Approaches are needed for therapy of the severe acute respiratory syndrome from SARS-CoV-2 coronavirus (COVID-19). Interfering with the interaction of viral antigens with the angiotensin converting ...enzyme 2 (ACE-2) receptor is a promising strategy by blocking the infection of the coronaviruses into human cells. We have implemented a novel protein engineering technology to produce a super-potent tetravalent form of ACE2, coupled to the human immunoglobulin γ1 Fc region, using a self-assembling, tetramerization domain from p53 protein. This high molecular weight Quad protein (ACE2-Fc-TD) retains binding to the SARS-CoV-2 receptor binding spike protein and can form a complex with the spike protein plus anti-viral antibodies. The ACE2-Fc-TD acts as a powerful decoy protein that out-performs soluble monomeric and dimeric ACE2 proteins and blocks both SARS-CoV-2 pseudovirus and SARS-CoV-2 virus infection with greatly enhanced efficacy. The ACE2 tetrameric protein complex promise to be important for development as decoy therapeutic proteins against COVID-19. In contrast to monoclonal antibodies, ACE2 decoy is unlikely to be affected by mutations in SARS-CoV-2 that are beginning to appear in variant forms. In addition, ACE2 multimeric proteins will be available as therapeutic proteins should new coronaviruses appear in the future because these are likely to interact with ACE2 receptor.
Infection by the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) causes COVID-19 disease. Therapeutic antibodies are being developed that interact with the viral spike proteins to limit ...viral infection of epithelium. We have applied a method to dramatically improve the performance of anti-SARS-CoV-2 antibodies by enhancing avidity through multimerization using simple engineering to yield tetrameric antibodies. We have re-engineered six anti-SARS-CoV-2 antibodies using the human p53 tetramerization domain, including three clinical trials antibodies casirivimab, imdevimab and etesevimab. The method yields tetrameric antibodies, termed quads, that retain efficient binding to the SARS-CoV-2 spike protein, show up to two orders of magnitude enhancement in neutralization of pseudovirus infection and retain potent interaction with virus variant of concern spike proteins. The tetramerization method is simple, general and its application is a powerful methodological development for SARS-CoV-2 antibodies that are currently in pre-clinical and clinical investigation.
Deep generative models are a class of techniques that train deep neural networks to model the distribution of training samples. Research has fragmented into various interconnected approaches, each of ...which make trade-offs including run-time, diversity, and architectural restrictions. In particular, this compendium covers energy-based models, variational autoencoders, generative adversarial networks, autoregressive models, normalizing flows, in addition to numerous hybrid approaches. These techniques are compared and contrasted, explaining the premises behind each and how they are interrelated, while reviewing current state-of-the-art advances and implementations.
Resolving the genealogy of life-the phylogenetic relationships that describe the evolutionary history of species-remains one of the great challenges of systematic biology. The recent proliferation of ...DNA sequencing technologies has sparked a rapid increase in the volume of genetic data being applied to phylogenetic studies. Single nucleotide polymorphism (SNP) data, ubiquitous genetic markers once considered reserved for population genetic studies, are now being applied in phylogenetics research at deep evolutionary timescales. The potential for SNPs to resolve contentious phylogenetic problems while researchers also investigate population demographics is promising, yet serious challenges remain with respect to data collection, assembly, modeling, and analysis. The low cost and ease of collecting SNPs suggest that they will remain an important source of genetic information for inferring phylogenies across time periods ranging from the Anthropocene to the Cretaceous.
Pancreatic cancer sufferers are faced with a dire prognosis in part because no curative chemotherapy regimen exists. The first-line pancreatic cancer combination therapy of folinic acid, ...5-fluorouracil, irinotecan and oxaliplatin (FOLFIRINOX) comes with significant toxicity and is only tolerable by some patients. Some improvement of the therapeutic index of existing chemotherapies may be achieved by encapsulation of chemotherapies inside nanoparticles (NPs) which have distinct physicochemical characteristics in comparison to free drugs. This enables a host of therapeutically relevant benefits such as avoidance of poorly tolerated solvents (e.g. Cremophor-EL in the case of paclitaxel), overcoming cellular drug efflux, improved pharmacokinetics and exploitation of the enhanced permeability and retention effect. Through these benefits several NPs are approved for use against pancreatic and other cancers. Despite this, there is great scope for improvement of their therapeutic properties in the second generation of nanomedicines. One area of interest is surface modification of the NPs with ligands of specific affinity to improve tumour specific localisation/internalisation and/or produce a therapeutically beneficial effect at the tumour. This concept is explored in the following thesis through conjugation of shark-derived immunoglobulin fragments known as Variable New Antigenic Receptors (VNARs) to the surface of polymeric NPs. This is a novel use of VNARs for which they have many advantageous properties including high physicochemical stability, small size, and low chemical complexity while still maintaining the ability to bind a diverse range of epitopes with high affinity. The possibility of multiple functional groups being available for targeting ligand conjugation is explored in the first chapter of this work. Polymers bearing vinyl sulfone or sulfonyl fluoride groups were