Practical Applications

We work on a lot of different projects and research topics here at the Institute for Protein Design, but how does that relate to you and the world outside of the labs? It may connect in more ways than you think! From celiac to cancer, we are pushing the limits of what proteins can do to help meet the challenges of the modern world. Below, you will find short descriptions of several of our areas of focus.

Anti-Flu and Flu Diagnostics

In 2011 we described a general computational method for designing proteins that bind a surface patch of interest on a target macromolecule. The method was used to design mini-proteins that bind a conserved surface patch on the stem of the influenza hemagglutinin (HA) from the 1918 H1N1 pandemic virus [14]. We have gone on to greatly improve these anti-flu binding proteins and this year we published results demonstrating the safety and efficacy of intra-nasal doses of our “Flu Binders” that protect rodents from exposure to lethal amounts of the flu virus [1]. Notably, we have designed our anti-flu viral proteins to be small, stable, and to formulate them for aerosol delivery to the lungs by intranasal routes (via the nose), a less immunogenic route of delivery than by needle injection. Our anti-flu proteins delivered repeatedly to rodent lungs have proven to be safe, to protect animals from lethal doses of flu virus, and not to result in any significant immune response to the proteins [1].

We have also incorporated the “Flu Binder” proteins targeting flu HA as diagnostic reagents into low cost nitrocellulose paper membranes diagnostic test strips.   Our computationally designed Flu Binders provide improved performance as an influenza assay compared to a traditional antibody-based capture systems [2]. Recent breakthroughs in our paper based diagnostic reagents allow us to detect less than 100 flu virus particles in a single nasal swab.

Anti-Viral and Anti-Cancer

We have also designed a hyper-stable mini-protein inhibitor of an Epstein-Barr viral (EBV) Bcl-2 protein, which induces apoptosis (promotes cell death) in infected cells [3]. When these proteins were delivered with an antibody-targeted intracellular delivery carrier, the suppressed tumor growth and extended survival in a xenograft disease model of EBV-positive human lymphoma. Hence these designed anti-EBV “BINDI” peptides are also capable of killing the cancerous forms of EBV infected cells.

Celiac Disease

In 2011, a team of our UW undergraduates won the grand championship prize at the annual International Genetically Engineered Machine (iGEM) competition for students interested in the field of synthetic biology. Their award winning project started with the goal of finding a cure for celiac disease, which one of their friends was suffering from. Specifically, we used computational design to re-engineer the specificity of a protease enzyme so that it would specifically break down gluten in the harsh acid conditions of the stomach [6]. In 2015, our team published the characterization of the most advanced version of KumaMax, “Kuma030”, which specifically cleaves gluten proteins at sites that are known to cause an improper immune reaction in the small intestine of people who suffer from celiac disease.   Kuma030 has high enough activity in stomach conditions to ensure that a small pill taken before a meal, can completely break down any accidentally ingested gluten in the stomach by before it can get to the small intestine where it would otherwise cause the unwanted inflammation in celiac disease patients. The Kuma030 is now being developed by our spin out company PvP Biologics as an oral therapy for celiac disease.

Anti-Cancer and Immune Silencing

The small size of our designed proteins is one of the design features that enables their stealth with respect to the immune system. In addition, we have been applying computational design strategies to reduce their immunogenicity at the amino acid sequence level. In 2014 we were the first to demonstrate that computational design can be used to eliminate “T-cell epitopes” from the protein (by altering the amino acid sequence) without affecting structure or function [15]. This immune silencing approach enabled collaborators at the National Cancer Institute to improve an immunotoxin used to treat cancer. The new designed immunotoxins maintained good activity, stability, and antitumor activity. Their reduced immunogenicity will enable more effective cancer therapy, because more treatment cycles can be given before an immune response is mounted to neutralize the therapy [4].

Vaccines and Epitope Design

Elicitation of an effective immune response (antibodies) against targets that are cryptic or transient in their native context has been a grand challenge for vaccine design. In 2010 we proved that Rosetta designed protein sequences resembling parts of the AIDS/HIV virus, ‘designed epitopes’, can take on shapes that elicit the production of neutralizing antibodies to HIV in when they are injected into animals as vaccine formulations (immunizations) [7, 9]. In 2014, we successfully applied these epitope design methods to create a novel epitope for immunization against the respiratory syncytial virus (RSV), a particularly deadly virus to infants and the elderly [8].

Biosensors

In 2013 we succeeded in reporting, for the first time, a general computational method for designing pre-organized and shape complementary small-molecule-binding sites. We generated protein binders to the steroid drug digoxigenin (DIG), the natural cardiac glycoside of digitalis found in the flowers of foxglove plants, often given to cardiac patients with atrial fibrillation or heart failure [11]. In 2014 we went on to show that the “DIG binder” could be used as a sensitive digoxigenin sensor [10] for point-of-care diagnostic applications, and also as a sensor in cells [12, 13].

