Algorithm-Based Liquid Formulation Development Including a DoE Concept Predicts Long-Term Viral Vector Stability.

An Antibody Delivery System for Regulated Expression of Therapeutic Levels of Monoclonal Antibodies In Vivo

An Antibody Delivery System for Regulated Expression of Therapeutic Levels of Monoclonal Antibodies In Vivo

Strategies to Modulate Immune Responses: A New Frontier for Gene Therapy

Plants have a demonstrated potential for large-scale, rapid production of recombinant proteins for diverse product applications, including subunit vaccines and monoclonal antibodies. In this field, the accent has recently shifted from the engineering of "edible" vaccines based on stable expression of target protein in transgenic or transplastomic plants to the development of purified formulated vaccines that are delivered via injection. The injectable vaccines are commonly produced using transient expression of target gene delivered into genetically unmodified plant host via viral or bacterial vectors. Most viral vectors are based on plant RNA viruses, where nonessential sequences are replaced with the gene of interest. Utilization of viral hybrids that consist of genes and regulatory elements of different virus species, or transcomplementation systems (vector/transgene) had a substantial impact on the level of target protein expression. Development and introduction of agroviral hybrid vectors that combine genetic elements of bacterial binary plasmids and plant viral vectors, and agroinfiltration as a tool of the vector delivery have resulted in significant progress in large-scale production of recombinant vaccines and monoclonal antibodies in plants. This article presents an overview of plant hybrid viral vector expression systems developed so far.

Viruses are widely modified and used as gene delivery vectors for various applications in science and therapeutics. To this end, my thesis focuses on modifying the recombinant adeno-associated viral (rAAV) vectors that are identified as a safer choice for cargo delivery compared to other known viral vectors. They are widely used in the scientific communities, have seen promising outcomes in gene therapy clinical trials, and as of today have three products approved to use in humans. However, the natural repertoire of rAAVs have broad tropism when delivered systemically, and there is room for further improvement on the efficiency and specificity, especially for gene delivery in the central nervous system (CNS). The prior work done in Dr. Gradinaru lab addresses the issue by using a directed evolution approach called CREATE, Cre recombination-based AAV targeted evolution, to identify AAV-PHP.B and AAV-PHP.eB capsids, which broadly transduce the CNS (Deverman et al, 2016; Chan et al, 2017). CREATE selects for functional lox-flipped viral DNA that crosses the blood-brain barrier (BBB) and successfully transduces a specific nerve cell-type expressing Cre, thereby applying a strong selection pressure. However, the method is limited by its ability to identify a handful of enriched variants, and may also be prone to false positives resulting from experimental biases. The effort to fully understand the selection landscape, and to select for capsids that are not just efficient towards a cell-type but also specific towards it, led to the development of Multiplexed-CREATE (M-CREATE). M-CREATE allows parallel positive selections across different cell-types of interest, enables post-hoc negative selections across off-targets using a next-generation sequencing (NGS) based capsid recovery, and retains the principles of Cre-dependent functional recovery from CREATE. The method has a synthetic library generation approach to minimize biases within selection rounds, a variant replicate feature to identify the signal versus noise within a biological system, and an analysis pipeline to group families of enriched variants based on amino acid motifs, all of which together increases the confidence in the outcome and the throughput from a single experiment. Selections across brain endothelial cells, neurons, and astrocytes yielded several AAV-PHP.B-like variants that broadly transduce the CNS, AAV-PHP.V variants that can efficiently transduce the vascular cells forming the BBB, a AAV-PHP.N variant that transduces neurons with greater specificity, and AAV-PHP.C variants that cross the BBB without murine strain specificity across tested strains. The AAV-PHP.C variants have different amino acid motifs compared to the AAV-PHP.Bs that have been previously shown to have limited CNS transduction across some mouse strains due to its interaction with the strain specific host cell surface receptor, ly6a, a homolog of which is not found in humans. (Hordeaux et al, 2018, Hordeaux et al, 2019; Huang et al, 2019; Batista et al, 2019) Therefore AAV-PHP.Cs offer some hope towards translation across other species. In summary, the M-CREATE methodology turns out to be a high-confidence, robust selection platform to yield several novel viral capsids for use in neuroscience and potential gene therapy related applications.

312. Clearance of Helper Adenovirus by an AAV1 Clinical Vector Manufacturing Process

Formulating monoclonal antibodies as powders for reconstitution at high concentration using spray-drying: Trehalose/amino acid combinations as reconstitution time reducing and stability improving formulations

