Epivolve: A Protocol for Site-Directed Antibodies

Abstract

Researchers can often successfully generate antibodies to predicted epitopes. Especially when the epitopes are on the surface of a protein or in a hydrophilic loop. But it is difficult to direct recombinant antibodies to bind either to- or near a specific amino acid on a protein or peptide. We have developed a unique immune-targeting strategy, that we call "Epivolve," that enables us to make site-specific antibodies (Abs). Epivolve technology leverages a highly immunogenic modified amino acid that acts as a "pseudo-hapten" immuno-target and takes advantage of Ab affinity maturation technologies to make high-affinity site-specific antibodies. Epivolve functions by the evolution of an Ab paratope to either synonymous or especially non-synonymous amino acid (aa) binding. Here we describe the use of Epivolve technology in phage display and the protocols for developing site-specific antibodies.

Keywords: Antibody; Biopanning; Epivolve; Phage display; Site-directed; Site-specific.

Fig. 1

Illustration the principles of Epivolve technology. An important residue (the Epivolve targeted site) on the target protein is chosen. A 10–15-mer peptide of the target sequence will then be synthesized with the modification on the Epivolve targeted amino acid, named mod1-peptide. This mod1-peptide is biopanned using a naïve phage display library to generate a mod1-specific Ab. The identified mod1-specific Abs are then mutagenized using error-prone PCR to generate a Discovery Maturation (DisMat) library. The DisMat library is then biopanned against the NAT peptide (or full-length protein if available) to identify anti-NAT (or anti-full-length protein) clonotypes. The resulting anti-NAT Abs can be further engineered for increased binding affinities using Affinity Maturation (AffMat)

Fig. 2

In vitro Epivolve workflow Developing site-specific Abs using Epivolve technology by in vitro phage display can be illuminated as 2 phases (Discovery and DisMat) and an optional third phase (AffMat). In Phase I, discovery biopanning against a mod1-peptide is used to identify mod1-specific scFvs. In phase II (DisMat), anti-mod1 specific scFvs from phase I are used as the template for Error-Prone PCR to make DisMat libraries. The DisMat library is then biopanned against a NAT peptide or full-length protein to identify anti-NAT clones. In the optional Phase III (AffMat), anti-NAT clones are subjected to a second round of Error-Prone PCR to make AffMat libraries. These libraries are then used to develop high-affinity and high-specificity anti-NAT peptide or an-NAT full-length protein Abs, which will be validated by the appropriate applications such as by MILKSHAKE Western-blot [25], ELISA, and other functional assays. The blue star indicates where there are several steps in between the two steps shown, which can include phage supernatant ELISA, scFv protein expression and purification with details we have published [2, 19]. Typical result of Ab^#1 is shown in Fig. 3a. Typical results of Ab^#2 and Ab^#3 are shown in Fig. 3b and Fig. 3c

Fig. 3

Simulated examples of scFv protein or full IgG protein titration ELISA results of site-specific antibodies discovered by Epivolve (a) Mod1-specific scFv or IgG protein titration ELISA results from anti-mod1 discovery phage display biopanning in phase I. A typical anti-Mod1 Ab will demonstrate specific binding to the mod1 peptide, and absence of binding to the NAT-biotin-peptide, or NAT-full-length protein, or scrambled mod1 peptide. (b) and (c) are scFv or IgG protein titration ELISA results of two different clonotypes of final anti-NAT Abs: (b) a mod1-independent Ab which demonstrates binding to mod1-peptide, NAT-peptide, NAT-full-length protein, and absence of binding to scrambled Mod1 peptide; (c) a NAT-specific Ab which demonstrates binding to NAT-peptide, NAT-full-length protein, and absence of binding to mod1-peptide and scrambled Mod1 peptide