Renal excretion of recombinant immunotoxins containing pseudomonas exotoxin

Abstract

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.

Figure 1.

BL22 in urine of patients BH31 (A) and BH36 (B). 1.6 mL aliquots of urine from the indicated hours after end of 30 min infusion of BL22 (30 μg/kg) were subjected to TCA precipitation, and 15 μg of total protein/lane was added (except 3.75 μg at 1–4 h in A).

Figure 2.

Degradation of BL22 and LMB-2 by normal urine. Immuno-blots show BL22 incubated at a final concentration of 1 μg/mL for 1 h with urine at the indicated pH from a normal donor (A), and CLL patients BC01 (B) and CL10 (C). Controls include 2 ng of BL22 and PE35. Coomassie-stained gels in D-F include urine alone at pH 7 (first lane) followed by 2 μg BL22 added to normal urine incubated at 37 °C for the indicated time periods at the indicated pH. LMB-2 was incubated similarly in G–I and the indicated control lanes included LMB-2, BL22, and PE35.

Figure 3.

Characterization of proteolytic products of BL22 and LMB-2 in urine. BL22 or LMB-2 at 200 μg/mL was incubated with normal urine for 16 h at 37 °C at pH 7, and 2 μg was added to lane 4 or 5, respectively. Control molecules included 1 μg of LMB- 2 (63 kDa, lanes 1 and 6), BL22 (51 kDa + 12 kDa, lanes 2 and 7), and PE35 (35 kDa, lanes 3 and 8).

Figure 4.

ADP-Ribosylation activity of urine-cleaved BL22 and LMB-2. LMB-2 (white) or BL22 (black) was tested for ADP-ribosylation activity using the indicated amounts in μg. As indicated, LMB-2 and BL22 were each mixed with urine for either 0 or 24 h at 37 °C, and the final 2 lanes (gray) show PBS and urine alone, respectively.

Figure 5.

Model for urine protease fragments of BL22 and LMB-2. Fv and toxin fragments are shown in red and blue, respectively. Fragments labeled A–G represent bands on SDS-PAGE (Figure 3) except for bands D and G, which are too small.