Implications of available design space for identification of non-immunogenic protein therapeutics.

Immunogenicity/antibody responses are major issues for parenteral proteins and nanotherapeutics (nanovectors, diagnostics, theranostics, etc.), and robust antibody responses require T-helper epitopes. T-helper epitopes consist of specific amino acids at specific positions (anchor positions) in immunogens which contact the major histocompatibility complex (MHC), provide most of the energy for MHC binding and constitute the binding motif for the corresponding MHC alleles. We developed an algorithm that considers motifs to design vaccines lacking unwanted T-cell epitopes, and found numbers of such vaccines can be astronomical (Lee et al. 2009). The algorithm can be used to design reduced immunogenicity proteins, and numbers of predicted proteins are also immense. Reducing T-helper epitope content reduces protein immunogenicity, but the depth of mutagensis needed to eliminate immunogenicity is commonly assumed to be too great for retention of protein bioactivity. However, very deep, but successful substitution, insertion and deletion mutagenesis have been reported. These reports and design space the algorithm reveals suggest development of non-immunogenic therapeutics might be more feasible than commonly assumed.

Estimating design space available for polyepitopes through consideration of major histocompatibility complex binding motifs.

Major histocompatibility complex (MHC ) epitope presentation is needed for robust adaptive immune responses. Core peptide binding motifs for class I and class II MHC are 8-10 amino acids long, containing two or more "anchor" residues. These binding motifs define epitope anchor amino acid content and spacing, and knowledge of them has facilitated emergence of polyepitope vaccines. However, polyepitopes can exhibit "junctional epitopes" (neoepitopes interfering with vaccine function) resulting from juxtaposition of authentic epitopes. We have developed an algorithm for consideration of polyepitope sequence in light of MHC motifs to exhaustively identify all junctional-free polyepitope designs for any given set of authentic epitopes, and in so doing discovered that the number of such variants of any given polyepitope can be astronomically high. Our approach designs polyepitopes of any length, considers multiple MHC class I or class II motifs simultaneously and can be adapted to design variants of existing proteins with pre-selected epitope contents. We have also implemented the algorithm as a computer-based tool (CANVAC II), which we make available to interested parties. The vast diversity of junctional-free polyepitopes suggests that the number of potential T-helper epitope free protein variants may also be large, which may have implications for discovery of bioactive but non-immunogenic therapeutics.

Detection of clinically relevant levels of protein analyte under physiologic buffer using planar field effect transistors.

Electrochemical detection of protein binding at physiological salt concentration by planar field effect transistor platforms has yet to be documented convincingly. Here we report detection of streptavidin and clinically relevant levels of biotinylated monokine induced by interferon gamma (MIG) at physiological salt concentrations with AlGaN heterojunction field effect transistors (HFETs). The AlGaN HFETs are functionalized with a silane linker and analyte-specific affinity elements. Polarity of sensor responses is as expected from n-type HFETs to negatively and positively charged analytes. Sensitivity of the HFET sensors increases when salt concentration decreases, and the devices also exhibit dose-dependent responses to analyte. Detection of clinically relevant MIG concentrations at physiological salt levels demonstrates the potential for AlGaN devices to be used in development of in vivo biosensors.

Engineering functional protein interfaces for immunologically modified field effect transistor (ImmunoFET) by molecular genetic means.

The attachment and interactions of analyte receptor biomolecules at solid-liquid interfaces are critical to development of hybrid biological-synthetic sensor devices across all size regimes. We use protein engineering approaches to engineer the sensing interface of biochemically modified field effect transistor sensors (BioFET). To date, we have deposited analyte receptor proteins on FET sensing channels by direct adsorption, used self-assembled monolayers to tether receptor proteins to planar FET SiO2 sensing gates and demonstrated interface biochemical function and electrical function of the corresponding sensors. We have also used phage display to identify short peptides that recognize thermally grown SiO2. Our interest in these peptides is as affinity domains that can be inserted as translational fusions into receptor proteins (antibody fragments or other molecules) to drive oriented interaction with FET sensing surfaces. We have also identified single-chain fragment variables (scFvs, antibody fragments) that recognize an analyte of interest as potential sensor receptors. In addition, we have developed a protein engineering technology (scanning circular permutagenesis) that allows us to alter protein topography to manipulate the position of functional domains of the protein relative to the BioFET sensing surface.

Selection and characteristics of peptides that bind thermally grown silicon dioxide films.

Dodecapeptides with affinity for thermally grown silicon dioxide were isolated by phage display. Selectants had high histidine content, though distributions of histidine are distinct from reported silica particle-precipitating peptides. Our peptides will have utility as nanoscale affinity domains when inserted into proteins intended for deposition on thermal oxide surfaces/interfaces in micro/nanodevices.