5,856 research outputs found

    Pathophysiological role and therapeutic potential of extracellular vesicles in cancer

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    Extracellular vesicles (EVs) are nanosized lipid bilayer vesicles that are endogenously generated through various biogenesis pathways within most cellular entities. Subsequently, they are released into the extracellular milieu to facilitate intercellular communication. They are composed of diverse bioactive molecules with important roles in physiological and pathological states. Over the past few decades, the therapeutic potential of EVs has garnered significant interest in the drug delivery field. However, deepened understanding of EV biology and further technological advances are needed to bridge the gap between research and clinical translation. In this thesis, we address these challenges and investigate EVs as novel biomedical agents. EVs are crucial components of physiological processes and disease development. Sensitive visualisation techniques are needed to better understand their function as therapeutic agents. In paper I, a bioluminescent labelling system was developed to track EVs in vitro and in vivo. The system uses genetic modifications to enable the encapsulation of sensitive luciferase-variants in EVs. The system was used in vivo to enable highly sensitive detection of EV distribution pattern. Exogenously administered EVs were found to rapidly distribute within different organs, with a preference for the spleen, lung, and liver. In addition to endogenously engineered EVs for in vivo tracking, exogenously engineered EVs can be utilised as promising drug delivery platforms. However, cargo loading is often insufficient, requiring improved EV loading approaches. In paper II, we developed an optimised cargo loading method using electroporation. An optimised protocol was designed to load EVs with doxorubicin, which increased cargo loading, EV recovery, and drug potency by 190-fold over free doxorubicin. Owing to their potential to cross biological barriers, transport bioactive cargo, and targetability, EVs can be exploited as delivery vehicles for targeting of therapeutics. EVs were used as delivery vectors in paper III by coating their surfaces with an Fc domain-specific antibodybinding moiety. These Fc-EVs were then decorated with various IgG antibodies and targeted to cells of interest. In vitro and in vivo antibody targeting studies showed the broad potential of this technology for cancer therapy. The platform efficiently targeted EVs to cancer cells, including HER2 and PD-L1 positive cells. As proof of concept, Fc-EVs with PD-L1 antibody accumulate in tumour tissue and, when loaded with doxorubicin, reduce tumour burden, and increase survival in melanoma-bearing mice. Despite significant EV engineering advances, we have a limited understanding of the biology of tumour-derived extracellular vesicles (tEVs). In paper IV, we investigated the role of in vitrogenerated melanoma-derived EVs as indirect communicators in tumour-induced haematopoiesis dysregulation. The tEVs, which contain high levels of angiogenic factors like VEGF, osteopontin, and tissue factor, were found to cause splenomegaly, extramedullary haematopoiesis, expansion of splenic immature erythroid progenitors, reduced bone marrow cellularity, medullary expansion of granulocytic myeloid suppressor cells, and anaemia in syngeneic mice. These findings suggest that tEVs dysregulate haematopoiesis during the immune escape phase of cancer immunoediting, making them potential targets for overcoming immune evasion and restoring normal haematopoiesis. To summarise, the tools generated in this thesis, including the ability to detect EVs in vivo, effective cargo loading, display antibody binding moieties on EV surfaces for targeting, and understanding the pathophysiological role of tEVs, contribute to the advancement of EVs for biomedical purposes, and clinical translation down the line

    Towards detection of structurally-diverse glycated epitopes in native proteins : single-chain antibody directed to non-A1c epitope in human haemoglobin

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    Over 500 million people worldwide are affected by diabetes mellitus, a chronic disease that leads to high blood glucose levels and causes severe side effects. The predominant biological marker for diagnosis of diabetes is glycated haemoglobin (GHb). In human blood the predominant reducing sugar, glucose, irreversibly conjugates onto accessible amine groups within Hb. Most methods for diagnosis and monitoring of diabetes selectively detect N-terminal glycation at Val-1 on the β-globin chain, but not glycation at other sites. Detection of other glycated epitopes of GHb has the potential to provide new information on the extent, duration and timing of elevated glucose, facilitating personalised diagnosis and intelligent diabetic control. In this work, a new anti-GHb Fab antibody (Fab-1) specific for haemoglobin A1c (HbA1c) with nanomolar affinity was discovered via epitope-directed immunisation and phage display. A single chain variable fragment (scFv) antibody derived from Fab-1 retained affinity and specificity for HbA1c, and affinity was enhanced tenfold upon addition of an enhanced green fluorescent protein tag. Both the scFv and Fab-1 recognised an epitope within HbA1c that was distinct from β-Val-1, and our data suggest that this epitope may include glycation at Lys-66 in the β-globin chain. To our knowledge, this is the first report of an scFv/Fab anti-glycated epitope antibody that recognises a non-A1c epitope in GHb, and confirms that fructosamine attached to different, discrete glycation sites within the same protein can be resolved from one another by immunoassay. [Abstract copyright: Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.

