Engineered Antibody and Monobody Domains with T Cell Receptor-Like Selectivity for Tumor Associated Peptide-MHC Antigens

Monoclonal antibody (mAb)-based therapeutics have established themselves as meaningful components of the treatment paradigm for a variety of tumors. However, since the approval of rituximab in 1997 as the first mAb-based therapy for cancer, there has been a paucity of novel, validated cancer targets for therapeutic intervention by mAbs. In effect, numerous challenges lie in the discovery of suitable extracellular or transmembrane antigens that permit the differentiation of tumor from healthy tissue. The adaptive immune system, though, mediates recognition of foreign antigens derived from the intracellular proteome by T cell receptor (TCR) binding to peptide-loaded major histocompatibility complex (pMHC) molecules. Because cancer is associated with large-scale alterations in the genome, there are a vast number of novel epitopes presented to the adaptive immune system. Although natural TCRs have exquisite functionality in distinguishing these foreign epitopes, and several tumor-reactive TCRs have, in fact, been characterized, the molecules themselves are poorly developable as therapeutic candidates. Thus, in order to enable TCR-like binding of a broader class of protein agents, this study explores the transfer of TCR binding domains to other mAb-based scaffolds, including the fibronectin-derived Fn3 and the IgG-derived 4D5 scaffolds. By using a combination of rational design and directed evolution to guide binding domain transfer, evidence for TCR-like binding was demonstrated for several engineered molecules. In addition to conferring binding functionality, the grafted TCR domains had a deleterious effect on the biophysical properties of these inherently robust protein scaffolds. Thus, this work provides novel insight into the objective of developing mAb-based agents with TCR-like binding specificity for pMHC antigens, informing future efforts to target the abundance of intracellular tumor epitopes

Frontiers in microfluidics, a teaching resource review

This is a literature teaching resource review for biologically inspired microfluidics courses or exploring the diverse applications of microfluidics. The structure is around key papers and model organisms. While courses gradually change over time, a focus remains on understanding how microfluidics has developed as well as what it can and cannot do for researchers. As a primary starting point, we cover micro-fluid mechanics principles and microfabrication of devices. A variety of applications are discussed using model prokaryotic and eukaryotic organisms from the set of bacteria (Escherichia coli), trypanosomes (Trypanosoma brucei), yeast (Saccharomyces cerevisiae), slime molds (Physarum polycephalum), worms (Caenorhabditis elegans), flies (Drosophila melangoster), plants (Arabidopsis thaliana), and mouse immune cells (Mus musculus). Other engineering and biochemical methods discussed include biomimetics, organ on a chip, inkjet, droplet microfluidics, biotic games, and diagnostics. While we have not yet reached the end-all lab on a chip, microfluidics can still be used effectively for specific applications

Combining phage display with SMRTbell next-generation sequencing for the rapid discovery of functional scFv fragments

Phage display technology in combination with next-generation sequencing (NGS) currently is a state-of-the-art method for the enrichment and isolation of monoclonal antibodies from diverse libraries. However, the current NGS methods employed for sequencing phage display libraries are limited by the short contiguous read lengths associated with second-generation sequencing platforms. Consequently, the identification of antibody sequences has conventionally been restricted to individual antibody domains or to the analysis of single domain binding moieties such as camelid VHH or cartilaginous fish IgNAR antibodies. In this study, we report the application of third-generation sequencing to address this limitation. We used single molecule real time (SMRT) sequencing coupled with hairpin adaptor loop ligation to facilitate the accurate interrogation of full-length single-chain Fv (scFv) libraries. Our method facilitated the rapid isolation and testing of scFv antibodies enriched from phage display libraries within days following panning. Two libraries against CD160 and CD123 were panned and monitored by NGS. Analysis of NGS antibody data sets led to the isolation of several functional scFv antibodies that were not identified by conventional panning and screening strategies. Our approach, which combines phage display selection of immune libraries with the full-length interrogation of scFv fragments, is an easy method to discover functional antibodies, with a range of affinities and biophysical characteristics