References
  1. Koday MT, Nelson J, Chevalier A, Koday M, Kalinoski H, Stewart L, Carter L, Nieusma T, Lee PS, Ward AB, Wilson IA, Dagley A, Smee DF, Baker D, Fuller DH: A Computationally Designed Hemagglutinin Stem-Binding Protein Provides In Vivo Protection from Influenza Independent of a Host Immune Response. PLoS pathogens 2016, 12(2):e1005409. PMC#
  2. Holstein CA, Chevalier A, Bennett S, Anderson CE, Keniston K, Olsen C, Li B, Bales B, Moore DR, Fu E, Baker D, Yager P: Immobilizing affinity proteins to nitrocellulose: a toolbox for paper-based assay developers. Anal Bioanal Chem 2016, 408(5):1335-1346. PMC#
  3. Procko E, Berguig GY, Shen BW, Song Y, Frayo S, Convertine AJ, Margineantu D, Booth G, Correia BE, Cheng Y, Schief WR, Hockenbery DM, Press OW, Stoddard BL, Stayton PS, Baker D: A computationally designed inhibitor of an epstein-barr viral bcl-2 protein induces apoptosis in infected cells. Cell 2014, 157(7):1644-1656. PMC#4079535
  4. Mazor R, Eberle JA, Hu X, Vassall AN, Onda M, Beers R, Lee EC, Kreitman RJ, Lee B, Baker D, King C, Hassan R, Benhar I, Pastan I: Recombinant immunotoxin for cancer treatment with low immunogenicity by identification and silencing of human T-cell epitopes. Proceedings of the National Academy of Sciences of the United States of America 2014, 111(23):8571-8576. PMC#4060717
  5. Wolf C, Siegel JB, Tinberg C, Camarca A, Gianfrani C, Paski S, Guan R, Montelione G, Baker D, Pultz IS: Engineering of Kuma030: A Gliadin Peptidase That Rapidly Degrades Immunogenic Gliadin Peptides in Gastric Conditions. Journal of the American Chemical Society 2015, 137(40):13106-13113. PMC#
  6. Gordon SR, Stanley EJ, Wolf S, Toland A, Wu SJ, Hadidi D, Mills JH, Baker D, Pultz IS, Siegel JB: Computational design of an alpha-gliadin peptidase. Journal of the American Chemical Society 2012, 134(50):20513-20520. PMC#3526107
  7. Ofek G, Guenaga FJ, Schief WR, Skinner J, Baker D, Wyatt R, Kwong PD: Elicitation of structure-specific antibodies by epitope scaffolds. Proceedings of the National Academy of Sciences of the United States of America 2010, 107(42):17880-17887. PMC#PMC2964213
  8. Correia BE, Bates JT, Loomis RJ, Baneyx G, Carrico C, Jardine JG, Rupert P, Correnti C, Kalyuzhniy O, Vittal V, Connell MJ, Stevens E, Schroeter A, Chen M, Macpherson S, Serra AM, Adachi Y, Holmes MA, Li Y, Klevit RE et al: Proof of principle for epitope-focused vaccine design. Nature 2014, 507(7491):201-206. PMC#PMC4260937
  9. Correia BE, Ban YE, Holmes MA, Xu H, Ellingson K, Kraft Z, Carrico C, Boni E, Sather DN, Zenobia C, Burke KY, Bradley-Hewitt T, Bruhn-Johannsen JF, Kalyuzhniy O, Baker D, Strong RK, Stamatatos L, Schief WR: Computational design of epitope-scaffolds allows induction of antibodies specific for a poorly immunogenic HIV vaccine epitope. Structure 2010, 18(9):1116-1126. PMC#
  10. Griss R, Schena A, Reymond L, Patiny L, Werner D, Tinberg CE, Baker D, Johnsson K: Bioluminescent sensor proteins for point-of-care therapeutic drug monitoring. Nature chemical biology 2014, 10(7):598-603. PMC#
  11. Tinberg CE, Khare SD, Dou J, Doyle L, Nelson JW, Schena A, Jankowski W, Kalodimos CG, Johnsson K, Stoddard BL, Baker D: Computational design of ligand-binding proteins with high affinity and selectivity. Nature 2013, 501(7466):212-216. PMC#3898436
  12. Taylor ND, Garruss AS, Moretti R, Chan S, Arbing MA, Cascio D, Rogers JK, Isaacs FJ, Kosuri S, Baker D, Fields S, Church GM, Raman S: Engineering an allosteric transcription factor to respond to new ligands. Nature methods 2016, 13(2):177-183. PMC#
  13. Feng J, Jester BW, Tinberg CE, Mandell DJ, Antunes MS, Chari R, Morey KJ, Rios X, Medford JI, Church GM, Fields S, Baker D: A general strategy to construct small molecule biosensors in eukaryotes. eLife 2015, 4. PMC#PMC4739774
  14. Fleishman SJ, Whitehead TA, Ekiert DC, Dreyfus C, Corn JE, Strauch EM, Wilson IA, Baker D: Computational design of proteins targeting the conserved stem region of influenza hemagglutinin. Science 2011, 332(6031):816-821. PMC#3164876
  15. King C, Garza EN, Mazor R, Linehan JL, Pastan I, Pepper M, Baker D: Removing T-cell epitopes with computational protein design. Proceedings of the National Academy of Sciences of the United States of America 2014, 111(23):8577-8582. PMC#4060723