High-level expression of chimeric antibodies using adapted cDNA variable region cassettes.
A rapid and generally applicable method for the modification of immunoglobulin cDNAs was developed so that the variable (V) regions could be expressed as cassettes, together with a variety of constant regions. Murine cDNAs were isolated, sequenced and the V regions joined to short oligonucleotides providing both splice donor sites and unique restriction sites for insertion into an expression vector. Using this strategy we have expressed the V regions of several murine antibodies, together with the human gamma 1 constant region. Although most of these chimeric antibodies were readily expressed, one murine light-chain cDNA sequence could not be expressed in transfected hybridoma cells. Reconstruction experiments indicate that the sequence created by the fusion of the murine leader and variable region blocked expression at the level of RNA accumulation. The methods described, as well as the potential problems of expression, are applicable to both traditional cDNA fragments and those obtained by in vitro amplification techniques.
Gillies
S D
SD
Damon Biotech, Inc., Needham Heights, MA 02194.
Lo
K M
KM
Wesolowski
J
J
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000235:genetics*; D000918:Antibody Specificity; D001483:Base Sequence; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics; D004274:DNA, Recombinant; D005786:Gene Expression Regulation; D005803:Genes, Immunoglobulin; D006801:Humans; D007135:Immunoglobulin Variable Region / Q000235:genetics; D007142:Immunoglobulin gamma-Chains / Q000235:genetics; D007145:Immunoglobulin kappa-Chains / Q000235:genetics; D051379:Mice; D008969:Molecular Sequence Data; D021382:Protein Sorting Signals / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics
D000906:Antibodies; D004274:DNA, Recombinant; D007135:Immunoglobulin Variable Region; D007142:Immunoglobulin gamma-Chains; D007145:Immunoglobulin kappa-Chains; D021382:Protein Sorting Signals; D012333:RNA, Messenger; D004247:DNA