pt/BS-CTRM/0051/2008, PTDC/EBB-BIO/102163/2008, PTDC/EBBBIO/ 098961/2008, PTDC/EBB-BIO/118317/2010 and doctoral grant SFRH/ BD/62475/2009, and Fundação Calouste Gulbenkian