No description available.

No description available.

The antibody response has evolved under constant pressure to recognize common pathogens and also remain adaptable to novel threats. Given the limited size of the germline antibody repertoire, adaptability requires that some antibodies must be polyspecific for multiple distinct antigens. Despite the profound importance of polyspecificity in the antibody response, the structural features that allow it are not well understood. Antibodies raised against glycoconjugates of Chlamydiaceae LPS oligosaccharides of the inner-core sugar Kdo (3-deoxy-d-manno-oct-2-ulosonic acid) have been shown to cross-react with several inner-core oligosaccharides through conserved recognition of single Kdo residues in a germline-encoded pocket, with additional sugars accommodated by flexible side-chains. Two of these antibodies, S25-2 and S25-39, were observed to bind several Kdo oligosaccharides with an identical binding site conformation, but adopted unique conformations of the heavy chain complementarity determining region loop 3 (CDR H3) in the absence of ligand. Conformational flexibility of germline antibodies is believed to facilitate polyspecificity by generating multiple unique binding sites in a single antibody. This thesis research further explores the conformational flexibility of the antibodies S25-2 and S25-39 to gain insight into mechanisms of antigen recognition and how this feature may allow polyspecificity. This was achieved first by solving structures of S25-39 from crystals grown in unique conditions to observe alternate CDR H3 conformations, and second by designing synthetic Kdo-based antigens so as both to inhibit interaction with the previously observed liganded conformation of S25-2 and S25-39 and to be accommodated by their observed unliganded conformations. These structures reveal an unprecedented level of structural diversity of CDR H3, notably including the exact ‘liganded’ conformation in the absence of ligand. This is the first direct structural evidence that CDR H3 can exist in a conformational equilibrium with antigen binding through a selection mechanism, as opposed to induced fit where antigen causes the observed conformational change. Definitive evidence for binding the synthetic antigens was not obtained, however the resulting structures revealed several additional unique conformations of CDR H3 suggesting that ligands can alter conformational equilibria during crystallization. A unique conformation was also observed with CDR H3 coordinating multiple iodide ions, revealing another potential source of polyspecificity with unique binding paratopes generated by ion coordination. Finally, the unparalleled level of conformational diversity observed for these antibodies highlights the challenges of antibody structure classification and prediction, and stresses the need for additional in-depth studies of conformational diversity and binding mechanisms to advance these fields for therapeutic application. This is the first targeted structural study of flexibility in antibodies and provides insight into their conformational dynamics and antigen-binding mechanisms. These are of fundamental importance in understanding antibody structure and function, a critical consideration in practical applications such as modelling and design of therapeutic or diagnostic antibodies. / Graduate / 2019-11-27

<p>The most common primary malignant brain tumor, glioblastoma, is uniformly fatal. Current therapy provides only incremental benefits in survival and is often incapacitating owing to limits defined by nonspecific toxicity. By contrast, immunotherapy offers a particularly promising approach, and has the theoretical potential to target and eliminate malignant cells with unprecedented specificity. The goal of this dissertation is to apply recombinant technologies to develop a new immune-based therapy for patients with malignant glioma. This work will span the design, production, and preclinical testing of a novel bispecific antibody designed to redirect T cells against a tumor-specific mutant of the epidermal growth factor receptor, EGFRvIII.</p><p>Chapters 1 and 2 will provide an overview of broad topics in antitumor immunotherapy and immune biology, with special focus on concepts as they relate to tumors of the central nervous system. In addition, the history and current state of bispecific antibodies, particularly those of the bispecific T-cell engager (BiTE) subclass, as well as their potential role in the treatment of malignant disease, will be considered in detail. Data presented in Chapter 3 will describe our approach to generating novel bispecific tandem single-chain antibody reagents, while experiments in Chapter 4 will demonstrate the capacity of one of these molecules, an EGFRvIII-specific BiTE, to achieve antitumor efficacy both <italic>in vitro</italic> and <italic>in vivo</italic> using murine models of glioma. Addressing a major barrier to the translation of immune therapies for cancer, chapter 5 will establish a potential role for BiTEs in overcoming cell-mediated immune suppression associated with malignant disease. Lastly, Chapter 6 and 7 will report on emerging areas of study, including the use of syngeneic, transgenic murine systems, and strategies by which BiTEs may be propelled rapidly into early phase clinical trials. </p><p>In summary, separating BiTEs from other available immunotherapeutic approaches, our work in this field suggests that BiTEs are (1) highly-specific molecules that greatly reduce the risk of toxicity, (2) have the ability to penetrate the blood-brain barrier and accumulate in intracerebral tumors, and (3) may potentially overcome multiple mechanisms of immunosuppression present in patients with glioblastoma. Together, these studies have the potential to improve the clinical management of patients with glioblastoma through the generation of a novel therapeutic.</p> / Dissertation

