Andrew James Williams

A fully human antibody neutralising biologically active human TGFβ2 for use in therapy

Phage display provides a methodology for obtaining fully human antibodies directed against human transforming growth factor-beta (TGFbeta) suitable for the treatment of fibrotic disorders. The strategy employed was to isolate a human single chain Fv (scFv) fragment that neutralises human TGFbeta2 from a phage display repertoire, convert it into a human IgG4 and then determine its TGFbeta binding and neutralisation properties and its physical characteristics. Several scFv fragments binding to TGFbeta2 were isolated by panning of an antibody phage display repertoire, and subsequent chain shuffling of the selected V(H) domains with a library of V(L) domains. The three most potent neutralising antibodies were chosen for conversion to IgG4 format. The IgG4 antibodies were ranked for their ability to neutralise TGFbeta2 and the most potent, 6B1 IgG4, was chosen for further characterisation. 6B1 IgG4 has a high affinity for TGFbeta2 with a dissociation constant of 0.89 nM as determined using the BIAcore biosensor and only 9% cross-reactivity with TGFbeta3 (dissociation constant, 10 nM). There was no detectable binding to TGFbeta1. 6B1 IgG4 strongly neutralises (IC50 = 2 nM) the anti-proliferative effect of TGFbeta2 in bioassays using TF1 human erythroleukaemia cells. Similarly, there was strong inhibition of binding of TGFbeta2 to cell surface receptors in a radioreceptor assay using A549 cells. 6B1 IgG4 shows no detectable cross-reactivity with related or unrelated antigens by immunocytochemistry or ELISA. The 6B1 V(L) domain has entirely germline framework regions and the V(H) domain has only three non-germline framework amino acids. This, together with its fully human nature, should minimise any potential immunogenicity of 6B1 IgG4 when used in therapy of fibrotic diseases mediated by TGFbeta2.

Selection of phage antibodies to surface epitopes of Phytophthora infestans

Antibodies specific for surface-exposed epitopes on germlings of the plant pathogen, Phytophthora infestans, were isolated from a diverse phage library displaying single-chain Fv (scFv) antibody fragments. The library was subpanned against external soluble components released from mycelia, sporangia and germlings and a discrete population of phage antibodies isolated. Binding of monoclonal phage antibodies was demonstrated by enzyme-linked immunosorbent assay (ELISA) and diversity was established by BstNI restriction enzyme digest patterns. Antibodies were subcloned as fusions at the C-terminus of maltose binding protein (MBP) and expressed as soluble proteins in Escherichia coli. These antibody fusion proteins bound to P. infestans germlings and to mycelial homogenates from various Phytophthora species. The binding activities to mycelial homogenates of fungal species not belonging to the order Peronosporales were substantially lower. Several phage-displayed scFvs were used in conjunction with fluorescently labelled antiphage antibody to visualise the distribution of their cognate epitopes on the surface of the germlings. The combination of procedures developed here with Phytophthora demonstrates the potential of phage antibody technology in isolating antibodies to cell surface and external soluble components of pathogens, some of which may play a role in host/pathogen interactions.

Characterization of a unique human single-chain antibody isolated by phage-display selection on membrane-bound mosquito midgut antigens.

The insect midgut is the primary site for food digestion, as well as for vector-borne pathogen infection into the invertebrate host. Accordingly, antigens of this critical insect organ are targets for anti-vector vaccines, insecticidal toxins, and transmission-blocking vaccines. We used midgut proteins of the African malaria vector mosquito Anopheles gambiae to select single-chain human antibody fragments (scFv) from a high-diversity, phage-displayed library. Using a phage-display selection method on western-blotted antigens, we selected an unusual truncated scFv clone, consisting of a heavy-chain only, which binds to An. gambiae midgut tissue. This clone binds a spectrum of mosquito antigens from the midgut and other mosquito tissues, as well as various mammalian glycoproteins, but binding was reduced when these glycoproteins were enzymatically deglycosylated. We also observed that this clone preferentially binds the lumenal midgut surface. Furthermore, antigen binding by our selected scFv was limited by competition with increasing concentrations of certain soluble carbohydrates, most dramatically by galactose and N-acetyl glucosamine. Our results show that the cognate epitope of this scFv is a carbohydrate moiety. This paper describes a phage-display selection of antibody fragments on mosquito midgut tissue and it also describes a method for phage-display selection on membrane-immobilized heterogeneous antigens. These selection methods resulted in the isolation of a novel, truncated, carbohydrate-binding human antibody fragment from a naive phage-display library.

Tagging Bloodmeals with Phagemids Allows Feeding of Multiple-Sample Arrays to Single Cages of Mosquitoes (Diptera: Culicidae) and the Recovery of Single Recombinant Antibody Fragment Genes from Individual Insects

Abstract A recombinant single-chain variable-region human antibody fragment (scFv) was expressed inEscherichia coli,extracted in hypertonic sucrose, mixed directly with blood and fed toAnopheles gambiaeGiles mosquitoes. WhenE. colicontaining the phagemids that encode these scFv were included in bloodmeals, phagemids could be recovered from the mosquito midgut for up to 3 d after feeding. Furthermore, large arrays of such gene-tagged scFv-containing bloodmeals could be fed to cages of mosquitoes using microtiter plates. Arrays of phagemids with and without an antibody insert were fed to single cages of mosquitoes to test whether individual mosquitoes fed from single wells of such arrays. Phagemids were recovered from 95% of blood-fed females and >80% of these phagemids were monoclonal. Therefore, it is possible to feed multiple sample arrays of recombinant proteins to single cages of mosquitoes and to recover the genetic material that encodes for only one of the array elements from individual mosquitoes. This demonstration indicates that multiple-sample feeding and recovery strategies are feasible and may represent a viable strategy for future rapid screening of biologically active genes, gene products or microorganisms in live arthropods.