toxins Review Basics of Antibody Phage Display Technology Line Ledsgaard 1, Mogens Kilstrup 1 ID , Aneesh Karatt-Vellatt 2, John McCafferty 2 and Andreas H. Laustsen 1,* ID 1 Department of Biotechnology and Biomedicine, Technical University of Denmark, Kongens Lyngby DK-2800, Denmark; [email protected] (L.L.); [email protected] (M.K.) 2 IONTAS Ltd., Cambridgeshire CB22 3EG, United Kingdom; [email protected] (A.K.-V.); [email protected] (J.M.) * Correspondence: [email protected]; Tel.: +45-2988-1134 Received: 31 May 2018; Accepted: 8 June 2018; Published: 9 June 2018 Abstract: Antibody discovery has become increasingly important in almost all areas of modern medicine. Different antibody discovery approaches exist, but one that has gained increasing interest in the field of toxinology and antivenom research is phage display technology. In this review, the lifecycle of the M13 phage and the basics of phage display technology are presented together with important factors influencing the success rates of phage display experiments. Moreover, the pros and cons of different antigen display methods and the use of naïve versus immunized phage display antibody libraries is discussed, and selected examples from the field of antivenom research are highlighted. This review thus provides in-depth knowledge on the principles and use of phage display technology with a special focus on discovery of antibodies that target animal toxins. Keywords: antibody discovery; recombinant antivenom; phage display; M13 phage; toxinology Key Contribution: Antibody phage display technology is thoroughly presented and discussed in relation to antivenom research and toxinology. 1. Introduction With the recent inclusion of snakebite envenoming on the World Health Organization’s list of Neglected Tropical Diseases [1], focus on both prevention and treatment of this infliction has increased. This creates renewed hope for snakebite victims worldwide and could potentially lead to a mobilization of scientific efforts toward the development of novel snakebite envenoming therapies. Several different avenues aimed at bringing innovation into the field of snakebite antivenoms have been pursued, including medicinal chemistry approaches, novel immunization techniques, and the use of biotechnological strategies [2–5]. One promising approach seems to be the use of human IgG antibodies [6] and/or camelid antibody fragments [7,8], as these molecules can be used to develop recombinant antivenoms with high efficacy and safety due to their compatibility with the human immune system [2]. Moreover, these therapeutic proteins could be manufactured cost-competitively using modern cell cultivation methods employed for large scale production [6,9]. To discover and develop antibodies, different techniques can be harnessed. One approach is phage display selection [10], which is a robust, easy-to-perform, and inexpensive method by which specific antigen binders are selected from large combinatorial libraries containing billions of antibody fragments. As antibody phage display is gaining increasing interest in the field of toxinology, the intention with this review is to provide both basic and more advanced knowledge on the underlying science behind the technology and the lifecycle of the M13 phage. Toxins 2018, 10, 236; doi:10.3390/toxins10060236 www.mdpi.com/journal/toxins Toxins 2018, 10, 236 2 of 15 Toxins 2018, 10, x FOR PEER REVIEW 2 of 15 2. The M13 Bacteriophage 2. TheCentral M13 to Bacteriophage phage display technology is the biology of the bacteriophage used to display antibodies. DifferentCentral bacteriophage to phage systems display cantechnology be utilized is forthe phage biology display, of the including bacteriophage the T4, used lambda, to display as well as theantibodies. filamentous Different M13 bacteriophage bacteriophage [11 systems]. These can different be utilized phage for systems phage eachdisplay, have including their benefits the T4, and drawbacks.lambda, as However, well as the primarily filamentous the M13M13 phagebacteriophage has been [11] utilized. These extensively different phage in recent systems times, each and have to the besttheir of ourbenefits knowledge, and drawbacks. this is the However, only phage primarily system the that M13 has phage been exploredhas been withinutilized toxinology extensively [ 2in,12 ]. Therefore,recent times, this reviewand to the will best focus of our on knowledge, antibody phage this is display the only techniques phage system utilizing that has this been specific explored phage system.within Beforetoxinology giving [2,12] an. in-depth Therefore, description this review of will the stepsfocus involvedon antibody in phagephage displaydisplay experiments,techniques anutilizing introduction this specific to the wild-type phage system. M13 phageBefore isgiving provided. an in-depth description of the steps involved in phageThe display M13 phage experiments, [13] belongs an introduction to a group to the of filamentouswild-type M13 phages phage collectivelyis provided. referred to