Abstract
Many diseases are mediated by targets that are not amenable to conventional small-molecule drug approaches. While antibody-based drugs have undeniable utility, peptides of the 1–9 kDa size range (10–80 amino acids) have drawn interest as alternate drug scaffolds This is born of a desire to identify compounds with the advantages of antibody-based therapeutics (affinity, potency, specificity, and ability to disrupt protein:protein interactions) without all of their liabilities (large size, expensive manufacturing, and necessity of humanization). Of these alternate scaffolds, cystine-dense peptides (CDPs) have several specific benefits. Due to their stable intra-chain disulfide bridges, CDPs often demonstrate resistance to heat and proteolysis, along with low immunogenicity. These properties do not require chemical modifications, permitting CDP screening by conventional genetic means. The cystine topology of a typical CDP requires an oxidative environment, and we have found that the mammalian secretory pathway is most effective at allowing diverse CDPs to achieve a stable fold. As such, high-diversity screens to identify CDPs that interact with targets of interest can be efficiently conducted using mammalian surface display. In this protocol, we present the theory and tools to conduct a mammalian surface display screen for CDPs that bind with targets of interest, including the steps to validate binding and mature the affinity of preliminary candidates. With these methods, CDPs of all kinds can be brought to bear against targets that would benefit from a peptide-based intervention.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Dang CV (2012) MYC on the path to cancer. Cell 149:22–35
Pylayeva-Gupta Y, Grabocka E, Bar-Sagi D (2011) RAS oncogenes: weaving a tumorigenic web. Nat Rev Cancer 11:761–774
Harvey KF, Zhang X, Thomas DM (2013) The Hippo pathway and human cancer. Nat Rev Cancer 13:246–257
Soto C (2003) Unfolding the role of protein misfolding in neurodegenerative diseases. Nat Rev Neurosci 4:49–60
Kinch MS (2015) An overview of FDA-approved biologics medicines. Drug Discov Today 20:393–398
Brüggemann M, Osborn MJ, Ma B et al (2015) Human antibody production in transgenic animals. Arch Immunol Ther Exp 63:101–108
Trenevska I, Li D, Banham AH (2017) Therapeutic antibodies against intracellular tumor antigens. Front Immunol 8:1001
Li Z, Krippendorff B-F, Sharma S et al (2016) Influence of molecular size on tissue distribution of antibody fragments. MAbs 8:113–119
Tabrizi M, Bornstein GG, Suria H (2010) Biodistribution mechanisms of therapeutic monoclonal antibodies in health and disease. AAPS J 12:33–43
Almagro JC, Daniels-Wells TR, Perez-Tapia SM et al (2017) Progress and challenges in the design and clinical development of antibodies for cancer therapy. Front Immunol 8:1751
Xenopoulos A (2015) A new, integrated, continuous purification process template for monoclonal antibodies: process modeling and cost of goods studies. J Biotechnol 213:42–53
Löfblom J, Frejd FY, Ståhl S (2011) Non-immunoglobulin based protein scaffolds. Curr Opin Biotechnol 22:843–848
Weidle UH, Auer J, Brinkmann U et al (2013) The emerging role of new protein scaffold-based agents for treatment of cancer. Cancer Genomics Proteomics 10:155–168
Beck A, Wurch T, Bailly C et al (2010) Strategies and challenges for the next generation of therapeutic antibodies. Nat Rev Immunol 10:345–352
Simeon R, Chen Z (2018) In vitro-engineered non-antibody protein therapeutics. Protein Cell 9:3–14
Veiseh M, Gabikian P, Bahrami S-B et al (2007) Tumor paint: a chlorotoxin:Cy5.5 bioconjugate for intraoperative visualization of cancer foci. Cancer Res 67:6882–6888
Shahbazzadeh D, Srairi-Abid N, Feng W et al (2007) Hemicalcin, a new toxin from the Iranian scorpion Hemiscorpius lepturus which is active on ryanodine-sensitive Ca2+ channels. Biochem J 404:89–96
Gurrola GB, Capes EM, Zamudio FZ et al (2010) Imperatoxin a, a cell-penetrating peptide from scorpion venom, as a probe of Ca2+-release channels/ryanodine receptors. Pharmaceuticals (Basel) 3:1093–1107
Koday MT, Nelson J, Chevalier A et al (2016) A computationally designed hemagglutinin stem-binding protein provides in vivo protection from influenza independent of a host immune response. PLoS Pathog 12:e1005409
Berger S, Procko E, Margineantu D et al (2016) Computationally designed high specificity inhibitors delineate the roles of BCL2 family proteins in cancer. elife 5:pii: e20352
