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Generation and Characterization of a Recombinant/Chimeric B72.3 (Human ₁)

Laboratory of Tumor Immunology and Biology, National Cancer Institute, Bethesda, Maryland 20892 [P. H., S. K., D. C., F. J. P., P. H. H., M. R., M. F., R. C., J. S.]; Celltech Limited, Berkshire, England [G. Y., M. B., N. W., D. K.]; and Medical Research Division, American Cyanamid Company, Lederle Laboratories, Pearl River, New York 10965 [C. C. L., D. W. M.]

We report here the generation and characterization of a recombinant/chimeric construct of murine ₁ monoclonal antibody (MAb) B72.3, containing the murine variable region and a human ₁ constant region [designated cB72.3(₁)]. cB72.3(₁) was generated by first isolating functionally rearranged V_H and V_L 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 V_L and V_H regions of B72.3 into unique sites of expression vectors which contain sequences encoding constant regions of human and 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(₁) at a level of 10–20 µg/ml. Reciprocal competition radioimmunoassays demonstrated that cB72.3(₁), a previously described cB72.3(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(₁) and to the nB72.3. Immunochemical studies of the nB72.3, cB72.3(₄), and cB72.3(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(₁). Antibody-dependent cell-mediated cytotoxicity experiments using human lymphokine-activated killer effector cells indicated better tumor cell killing by the cB72.3(₁) than the nB72.3 or cB72.3(₄). Dual label studies of coinjected cB72.3(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(₁), cB72.3(₄), and nB72.3 in mice, showed that the nB72.3 ß phase of clearance was slower than that of the other MAb forms. However, when the pharmacokinetic patterns of these three MAb forms were analyzed in monkeys, the cB72.3(₁) and the nB72.3 showed similar clearance curves, while the cB72.3(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(₁) may serve as a potentially useful diagnostic and/or therapeutic reagent.

¹ To whom request for reprints should be addressed, at the Laboratory of Tumor Immunology and Biology, National Cancer Institute, Building 10, Room 8B07, Bethesda, MD 20892.

Received 1/22/90. Accepted 10/22/90.