The L49 (IgG1) monoclonal antibody binds to p97 (melanotransferrin), a tumor-selective antigen that is expressed on human melanomas and carcinomas. A recombinant fusion protein, L49-sFv-bL, that contains the antibody binding regions of L49 fused to the Enterobacter cloacae r2-1 beta-lactamase (bL) was constructed, expressed, and purified to homogeneity in an Escherichia coli soluble expression system. The variable regions of L49 were cloned by reverse transcription-polymerase chain reaction from L49 hybridoma mRNA using signal sequence and constant region primers. Construction of the gene encoding L49-sFv-bL was accomplished by hybridization insertion of VH, VL, and sFv linker sequences onto a pET phagemid template containing the bL gene fused to the pelB leader sequence. Optimal soluble expression of L49-sFv-bL in E. coli was found to take place at 23 degrees C with 50 microM isopropyl beta-D-thiogalactopyranoside induction and the use of the nonionic detergent Nonidet P-40 for isolation from the bacteria. Construction and expression of a soluble form of the p97 antigen in Chinese hamster ovary cells allowed affinity-based methods for analysis and purification of the fusion protein. Surface plasmon resonance, fluorescent activated cell sorting, and Michaelis-Menten kinetic analyses showed that L49-sFv-bL retained the antigen binding capability of monovalent L49 as well as the enzymatic activity of bL. In vitro experiments demonstrated that L49-sFv-bL bound to 3677 melanoma cells expressing the p97 antigen and effected the activation of 7-(4-carboxybutanamido)cephalosporin mustard (CCM), a cephalosporin nitrogen mustard prodrug. On the basis of these results, L49-sFv-bL was injected into nude mice with subcutaneous 3677 tumors, and localization was determined by measuring bL activity. Tumor to blood conjugate ratios of 13 and 150 were obtained 4 and 48 h post conjugate administration, respectively, and the tumor to liver, spleen, and kidney ratios were even higher. A chemically produced L49-Fab'-bL conjugate yielded a much lower tumor to blood ratio (5.6 at 72 h post administration) than L49-sFv-bL. Therapy experiments established that well-tolerated doses of L49-sFv-bL/CCM combinations resulted in cures of 3677 tumors in nude mice. The favorable pharmacokinetic properties of L49-sFv-bL allowed prodrug treatment to be initiated 12 h after the conjugate was administered. Thus, L49-sFv-bL appears to have promising characteristics for site-selective anticancer prodrug activation.

We demonstrate that murine myeloma cells can efficiently mediate homologous recombination. The murine myeloma cell line J558L was shown to appropriately recombine two transfected DNA molecules in 30% of cells that received and integrated intact copies ofboth molecules. This activity was then exploited to direct major reconstructions of an endogenous locus within a hybridoma cell line. Production of antigenspecific chimeric heavy chain was achieved by targeting the human IgG1 heavy chain constant region (Cyi) exons to the genomic heavy chain locus of a hybridoma cell line secreting antibody specific for a human tumor-associated antigen. The frequency of productive genomic recombinations was 1 in 200 transfectants, with accumulation of the chimeric protein reaching >20 ,ug/ml in culture supernatants.