A monoclonal antibody with anti-D-like activity in murine immune thrombocytopenia requires Fc domain function for immune thrombocytopenia ameliorative effects.
BACKGROUND
The mechanism of action of anti-D in ameliorating immune thrombocytopenia (ITP) remains unclear. The monoclonal antibody (MoAb) Ter119, which targets murine red blood cells (RBCs), has been shown to mimic the effect of anti-D in improving antibody-mediated murine ITP. The mechanism of Ter119-mediated ITP amelioration, especially the role of the antigen-binding and Fc domains, remains untested. A functional Fc domain is crucial for many therapeutic MoAb activity; therefore, the requirement of Ter119 Fc domain in ITP amelioration is investigated using outbred CD-1 mice.


METHODS
Ter119 variants, including Ter119 F(ab')2 fragments, deglycosylated Ter119, and afucosylated Ter119, were generated to test their effect in ameliorating antibody-induced murine ITP. In vivo inhibition of FcγRIII and FcγRIIB was achieved using the Fab fragment of the FcγRIII/FcγRIIB-specific MoAb 2.4G2.


RESULTS
Ter119 F(ab')2 fragments and deglycosylated Ter119 were unable to ameliorate murine ITP or mediate phagocytosis of RBCs by RAW264.7 macrophages in vitro. Inhibition of FcγRIII and FcγRIIB, as well as Ter119 defucosylation, do not affect Ter119-mediated ITP amelioration.


CONCLUSIONS
The Fc domain of Ter119, as well as its Fc glycosylation, is required for Ter119-mediated ITP amelioration. Moreover, both Fc and Fc glycosylation are required for Ter119-mediated phagocytosis in vitro. These findings demonstrate the importance of the Fc domain in a therapeutic MoAb with anti-D-like activity.
Canadian Blood Services, Ottawa, Ontario, Canada.|Department of Laboratory Medicine, the Keenan Research Centre, Li Ka Shing Knowledge Institute, St Michael's Hospital, Toronto, Ontario, Canada.
Xiaojie
X
Yu
Department of Laboratory Medicine, the Keenan Research Centre, Li Ka Shing Knowledge Institute, St Michael's Hospital, Toronto, Ontario, Canada.|Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, Canada.
Melissa
M
Menard
Department of Biology & Biochemistry, University of Bath, Bath, United Kingdom.|Department of Biochemistry, Oxford Glycobiology Institute, University of Oxford, Oxford, United Kingdom.
Gemma
G
Seabright
Department of Biochemistry, Oxford Glycobiology Institute, University of Oxford, Oxford, United Kingdom.
Max
M
Crispin
Canadian Blood Services, Ottawa, Ontario, Canada.|Department of Laboratory Medicine, the Keenan Research Centre, Li Ka Shing Knowledge Institute, St Michael's Hospital, Toronto, Ontario, Canada.|Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, Canada.
Alan H
AH
Lazarus
D000818:Animals; D021461:Animals, Outbred Strains; D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology* / Q000627:therapeutic use; D002478:Cells, Cultured; D004195:Disease Models, Animal; D005260:Female; D006031:Glycosylation; D007140:Immunoglobulin Fab Fragments / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology / Q000627:therapeutic use; D007141:Immunoglobulin Fc Fragments / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology* / Q000627:therapeutic use; D007518:Isoantibodies / Q000737:chemistry / Q000494:pharmacology*; D051379:Mice; D008810:Mice, Inbred C57BL; D017434:Protein Structure, Tertiary / Q000502:physiology; D016553:Purpura, Thrombocytopenic, Idiopathic / Q000276:immunology / Q000473:pathology / Q000628:therapy*; D018029:Rho(D) Immune Globulin
D000911:Antibodies, Monoclonal; D007140:Immunoglobulin Fab Fragments; D007141:Immunoglobulin Fc Fragments; D007518:Isoantibodies; C061961:RHO(D) antibody; D018029:Rho(D) Immune Globulin