as Ff phagesThe [14]. M13 The phage Ff phages [13] onlybelongs infect to Escherichiaa group of coli filamentousstrains that phages express collect the Fively pilus referred as the adsorptionto as Ff ofphages the phage [14] to. The the Ff bacterium phages only requires infect Escherichia binding of coli a strains phage that coat express protein the to F the pilus tip as of the the adsorption F pilus [15 ]. Theof M13the phage phage to is the neither bacterium temperate requires nor binding lytic. Instead, of a phage the phagecoat protein establishes to the atip chronic of the infectionF pilus [15] in. its The M13 phage is neither temperate nor lytic. Instead, the phage establishes a chronic infection in its host, where it continuously releases new phages. The phage contains a genome of single-stranded host, where it continuously releases new phages. The phage contains a genome of single-stranded DNA (ssDNA) with a length of 6407 bp [16] that consists of nine genes encoding 11 different proteins. DNA (ssDNA) with a length of 6407 bp [16] that consists of nine genes encoding 11 different proteins. Five of these proteins are coat proteins, and the remaining six proteins are involved in replication Five of these proteins are coat proteins, and the remaining six proteins are involved in replication and assembly of the phage. The M13 phage has a length of 900 nm and a width of 6.5 nm [17]. and assembly of the phage. The M13 phage has a length of 900 nm and a width of 6.5 nm [17]. The Themost most abundant abundant of the of the coat coat proteins proteins is the is thecapsid capsid protein protein G8P, G8P,which which forms forms an envelope an envelope around around the thechromosome chromosome consisting consisting of ofapproximately approximately 2700 2700 protein protein units units (Figure (Figure 1).1 ).The The remaining remaining four four coat coat proteins,proteins, G3P, G3P, G6P, G6P, G7P, G7P, and and G9P, G9P, are are each each present present inin approximatelyapproximately five five copies. copies. Information Information on on the the genesgenes and and proteins proteins of of the the M13 M13 phage phage is is listedlisted inin TableTable1 1.. G 8P G 3P G 7P G 9P + ssD N A G 6P FigureFigure 1. 1.Schematic Schematic representation representation ofof thethe M13M13 bacteriophage, which which is is a afilamentous filamentous phage phage carrying carrying a single-strandeda single-stranded DNA DNA (ssDNA) (ssDNA)chromosome. chromosome. The The genome genome contains contains nine nine genes, genes, which which encode encode 11 11 proteins.proteins. Five Five of of these these proteins proteins are are coat coat proteins proteins (G3P, (G3P, G6P, G6P G7P,, G7P, G8P G8P, and and G9P), G9P), while while the the remaining remainingsix proteinssix proteins are used are used for replication for replication of the of genome,the genome, assembly assembly of theof the phage, phage, and and phage phage extrusion. extrusion. Table 1. Gene name, protein name, protein size, and the function of the genes carried by the M13 Table 1. Gene name, protein name, protein size, and the function of the genes carried by the M13 phage [16]. phage [16]. GeneGene Name Name ProteinProtein Name Name (Abbreviation) (Abbreviation) Size (kDa)Size (kDa) FunctionFunction GeneGene 1 protein 1 protein (G1P) (G1P) 39.639.6 AssemblyAssembly I I GeneGene 11 protein 11 protein (G11P) (G11P) 12.412.4 AssemblyAssembly Replication-associatedReplication-associated protein protein (G2P) (G2P) 46.246.2 ReplicationReplication II II GeneGene 10 protein 10 protein (G10P) (G10P) 12.712.7 ReplicationReplication Coat protein IIIIII AttachmentAttachment protein protein (G3P) (G3P) 44.744.7 Coat protein Adsorption and extrusion Adsorption and extrusion IV IV VirionVirion export exp proteinort protein (G4P) (G4P) 45.945.9 AssemblyAssembly and and extrusion extrusion V V DNA-bindingDNA-binding protein protein (G5P) (G5P) 9.79.7 ReplicationReplication Coat protein VI VI HeadHead virion virion protein protein (G6P) (G6P) 12.412.4 Coat protein Infection and budding Infection and budding VII Tail virion protein (G7P) 3.6 Coat protein Assembly and budding Coat protein VII Tail virion protein (G7P) 3.6 VIII Capsid protein (G8P) 7.6Assembly Coat protein and budding IXVIII Tail virionCapsid protein protein (G9P) (G8P) 3.77.6 Coat protein AssemblyCoat protein and budding Toxins 2018, 10, x FOR PEER REVIEW 3 of 15 Toxins 2018, 10, 236 3 of 15 Coat protein IX Tail virion protein (G9P) 3.7 Assembly and budding The first stage of the M13 infection is the adsorption process, which takes place through binding The first stage of the M13 infection is the adsorption process, which takes place through binding of the N2 domain of the G3P coat protein to the tip of a F pilus on the surface of E. coli hosts of the N2 domain of the G3P coat protein to the tip of a F pilus on the surface of E. coli hosts (Figure (Figure2)[ 18–20]. Under normal conditions, “male” E. coli cells (F+, containing F-pili) appear to trawl 2) [18–20]. Under normal conditions, “male” E.