In recent years, the demand for antibodies for therapeutic purposes has increased [1]. To cope with this demand, some technologies have been adapted to generate and improve these antibodies [2,3]. Two of these methods are phage display [4,5] and directed evolution [6,7]. These technologies have allowed the generation and improvement of different antibodies, which now reach affinities similar to those of a secondary immunological response [3]. Depending on the purpose for which the antibody fragments are intended, several expression formats have been developed [8] This study describes the construction of a library of single-chain antibody fragments (scFvs) from a single human donor by individual amplification of all heavy and light variable domains (1.1 · 10 8 recombinants). The library was panned using the phage display technique, which allowed selection of specific scFvs (3F and C1) capable of recognizing Cn2, the major toxic component of Centruroides noxius scorpion venom. The scFv 3F was matured in vitro by three cycles of directed evolution. The use of stringent conditions in the third cycle allowed the selection of several improved clones. The best scFv obtained (6009F) was improved in terms of its affinity by 446-fold, from 183 nm (3F) to 410 pm. This scFv 6009F was able to neutralize 2 LD 50 of Cn2 toxin when a 1 : 10 molar ratio of toxin-to-antibody fragment was used. It was also able to neutralize 2 LD 50 of the whole venom. These results pave the way for the future generation of recombinant human antivenoms.Abbreviations CDR, complementarity determining region; Cn2, toxin from Centruroides noxius scorpion; scFv, single-chain antibody fragment; TEA, triethylamine; V H : heavy chain; V L , light chain.
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.
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