Phage library for generation of recombinant antibodies: novel approaches for diagnosis and therapy of infections caused by different Escherichia coli pathotypes

Abstract

Antibodies are produced by the immune response to foreign antigens, however this response is limited to an individual's immune system, and for this reason, synthetic antibodies enable applications beyond the extent of natural antibodies. Recombinant antibodies can be obtained from synthetic antibody libraries through the use of several technologies, among them phage display technology (Zhou et al. N. Biotechnol. 28: 448-452, 2011; Miersch & Sidhu, Methods, 57:486-498, 2012; Adams & Sidhu, Curr. Opin. Struct. Biol. 24:1-9, 2014). In phage display, billions of antibody fragments, such as Fabs and scFvs, are fused to phage coat proteins that get displayed on the surface of phage particles. These can then be screened for binding to a given target in high throughput (Sidhu & Fellouse, Nat. Chem. Biol. 2:682-688, 2006; Nelson & Sidhu, Methods Mol. Biol. 899: 27-41, 2012; Rajan & Sidhu, Methods Enzymol. 502: 3-23, 2012). The synthetic antibodies generated in this manner can be subcloned and produced in vitro in different antibody formats adapted to the application, and they can also be optimized by further molecular engineering to obtain antibodies with high affinity and high specificity for the target.Toxins are defined as any substance capable of causing damage to organisms, and constitute an important part of the virulence factors that mediate the harmful effects generated by pathogenic bacteria (Ivarsson et al. Angew. Chem. Int. 51:4024-4045, 2012). The same bacteria may present different classes of toxins, for example, pathogenic Escherichia coli (E. coli) secrete several toxins among their pathotypes. Shiga toxin-producing E. coli produce the AB5 type Stx toxins, for which the receptor is GB3; enteropathogenic and enterohemorrhagic E. coli produce the EspB/EspD toxin, responsible for the formation of pores in the membrane through which proteins are translocated by T3SS; enterotoxigenic E. coli produce a heat-labile toxin, also of the AB5 type, recognized by the GM1 receptor, and a heat-stable toxin capable of activating guanylate cyclase; and uropathogenic E. coli produce hemolysin, which forms pores in the host membrane, as well as cytotoxic necrotizing factor, a toxin capable of activating GTPases and altering the structure of the cytoskeleton (Lemichez et al. Mol. Microbiol. 24:1061-1070, 1997; Kaper et al. Nat. Rev. Microbiol. 2: 123-140, 2004; Ruiz-Perez & Nataro, Cell. Mol. Life Sci. 71:745-770, 2014). These different E. coli pathotypes are responsible for distinct pathologies with economic and social burden in both developing and industrialized countries. Over the years, our research group at the Bacteriology Lab of the Butantan Institute has successfully obtained antibodies (polyclonal and monoclonal) against E. coli toxins, as well as developed and standardized diagnostic methods for epidemiologically important pathotypes of diarrheagenic E. coli. In order to overcome the use of animals and cell culture traditionally used in antibody production, we have initiated a collaboration with the Sidhu group to implement recombinant antibody technology in our laboratory. Thus far, we obtained recombinant antibodies against Stx toxins in two formats (scFv and Fab) (Luz et al. PLoS One 10:3, 2015; Luz et al. Antibodies 7:9, 2018). The scFv fragments are a bacteria-produced tool for use in a rapid diagnosis test, providing an alternative for STEC diagnosis. The Fab, a human monovalent molecule produced in bacteria, is able to neutralize the cytotoxicity of Stx in vitro. Therefore, the present study aims to obtain additional recombinant antibodies molecules (stable, easy to produce and low cost) against other E. coli toxins. This project will contribute to the rapid generation of such molecules, leveraging the technology developed by the Sidhu group, which provide us with a rapid method to generate recombinant antibodies and will result in the implementation of phage display library technology in our laboratory. (AU)