Designing stem cell niches for differentiation and self-renewal

Mesenchymal stem cells, characterized by their ability to differentiate into skeletal tissues and self-renew, hold great promise for both regenerative medicine and novel therapeutic discovery. However, their regenerative capacity is retained only when in contact with their specialized microenvironment, termed the stem cell niche. Niches provide structural and functional cues that are both biochemical and biophysical, stem cells integrate this complex array of signals with intrinsic regulatory networks to meet physiological demands. Although, some of these regulatory mechanisms remain poorly understood or difficult to harness with traditional culture systems. Biomaterial strategies are being developed that aim to recapitulate stem cell niches, by engineering microenvironments with physiological-like niche properties that aim to elucidate stem cell-regulatory mechanisms, and to harness their regenerative capacity in vitro. In the future, engineered niches will prove important tools for both regenerative medicine and therapeutic discoveries

Custom-Designed Biohybrid Micromotor for Potential Disease Treatment

Micromotors are recognized as promising candidates for untethered micromanipulation and targeted cargo transport. Their future application is, however, hindered by the low efficiency of drug encapsulation and their poor adaptability in physiological conditions. To address these challenges, one potential solution is to incorporate micromotors with biological materials as the combination of functional biological entities and smart artificial parts represents a manipulable and biologically friendly approach. This dissertation focuses on the development of custom-designed micromotors combined with sperm and their potential applications on targeted diseases treatment. By means of 2D and 3D lithography methods, microstructures with complex configurations can be fabricated for specific demands. Bovine and human sperm are both for the first time explored as drug carriers thanks to their high encapsulation efficiency of hydrophilic drugs, their powerful self-propulsion and their improved drug-uptake relying on the somatic-cell fusion ability. The hybrid micromotors containing drug loaded sperm and constructed artificial enhancements can be self-propelled by the sperm flagella and remotely guided and released to the target at high precision by employing weak external magnetic fields. As a result, micromotors based on both bovine and human sperm show significant anticancer effect. The application here can be further broadened to other biological environments, in particular to the blood stream, showing the potential on the treatment of blood diseases like blood clotting. Finally, to enhance the treatment efficiency, in particular to control sperm number and drug dose, three strategies are demonstrated to transport swarms of sperm. This research paves the way for the precision medicine based on engineered sperm-based micromotors

Developing novel ways of studying motility in Schistosoma mansoni and its potential contribution towards inhibiting Schistosomiasis.

Schistosomiasis, caused by Schistosoma mansoni, is responsible for infecting approximately 200 million people worldwide, mostly from low-income and middle-income populations; it is a key neglected tropical parasitic disease, second only to malaria as the most devastating parasitic disease in the world. An infection is initiated when the cercarial form of the parasite is released from its intermediary invertebrate host, a Biomphalaria snail, into the surrounding fresh water. Cercariae are non-feeding, free swimming, extremely infectious, highly motile schistosomal stage with bifurcated tails and they penetrate the mammalian skin tail-first, thus infecting the human host. Post attachment, the cercariae sheds its tail and the resulting schistosomule continues to develop within the host circulatory system. The parasites travel to the hepatic system, where they transform into adult worms, mate and lay eggs, most of which are excreted through the host’s excretory system and the rest accumulating within the internal organs of the body. The spread of schistosomiasis relies heavily on the motility of the cercariae before human infection, as well as the movement of the schistosomules through the human body, post infection. For my doctoral dissertation, I have focused on the aspect of motility of the S.mansoni worms pre and post infection. The first part of my research deals with the design and development of a sensitive, simple, cheap biological assay i.e. a microfluidic platform to study the movement of the schistosomules as they travel through the host circulatory system. The complete navigation and the kinetics of the movement of the juvenile worms through the convoluted pulmonary, blood and hepatic vessels within the host remains largely unexplored. We believe that this novel approach will provide a highly efficient method for screening potential anti-schistosomal compounds and improving motility assays. The second part of my research concentrates on the qualitative and quantitative proteomic analysis of the cercarial tails and cercarial bodies. Using mechanical separation of cercarial tails and bodies, and mass spectrometric analyses, we have identified a total of 945 proteins in the combined cercarial proteome from 4 independent samples: 791 proteins in the cercarial tails and 645 proteins from the somule bodies. Gene oncology analysis was conducted on the obtained proteomic data, and the peptide hits were classified based on molecular function, biological function and subcellular location. In conclusion, I believe that by preventing the motility of the parasitic worms at different stages of the life cycle is a novel, previously unexplored route for investigating potential drug targets which could interrupt the spread of the disease