    Viability of calcinated wastepaper sludge ash geopolymer in the treatment of road pavement subgrade materials

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    Problematic ground conditions constituted by weak or expansive clays are commonly encountered in construction projects and require some form of chemical treatment such as lime and cement to re-engineer their performance. However, in the light of the adverse effects of these traditional additives on the climate, alternative eco-friendlier materials are now sourced. In the current study, the viability of calcinated wastepaper sludge ash geopolymer in enhancing the engineering behaviour of a problematic site condition is evaluated. A highly expansive clay (HEC) constituted with a blend of kaolinite and bentonite clays is treated with calcinated wastepaper sludge ash (CPSA) geopolymer. Activation of the precursor is actualised at room temperature using a combination of NaOH and Na2SiO3 at various activator to soil+binder ratios (AL/P), and molarity (M). The mechanical, microstructural, and mineralogical characteristics of the treated clay were investigated through unconfined compressive strength (UCS), swell, water absorption, SEM, and EDX analysis. The performance of the stabilised samples was then compared with the requirements for road subgrade and subbase materials and that of OPC and lime-GGBS treatment. The results showed that CPSA-geopolymer enhanced the engineering properties of the treated clay better than traditional binders (OPC and Iime-GGBS). UCS improvement of 220% was observed in the CPSA-stabilised soil over that of OPC-treated ones, while the swell potential and water absorption were drastically reduced by over 95 and 97% respectively after 28-day soaking. The SEM and EDX results showed improved crystallisation of earth-metal-based cementitious flakes (NASH) with increasing CPSA, molarity, and AL/P ratios, which enhanced the inter-particle bonds with simultaneous reduction in porosity. The modified characteristics of the stabilised materials meet the requirements for pavement subgrades. Further, the equivalent carbon emission (CO2-e) from the stabilised materials were also evaluated and compared with that of traditional binders. The results also showed that CPSA-geopolymer had lower CO2-e at higher subgrade strengths than OPC, making it more eco-friendly. Therefore, wastepaper sludge, a common landfill waste from paper recycling is a viable geopolymer precursor that could be utilised in enhancing the engineering properties of subgrade and sub-base materials for road and foundation construction

    Design and Synthesis of a Novel Class of RNA Polymerase I Inhibitors

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    RNA Polymerase I (Pol I) is one of three DNA-dependent RNA polymerases and is responsible for transcription of the 47S ribosomal RNA (rRNA) precursor. The 47S rRNA transcript is subsequently processed to release the 18S, 5.8S, and 28S rRNAs which are assembled into ribosomes. Pol I transcription serves as the rate-limiting step in ribosome biogenesis, accounting for up to 60% of active transcription in eukaryotic cells, directly influencing protein accumulation, cell growth, and cell division. Many types of cancers exhibit dysregulated rates of Pol I transcription, reflecting a need for increased ribosome synthesis to generate proteins to sustain heightened growth rates. Cancer cells may be selectively vulnerable to agents that inhibit Pol I transcription, providing an attractive therapeutic strategy for cancer treatment. BMH-21 is the first specific and selective Pol I inhibitor and does so by intercalating into GC-rich rDNA and creating a transcription block, leading to the ubiquitination and proteasomal degradation of the large catalytic subunit, RPA194. Notably, it accomplishes this independently of p53 and without eliciting a DNA damage response. BMH-21 is the first of only a small number of compounds to exhibit the RPA194 degradation phenotype, and a quantitative cell-based assay has been developed to measure the extent of RPA194 degradation caused by compound treatment. The primary goal of this work is to design and synthesize small molecule inhibitors of Pol I, with a focus on determining key pharmacophores and generating structure-activity relationship (SAR) data. SAR studies revealed key pharmacophores, but also showed that activity was limited within narrow chemical space. Further SAR efforts, summarized by this work, have produced additional scaffolds as well as addressed some off-target activity while maintaining desired RPA194 degradation potency. In collaboration with Evotec, the RPA194 degradation assay was translated from 96-well plate format to 384-well plate format to facilitate high-throughput screening (HTS) efforts, providing the opportunity to discover new Pol I inhibitors and to generate new SARs. Efforts were made to transform BMH-21 into a chemical probe to gain structural insight about its binding interactions and to identify its molecular target, providing rationale for the improved design of future compounds

    Bispecific killer cell engager with high affinity and specificity toward CD16a on NK cells for cancer immunotherapy

