Polymerase chain reaction facilitates the cloning, CDR-grafting, and rapid expression of a murine monoclonal antibody directed against the CD18 component of leukocyte integrins.
Two novel approaches of recombinant PCR technology were employed to graft the complementarity determining regions from a murine monoclonal antibody (mAb) onto human antibody frameworks. One approach relied on the availability of cloned human variable region templates, whereas the other strategy was dependent only on human variable region protein sequence data. The transient expression of recombinant humanized antibody was driven by the adenovirus major late promoter and was detected 48 hrs post-transfection into non-lymphoid mammalian cells. The application of these new approaches enables the expression of a recombinant humanized antibody just 6 weeks after initiating the cDNA cloning of the murine mAb.
Daugherty
B L
BL
Department of Cellular and Molecular Biology, Merck Sharp and Dohme Research Laboratories, Rahway, NJ 07065.
DeMartino
J A
JA
Law
M F
MF
Kawka
D W
DW
Singer
I I
II
Mark
G E
GE
D000818:Animals; D000911:Antibodies, Monoclonal / Q000235:genetics* / Q000276:immunology; D015703:Antigens, CD / Q000276:immunology*; D001483:Base Sequence; D001666:Binding Sites, Antibody / Q000235:genetics; D018821:CD18 Antigens; D002478:Cells, Cultured; D003001:Cloning, Molecular / Q000379:methods*; D004247:DNA; D015870:Gene Expression; D005803:Genes, Immunoglobulin; D005822:Genetic Vectors; D006801:Humans; D007143:Immunoglobulin Heavy Chains / Q000235:genetics; D007147:Immunoglobulin Light Chains / Q000235:genetics; D016023:Integrins / Q000276:immunology*; D007962:Leukocytes / Q000276:immunology; D051379:Mice; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology; D014162:Transfection
D000911:Antibodies, Monoclonal; D015703:Antigens, CD; D018821:CD18 Antigens; D007143:Immunoglobulin Heavy Chains; D007147:Immunoglobulin Light Chains; D016023:Integrins; D011993:Recombinant Fusion Proteins; D004247:DNA