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An In Vitro Comparative Immunogenicity Assessment (IVCIA) assay was evaluated as a tool for predicting the potential relative immunogenicity of biotherapeutic attributes. Peripheral blood mononuclear cells from up to 50 healthy naïve human donors were monitored up to 8 days for T-cell proliferation, the number of IL-2 or IFN-γ secreting cells, and the concentration of a panel of secreted cytokines. The response in the assay to 10 monoclonal antibodies was found to be in agreement with the clinical immunogenicity, suggesting that the assay might be applied to immunogenicity risk assessment of antibody biotherapeutic attributes. However, the response in the assay is a measure of T-cell functional activity and the alignment with clinical immunogenicity depends on several other factors. The assay was sensitive to sequence variants and could differentiate single point mutations of the same biotherapeutic. Nine mAbs that were highly aggregated by stirring induced a higher response in the assay than the original mAbs before stirring stress, in a manner that did not match the relative T-cell response of the original mAbs. In contrast, mAbs that were glycated by different sugars (galactose, glucose, and mannose) showed little to no increase in response in the assay above the response to the original mAbs before glycation treatment. The assay was also used successfully to assess similarity between multiple lots of the same mAb, both from the same manufacturer and from different manufacturers (biosimilars). A strategy for using the IVCIA assay for immunogenicity risk assessment during the entire lifespan development of biopharmaceuticals is proposed.

Purpose Determine the effect of minute quantities of sub-visible aggregates on the in vitro immunogenicity of clinically relevant protein therapeutics. Methods Monoclonal chimeric (rituximab) and humanized (trastuzumab) antibodies were subjected to fine-tuned stress conditions to achieve low levels (<3% of total protein) of sub-visible aggregates. The effect of stimulating human dendritic cells (DC) and CD4 + T cells with the aggregates was measured in vitro using cytokine secretion, proliferation and confocal microscopy. Results Due to its intrinsic high clinical immunogenicity, aggregation of rituximab had minimal effects on DC activation and T cell responses compared to monomeric rituximab. However, in the case of trastuzumab (low clinical immunogenicity) small quantities of aggregates led to potent CD4 + T cell proliferation as a result of strong cytokine and co-stimulatory signals derived from DC. Consistent with this, confocal studies showed that stir-stressed rituximab was rapidly internalised and associated with late endosomes of DC. Conclusions These data link minute amounts of aggregates with activation of the innate immune response, involving DC, resulting in T cell activation. Thus, when protein therapeutics with little or no clinical immunogenicity, such as trastuzumab, contain minute amounts of sub-visible aggregates, they are associated with significantly increased potential risk of clinical immunogenicity.

Anti-CD52 therapy has been shown to be effective in the treatment of a number of B cell malignancies, hematopoietic disorders and autoimmune diseases (including rheumatoid arthritis and multiple sclerosis); however the current standard of treatment, the humanized monoclonal antibody alemtuzumab, is associated with the development of anti-drug antibodies in a high proportion of patients. In order to address this problem, we have identified a novel murine anti-CD52 antibody which has been humanized using a process that avoids the inclusion within the variable domains of non-human germline MHC class II binding peptides and known CD4+ T cell epitopes, thus reducing its potential for immunogenicity in the clinic. The resultant humanized antibody, ANT1034, was shown to have superior binding to CD52 expressing cells than alemtuzumab and was more effective at directing both antibody dependent and complement dependent cell cytotoxicity. Furthermore, when in the presence of a cross-linking antibody, ANT1034 was more effective at directly inducing apoptosis than alemtuzumab. ANT1034 also showed superior activity in a SCID mouse/human CD52 tumour xenograft model where a single 1 mg/Kg dose of ANT1034 led to increased mouse survival compared to a 10 mg/Kg dose of alemtuzumab. Finally, ANT1034 was compared to alemtuzumab in in vitro T cell assays in order to evaluate its potential to stimulate proliferation of T cells in peripheral blood mononuclear cells derived from a panel of human donors: whereas alemtuzumab stimulated proliferation in a high proportion of the donor cohort, ANT1034 did not stimulate proliferation in any of the donors. Therefore we have developed a candidate therapeutic humanized antibody, ANT1034, that may have the potential to be more efficacious and less immunogenic than the current standard anti-CD52 therapy.