Small stable protein domains are attractive scaffolds for engineering affinity proteins due to their high tolerance to mutagenesis without loosing structural integrity. The albuminbinding domain is a 5 kDa three-helix bundle derived from the bacterial receptor Protein G with low-nanomolar affinity to albumin. In this thesis, the albumin-binding domain is explored as a scaffold for engineering novel affinity proteins with the possible benefit of combining a prolonged serum half-life with specific targeting in a single small scaffold protein. Previously, a library was created by randomizing surface-exposed residues in order to engineer affinity to a new target antigen in addition to the inherent albumin affinity. Here, phage display selections were separately performed against the tumor antigens ERBB2 and ERBB3. The ERBB3 selection resulted in a panel of candidates that were found to have varying affinities to ERBB3 in the nanomolar range, while still retaining a high affinity to albumin. Further characterization concluded that the clones also competed for binding to ERBB3 with the natural activating ligand Heregulin. The selections against ERBB2 resulted in sub-nanomolar affinities to ERBB2 where the binding site was found to overlap with the antibody Trastuzumab. The binding sites on ABD to albumin and either target were found in both selections to be mutually exclusive, as increased concentrations of albumin reduced the level of binding to ERBB2 or ERBB3. An affinity-matured ERBB2 binder, denoted ADAPT6, which lacked affinity to albumin was evaluated as a radionuclide-labeled imaging tracer for diagnosing ERBB2-positive tumors. Biodistribution studies in mice showed a high renal uptake consistent with affinity proteins in the same size range and the injected ADAPT quickly localized to the implanted tumor. High contrast images could be generated and ERBB2-expressing tissue could be distinguished from normal tissue with high contrast, demonstrating the feasibility of the scaffold for use as diagnostic tool. In a fourth study, affinity maturation strategies using staphylococcal cell-surface display were evaluated by comparing two replicate selections and varying the stringency. A sub-nanomolar target concentration was concluded to be inappropriate for equilibrium selection as the resulting output was highly variable between replicates. In contrast, equilibrium sorting at higher concentrations followed by kinetic-focused off-rate selection resulted in high output overlap between attempts and a clear correlation between affinity and enrichment. / <p>QC 20160510</p>

No description available.

This report reviews the field of bispecific antibodies, artificially engineered antibodies thathave the ability to bind two or more different antigen simultaneously. Historical as well asrecently developed techniques are demonstrated, together with formats in preclinical andclinical development. We studied the field with the future needs of the developers in mind,when it comes to the processes and tools that can be offered by GE Healthcare BiosciencesAB. The development of bispecific antibodies gave rise to new challenges and product-relatedimpurities, which are handled by various methods. We argue for, based on the formats inclinical and preclinical development, that the methods already used to purify monospecificantibodies remain the most successful methods for the purification of bispecific antibodies.This, together with the design strategies that resolve the initial bottle-necks, ensures that theneeds of the developers are met to the same extent as for monoclonal antibodies. The methodsand formats demonstrated here do not represent all that are available or under trial.

The functions of the immune system include immunosurveillance of transformed cells, i.e., the ability to eliminate these cells before they become harmful to the organism. If the transformed cells succeed to escape the immune system surveillance, an oncological disease develops. The tumour immunotherapy aims to stimulate the immune system mechanisms to fight against the tumour. Lately, there's an interest in using NK cells in the immunotherapy of tumours. These cells appertain to the innate immune system and participate in immunosurveillance. When an NK cell encounters a target cell, its activation depends on the integration of signals from the surface activating and inhibiting receptors which bind ligands on the surface of the target cell. Upon activation, NK cell exhibits a cytotoxic response against the target cell. The use of NK cells in immunotherapy includes, among others, the testing of bispecific fusion proteins which can bind a tumour surface antigen by one part and NK cell activating receptor by the other part. Thus, these fusion proteins mediate a contact between both cells and trigger the cytotoxic response. This work presents a preparation of bispecific fusion proteins which consist of an activating ligand MICA (for the receptor NKG2D) or B7H6 (for the receptor NKp30), and a nanobody...