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    IntroductionThe Fc region of monoclonal antibodies (mAbs) interacts with the CD16a receptor on natural killer (NK) cells with “low affinity” and “low selectivity”. This low affinity/selectivity interaction results in not only suboptimal anticancer activity but also induction of adverse effects. CD16a on NK cells binds to the antibody-coated cells, leading to antibody-dependent cell-mediated cytotoxicity (ADCC). Recent clinical data have shown that the increased binding affinity between mAb Fc region and CD16a receptor is responsible for significantly improved therapeutic outcomes. Therefore, the objective of this study was to develop a bispecific killer cell engager (BiKE) with high affinity and specificity/selectivity toward CD16a receptor for NK cell-based cancer immunotherapy.MethodsTo engineer BiKE, a llama was immunized, then high binding anti-CD16a and anti-HER2 VHH clones were isolated using phage display. ELISA, flow cytometry, and biolayer interferometry (BLI) data showed that the isolated anti-CD16a VHH has high affinity (sub-nanomolar) toward CD16a antigen without cross-reactivity with CD16b-NA1 on neutrophils or CD32b on B cells. Similarly, the data showed that the isolated anti-HER2 VHH has high affinity/specificity toward HER2 antigen. Using a semi-flexible linker, anti-HER2 VHH was recombinantly fused with anti-CD16a VHH to create BiKE:HER2/CD16a. Then, the ability of BiKE:HER2/CD16a to activate NK cells to release cytokines and kill HER2+ cancer cells was measured. As effector cells, both high-affinity haNK92 (CD16+, V176) and low-affinity laNK92 (CD16+, F176) cells were used.Results and discussionThe data showed that the engineered BiKE:HER2/CD16a activates haNK92 and laNK92 cells to release cytokines much greater than best-in-class mAbs in the clinic. The cytotoxicity data also showed that the developed BiKE induces higher ADCC to both ovarian and breast cancer cells in comparison to Trazimera™ (trastuzumab). According to the BLI data, BiKE:HER2/CD16 recognizes a different epitope on CD16a antigen than IgG-based mAbs; thus, it provides the opportunity for not only monotherapy but also combination therapy with other antibody drugs such as checkpoint inhibitors and antibody-drug conjugates. Taken together, the data demonstrate the creation of a novel BiKE with high affinity and specificity toward CD16a on NK cells with the potential to elicit a superior therapeutic response in patients with HER2+ cancer than existing anti-HER2 mAbs

    Raveling Performance of Conventional and Rubberized Chip Seal under Field and Laboratory Traffic Loading

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    Raveling, or the Loss of Surface Aggregate, is a Major Concern in Chip Seal Pavement. Previous Studies Have Used the Standard Sweep, Vialit, and Pennsylvania Tests to Evaluate Raveling, But These Tests Do Not Replicate the Stresses of Rolling Vehicle Tires on Chip Seal and Have Produced Inconsistent Results. Additionally, These Tests Mainly Evaluate Early-Stage Raveling Due to their Short Curing Time. in This Study, a Small-Wheel Traffic Simulation Device (SWTS) Was Used to Apply Similar Stresses as Rolling Vehicle Tires and Produce Comparable Raveling Results. This Was Confirmed through a 13-Month Field Observation of Chip Seal, Where the Laboratory and Field Data Were Compared and Correlated. Both Conventional (Mineral Aggregate) and Eco-Friendly Rubberized Chip Seal with 25%, 50%, and 100% Crumb Rubber as Aggregate Were Tested in the Laboratory and Field. the Effects of Different Aggregate Types, Binder Application Rates, and Crumb Rubber Contents Were Studied. the Results Showed that Each Wheel Application by SWTS is Equivalent to 2.5 to 3 Passenger Car Passes in the Field. It Was Also Found that the Majority of Macrotexture Loss Occurred in the First 50,000 Passenger Car Equivalent Load. Up to 50% Replacement of Mineral Aggregate with Crumb Rubber Did Not Significantly Affect the Raveling Resistance But using More Than 50% Rubber Impaired the Raveling Resistance. using Crumb Rubber Instead of Mineral Aggregate Also Significantly Reduced Tire Wear and Microplastic Emissions from Vehicle Tires. Moreover, It Was Found that Pennsylvania and Vialit Tests May Not Be Suitable for Assessing Aggregate Retention under Traffic Load

    Use of TCR antibody fusion proteins as bispecific agents for NK and T cell-mediated immunotherapy