Janda CY, Dang LT, You C et al (2017) Surrogate Wnt agonists that phenocopy canonical Wnt and β-catenin signalling. Nature 545:234–237
Moore SJ, Cochran JR (2012) Engineering knottins as novel binding agents. Methods Enzymol 503:223–251
Gates ZP, Vinogradov AA, Quartararo AJ et al (2018) Xenoprotein engineering via synthetic libraries. Proc Natl Acad Sci 115:E5298–E5306
Herzig V, King G, Herzig V et al (2015) The cystine knot is responsible for the exceptional stability of the insecticidal spider toxin ω-hexatoxin-Hv1a. Toxins (Basel) 7:4366–4380
Kikuchi K, Sugiura M, Kimura T (2015) High proteolytic resistance of spider-derived inhibitor cystine knots. Int J Pept 2015:537508
Correnti CE, Gewe MM, Mehlin C et al (2018) Screening, large-scale production and structure-based classification of cystine-dense peptides. Nat Struct Mol Biol 25(3):270–278
Craik DJ, Clark RJ, Daly NL (2007) Potential therapeutic applications of the cyclotides and related cystine knot mini-proteins. Expert Opin Investig Drugs 16:595–604
Maillère B, Mourier G, Hervé M et al (1995) Immunogenicity of a disulphide-containing neurotoxin: presentation to T-cells requires a reduction step. Toxicon 33:475–482
Corbi-Verge C, Garton M, Nim S et al (2017) Strategies to develop inhibitors of motif-mediated protein-protein interactions as drug leads. Annu Rev Pharmacol Toxicol 57:39–60
Angelini A, Chen TF, de Picciotto S et al (2015) Protein engineering and selection using yeast surface display. Methods Mol Biol 1319:3–36
Chen I, Dorr BM, Liu DR (2011) A general strategy for the evolution of bond-forming enzymes using yeast display. Proc Natl Acad Sci U S A 108:11399–11404
Bowers PM, Horlick RA, Neben TY et al (2011) Coupling mammalian cell surface display with somatic hypermutation for the discovery and maturation of human antibodies. Proc Natl Acad Sci 108:20455–20460
Boder ET, Wittrup KD (1997) Yeast surface display for screening combinatorial polypeptide libraries. Nat Biotechnol 15:553–557
Kintzing JR, Cochran JR (2016) Engineered knottin peptides as diagnostics, therapeutics, and drug delivery vehicles. Curr Opin Chem Biol 34:143–150
Crook ZR, Sevilla GP, Friend D et al (2017) Mammalian display screening of diverse cystine-dense peptides for difficult to drug targets. Nat Commun 8:2244
Langmead B, Salzberg SL (2012) Fast gapped-read alignment with Bowtie 2. Nat Methods 9:357–359. https://doi.org/10.1038/nmeth.1923
Bandaranayake AD, Correnti C, Ryu BY et al (2011) Daedalus: a robust, turnkey platform for rapid production of decigram quantities of active recombinant proteins in human cell lines using novel lentiviral vectors. Nucleic Acids Res 39:e143–e143
McCullum EO, Williams BAR, Zhang J et al (2010) Random mutagenesis by error-prone PCR. Methods Mol Biol 634:103–109
Lemmon G, Meiler J (2012) Rosetta ligand docking with flexible XML protocols. Methods Mol Biol 819:143–155
Addgene: lentivirus production protocol https://www.addgene.org/protocols/lentivirus-production/
Rouillard JM, Lee W, Truan G et al (2004) Gene2Oligo: oligonucleotide design for in vitro gene synthesis. Nucleic Acids Res 32:176–180
Gibson DG, Young L, Chuang RY et al (2009) Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat Methods 6:343–345
Acknowledgments
The author thanks Colin Correnti, Roland Strong, and Ashok Bandaranayake for helpful discussions as the manuscript was prepared. The Fred Hutch Shared Resources, particularly the Flow Cytometry and Genomics facilities, were instrumental to data generation. The author also thanks Shelli Morris and Chris Mehlin for their help in editing the manuscript. This work was funded by NIH Grants R01CA114567 (J.M.O.) and R01CA155360 (J.M.O.); A Washington Research Foundation Innovation Fellowship through the University of Washington Institute for Protein Design; NIH Fellowship T32AG00005740 (Z.R.C.); and Project Violet (www.projectviolet.org).
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2020 Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Crook, Z.R., Sevilla, G.P., Mhyre, A.J., Olson, J.M. (2020). Mammalian Surface Display Screening of Diverse Cystine-Dense Peptide Libraries for Difficult-to-Drug Targets. In: Zielonka, S., Krah, S. (eds) Genotype Phenotype Coupling. Methods in Molecular Biology, vol 2070. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9853-1_21
Download citation
DOI: https://doi.org/10.1007/978-1-4939-9853-1_21
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-4939-9852-4
Online ISBN: 978-1-4939-9853-1
eBook Packages: Springer Protocols