Production of High-Affinity Human Monoclonal Antibody Fab Fragments to the 19-Kilodalton C-Terminal Merozoite Surface Protein 1 ofPlasmodium falciparum

Abstract

A combinatorial immunoglobulin gene library was constructed from peripheral blood lymphocytes of eight patients infected with Plasmodium falciparum and was screened for the production of human monoclonal antibody Fab fragments to the C-terminal 19-kDa fragment of P. falciparum merozoite surface protein 1 (MSP-1 19 ). Three Fab clones recognized recombinant MSP-1 19 under nonreducing conditions. Indirect immunofluorescence microscopy demonstrated that three Fab clones stained the surfaces of late trophozoites/ schizonts and merozoites of the FCR3 and 3D7 strains, suggesting the Fabs' reactivities to a conserved epitope. Sequence analysis of the heavy-chain genes revealed that the closest germ line V segments were VH1-8 and VH7-81, with 91% to 98% homology. The closest germ line D segment was D3-10, and the closest germ line J segment was JH4 or JH5, with 90% to 97% homology. In the light-chain genes, the closest germ line V segment was A27 for the J2, J4, and J5 segments. The dissociation constants of these Fab fragments for recombinant MSP-1 19 ranged from 1.09 ؋ 10 ؊9 to 2.66 ؋ 10 ؊9 M. The binding of the three Fab fragments to MSP-1 19 was competitively inhibited by the anti-MSP-1 19 mouse monoclonal antibody 12.8, which inhibits erythrocyte invasion by merozoites. However, the human Fab fragment with the highest affinity did not inhibit in vitro growth of P. falciparum. This is the first report of gene analysis and bacterial expression of human monoclonal antibodies to P. falciparum MSP-1 19 . The combinatorial immunoglobulin gene library derived from malaria patients provides a potential tool for producing high-affinity human antibodies specific for P. falciparum.

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

“…The amino acid sequences of CDRs in the heavy and light chains of the Fab with Ile 97 -Leu (Pf25-L97L) were identical to those of Pf227 which we previously produced (Cheng et al 2007). However, the affinity of Pf25-L97L (K D =9.36×10 −10 M) was higher than that of Pf227 (K D = 2.66×10 −9 M), which may be due to the presence of different amino acid sequences in the framework of these Fabs.…”

“…SDS-PAGE of the purified proteins demonstrated two bands with apparent molecular masses of 24 and 25 kDa under reduced conditions (data not shown). The reactivity of these purified (Cheng et al 2007) Fabs to MSP-1 19 in an ELISA was comparable with that of Pf25. In contrast, these Fabs showed no reactivity with two other recombinant proteins prepared in E. coli, E. histolytica C-Igl and T. gondii P30, indicating that the modified Fabs remain specific for MSP-1 19 .…”

“…In the Kabat numbering system, CDR3 of the heavy and light chains of Pf25 are the amino acid segments from positions 99 to 108 and 90 to 98, respectively (Cheng et al 2007). The corresponding amino acid sequences of the heavy and light chains are Val-Gly-Val-Trp-Phe-GlyGlu-Leu-Trp-Tyr and Gln-Gln-Tyr-Gly-Ser-Ser-Pro-IleThr, respectively.…”

“…Recombinant MSP-1 19 prepared as previously described was used for screening of Fab clones (Cheng et al 2007). Each well of a Nunc MaxiSorp™ high-protein-binding capacity ELISA plate (Nunc, Roskilde, Denmark) was coated with 0.1 μg/well of MSP-1 19 overnight at 4°C.…”

“…We recently produced three human monoclonal antibody Fab fragments specific for MSP-1 19 in Escherichia coli using combinatorial immunoglobulin gene libraries prepared from peripheral lymphocytes of eight patients infected with P. falciparum (Cheng et al 2007). In antibody molecules, the complementarity-determining regions (CDRs), and especially CDR3 of the heavy and light chains, are responsible for high-affinity binding with an antigen.…”