Most protein therapeutics have the potential to induce undesirable immune responses in patients. Many patients develop anti-therapeutic antibodies, which can affect the safety and efficacy of the therapeutic protein, particularly if the response is neutralizing. There are a variety of factors that influence the immunogenicity of protein therapeutics and, in particular, the presence of B- and T-cell epitopes is considered to be of importance. In silico tools to identify the location of both B- and T-cell epitopes and to assess the potential for immunogenicity have been developed, and such tools provide an alternative to more complex in vitro or in vivo immunogenicity assays. This article reviews computational epitope prediction methods and also the use of manually curated databases containing experimentally derived epitope data. However, due to the complexities of the molecular interactions involved in epitope recognition by the immune system, the heterogeneity of key proteins in human populations and the adaptive nature of the immune response, in silico methods have not yet achieved a level of accuracy that enables them to be used as stand-alone tools for predicting clinical immunogenicity. Computational methods, particularly with regard to T-cell epitopes, only consider a limited number of events in the process of epitope formation and therefore routinely over-predict the number of epitopes within a molecule. Epitope databases such as the Immune Epitope Database (IEDB) and the proprietary T Cell Epitope Database™ (TCED™) have reached a size and level of organization that increases their utility; however, they are not exhaustive. These methods have greatest utility as an adjunct to in vitro assays where they can be used either to reduce the amount and complexity of the in vitro screening, or they can be used as tools to analyze the sequence of the identified epitope in order to locate amino acids critical for its properties.

\"Where this book is exceptional is that the reader will not just learn how LTE works but why it works\" Adrian Scrase, ETSI Vice-President, International Partnership Projects Following on the success of the first edition, this book is fully updated, covering the latest additions to LTE and the key features of LTE-Advanced. This book builds on the success of its predecessor, offering the same comprehensive system-level understanding built on explanations of the underlying theory, now expanded to include complete coverage of Release 9 and the developing specifications for LTE-Advanced. The book is a collaborative effort of more than 40 key experts representing over 20 companies actively participating in the development of LTE, as well as academia. The book highlights practical implications, illustrates the expected performance, and draws comparisons with the well-known WCDMA/HSPA standards. The authors not only pay special attention to the physical layer, giving an insight into the fundamental concepts of OFDMA-FDMA and MIMO, but also cover the higher protocol layers and system architecture to enable the reader to gain an overall understanding of the system. Key New Features: Comprehensively updated with the latest changes of the LTE Release 8 specifications, including improved coverage of Radio Resource Management RF aspects and performance requirements. Provides detailed coverage of the new LTE Release 9 features, including: eMBMS, dual-layer beamforming, user equipment positioning, home eNodeBs / femtocells and pico cells and self-optimizing networks. Evaluates the LTE system performance. Introduces LTE-Advanced, explaining its context and motivation, as well as the key new features including: carrier aggregation, relaying, high-order MIMO, and Cooperative Multi-Point transmission (CoMP). Includes an accompanying website containing a complete list of acronyms related to LTE and LTE-Advanced, with a brief description of each This book is an invaluable reference for all research and development engineers involved in implementation of LTE or LTE-Advanced, as well as graduate and PhD students in wireless communications. Network operators, service providers and R&D managers will also find this book insightful\"-- \"LTE -- The UMTS Long Term Evolution, Second Edition builds on the success of its predecessor, offering an updated treatment of the new LTE standard\"--

Clinical studies show that immunogenicity observed against therapeutic proteins can limit efficacy and reduce the safety of the treatment. It is therefore beneficial to be able to predict the immunogenicity of therapeutic proteins before they enter the clinic. Studies using deimmunized proteins have highlighted the importance of T-cell epitopes in the generation of undesirable immunogenicity. In silico, in vitro, ex vivo and in vivo methods have therefore been developed that focus on identification of CD4+ T-cell epitopes in the sequence of therapeutic proteins. A case study of existing therapeutic proteins is presented to review these different approaches in order to assess their utility in predicting immunogenic potential.

Interferon-α (IFN-α), in conjunction with ribavirin, is the current standard for the treatment of chronic hepatitis C virus (HCV) infection. This treatment requires frequent dosing, with a significant risk of the development of anti-IFN-α neutralizing antibodies that correlates with lack of efficacy or relapse. We have developed an IFN-α linked to the Fc region of human IgG1 for improved half-life and less frequent dosing. We have also identified, using a human T cell proliferation assay, three regions of IFN-α2b that are potentially immunogenic, and a variant containing a total of six mutations within these regions was made. This variant was made as a fusion to Fc either with or without a flexible linker between the fusion partners. Both configurations of the variant were less active than native IFN-α alone, although the variant containing the flexible linker had in vitro antiviral activity within the range of other modified IFN-αs currently in clinical use. Peptides spanning the modified regions were tested in T cell proliferation assays and found to be less immunogenic than native controls when using peripheral blood mononuclear cells (PBMCs) from both healthy individuals and HCV-infected patients who had been treated previously with IFN-α2b.

