title: Antibody C region influences TGN1412-like functional activity in vitro.
abstract: The unexpected outcome of the clinical trial of the superagonistic CD28 mAb TGN1412 (IgG4κ) continues to stimulate interest. We show that TGN1412 binds similarly to human and cynomolgus macaque FcγR, eliminating the possibility that differences in Fc-mediated interactions with FcγR contributed to the failure of preclinical testing in macaques to predict toxicity in humans. The influence of the Fc domain and C region structure on the in vitro functional activity of TGN1412 was investigated using F(ab')(2) and Fab fragments derived from TGN1412 recovered from the trial and recombinant TGN1412 subclass variants and mutants. Superagonistic activity, as measured by cytokine release and proliferation, was assessed by exposing PBMCs to immobilized mAbs/fragments or to aqueous mAbs/fragments in the presence of HUVEC monolayers. Removing the Fc generally curtailed or abolished PBMC activation. However, eliminating detectable FcγR-binding of the IgG4 by mutation (L235E) did not abrogate activity. Stabilizing the "wild-type" IgG4 hinge (S228P) enhanced activity without increasing FcγR binding, which could only partially be explained by inhibition of Fab arm-exchange. Subclass switching the IgG4 mAb to IgG1 decreased activity, whereas switching to IgG2 markedly increased activity. We conclude that the C region strongly influences in vitro CD28-mediated superagonistic signaling. Superagonism requires an intact Fc, as shown by the absence of activity of TGN1412 Fab and F(ab')(2) fragments, but, notably, appears to be relatively independent of FcγR-binding properties. We propose that the Fc, potentially through restricting flexibility, maintains a favorable V region conformation to allow superagonistic activity. These findings have important implications for Ab design strategies.
authors.affiliation: National Institute for Biological Standards and Control, Potters Bar, Hertfordshire EN6 3QG, United Kingdom.
authors.forename: Christina
authors.initials: C
authors.lastname: Ball
authors.forename: Bernard
authors.initials: B
authors.lastname: Fox
authors.forename: Simon
authors.initials: S
authors.lastname: Hufton
authors.forename: Giles
authors.initials: G
authors.lastname: Sharp
authors.forename: Stephen
authors.initials: S
authors.lastname: Poole
authors.forename: Richard
authors.initials: R
authors.lastname: Stebbings
authors.forename: David
authors.initials: D
authors.lastname: Eastwood
authors.forename: Lucy
authors.initials: L
authors.lastname: Findlay
authors.forename: Paul W H I
authors.initials: PW
authors.lastname: Parren
authors.forename: Robin
authors.initials: R
authors.lastname: Thorpe
authors.forename: Adrian
authors.initials: A
authors.lastname: Bristow
authors.forename: Susan J
authors.initials: SJ
authors.lastname: Thorpe
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000502:physiology*; D061067:Antibodies, Monoclonal, Humanized / Q000378:metabolism* / Q000502:physiology; D001666:Binding Sites, Antibody / Q000276:immunology; D049109:Cell Proliferation; D002478:Cells, Cultured; D017321:Clinical Trials, Phase I as Topic; D018920:Coculture Techniques; D016207:Cytokines / Q000378:metabolism; D061307:Human Umbilical Vein Endothelial Cells; D006801:Humans; D007127:Immunoglobulin Constant Regions / Q000737:chemistry / Q000378:metabolism / Q000502:physiology*; D007140:Immunoglobulin Fab Fragments / Q000502:physiology; D007141:Immunoglobulin Fc Fragments / Q000502:physiology; D007963:Leukocytes, Mononuclear / Q000276:immunology / Q000378:metabolism; D008252:Macaca fascicularis; D011485:Protein Binding / Q000276:immunology; D017452:Receptors, IgG / Q000378:metabolism / Q000502:physiology; D011994:Recombinant Proteins / Q000378:metabolism
chemical_list: D000911:Antibodies, Monoclonal; D061067:Antibodies, Monoclonal, Humanized; D016207:Cytokines; D007127:Immunoglobulin Constant Regions; D007140:Immunoglobulin Fab Fragments; D007141:Immunoglobulin Fc Fragments; D017452:Receptors, IgG; D011994:Recombinant Proteins; C509303:TGN-1412