BIOLOGICALLY-ACTIVE INTERLEUKIN 2-RICIN A-CHAIN FUSION PROTEINS MAY REQUIRE INTRACELLULAR PROTEOLYTIC CLEAVAGE TO EXHIBIT A CYTOTOXIC EFFECT

DNA fusions encoding chimeric proteins in which human interleukin 2 (I L2) was fused to the A subunit of the plant cytotoxin ricin (RA) have been expressed in Xenopus oocytes. The constructs contained N-terminal IL2 and C-terminal RA, or N-terminal RA and C-terminal IL2. In the ex pressed chimeric proteins, the IL2 and RA moieties were joined by a pe ptide sequence containing a proteolytic cleavage site. Two proteolytic ally-sensitive peptide sequences were utilized; a peptide that forms t he trypsin-sensitive disulfide-bonded loop in diphtheria toxin (DT) or a synthetic peptide containing the factor Xa recognition site in a se quence flanked by two cysteine residues. In an in vitro cell free syst em the RA component was biologically active in all chimeric proteins p roduced since it specifically depurinated 28S ribosomal RNA. Proteolyt ic cleavage of the chimeras with either trypsin or factor Xa as approp riate separated the IL2 and RA moieties, but they did not remain coval ently linked by a disulfide bond. Because of this, the cytotoxicity of protease-treated chimeras could not be assessed. Chimeras not pretrea ted with factor Xa but which contained the factor Xa target sequence w ere not cytotoxic to CTLL-2 cells. Rather, these molecules had a stimu latory effect that was ascribed to the IL2 moiety. In contrast, recomb inant chimeric toxins containing the DT loop sequence were cytotoxic t o CTLL-2 cells. Taken together the data suggest that RA-containing chi meras require intracellular proteolytic cleavage to release the RA moi ety to render them cytotoxic to target cells.