Generation and characterization of a recombinant/chimeric B72.3 (human gamma 1).
We report here the generation and characterization of a recombinant/chimeric construct of murine gamma 1 monoclonal antibody (MAb) B72.3, containing the murine variable region and a human gamma 1 constant region [designated cB72.3(gamma i)]. cB72.3(gamma 1) was generated by first isolating functionally rearranged VH and VL genes of B72.3 from partial genomic libraries in phage vectors. Construction of mouse-human chimeric heavy and light chain genes was performed by inserting restriction fragments carrying VL and VH regions of B72.3 into unique sites of expression vectors which contains sequences encoding constant regions of human kappa and gamma 1, respectively. The expression constructs were subsequently electroporated into SP2/0 cells. The transfected SP2/0 murine cell line has been shown to synthesize cB72.3(gamma 1) at a level of 10-20 micrograms/ml. Reciprocal competition radioimmunoassays demonstrated that cB72.3(gamma 1), a previously described cB72.3(gamma 4), and native B72.3 (designated nB72.3) competed similarly. A rat anti-idiotype MAb made against nB72.3 was shown to bind equally well to cB72.3(gamma 1) and to the nB72.3. Immunochemical studies of the nB72.3, cB72.3(gamma 4), and cB72.3(gamma 1) revealed slight differences in size among the three MAb forms on sodium dodecyl sulfate gels and revealed a higher isoelectric point for the cB72.3(gamma 1). Antibody-dependent cell-mediated cytotoxicity experiments using human lymphokine-activated killer effector cells indicated better tumor cell killing by the cB72.3(gamma 1) than the nB72.3 or cB72.3(gamma 4). Dual label studies of coinjected cB72.3(gamma 1) and nB72.3 revealed that both MAbs could efficiently localize human tumor xenografts in athymic mice. Pharmacokinetic studies, analyzing the blood clearance of cB72.3(gamma 1), cB72.3(gamma 4), and nB72.3 in mice, showed that the nB72.3 beta phase of clearance was slower than that of other MAb forms. However, when the pharmacokinetic patterns of these three MAbs forms were analyzed in monkeys, the cB72.3(gamma 1) and the nB72.3 showed similar clearance curves, while the cB72.3(gamma 4) showed a much slower plasma clearance. In view of the binding properties of nB72.3 and its ability to localize a range of carcinomas in clinical trials, the studies reported here demonstrate that the cB72.3(gamma 1) may serve as a potentially useful diagnostic and/or therapeutic reagent.
Hutzell
P
P
Laboratory of Tumor Immunology and Biology, National Cancer Institute, Bethesda, Maryland 20892.
Kashmiri
S
S
Colcher
D
D
Primus
F J
FJ
Hand
P H
PH
Roselli
M
M
Finch
M
M
Yarranton
G
G
Bodmer
M
M
Whittle
N
N
D000818:Animals; D000911:Antibodies, Monoclonal / Q000235:genetics* / Q000276:immunology / Q000493:pharmacokinetics; D000912:Antibodies, Neoplasm / Q000235:genetics* / Q000276:immunology / Q000493:pharmacokinetics; D000918:Antibody Specificity; D000920:Antibody-Dependent Cell Cytotoxicity; D001483:Base Sequence; D001667:Binding, Competitive; D002277:Carcinoma / Q000276:immunology*; D002678:Chimera; D003001:Cloning, Molecular; D015322:Gene Rearrangement, B-Lymphocyte; D005803:Genes, Immunoglobulin; D005822:Genetic Vectors; D007526:Isoelectric Point; D008252:Macaca fascicularis; D051379:Mice; D008819:Mice, Nude; D011993:Recombinant Fusion Proteins; D014162:Transfection
D000911:Antibodies, Monoclonal; D000912:Antibodies, Neoplasm; D011993:Recombinant Fusion Proteins