obtained or synthesised, respectively. Both functional groups were incorporated within amphiphilic block copolymers which had not been previously investigated for protein conjugation. Vinyl sulfone or sulfonyl fluoride functionalised polymers were blended with PLGA in separate formulations and C-terminal Cys bearing anti Delta-like ligand 4 (DLL4) VNARs conjugated to the resultant NPs. Their binding capabilities were then examined which, while functional, were not as potent as a more routine maleimide PLGA-PEG NP approach. Secondly, the potential of anti-DLL4 VNAR conjugated NPs in pancreatic ductal adenocarcinoma (PDAC) relevant models was investigated. Anti-DLL4 VNARs were covalently conjugated to maleimide functionalised PLGA-PEG NPs to produce conjugates with physicochemical characteristics amenable to therapeutic use and with high specific DLL4 binding affinity. Anti-DLL4 VNAR conjugated NPs were found to preferentially associate with DLL4 expressing PDAC cell lines and showed an anti-angiogenic effect in endothelial cells. This latter effect was enhanced by multimerization of VNARs on the NP surface indicating a distinct advantage of the VNAR conjugated NP concept. Finally, the use of an anti-human serum albumin (HSA) VNAR to control the formation of the NP protein corona in biological media was demonstrated. Formation of a stable HSA corona on NPs then influenced the identity of further corona formation as shown by mass spectrometry. Coronal control also influenced NP cellular interaction, with increased fluorescent NP association and increased camptothecin (CPT) encapsulated NP cytotoxicity observed against PANC-1 cell lines. A pilot in vivo biodistribution study was also performed with fluorescent NPs indicating a different biodistribution profile to naïve control VNAR conjugated NPs and warranting further investigation. In summary this work within this thesis exemplifies the surface modification of polymeric NPs with VNARs and characterises the therapeutic potential of the VNAR NP conjugates for pancreatic cancer therapy.
The SARS-CoV-2 receptor angiotensin converting enzyme 2 (ACE2) was previously engineered into a high affinity tetravalent format (ACE2-Fc-TD) that is a potential decoy protein in SARS-CoV-2 ...infection.We report that this protein shows greatly enhanced binding to SARS-CoV-2 spike proteins of the SARS-CoV-2 variants of concern B.1.1.7 (alpha variant, originally isolated in the United Kingdom) and B.1.351 (beta variant, originally isolated in South Africa) with picomolar compared with nanomolar Kd values. In addition, ACE2-Fc-TD displays greater neutralization of SARS-CoV-2 pseudotype viruses compared to a dimeric ACE2-Fc, with enhanced activity on variant B.1.351. This tetrameric decoy protein would be a valuable addition to SARS-CoV-2 therapeutic approaches, especially where vaccination cannot be used but also should there be any future coronavirus pandemics.
•Soluble ACE2 coronavirus receptor neutralizes SARS-CoV-2 infection.•SARS-CoV-2 neutralization is enhanced by tetramerization of ACE2.•Tetrameric ACE2 is more potent than a dimeric form.•SARS-CoV-2 variants of concern are neutralized by tetrameric ACE2.•Tetrameric ACE2 decoy protein could be used in future, new coronavirus pandemics.
Whilst there is an extensive body of preclinical nanomedicine research, translation to clinical settings has been slow. Here we present a novel approach to the targeted nanoparticle (NP) concept: ...utilizing both a novel targeting ligand, VNAR (Variable New Antigen Receptor), a shark-derived single chain binding domain, and an under-investigated target in delta-like ligand 4 (DLL4). We describe the development of an anti-DLL4 VNAR and the site-specific conjugation of this to poly(lactic-co-glycolic) acid PEGylated NPs using surface maleimide functional groups. These nanoconjugates were shown to specifically bind DLL4 with high affinity and were preferentially internalized by DLL4-expressing pancreatic cancer cell lines and endothelial cells. Furthermore, a distinct anti-angiogenic effect endowed by the anti-DLL4 VNAR was evident in in vitro tubulogenic assays. Taken together these findings highlight the potential of anti-DLL4 targeted polymeric NPs as a novel therapeutic approach in pancreatic cancer.
The continued presence and importance of Christian moral values in our daily lives, coupled with the fact that faith in Christianity is in continual decline, raises the question as to why having lost ...faith in Christianity, we have also not lost faith in our Christian moral values. This question is also indicative of a more pressing phenomenon: not only have we maintained our faith in Christian values, we fail to see that the widespread collapse of Christianity should affect this faith. To tackle this latter phenomenon, I claim, we have to pose the Nietzschean question of the value of our moral values, so as to see that this value can be a possible object of questioning. In chapter one, I consider different approaches found in the history of moral philosophy that look like potential candidates for this task. I argue that, ultimately, the task requires simultaneously taking our familiarity with Christian moral values as both sui generis and a questionable phenomenon. In chapter two, I articulate in detail the sui generis nature of this familiarity with moral values,in terms of the phenomena of habituation and sedimentation. In chapter three, I consider the possibility of estrangement that is built into our familiarity with moral values, by focusing on the role of cognition. I demonstrate how cognition, in the form of self-consciousness, can disrupt the sedimented, habituated nature of our moral values through a form of ironic disruption. In chapter four, I develop this account by considering the possibility of an appeal to an alternative moral outlook. To do so, I draw upon the structural isomorphism that is present between the process of estrangement and a rite of passage.
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