Synthetic antibody libraries focused towards peptide ligands

Abstract

Synthetic antibody libraries have proven immensely useful for the de novo isolation of antibodies without the need for animal immunization. Recently, focused libraries designed to recognize particular classes of ligands, such as haptens or proteins, have been employed to facilitate the selection of high-affinity antibodies. Focused libraries are built using V regions encoding combinations of canonical structures that resemble the structural features of antibodies that bind the desired class of ligands and sequence diversity is introduced at residues typically involved in recognition. Here we describe the generation and experimental validation of two different single-chain antibody variable fragment libraries that efficiently generate binders to peptides, a class of molecules that has proven to be a difficult target for antibody generation. First, a human anti-peptide library was constructed by diversifying a scaffold: the human variable heavy chain (V(H)) germ line gene 3-23, which was fused to a variant of the human variable light chain (V(L)) germ line gene A27, in which L1 was modified to encode the canonical structure found in anti-peptide antibodies. The sequence diversity was introduced into 3-23 (V(H)) only, targeting for diversification residues commonly found in contact with protein and peptide antigens. Second, a murine library was generated using the antibody 26-10, which was initially isolated based on its affinity to the hapten digoxin, but also binds peptides and exhibits a canonical structure pattern typical of anti-peptide antibodies. Diversity was introduced in the V(H) only using the profile of amino acids found at positions that frequently contact peptide antigens. Both libraries yielded binders to two model peptides, angiotensin and neuropeptide Y, following screening by solution phage panning. The mouse library yielded antibodies with affinities below 20 nM to both targets, although only the V(H) had been subjected to diversification.

Figure 1. Structure of the Hu:anti-pep and M:anti-pep V_H libraries

(a) Connolly surface using a 1.7-Å ratio probe of molecular models of Hu:anti-peptide and M:anti-peptide, on the right and left respectively. At the top, side view of the models. The structures in the bottom present the antigen-binding site seen from the antigen perspective. The invariant V_L chains are colored in brown. V_H scaffolds are colored in gray. Within the antigen-binding site, green represents fully randomized positions, i.e. those incorporating all 20 amino acids. Positions diversified to Tyr, Asp, Ala, and Ser are in blue. Positions diversified to incorporate the most common amino acids at each position found in an alignment of anti-peptide antibodies in the Kabat database (see Table 1 for details) are in red. The models were built in the Homology module of Insight II (Accerlys) following standard procedures. For Hu:anti-peptide, the coordinates with PDB code 1mcp and PDB code 2igf were used as template to built V_L and V_H, respectively. The coordinates with PDB code 1igi (Jeffrey et al., 1993) were used as template for modeling M:anti-pepide. The side-chains of residues variegated in the libraries were modeled and are shown as Ala residues. (b) Sequence of the V_H chains showing positions variegated in the libraries following the same color code as Figure 1a, except for position 94 in Hu: anti-peptide, which is colored in pink. It is not shown in the model, as it is buried in the protein. Numbering and gaps follow Chothia's convention.

Figure 2. Sequence of L1 in A27mdJκ1

The HV loop L1 of germline V_L gene B3, with a long canonical structure, was grafted onto germline V_κ gene A27. L1 of both genes A27 and B3 share a key canonical structural determining residue (V29, underlined in B3). Sequence identities are indicated with vertical bars.