Structural insight into how an anti-idiotypic antibody against D3H44 (anti-tissue factor antibody) restores normal coagulation.
6A6 is a murine monoclonal antibody raised against the humanized anti-tissue factor antibody D3H44. 6A6 is able to completely neutralize the anticoagulant activity of D3H44 in tissue factor-dependent functional assays, such as endotoxin-induced whole blood clotting, prothrombin time, as well as factor X and factor IX activation. ELISA-type assays further showed that 6A6 binds to an epitope with critical determinants on the V(L) domain of D3H44. The possibility that the anti-idiotypic 6A6 might carry an "internal image" of the original antigen (tissue factor) was examined using the X-ray structure of the 6A6-Fab/D3H44-Fab complex determined at 2.5A resolution. We find that 6A6 structurally mimics tissue factor only so far as it combines with the antigen recognition surface of D3H44. While 6A6 contacts both V(L) and V(H) domains of D3H44, as does tissue factor, there is more contact with the D3H44 V(L) domain and less with the D3H44 V(H) domain relative to the tissue factor contacts on D3H44. Additionally, there is an almost total lack of correspondence between 6A6 and tissue factor at the level of amino acid side-chain functional groups. Despite the fact that both tissue factor and 6A6 are composed largely of beta-sheets, they present fundamentally different elements of secondary structure to D3H44; tissue factor presents beta-sheets edge-on, while 6A6 uses mostly loops. Finally, the finding that 6A6 competes with tissue factor for D3H44 binding raises the possibility of using 6A6 as an antidote for D3H44 anticoagulant therapy. To this end, we constructed a chimeric murine/human 6A6-Fab, which effectively neutralized D3H44 and fully restored tissue factor function in enzymatic assays.
Eigenbrot
Charles
C
Department of Protein, Genentech Inc, South San Francisco, CA 94080, USA. eigenbrot@gene.com
Meng
Y Gloria
YG
Krishnamurthy
Rajeswari
R
Lipari
Michael T
MT
Presta
Leonard
L
Devaux
Brigitte
B
Wong
Terence
T
Moran
Paul
P
Bullens
Sherron
S
Kirchhofer
Daniel
D
D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry*; D000941:Antigens / Q000737:chemistry; D018360:Crystallography, X-Ray; D004306:Dose-Response Relationship, Immunologic; D004731:Endotoxins / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes; D005164:Factor IX / Q000378:metabolism; D005170:Factor X / Q000378:metabolism; D006801:Humans; D007128:Immunoglobulin Fragments / Q000737:chemistry; D051379:Mice; D008958:Models, Molecular; D011485:Protein Binding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011517:Prothrombin Time; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D013925:Thromboplastin / Q000737:chemistry / Q000276:immunology*
D000911:Antibodies, Monoclonal; D000941:Antigens; D004731:Endotoxins; D000939:Epitopes; D007128:Immunoglobulin Fragments; D011993:Recombinant Fusion Proteins; D005164:Factor IX; D005170:Factor X; D013925:Thromboplastin