Effect of VK framework-1 glycosylation on the binding affinity of lymphoma-specific murine and chimeric LL2 antibodies and its potential use as a novel conjugation site.
A potential asparagine (Asn)-linked glycosylation site was identified in the VK FRI sequence of an anti-B lymphoma monoclonal antibody (MAb), LL2.SDS-PAGE analysis and endo-F treatment of both murine and chimeric LL2 antibodies indicated that this site was glycosylated; however, no differences in the binding affinity to Raji cells were observed between the native murine LL2 and the endo-F-deglycosylated murine LL2 antibodies. Elimination of the glycosylation site from the chimeric LL2 antibody was accomplished by an Asn to Gln mutation in the tri-acceptor site found in the light chain. The resultant aglycosylated chimeric LL2 exhibited a similar Raji cell binding affinity to that of the glycosylated form. The results are in agreement with computer modeling studies which suggested the lack of interactions between the oligosaccharide moiety and the CDRs. The finding is interesting because it enables a wider choice of human framework sequences, which in most cases do not have a corresponding glycosylation site, for the humanization of the LL2 VK domain, as well as a greater latitude of host expression systems. Most importantly, the LL2 VK carbohydrate moiety might be used as a novel conjugation site for drugs and radionuclides without compromising the immunoreactivity of the antibody.
Leung
S O
SO
Immunomedics, Inc., Morris Plains, NJ 07950.
Dion
A S
AS
Pellegrini
M C
MC
Losman
M J
MJ
Grebenau
R C
RC
Goldenberg
D M
DM
Hansen
H J
HJ
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry* / Q000235:genetics; D000912:Antibodies, Neoplasm / Q000737:chemistry*; D000915:Antibody Affinity; D001216:Asparagine / Q000737:chemistry; D001483:Base Sequence; D001666:Binding Sites, Antibody; D006031:Glycosylation; D006801:Humans; D007074:Immunoglobulin G / Q000737:chemistry; D007135:Immunoglobulin Variable Region / Q000737:chemistry*; D007145:Immunoglobulin kappa-Chains / Q000737:chemistry; D000922:Immunotoxins / Q000737:chemistry; D016393:Lymphoma, B-Cell / Q000276:immunology*; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D011993:Recombinant Fusion Proteins / Q000737:chemistry
D000911:Antibodies, Monoclonal; D000912:Antibodies, Neoplasm; D007074:Immunoglobulin G; D007135:Immunoglobulin Variable Region; D007145:Immunoglobulin kappa-Chains; D000922:Immunotoxins; D011993:Recombinant Fusion Proteins; D001216:Asparagine