We have studied the initial events in the cytotoxic pathway of PE and PE chimeric toxins. We have also focused on the translocating activity of domains II and III of PE. Low density lipoprotein receptor-related protein (LRP) was shown to be the surface receptor for Pseudomonas exotoxin (PE). Receptor-negative cells were toxin resistant but sensitive to a chimeric toxin that entered cells via the transferrin receptor. Furin is the cellular enzyme that cleaves PE. We showed that furin also cleaves PE chimeric toxins such as TGFalphaPE38, but not a mutant form with a glycine in place of the P1 arginine. Furine- mediated cleavage of TGFalphaPE38 had a broader pH (acidic) optimum than that observed with native PE. In collaboration with M. Lord, PE- RTA chimerics were constructed whereby domain III of PE was replaced by ricin A chain. Based on the use of various PE mutants, the translocation of these chimerics was shown to be mediated by PE sequences and not by ricin sequences. Immunotoxins to CD19 and CD22 were constructed. Disulfide-linked toxins were more cytotoxic than the corresponding thioether linked toxins, implying that the cellular cleavage of PE38 within target cells was a rate-limiting step. The variable regions of the light and heavy chains of RFB4 (an anti-CD22 antibody) were cloned, sequenced and arranged to form an FvPE40 immunotoxin. This was cytotoxic for antigen-positive B cells. Antitumor activity of RFB4 immunotoxins was also demonstrated. HIV sequences from the V3 loop of gp120 have been introduced into the Ib region of native PE. These sequences were delivered to the cytosol. This constitutes the first step in the generation of candidate vaccines for the generation of cytotoxic T cells to HIV.
