Human IgG1 Cgamma1 domain is crucial for the bioactivity of the engineered anti-CD20 antibodies.
In this study, we discussed the necessity of human IgG1 Cgamma1 domain for recombinant antibody using computer-aided homology modeling method and experimental studies. The heavy (VH) and light (VL) chain variable regions of 1-28, a murine IgM-type anti-CD20 mAb, were ligated by linker peptide (Gly4Ser)3 to form the single-chain Fv fragment (scFv). Then, the engineered antibody (LH1-3) was generated by fusing scFv with the entire IgG1 heavy constant regions. The 3-D structure of LH1-3 was modeled using computer-aided homology modeling method and the binding activity of LH1-3 was evaluated theoretically. Compared to the 3-D structure of the Fv fragment of the parent antibody, the conformation of the active pocket of LH1-3 was remained because of the rigid support of Cgamma1. Further experimental results of flow cytometry showed that the engineered anti-CD20 antibody possessed specifically binding activity to CD20-expressing target cells. The anti-CD20 antibody fragments could also mediate complement-dependent cytotoxicity (CDC) of human B-lymphoid cell lines. Our study highlights some interests and advantages of a methodology based on the homology modeling and analysis of molecular structural properties.
Geng
Shusheng
S
College of Life Science, Hebei University, Baoding, China.
Feng
Jiannan
J
Li
Yan
Y
Kang
Xianjiang
X
Sun
Yingxun
Y
Gu
Xin
X
Huang
Ying
Y
Chang
Hong
H
Shen
Bei Fen
BF
D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D000915:Antibody Affinity; D000918:Antibody Specificity; D000920:Antibody-Dependent Cell Cytotoxicity; D018951:Antigens, CD20 / Q000276:immunology* / Q000378:metabolism; D001666:Binding Sites, Antibody; D005434:Flow Cytometry; D006801:Humans; D021621:Imaging, Three-Dimensional; D007127:Immunoglobulin Constant Regions / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D007074:Immunoglobulin G / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D007075:Immunoglobulin M / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D007135:Immunoglobulin Variable Region / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D007142:Immunoglobulin gamma-Chains / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D019169:Jurkat Cells; D051379:Mice; D008958:Models, Molecular; D011487:Protein Conformation; D015202:Protein Engineering; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D013329:Structure-Activity Relationship
D000911:Antibodies, Monoclonal; D018951:Antigens, CD20; D007127:Immunoglobulin Constant Regions; D007074:Immunoglobulin G; D007075:Immunoglobulin M; D007135:Immunoglobulin Variable Region; D007142:Immunoglobulin gamma-Chains; D011993:Recombinant Fusion Proteins