Renal excretion of recombinant immunotoxins containing pseudomonas exotoxin

Recombinant immunotoxins BL22 (CAT-3888) and LMB-2, composed of Fv fragments of anti-CD22 and CD25 MAbs, respectively, have produced major responses in patients with hematologic malignancies, and are also associated with renal toxicity, particularly with BL22. Characterization of the renal excretion of recombinant immunotoxins, which have 2-4 h half-lives in plasma, has not been reported in humans. To study the renal excretion of recombinant immunotoxins, urine from patients treated with BL22 was collected and the recombinant protein visualized after trichloroacetic acid (TCA) precipitation or anion exchange chromatography. BL22 viewed by immunoblot was found in the urine of patients within 8 h after dosing as an intact protein, and progressively degraded to fragments of <20 kDa within 1 day. We studied the stability of BL22 and LMB-2 added to urine at different time points and pH. When exposed to urine ex vivo, BL22 time-dependent proteolysis was similar to that observed in treated patients. By N-terminal sequencing, proteolysis was documented at positions 348-349 and 350-351 of BL22, and 339-340 and 341-342 of LMB-2, and other proteolytic sites were observed as well. Our data suggest that BL22 is excreted into the urine in a potentially cytotoxic form, even after its plasma level declines, and may remain intact long enough to cause renal toxicity.