High efficiency reconstitution of a human-mouse chimeric Fab of CAb-1 antibody specific to human colon cancer.
Monoclonal antibody (mAb) has been widely applied in the treatment of human diseases, especially in malignant tumours. However, most antibodies produced in mouse by hybridoma technology might induce severe human anti-mouse reactions. We had reported a murine mAb CAb-1 of therapeutic interest for its specifically binding to a cell surface glycoprotein of human colon cancer. Here, we attempted to generate a reconstituted human-mouse chimeric Fab (cFab) of CAb-1 in vitro to reduce its antigenicity and increase its capacity of penetration. First, the genes of heavy and light chain variable region (VH, VL) of CAb-1 were cloned. Then, the chimeric light chain (cL) and Fd (cFd) were constructed and expressed in Escherichia coli. Finally, the reconstituted cFab was obtained by gradient dialysis of the mixture of cFd and cL. SDS-PAGE and western blot analysis showed the reconstituted cFab with a recovery rate of 70.2% when the initial total concentration of cL and cFd proteins to be 100 microg/ml. The reconstituted cFab maintained the affinity and specificity to colon cancer cells compared with its parental antibody as determined by immunostaining analysis, FACS and ELISA. Our results established a foundation for further application of the cFab in diagnosis and treatment of colon cancer.
Yang
X-M
XM
Cell Engineering Research Centre & Department of Cell Biology, State Key Laboratory of Cancer Biology, Fourth Military Medical University, Xi'an, China.
Xing
J-L
JL
Liao
C-G
CG
Yao
X-Y
XY
Li
Y
Y
Chen
Z-N
ZN
D000818:Animals; D000911:Antibodies, Monoclonal / Q000235:genetics* / Q000276:immunology*; D000918:Antibody Specificity; D000951:Antigens, Neoplasm / Q000276:immunology*; D015153:Blotting, Western; D002678:Chimera; D003001:Cloning, Molecular; D003110:Colonic Neoplasms / Q000276:immunology*; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D005434:Flow Cytometry; D005813:Genes, Synthetic; D005822:Genetic Vectors; D006801:Humans; D006825:Hybridomas; D007140:Immunoglobulin Fab Fragments / Q000235:genetics* / Q000276:immunology*; D051379:Mice; D020133:Reverse Transcriptase Polymerase Chain Reaction
D000911:Antibodies, Monoclonal; D000951:Antigens, Neoplasm; D007140:Immunoglobulin Fab Fragments