Design and validation of a synthetic VH repertoire with tailored diversity for protein recognition

Abstract

Previous studies have indicated differences in the specificity-determining residues (SDRs) of antibodies that recognize haptens, peptides, or proteins. Here, we designed a V(H) repertoire based on the human scaffold 3-23/J(H)4 and diversification of high and medium-usage SDRs of anti-protein and anti-peptide antibodies. The repertoire was synthesized by overlapping polymerase chain reaction (PCR) and combined with the V(L) chain of the anti-hen egg-white lysozyme (HEL) antibody D1.3. The resulting chimeric single-chain Fv fragments (scFvs) phage-displayed library was panned in HEL-coated immunotubes. After two rounds of selection under non-stringent conditions, that is, trypsinization after 2 h of incubation at room temperature, 63 of 167 clones analyzed (38%) were found to express scFvs specific to HEL. Twenty clones were characterized by DNA sequencing resulting in 10 unique scFvs. Interestingly, the panel of unique scFvs was highly diverse, with V(H) sequences differing in 16 of the 17 positions variegated in the repertoire. Thus, diverse chemico-physical and structural solutions were selected from the library, even when the V(H) repertoire was constrained by the V(L) chain of D1.3 to yield binders against a definite region of HEL surface. The more often selected scFvs, namely H6-1 and B7-1, which differed in eight SDRs, showed levels of expression in E. coli TG1 strain, 6 and 10 times higher than the parental D1.3 Fv fragment, respectively. Dissociation constants (K(Ds)) measured in the BIAcore were 11 and 6.6 nM for H6-1 and B7-1, respectively. These values compared well to the K(D) of 4.7 nM measured for D1.3, indicating that the V(H) repertoire here designed is a valuable source of diverse, well-expressed and high affinity V(H) domains.

Protein Design through Systematic Catalytic Loop Exchange in the (β/α)8 Fold

“…46,47 Furthermore, in the antibody system, the loop repertoire is itself limited, suggesting the efficiency of employing modular exchange as opposed to randomization. 48 Third, in enzymes with the TIM barrel fold, the active site is always composed of loops and residues in the same face of the protein architecture.…”

An Ultra-specific Avian Antibody to Phosphorylated Tau Protein Reveals a Unique Mechanism for Phosphoepitope Recognition

“…These analyses have led to the recognition that the structural topography and chemical content of the paratope can be distinctly different between antibodies, depending on the size and nature of the antigen (63). Such structural insights have been critical to the adoption of the "designer compound libraries" (64) approach in antibody drug discovery, leading to the creation of synthetic antibody libraries that are tailored to recognize therapeutically or diagnostically relevant proteins (40), haptens (39), or peptides (41). The simplified generation of high-affinity recombinant antibodies to phosphoepitopes, as described here, will improve our ability to provide further structural insights and may potentiate phosphoprotein-targeted designer libraries in the future.…”

“…It may also inform fundamental comparative immunogenetics and antibody structure-function relationships. In the future, these studies could ameliorate the need for animal immunization by providing sufficient data to create designer, fully synthetic, phospho-biased antibody repertoires, as recently described for libraries biased toward the recognition of haptens (39), proteins (40), and peptides (41).…”

De Novo Selection of High-Affinity Antibodies from Synthetic Fab Libraries Displayed on Phage as pIX Fusion Proteins

“…11,14 These libraries have generated specific and high-affinity human antibodies against a wide array of structurally and chemically diverse antigens. More recently, an observed correlation between the type of antigen and the anatomy of the antigen-binding site has been applied to design focused libraries for biased isolation of antibodies to haptens, 16 proteins, 17 and peptides. 18 Phage coat protein III (pIII) has been the fusion partner of choice for display of antibody libraries.…”