Immunoglobulin complementarity-determining region grafting by recombinant polymerase chain reaction to generate humanised monoclonal antibodies.
We describe an approach to rapidly generate humanised monoclonal antibodies by grafting rodent complementarity-determining regions onto human immunoglobulin frameworks using recombinant polymerase chain reaction (PCR) methodology. The approach was applied to grafting a rat complementarily-determining region onto a human framework and amplifying the entire humanised heavy chain. The terminal oligodeoxyribonucleotide primers incorporated restriction sites to allow forced cloning into plasmid vectors for sequencing and expression. No nucleotide errors were introduced into the 1463-bp sequence even after sequential applications of PCR.
Lewis
A P
AP
Department of Cell Biology, Wellcome Research Laboratories, Beckenham, Kent, U.K.
Crowe
J S
JS
D000818:Animals; D000911:Antibodies, Monoclonal / Q000235:genetics*; D001483:Base Sequence; D003001:Cloning, Molecular; D004077:Digoxin / Q000276:immunology; D006801:Humans; D007074:Immunoglobulin G / Q000235:genetics; D007143:Immunoglobulin Heavy Chains / Q000235:genetics*; D007135:Immunoglobulin Variable Region / Q000235:genetics*; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods; D015202:Protein Engineering; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000235:genetics*
D000911:Antibodies, Monoclonal; D007074:Immunoglobulin G; D007143:Immunoglobulin Heavy Chains; D007135:Immunoglobulin Variable Region; D011993:Recombinant Fusion Proteins; D004077:Digoxin