Improved Affinity of a Human Anti-Entamoeba histolyticaGal/GalNAc Lectin Fab Fragment by a Single Amino Acid Modification of the Light Chain

Abstract

We previously produced, in Escherichia coli, a human monoclonal antibody Fab fragment, CP33, specific for the galactose-and N-acetyl-D-galactosamine-inhibitable lectin of Entamoeba histolytica. To prepare antibodies with a higher affinity to the lectin, recombination PCR was used to exchange Ser 91 Amebiasis caused by infection with the intestinal protozoan parasite Entamoeba histolytica is a notable parasitic disease in both developing and developed countries. It has been estimated that 50 million people develop amebic colitis and extraintestinal abscesses, resulting in up to 110,000 deaths annually (18). The development of immunoprophylaxis and accurate diagnostic tools is important for the control of amebiasis. The application of monoclonal antibodies is a promising avenue of research for improvement in diagnosis.We recently produced several human monoclonal antibody Fab fragments specific for E. histolytica in Escherichia coli by use of combinatorial immunoglobulin gene libraries constructed from the peripheral lymphocytes of a patient with an amebic liver abscess and from an asymptomatic cyst passer (1,14,17). One of the Fab clones, CP33, derived from the asymptomatic cyst passer, recognized the cysteine-rich domain of the heavy subunit of the galactose-and N-acetyl-D-galactosamineinhibitable (Gal/GalNAc) lectin (12) of E. histolytica (17). This clone exhibited neutralizing activities to amebic adherence and to erythrophagocytosis. Furthermore, we produced the Fab fragment fused with alkaline phosphatase for diagnostic purposes (16).Recombinant antibody technology makes it possible to introduce site-directed or random mutations in the original antibody gene (3-5, 13, 19). Residues in the complementaritydetermining region (CDR), especially in CDR3 of both the heavy and light chains of antibody, are considered responsible for high-energy interactions with antigen. Therefore, mutations at these residues will likely abolish antigen binding. However, an increased affinity may also occur by mutation if the native residue exhibits a negative effect on the interaction. In the Kabat numbering system, CDR3 of the light chain is the amino acid segment from position 89 to 97 (6, 20). The corresponding amino acid residues in CP33 were GlnGlnSerTyrSer ThrProArgThr (17). When an additional 13 light chains which constitute antilectin Fabs with the heavy chain of CP33 were analyzed, high variability was observed at positions 91, 92, 94, and 96 (17). As a first step in the affinity maturation of human antibodies to E. histolytica, we attempted to modify Fab clone CP33 by single-amino-acid substitutions of Ser 91 and Arg 96 in the light chain. MATERIALS AND METHODSSite-directed mutagenesis. Site-directed mutagenesis in the light chain gene of CP33 (17) was performed by recombination PCR (7). The plasmid vector pFab1-His2, containing the light and the Fd region of the heavy chain genes, was amplified by using two sets of primers, CP33L-S 91 X-For (5Ј-CAACTTACTAC TGTCAACAGNNNTACAGTAC-3Ј, where N is any nucleotide) and CP33L-...

Hybridoma Passage In Vitro May Result in Reduced Ability of Antimannan Antibody To Protect against Disseminated Candidiasis

“…Although these changes comprise part of the first hypervariable region for CDR1, which is involved in antigen recognition and may affect epitope affinity (43), the antibodies we studied have the same epitope binding characteristics and similar relative affinities (estimated by the 50% inhibitory concentration), which rules out loss of protective activity due to affinity changes. Additionally, as discussed above, changes in glycosylation sites do not account for differences in C3 activation kinetics.…”

Modification of a human monoclonal antibody Fab fragment specific for Plasmodium falciparum 19-kDa C-terminal merozoite surface protein 1 by site-directed mutagenesis

“…In our previous study on Fab fragments specific to E. histolytica lectin, a Fab with a single amino acid substitution at position 91 in the light chain exhibited about fivefold higher affinity than the original Fab (Tachibana et al 2004b). However, such a marked improvement in binding affinity was not observed in the current study.…”

“…Therefore, it is possible to maturate affinity by chain shuffling and site-directed mutagenesis in recombinant antibody technology (Winkler et al 2000;Dong et al 2003). We have also demonstrated that single amino acid substitution in CDR3 of the light chain can improve the binding affinity of an anti-Entamoeba histolytica human Fab fragment (Tachibana et al 2004b). In this study, we substituted single amino acid residues in either the heavy or light chain to improve affinity of the original antibody.…”

Development of the dual-vector system-III (DVS-III), which facilitates affinity maturation of a Fab antibody via light chain shuffling

“…Unfortunately, however, it seems rather difficult to isolate high affinity antibodies at nanomolar ranges from a naïve Ig repertoire [4], and it is generally acknowledged that lengthy antibody gene manipulations, including site-directed (non-stochastic) or random mutations (stochastic) in the original H and/or L chain antibody genes [5,6,7], are usually followed to improve the relative affinities of antibodies of naïve origin [8]. L chain optimization is an attractive and convenient alternative for improving antibody affinities because it has been known that L chain gene arrangement in peripheral B cells contributes the affinity maturation of antibodies [9], and even a single amino acid modification of the L chain can result in a 5-fold increase of binding affinity for the anti-Gal/GalNAc lectin Fab fragment [10].…”