Bacterial Mechanosensitive Channel of Large Conductance (MscL) in Mammalian Cells for Novel Mechanobiology Applications

Mechanobiology, a relatively young field that centers on how external physical forces on cells or tissues and their intrinsic mechanical properties can influence physiology and disease, has become a pillar in cell biology. Indeed, cells experience a myriad of external, mechanical stimuli such as shear stress, stretch, substrate and matrix rigidity, surface topography, compression, and inter-cellular junction forces. Mechanosensors on the cell surface interfacing with the external environment (e.g., receptors, mechanosensitive ion channels, focal adhesions) and within the cell (e.g. the cytoskeleton) can sense, transmit, and amplify these inputs. This results in a cascade of intracellular biochemical signaling that leads to altered gene expression, protein expression, and finally, altered cell behavior and function. This process is known as mechanotransduction. Mechanotransduction at the cellular-scale has perceptible, large-scale implications such as proper organism development, our ability to sense sound and touch, function and homeostasis of organ systems, and disease progression. Previous work in mechanobiology has focused on investigating or capitalizing on native, endogenous mechanotransduction. This dissertation work proposes a relatively unexplored frontier in mechanobiology: exogenous mechanotransduction, by demonstrating a novel approach towards achieving signal transduction and mechanically driven behavior in cells through the introduction of exogenous mechanosensory components. Here we demonstrate how the functional expression of the E. coli membrane tension gated mechanosensitive channel of large conductance (MscL) in mammalian cells endows the cells with new mechano-sensing capabilities such as the activation of MscL in the plasma membrane through membrane tension resulting from (1) osmotic down-shock and (2) new interactions with native mechano-sensory components, as well as altered cell function such as (3) impairment of cell migration in metastasis in vivo and narrow, 3D confinement in vitro. The first major contribution in this thesis was to show that MscL can be expressed in mammalian cells, localize to cellular membranes, and responds to membrane tension via osmotic down-shock. The second contribution was demonstrating that the activation of the bacterial MS channel expressed in mammalian cells can be mediated through localized membrane stress that is dependent on the native actin-cytoskeleton. This was done by using acoustic tweezing cytometry (ATC) where acoustic excitation of microbubbles targeted to surface integrin receptors generated localized forces that robustly gated MscL. Impermeable, fluorescent dye uptake was used to report MscL activation; also showing that activated MscL can deliver large molecules into the cell. Lastly, we investigated the effect of MscL mechanotransduction on the cell function of migration in cancer metastasis and then more specifically, 3D-confinements. Our findings in our in vivo mouse model showed that there was a marked reduction in metastasis to the lung for MscL expressing cancer cells compared to controls. In vitro migration experiments using a biomimetic microfluidic device revealed that MscL activation due to 3D-confined migration could be responsible for the observed reduction in metastasis. We found that ~46% of MscL-expressing cancer cells that entered extremely narrow confinements of 30 µm2 cross-section had activated MscL and only 11% of these cells were able to fully enter the channel and migrate. Implications of this thesis are that MscL: (1) can be used as a molecular delivery tool for live-cells via mechanical stimulus; (2) can provide insight into the metastatic cascade and mechanobiology focused therapies; and (3) in mammalian cells can serve to study existing mechanotransduction or potentially engineer new mechanical properties and signaling in cells.PHDMechanical EngineeringUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttps://deepblue.lib.umich.edu/bitstream/2027.42/144021/1/heureaux_1.pd