title: Immunity against a therapeutic xenoprotein/Fc construct delivered by gene transfer is reduced through binding to the inhibitory receptor FcγRIIb.
abstract: BACKGROUND
  Therapeutic xenoproteins are immunogenic and can induce neutralizing antibodies. When delivered by intramuscular injection of a plasmid vector, this mimics classical DNA vaccination. To demonstrate this, we chose Exendin-4 (Ex4), which is a glucagon-like peptide-1 mimetic xenoprotein in clinical use for treating type 2 diabetes. We constructed an Ex4 and mouse immunoglobulin (Ig)G1-Fc fusion fragment (Ex4/Fc), and hypothesized that it would have minimal immunogenicity as a result of its capacity to bind the inhibitory Fc receptor FcγRIIb expressed by B lymphocytes.
  
  
  METHODS
  Plasmid vectors encoding Ex4/Fc constructs, with wild-type or mutant Fc, were injected intramuscularly into mice, and local electroporation was applied to enhance gene transfer. Gene transfer was performed in both wild-type and FcγRIIb knockout mice. Antibody production was detected in serum by an enzyme-linked immunosorbent assay.
  
  
  RESULTS
  Recombinant Ex4/Fc bound only to B cells expressing FcγRIIb. This binding was dependent on a motif in the Fc region, which we mutated to abolish binding (Ex4/Fcmut). Ex4 antibody was detected in mice treated with Ex4, as well as Ex4/Fcmut, but not in those treated with Ex4/Fc. Thus, wild-type Fc was associated with reduced immunogenicity. To confirm this was related to the presence of inhibitory Fc receptors, we also performed experiments in FcγRIIb-null mice. Mice lacking this receptor produced antibodies against all Ex4 constructs, including the wild-type Fc (Ex4/Fc).
  
  
  CONCLUSIONS
  The present study shows that inhibitory FcγRIIb receptors interacting with the wild-type IgG1-Fc reduce immunity against Ex4/Fc, suggesting an approach for reducing the immunogenicity of therapeutic proteins in the context of gene therapy.
authors.affiliation: Division of Endocrinology and Metabolism, Keenan Research Centre in the Li Ka Shing Knowledge Institute, St Michael's Hospital, Toronto, Ontario, Canada.
authors.forename: Yaming
authors.initials: Y
authors.lastname: Liang
authors.forename: Hongmin
authors.initials: H
authors.lastname: Qiu
authors.forename: Yelena
authors.initials: Y
authors.lastname: Glinka
authors.forename: Alan H
authors.initials: AH
authors.lastname: Lazarus
authors.forename: Heyu
authors.initials: H
authors.lastname: Ni
authors.forename: Gerald J
authors.initials: GJ
authors.lastname: Prud'homme
authors.forename: Qinghua
authors.initials: Q
authors.lastname: Wang
mesh_terms: D000818:Animals; D057134:Antibodies, Neutralizing / Q000097:blood / Q000276:immunology; D001402:B-Lymphocytes / Q000276:immunology / Q000378:metabolism; D003924:Diabetes Mellitus, Type 2 / Q000628:therapy; D000077270:Exenatide; D005786:Gene Expression Regulation; D018014:Gene Transfer Techniques; D015316:Genetic Therapy; D007004:Hypoglycemic Agents / Q000276:immunology*; D007141:Immunoglobulin Fc Fragments / Q000235:genetics* / Q000378:metabolism; D007273:Injections, Intramuscular; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D010455:Peptides / Q000235:genetics* / Q000276:immunology*; D010957:Plasmids / Q000008:administration & dosage / Q000235:genetics; D011485:Protein Binding; D017452:Receptors, IgG / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000276:immunology / Q000378:metabolism; D014161:Transduction, Genetic; D014688:Venoms / Q000235:genetics* / Q000276:immunology*
chemical_list: D057134:Antibodies, Neutralizing; D007004:Hypoglycemic Agents; D007141:Immunoglobulin Fc Fragments; D010455:Peptides; D017452:Receptors, IgG; D011993:Recombinant Fusion Proteins; D014688:Venoms; D000077270:Exenatide