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    Bispecific agents are a promising approach for cancer immunotherapy as they enable the redirection of cytotoxic lymphocytes towards tumor cells by targeting different structures on the tumor cell surface and triggering cytotoxic lymphocytes through agonistic binding to activating receptors. Targeted tumor antigens can be roughly divided into peptide antigens presented by major histocompatibility complex (MHC) class I molecules to antigen-specific T cell receptors and cell surface antigens recognized by antibodies. Peptides presented in the context of an MHC-I molecule mostly result from intracellular proteins, which make up the majority of the proteome, and thus serve as an highly attractive target for immunotherapeutic strategies. In contrast to TCR-transgenic T cells that express novel TCR specificities in the natural cellular context, the development of soluble TCR-based therapies is hampered due to the poor stability of recombinant TCRs and a generally significantly lower affinity in comparison to therapeutic antibodies. In the past years progress has been made to address these problems, which include different modifications improving construct stability and methods for TCR affinity maturation enabling the development and clinical application of first TCR-based bispecific recombinant fusion proteins that retarget T cells to tumors displaying intracellular antigens through MHC molecules. This study aimed to develop novel soluble bispecific TCR-based agents for the redirection of NK and T cells. To achieve potent bispecific mediators a bivalent immunoglobulin G (IgG)-like TCR-Fc fusion format was adapted. The ectodomain of the TCR V/C chain was fused to the hinge/Fc part of human IgG1 and the ectodomain of the TCR V/C chain was expressed as a second soluble protein in cis using a ribosomal skipping sequence. Efficient assembly of TCR and chains was facilitated by an additional artificial intermolecular disulfide bridge in the TCR constant domains. The natural intermolecular disulfide bonds of the human IgG1 hinge region enabled assembly of TCR-Fc fusion proteins to stable homodimers which could be successfully expressed by transient transfection of CHO-S producer cells. To enable NK cell redirection, specific mutations known to enhance FcRIIIa binding were introduced in the Fc fragment. Other investigated formats made use of single chain variable fragments (scFv) recognizing CD16 or NKp46 for NK cell redirection or binding CD3 for the redirection and activation of T cells. NK- and T-cell-binding scFv antibodies were analyzed after insertion at C-terminal end of TCR C or C, respectively. A cytomegalovirus (CMV) pp65 peptide/HLA-A*02:01-specific TCR sequence was used as a model system. To analyze the influence of TCR affinity, the wildtype TCR was compared to an affinity-maturated variant. The soluble IgG1-like TCRs showed a target-specific, affinity- and concentrationdependent binding as well as NK and T cell redirection and activation upon co-culture with peptide-pulsed or transfected cells. In particular, the TCR-NKp46-Fcenhanced and TCR-CD3-Fcaglycan constructs were found to be highly potent in redirecting NK and T cells, respectively. TCR-scFv-Ig fusion proteins efficiently elicited peptide antigen-specific activation of purified NK cells and T cells and induced cytotoxicity against different tumor targets. These results were further confirmed using low- and and high-affinity variants of three TCRs recognizing HLA-A*02:01-restricted gp100, MART-1 and NY-ESO-1 peptides, respectively, for the redirection of NK cells. TCRhigh aff. -NKp46-Fcenh constructs, however, failed to facilitate NK cytotoxicity against HLA-A2+ melanoma cell lines expressing the antigens of choice unless the cell lines were incubated with an excess of the cognate HLA-A2-binding peptide. Thus, the NK cell-engaging TCR-Fc fusion proteins constructs apparently were not sensitive enough to redirect and activate NK cells against melanoma cells presenting low quantities of naturally processed specific peptide/MHC-I complexes. In sum, in this work a panel of novel recombinant bispecific TCR-Fc and TCR-scFv-Fc fusion proteins were genetically engineered, produced and demonstrated to facilitate the activation and cytotoxicity of NK and T cells towards tumor cells expressing a model peptideMHC-I complex in sufficient quantities. Further investigations are required to investigate if the avidity of soluble TCR-Fc constructs can be sufficiently increased by multimerization approaches in order to overcome the sensitivity issues resulting from low abundance of peptide-MHC-I complexes and low-affinity TCRs

    Rapid escape of new SARS-CoV-2 Omicron variants from BA.2-directed antibody responses

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    In November 2021, Omicron BA.1, containing a raft of new spike mutations, emerged and quickly spread globally. Intense selection pressure to escape the antibody response produced by vaccines or severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection then led to a rapid succession of Omicron sub-lineages with waves of BA.2 and then BA.4/5 infection. Recently, many variants have emerged such as BQ.1 and XBB, which carry up to 8 additional receptor-binding domain (RBD) amino acid substitutions compared with BA.2. We describe a panel of 25 potent monoclonal antibodies (mAbs) generated from vaccinees suffering BA.2 breakthrough infections. Epitope mapping shows potent mAb binding shifting to 3 clusters, 2 corresponding to early-pandemic binding hotspots. The RBD mutations in recent variants map close to these binding sites and knock out or severely knock down neutralization activity of all but 1 potent mAb. This recent mAb escape corresponds with large falls in neutralization titer of vaccine or BA.1, BA.2, or BA.4/5 immune serum
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