Anti-CD52 therapy has been shown to be effective in the treatment of a number of B cell malignancies, hematopoietic disorders and autoimmune diseases (including rheumatoid arthritis and multiple sclerosis); however the current standard of treatment, the humanized monoclonal antibody alemtuzumab, is associated with the development of anti-drug antibodies in a high proportion of patients. In order to address this problem, we have identified a novel murine anti-CD52 antibody which has been humanized using a process that avoids the inclusion within the variable domains of non-human germline MHC class II binding peptides and known CD4+ T cell epitopes, thus reducing its potential for immunogenicity in the clinic. The resultant humanized antibody, ANT1034, was shown to have superior binding to CD52 expressing cells than alemtuzumab and was more effective at directing both antibody dependent and complement dependent cell cytotoxicity. Furthermore, when in the presence of a cross-linking antibody, ANT1034 was more effective at directly inducing apoptosis than alemtuzumab. ANT1034 also showed superior activity in a SCID mouse/human CD52 tumour xenograft model where a single 1 mg/Kg dose of ANT1034 led to increased mouse survival compared to a 10 mg/Kg dose of alemtuzumab. Finally, ANT1034 was compared to alemtuzumab in in vitro T cell assays in order to evaluate its potential to stimulate proliferation of T cells in peripheral blood mononuclear cells derived from a panel of human donors: whereas alemtuzumab stimulated proliferation in a high proportion of the donor cohort, ANT1034 did not stimulate proliferation in any of the donors. Therefore we have developed a candidate therapeutic humanized antibody, ANT1034, that may have the potential to be more efficacious and less immunogenic than the current standard anti-CD52 therapy.

Predicting groundwater flow and transport of contaminants in fractured rock is challenging due to the heterogeneity of hydraulic properties that are difficult to characterize using conventional hydraulic testing methods. Heterogeneity is often introduced by fracture aperture variability that creates preferential flow pathways also referred to as flow channeling. Ground-penetrating radar (GPR) has been used successfully for imaging fractures. This study investigates the polarization properties and capabilities of GPR signals, both amplitude and phase, for 3-D imaging of flow channeling in a discrete, subhorizontal fracture. Two separate field studies were conducted at the Altona Flat Rock test site in New York State. The first, conducted in 2010, used surface-based multi-polarization 3-D GPR to examine the effects of radar signal polarization for imaging a fresh-water saturated, millimeter scale subhorizontal fracture. Imaging of a horizontal reflection plane should be independent of radar signal polarization. However, amplitude variations as a function of wavefield orientation were observed along the subhorizontal fracture plane indicating that polarization effects are significant. Furthermore, it was shown that summation of two orthogonal parallel-polarized signals compensates adequately for the polarization effects and results in a more accurate image of the fracture. Therefore, for imaging of flow through a discrete fracture, multi-polarization GPR acquisition is necessary. The second investigation, conducted in 2011, utilized a multi-component, surface based GPR to monitor saline tracer flow through the same water-saturated fracture. The multi-component system allowed for simultaneous acquisition of orthogonal polarizations. The presence of saline tracer in the fracture resulted in an amplitude increase and phase decrease of the reflected GPR signal. Hydraulic dipole-flow tracer tests were used to generate flow between boreholes within the fracture of interest. A five-spot well configuration allowed control over the hydraulic gradient orientation. Various concentrations of saline tracer were utilized with hydraulic gradients oriented E-W and N-S, as well as along the natural gradient. GPR imaging of saline tracer results revealed a direct and narrow channelized flow path along the E-W orientation based on both amplitude and phase changes suggesting good well connectivity. The N-S dipole tests revealed greater tracer dispersion over a larger area suggesting poorer well connectivity. These results are supported by hydraulic tests conducted at the site. This work supports imaging tracer flow using GPR signal amplitude and presents, for the first time, imaging changes in flow channeling based on hydraulic gradient orientation and the use of GPR phase for imaging saline tracer distribution.

Interferon-alpha (IFN-alpha), in conjunction with ribavirin, is the current standard for the treatment of chronic hepatitis C virus (HCV) infection. This treatment requires frequent dosing, with a significant risk of the development of anti-IFN-alpha neutralizing antibodies that correlates with lack of efficacy or relapse. We have developed an IFN-alpha linked to the Fc region of human IgG1 for improved half-life and less frequent dosing. We have also identified, using a human T cell proliferation assay, three regions of IFN-alpha2b that are potentially immunogenic, and a variant containing a total of six mutations within these regions was made. This variant was made as a fusion to Fc either with or without a flexible linker between the fusion partners. Both configurations of the variant were less active than native IFN-alpha alone, although the variant containing the flexible linker had in vitro antiviral activity within the range of other modified IFN-alphas currently in clinical use. Peptides spanning the modified regions were tested in T cell proliferation assays and found to be less immunogenic than native controls when using peripheral blood mononuclear cells (PBMCs) from both healthy individuals and HCV-infected patients who had been treated previously with IFN-alpha2b.