AN IMMUNOTOXIN WITH INCREASED ACTIVITY AND HOMOGENEITY PRODUCED BY REDUCING THE NUMBER OF LYSINE RESIDUES IN RECOMBINANT PSEUDOMONAS EXOTOXIN

Pseudomonas exotoxin A (PE) is a protein composed of 613 amino acids arranged into three major, and one minor, domains. Immunotoxins (ITs) containing PE38, a mutant form of PE which lacks the cell binding domain (Ia, amino acids 1-252) and 16 amino acids from domain lb (amino acids 365-380), are extremely potent cytotoxic agents. which can cause a complete regression of various human carcinomas grown in nude mice. However, these ITs are a mixture of several different chemical forms since the coupling between the antibody and the toxin may occur between either the light or heavy chain of the antibody and one of the four primary amino groups present on the truncated toxin. To modify the toxin with heterobifunctional crosslinking reagents only at specific sites, we replaced lysines 590 and 606 with glutamines and lysine 613 with arginine (PE38QQR). We also added two different peptide sequences, each containing a lysine residue, at the N-terminus of PE38. In one of these the sequence is ANLAEEAFK (''Lys'' peptide), and in the other, the sequence is LQGTKLMAEE (''NLys'' peptide). The mutant toxins were coupled using a thioether linkage to monoclonal antibody B3 which recognizes an antigen present in large amounts on many human cancers. PE38QQR-containing recombinant toxins can only be linked to an antibody through the N-terminal methionine or the lysine within the peptide. B3-LysPE38QQR and B3-NLysPE38QQR were four times more cytotoxic to target cells than the corresponding B3-LysPE38 and B3-NLysPE38 ITs. Furthermore, the antitumor effect of B3-NLysPE38QQR was significantly greater than that of B3-NLysPE38. We conclude that B3-LysPE38QQR and B3-NLysPE38QQR are more active because they are more homogenous components with all the antibody coupled to the N-terminus of the toxin and not some to the C-terminus, producing ITs with very low cytotoxic activity.