Binding activity difference of anti-CD20 scFv-Fc fusion protein derived from variable domain exchange.
Two novel engineered antibody fragments binding to antigen CD20 were generated by fusing a murine IgM-type anti-CD20 single-chain Fv fragment (scFv) to the human IgG1 CH2 (i.e., Cgamma2) and CH3 (i.e., Cgamma3) domains with the human IgG1 hinge (i.e. Hgamma). Given the relationship between structure and function of protein, the 3-D structures of the two engineered antibody fragments were modeled using computer-aided homology modeling method. Furthermore, the relationship between 3-D conformation and their binding activity was evaluated theoretically. Due to the change of active pocket formed by CDRs, the HL23 (VH-Linker-VL-Hgamma-Cgamma2-Cgamma3) remained its activity because of its preserved conformation, while the binding activity of the LH23 (VL-Linker-VH-Hgamma-Cgamma2-Cgamma3) was impaired severely. Experimental studies by flow cytometry and fluorescence microscopy showed that HL23 possessed significantly superior binding activity to CD20-expressing target cells than LH23. That is to say, the order of variable regions could influence the binding activity of the fusion protein to CD20+ cell lines, which was in accordance with the theoretical results. The study highlights the potential relationship between the antibody binding activity and their 3-D conformation, which appears to be worthwhile in providing direction for future antibody design of recombinant antibody.
Geng
Shu Sheng
SS
College of Life Science, Hebei University, Baoding, Hebei, China.
Feng
Jiannan
J
Li
Yan
Y
Sun
Yingxun
Y
Gu
Xin
X
Huang
Ying
Y
Wang
Yugang
Y
Kang
Xianjiang
X
Chang
Hong
H
Shen
Beifen
B
D000818:Animals; D018951:Antigens, CD20 / Q000276:immunology* / Q000378:metabolism*; D001666:Binding Sites, Antibody / Q000235:genetics; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D006801:Humans; D007141:Immunoglobulin Fc Fragments / Q000235:genetics / Q000378:metabolism*; D007135:Immunoglobulin Variable Region / Q000235:genetics / Q000378:metabolism*; D019169:Jurkat Cells; D051379:Mice; D011485:Protein Binding / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*
D018951:Antigens, CD20; D007141:Immunoglobulin Fc Fragments; D007135:Immunoglobulin Variable Region; D011993:Recombinant Fusion Proteins