A fully human antibody neutralising biologically active human TGFbeta2 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.
Thompson
J E
JE
Cambridge Antibody Technology, The Science Park, Melbourn, Royston, Cambridgeshire, UK.
Vaughan
T J
TJ
Williams
A J
AJ
Wilton
J
J
Johnson
K S
KS
Bacon
L
L
Green
J A
JA
Field
R
R
Ruddock
S
S
Martins
M
M
Pope
A R
AR
Tempest
P R
PR
Jackson
R H
RH
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000276:immunology*; D003429:Cross Reactions; D006801:Humans; D007128:Immunoglobulin Fragments / Q000276:immunology*; D007074:Immunoglobulin G / Q000145:classification / Q000276:immunology; D008969:Molecular Sequence Data; D009500:Neutralization Tests; D016212:Transforming Growth Factor beta / Q000276:immunology*
D000911:Antibodies, Monoclonal; D007128:Immunoglobulin Fragments; D007074:Immunoglobulin G; D016212:Transforming Growth Factor beta; C055966:immunoglobulin Fv