Molecular cloning and chimerisation of an inhibitory anti-cathepsin B antibody and its expression in Chinese hamster ovary cells.
The lysosomal cysteine protease cathepsin B is one of several proteases that have been linked to tumour progression. Its increased expression and secretion in tumour cells may facilitate the degradation of extracellular matrix proteins, leading to tumour cell invasion and metastasis. Specific inhibitory monoclonal antibodies are a possible alternative to synthetic inhibitors as a therapeutic tool for cancer treatment. An inhibitory monoclonal antibody, which binds to an epitope near the active site of cathepsin B and inhibits its proteolytic activity, was prepared and its effect on invasion of ras-transformed MCF-10A neoT cells was tested in vitro. Here we present the nucleotide sequences of the heavy and light chains of the inhibitory antibody and compare them to the murine immunoglobulin germline sequences for possible somatic hypermutations. Since no harmful mutations were found, a mouse/human chimeric antibody was constructed by fusing murine V(H) and V(L) variable regions of the inhibitory antibody with human gamma 1 and K constant regions, respectively. Chinese hamster ovary K1 cells were co-transfected with expression vectors pcD-NA3L and pcDNA3H and the reactivity of the isolated chimeric antibody was tested by ELISA and Western blotting. We could demonstrate an inhibitory effect of the chimeric antibody.
Fan
Xiaohui
X
Department of Biochemistry and Molecular Biology, Jozef Stefan Institute, Jamova 39, SI-1 000 Ljubljana, Slovenia.
Kopitar-Jerala
Natasa
N
Premzl
Ales
A
Bestagno
Marco
M
Burrone
Oscar
O
Kos
Janko
J
D000595:Amino Acid Sequence; D000818:Animals; D019138:Antibodies, Blocking / Q000737:chemistry* / Q000494:pharmacology; D001483:Base Sequence; D015153:Blotting, Western; D016466:CHO Cells; D002401:Cathepsin B / Q000037:antagonists & inhibitors* / Q000235:genetics; D003001:Cloning, Molecular; D006224:Cricetinae; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000737:chemistry / Q000276:immunology; D005822:Genetic Vectors; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000494:pharmacology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection
D019138:Antibodies, Blocking; D000939:Epitopes; D011993:Recombinant Fusion Proteins; D002401:Cathepsin B