Protein engineering and in vitro selection systems are powerful methods to generate binding proteins. In nature, antibodies are the primary affinity proteins and their usefulness has led to a widespread use both in basic and applied research. By means of combinatorial protein engineering and protein library technology, smaller antibody fragments or alternative non-immunoglobulin protein scaffolds can be engineered for various functions based on molecular recognition. In this thesis, a 46 amino acid small albumin-binding domain derived from streptococcal protein G was evaluated as a scaffold for the generation of affinity proteins. Using protein engineering, the albumin binding has been complemented with a new binding interface localized to the opposite surface of this three-helical bundle domain. By using in vitro selection from a combinatorial library, bispecific protein domains with ability to recognize several different target proteins were generated. In paper I, a bispecific albumin-binding domain was selected by phage display and utilized as a purification tag for highly efficient affinity purification of fusion proteins. The results in paper II show how protein engineering, in vitro display and multi-parameter fluorescence-activated cell sorting can be used to accomplish the challenging task of incorporating two high affinity binding-sites, for albumin and tumor necrosis factor-alpha, into this new bispecific protein scaffold. Moreover, the native ability of this domain to bind serum albumin provides a useful characteristic that can be used to extend the plasma half-lives of proteins fused to it or potentially of the domain itself. When combined with a second targeting ability, a new molecular format with potential use in therapeutic applications is provided. The engineered binding proteins generated against the epidermal growth factor receptors 2 and 3 in papers III and IV are aimed in this direction. Over-expression of these receptors is associated with the development and progression of various cancers, and both are well-validated targets for therapy. Small bispecific binding proteins based on the albumin-binding domain could potentially contribute to this field. The new alternative protein scaffold described in this thesis is one of the smallest structured affinity proteins reported. The bispecific nature, with an inherent ability of the same domain to bind to serum albumin, is unique for this scaffold. These non-immunoglobulin binding proteins may provide several advantages as compared to antibodies in several applications, particularly when a small size and an extended half-life are of key importance. / <p>QC 20121122</p>

T cells are a cornerstone of the mammalian adaptive immune system. A range of T-cell subsets exist that can orchestrate the overall immune response to pathogens or cancers, either by directly killing infected cells or licensing other cells to do so. Dysregulation of this important process can result in immunodeficiency or autoimmunity. Although T cells have been studied extensively over many decades, the detailed mechanisms underlying T-cell activation remain to be fully resolved. This thesis describes the development of new optogenetic approaches for the modulation of T-cell signalling dynamics and the interrogation of key events in T-cell activation to help investigate this question. Optogenetics is a rapidly emerging technique whereby light can be used to control the spatial and temporal activation, or inactivation of signalling pathways at unprecedented resolution. The methods described in this work utilise the blue light-responsive LOV2 photo-domain from the common oat A. Sativa, which is the foundation of the both the ‘LOVTRAP’ and ‘TULIPs’ optogenetic toolkits. T-cell antigen receptor (TCR) microclusters arise early during the interaction between T cells and antigen presenting cells (APCs). These TCR signalling platforms contain the proteins necessary for sustained T-cell activation, yet the processes underlying their formation and dissociation are still not fully characterised as they have been difficult to investigate with current chemical and genetic manipulations of T cells. Using two optogenetics systems combining either LOVTRAP or TULIPs and the microcluster- scaffolding protein LAT (Linker for the Activation of T cells), it was possible to modulate early T-cell signalling events and measure functional outputs in real-time. Unfortunately, the biological limitations of these LAT-based systems meant that they could not be used to quantitatively investigate microcluster formation. However, in an alternative approach, a drug-inducible, light-controllable chimeric antigen receptor was successfully developed that yielded important new insights into the rapid rate of signal decay within the TCR signalling pathway and the temporal dynamics of T-cell activation over several timescales. T cell-dependent bispecific antibodies (TDBs) are a new class of immuno-therapeutics that can specifically direct a T-cell response towards tumours, by crosslinking the TCR complex to a surface- expressed target on the cancerous cells. However, their mechanism of action has not been studied in detail. The close apposition of the T cell and target cells driven by the TDB interaction can result in the steric exclusion of phosphatases, such as CD45, away from the TCR at the TDB-generated cell-cell interface due to their large, rigid extracellular domains. Using the myeloma-expressed antigen, FcRH5, it was found that membrane-proximal epitopes of FcRH5 drive more robust TCR clustering and increased CD45 exclusion than membrane distal epitopes, which strongly correlated with effective killing of the target cell. These findings have important implications for therapeutic design and implementation of TDBs.