MAb 4B7 neutralizes Pfs25 from Plasmodium falciparum.

Abstract: X-ray quality crystals of a Fab fragment from a transmission-blocking monoclonal antibody 4B7 (MAb 4B7) that was formed against a sexual stage protein Pfs25 of Plasmodium falciparum were grown as uncomplexed and peptide complexed forms. Because cleavage with pepsin or papain produced a non-homogeneous product, the whole immunoglobulin was crystallized (Stura et al. companion article1). Subsequently, a Fab fragment was obtained using the protease, elastase. The best complex crystals were obtained using cyclic peptide from the third EGF-like domain of Pfs25, where the N and C termini are joined with a covalent hydrogen-bond mimic. MAb 4B7 binds a linear peptide, however, the cyclic peptide shows a significant increase in binding affinity. An initial data set, complete to 3.0Å, has been collected from these complex crystals. The packing arrangement of the Fab in the crystals has been determined by molecular replacement and refinement of the structure is in progress. The genes for the variable domain of the Fab were cloned, sequenced and the primary amino acid sequence deduced. Knowledge of the three-dimensional structure of this Fab-peptide complex will be important in the understanding the mode by which this antibody recognizes and prevents transmission of the parasite.

34. Stura E.A., Kang, A.S, Stefanko, R.S., Calvo, J, Gaarder, K.L, Kaslow D.C., and Satterthwait, A.C. (1993) Crystallographic studies of transmission blocking antimalaria Fab 4B7 with cyclic and linear peptides from the Pfs25 protein of Plasmodium falciparum. American Journal of Tropical Medicine and Hygiene 49:Suppl.(137)-177.

Abstract: X-ray quality crystals of a transmission-blocking monoclonal antibody 4B7 (MAb 4B7) against a sexual stage protein Pfs25 of Plasmodium falciparum have been obtained. Both the intact immunoglobulin and an elastase produced Fab fragment have been crystallized both free and complexed with cyclic and linear peptides. While MAb 4B7 binds a linear peptide, cyclic peptides modeled on a predicted b-hairpin loop of the third EGF-like domain of Pfs25 bind better and are readily co-crystallized with the Fab. Several cyclic peptides and their linear counterparts have been synthesized. The affinity of MAb 4B7 for the different peptides varies widely. X-ray data have been collected from various peptide-complexed and free Fab crystals. The packing arrangement of the Fab in three independent crystal forms has been determined by molecular replacement and refinement of the structures is in progress. The genes for the variable domain of the Fab have been cloned, sequenced and the primary amino acid sequence deduced. The three-dimensional structure will aid in an understanding of the mode by which this antibody recognizes and prevents transmission of the parasite. The system presents unique opportunities to understand neutralization from the comparison between the bound forms of differently constrained cyclic peptides and the linear counterparts and from the comparison of the free and antigen-bound MAb. The study is being extended to include the structure determination of Pfs25 and its complex with MAb 4B7.

IgG 4B7 was crystallized in complex with a linear peptide

Abstract: Monoclonal antibody 4B7 is a neutralizing antibody against the protein Pfs25 from the sexual stage of the malaria parasite Plasmodium falciparum. Here we report the determination of the fine specificity of MAb 4B7 within the linear epitope of Pfs25 and the crystallization of the intact murine monoclonal antibody with peptides from the Pfs25 antigen. This study highlights the importance of ligands in the crystallization of proteins. In this case peptides have been used to modulate the solubility of the peptide-IgG complex and may have provided different or additional crystal contacts to create or enhance a crystalline reticulum not favoured by the uncomplexed IgG. Multiple crystal forms characterize this crystallization and the various peptides differing both in length and sequence have been used to investigate how small changes can have a strong effect on nucleation and crystal growth.

This work is important contribution to the field of Peptide Chemistry:

43. Stura E.A., Kaslow, D.C. and Satterthwait A.C. (1994) Crystallization of a neutralizing malaria immunoglobulin with linear and cyclic peptides In Peptides: Biology and Chemistry, Proceedings of the 13th American Peptide Symposium. Hodges, R., Smith, J.A. (eds.) ESCOM, Leiden, the Netherlands, pp 817.

63. Satterthwait, A.C., Cabezas, E., Calvo, J.C., Wu, J.X., Chen, S.Q., Kaslow, D.C., Livnah, O., and Stura, E.A., (1996) Constrained Synthetic Peptide Vaccines in ``Peptides: Chemistry, Structure & Biology'' Proceedings of the 14th American Peptide Symposium. Kaumaya P.T.P., Hodges, R.S. eds. pp. 772-773.

Abstract: A critical issue involves the development of a strategy for identifying constrained peptide vaccines. We systematically vary the size and cadence of natural peptide sequences constrained with hydrogen mimics while using neutralizing monoclonal antibodies to identify the best mimetic. Since every other amino acid in protein engages in a hydrogen bond and most hydrogen bonds in proteins are localized, many potential conformations can be screened. Here we report on the use of this strategy for the identification of a constrained peptide corresponding to an epitope on Pfs25, a protein found on malaria sexual stages and recognized by a neutralizing monoclonal antibody, Mab 4B7.