Anti-Tac(Fv)-PE40, a single chain antibody Pseudomonas fusion protein directed at interleukin 2 receptor bearing cells

Anti-Tac(Fv)-PE40 is a chimeric single chain immunotoxin in which anti-Tac variable heavy and light chains held together by a peptide linker are attached to PE40, a truncated form of Pseudomonas exotoxin. This molecule was shown to be extremely cytotoxic for interleukin 2 (IL2) receptor bearing cells in tissue culture (Chaudhary, V. K., Queen, C., Junghans, R. P., Waldmann, T. A., FitzGerald, D. J., and Pastan, I. (1989) Nature 339, 394-397). Here we describe various forms of anti-Tac(Fv)-PE40 protein in which the order of the variable domains of anti-Tac has been switched and also three different types of peptide linkers have been used. All these proteins were purified to near homogeneity and were found to have similar cytotoxic activities against various human cells expressing the p55 subunit of the IL2 receptor. Anti-Tac(Fv)-PE40 was also found to have a very potent suppressive activity against phytohemagglutinin-activated human lymphoblasts and in a human mixed lymphocyte reaction. Anti-Tac(Fv)-PE40 appeared in the blood rapidly in mice after intraperitoneal administration and could be detected in the blood for up to 8 h. Anti-Tac(Fv)-PE40 warrants evaluation as an anti-tumor and immunosuppressive agent in humans

A rapid method of cloning functional variable-region antibody genes in Escherichia coli as single-chain immunotoxins

We have devised a strategy based on polymerase chain reaction (PCR) for the rapid cloning of functional antibody genes as single-chain immunotoxins. RNA from a hybridoma producing an antibody (OVB3) that reacts with ovarian cancer cells was used as a template to make the first strand of a cDNA. Then a second strand was synthesized and amplified by using two sets of DNA primers that (i) hybridized to the ends of the light- and heavy-chain variable regions, (ii) encoded a linker peptide, and (iii) contained appropriate restriction enzyme sites for cloning. After 30 cycles of PCR, the DNA fragments containing sequences encoding the light- and heavy-chain variable regions were cloned into an Escherichia coli expression vector containing a portion of the Pseudomonas exotoxin gene. Clones encoding recombinant single-chain immunotoxins were expressed in E. coli and the protein product was assessed for its ability to bind to or kill cells bearing the OVB3 antigen. By using this approach it should be possible to rapidly clone the functional variable region sequences of many different antibodies from hybridoma RNA