Adeno-Associated Virus Vectors in Clinical Trials

The nuclear thermal rocket (NTR) provides a unique propulsion capability to planners/designers of future human exploration missions to the Moon and Mars. In addition to its high specific impulse (approximately 850-1000 s) and engine thrust-to-weight ratio (approximately 3-10), the NTR can also be configured as a 'dual mode' system capable of generating electrical power for spacecraft environmental systems, communications, and enhanced stage operations (e.g., refrigeration for long-term liquid hydrogen storage). At present the Nuclear Propulsion Office (NPO) is examining a variety of mission applications for the NTR ranging from an expendable, single-burn, trans-lunar injection (TLI) stage for NASA's First Lunar Outpost (FLO) mission to all propulsive, multiburn, NTR-powered spacecraft supporting a 'split cargo-piloted sprint' Mars mission architecture. Each application results in a particular set of requirements in areas such as the number of engines and their respective thrust levels, restart capability, fuel operating temperature and lifetime, cryofluid storage, and stage size. Two solid core NTR concepts are examined -- one based on NERVA (Nuclear Engine for Rocket Vehicle Application) derivative reactor (NDR) technology, and a second concept which utilizes a ternary carbide 'twisted ribbon' fuel form developed by the Commonwealth of Independent States (CIS). The NDR and CIS concepts have an established technology database involving significant nuclear testing at or near representative operating conditions. Integrated systems and mission studies indicate that clusters of two to four 15 to 25 klbf NDR or CIS engines are sufficient for most of the lunar and Mars mission scenarios currently under consideration. This paper provides descriptions and performance characteristics for the NDR and CIS concepts, summarizes NASA's First Lunar Outpost and Mars mission scenarios, and describes characteristics for representative cargo and piloted vehicles compatible with a reference 240 t-class heavy lift launch vehicle (HLLV) and smaller 120 t HLLV option. Attractive performance characteristics and high-leverage technologies associated with both the engine and stage are identified, and supporting parametric sensitivity data is provided. The potential for commonality of engine and stage components to satisfy a broad range of lunar and Mars missions is also discussed.

There is an increased interest from industry, academia and regulators for protein aggregates, subvisible and visible particles due to possible biological consequences, such as immunogenicity, altered bioactivity and modified pharmacokinetic profiles. Aggregates, subvisible and visible particles are important product instabilities, which might be present in every formulation of biotherapeutic products like monoclonal antibody solutions. Especially, the presence of subvisible particles in biotherapeutic products is currently a hot topic and it constantly gains more importance. The ultimate goal of this thesis is to develop tools and techniques in order to be able to characterize well-defined size fractions of proteinaceous subvisible particles with various desired oxidation profiles using in vivo transgenic mouse model. Up to now only a few articles were published and the available data from in vitro and in vivo experiments on aggregates and subvisible particles is often conflicting and fragmented, which impedes the development of sound conclusions. Moreover, complex mixtures of monomers, aggregates, particles and other degradants were used to draw conclusions. Only estimated values of protein particle density were used up to now in published studies although it is required to know the density of the measured particles in order to accurately calculate their dimensions and mass. The first aim of this thesis was therefore to develop a method to measure experimentally the protein particle density without extrapolation (Chapter 1). The density for commercially available standard beads (polystyrene, polymethacrylate and melamine) and a large bench of stressed proteinaceous samples was determined with the use of the resonant mass measurement instrument (RMM, Archimedes) and its ability to measure the buoyant mass of individual particles. Various fluids with increasing densities were implemented in order to determine the neutral buoyant mass where the particle density equals the fluid density. Chapter 2 reports the development of a process to isolate well-defined subvisible fractions using differential centrifugation for a model IgG1 antibody. The process to separate four fractions in the submicron and micrometer size range was developed and successfully optimized through the use of a design of experiments. The centrifugation technique was compared to an already published fractionation method using a preparative fluorescence-activated cell sorter. Efficiency, advantages and drawbacks for both methods were compared and discussed (Chapter 2). Oxidation profile of aggregates and subvisible particles seem to be an important attribute regarding induced biological consequences. That is why the next goal was to develop a method for selective oxidation of methionine and tryptophan residues in a model mAb in order to be able to delineate the effects and the contribution of individual protein modifications in the primary structure. This included a large set of experiments where different reaction conditions such as temperature of incubation, reaction time, type and concentration of oxidant (t-BHP, H2O2, AAPH) were evaluated in presence (or not) of a large excess of anti-oxidant (free amino acids) in order to protect the corresponding amino acid in a model antibody of the IgG1 subtype (Chapter 3). To complete the work, unfractionated materials and well-defined size fractions (with well-established oxidation profile) were prepared using the established tools (Chapter 1-3). Those samples were deeply characterized and injected subcutaneously into wild type and transgenic mice for immunization. Anti-drug antibody levels were measured following ELISA in order to assess the immunogenic potential of those preparations (Chapter 4).

Huntington’s disease (HD) is a devastating, genetic, neurodegenerative disease for which there is currently no effective therapy. The polyglutamine (polyQ) expansion that causes HD is in the first exon (HDx1) of huntingtin (Htt). However, other parts of the protein, including the 17 N-terminal amino acids (AAs) and two proline (polyP) repeat domains, modulate the toxicity of mutant Htt (mHtt). The role of the P-rich domain that is flanked by the polyP domains has not been explored. Using highly specific intracellular antibodies (intrabodies; iAbs), we tested various epitopes for their roles in mHDx1 toxicity, aggregation, localization and turnover. Three domains in the P-rich region (PRR) of HDx1 are defined by iAbs: MW7 binds the two polyP domains, and Happs 1 and 3, two new iAbs, bind the unique, P-rich epitope located between the two polyP epitopes. In cultured cells, we find that the three PRR-binding iAbs, as well as VL12.3, which binds an epitope in the N-terminal 17 AA segment, decrease the toxicity and aggregation of mHDx-1, but they do so by different mechanisms. The PRR-binding iAbs have no effect on Htt localization, but they cause a significant increase in the turnover rate of mHtt, which VL12.3 does not change. In contrast, expression of VL12.3 increases nuclear Htt. These results suggest that the PRR domain regulates mHtt stability and toxicity. Thus, compromising this pathogenic epitope by iAb binding represents a novel therapeutic strategy for treating HD. We have tested this hypothesis by delivering both VL12.3 and Happ1 to the brains of HD model mice using an AAV2/1 viral vector with a modified CBA promoter. VL12.3 treatment, while beneficial in a lentiviral model of HD, has no effect on the YAC128 HD model and actually increases severity of phenotype and mortality in the R6/2 HD model. In contrast, Happ1 treatment confers significant beneficial effects in assays of motor and cognitive deficits as well as in the neuropathology found in the lentiviral, R6/2, N171-82Q, YAC128 and BACH models of HD. These results indicate that increasing the turnover of mHtt using AAV-Happ1 gene therapy represents a highly specific and effective treatment possibility for HD.

Upon exposure to DNA damage the p53 tumor suppressor is accumulated and activated to stall cellular growth. For this to occur, p53 must be relieved from its major inhibitors, Mdm2 (Hdm2 in humans) and Mdmx (Mdm4; Hdmx in humans). A key mechanism controlling this relief is the post-translational modifications of p53 and its inhibitors. We have previously demonstrated that the stress-activated tyrosine kinase, c-Abl, contributes to the relief of p53 from Hdm2. Because Hdmx is the major inhibitor of p53 activity, the additional possibility that c-Abl protects p53 through targeting Hdmx was explored in this study. c-Abl was found to interact with and to phosphorylate Hdmx. This phosphorylation was enhanced in response to DNA damage. Importantly, we mapped the sites of phosphorylation to the p53 binding domain of Hdmx. One of these phosphorylations, on tyrosine 99, inhibited Hdmx interaction with p53. This inhibition is consistent with the predicted role of this residue in the interaction with p53 based on the crystal structure of the interaction site. Our results show that c-Abl not only targets Hdm2, but also Hdmx, which together contribute to p53 activation in response to DNA damage.

Corrigendum to “Long-term liquid storage and reproductive evaluation of an innovative boar semen extender (Formula12®) containing anon-reducing disaccharide and an enzymatic agent” [Anim. Reprod. Sci. 180 (2017) 10–16

Introduction: Monoclonal antibodies (mAB) are highly effective drugs for treatment of cancer and other diseases.Production and development of mAB require physicochemical characterization of several Critical Quality Attributes (CQA), including post-translational-modifications, disulfide bond and amino acid primary sequence mapping.In this study we aim to use design of experiments (DoE) to improve both sample preparation and nLC-MS/MS analysis by decreasing protein digestion time, artificial amino acid modifications, increasing protein sequence coverage and analysis throughput.We aim to develop an optimized nano LC method with in-house produced columns capable of characterizing CQA of mAB.Objectives: Optimization of protein coverage and relative quantification of PTM by nLC-MS/MS.Methodology: Nivolumab and pembrolizumab were used in this study.For DoE, a full factorial 2 2 or 2 4 with or without central point were used.For protein digestion protocol, different parameters were compared: time of digestion, digestion buffer and denaturation agent.Chromatographic conditions were tested, varying gradient and sample injected volume for both mAB peptide and disulfide bond mapping methods by nLC-MS/MS.The nLC-MS/MS was carried out on an Ultimate 3000 coupled with an Orbitrap Exploris 120.Peptides were sequenced using Biopharma Finder 5.1.Results: DoE is incredibly challenging for optimization of peptide mapping protocols.Several responses must be considered to compare different analysis conditions.Protein coverage, by itself, is a poor response for method evaluation.The use of guanidine HCl without desalting prior to digestion decreases digestion efficiency but increased light chain coverage.Digestion time of 1h reduces asparagine deamidation by over 30x in comparison to 18 h.CaCl 2 as a stabilizer for trypsin didn't increase the efficiency of protein digestion.Sequencing grade trypsin can be used for 1 hour at a temperature of 47 C.Other temperature conditions may be tested.The use of 50 mM Tris-HCl pH 8.0 as a digestion buffer instead of ammonium bicarbonate had no impact on the results of peptide mapping of nivolumab.The gradient of 120 min for separation of protein digest showed no advantage over 60 min gradient. Conclusion:A robust peptide mapping by LC-MS/MS method can be achieved with 60 min digestion and 60 min gradients with high protein coverage and little artificial modification of residues due to sample preparation using nLC and in house produced columns.

Physiological characteristics of epididymal spermatozoa from postmortem rams