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D A B d L - 2 (IL-2 Toxin) Selectively Inactivates High-Affinity IL-2 Receptor-Bearing Human Peripheral Blood Mononuclear Cells CORY A. WATERS," CATHERINE E. SNIDER," KYOGO ITOH,b LOUIS POISSON," TERRY B. STROM,' JOHN R. MURPHY,d AND JEAN C. NICHOLS" (ISeragen, Inc. 97 South Street P . O . Box 9 / 0 4 Hopkinton, Massachusetts 01748 bUniversity of Texas M.D. Anderson Cancer Center Houston, Texas 77030 "Haruard Medical School and Beth Israel Hospital Boston, Massachusetts 02215 dUniversity Hospital Boston University Medical Center Boston, Massachusetts 021 18 The diphtheria toxin-related IL-2 gene fusion protein, DAB4861L-2(IL-2 toxin), has been shown to be selectively cytotoxic to tumor cells which constitutively express high-affinity IL-2 receptors (IL-2R).I.* Because these studies underscore the potential utility of DAB4861L-2as a selective immunosuppressive agent in transplantation and autoimmunity, we further investigated the interaction between the fusion protein and resting or activated normal human peripheral blood mononuclear cells (PBMC). Equilibrium binding studies were performed in order to demonstrate binding of DAB4861L-2to the high-affinity IL-2R on PBMC activated for 72 h with phytohemagglutinin (PHA). Scatchard analysis of these data revealed an average of 1577 high-affinity sites per cell and a Kd of approximately 748 pM (TABLE1). rIL-2 interacted with a comparable number of sites per cell, but its Kd for high-affinity binding (28 pM) was nearly 25-fold more affinate. When 72-h PHA-activated PBMC were exposed to DAB4s61L-2,potent inhibition of (14C)-leucineincorporation was observed, typically occurring at doses 51 X M (FIG.1 ) . The cytotoxic action of DAB4861L-2was blocked by the addition of monoclonal antibodies to either the p7S or p5S subunits of the IL-2R as well as by rIL-2 itself. In addition, a Schild plot of rIL-2 blocking data was linear with a slope of unity, consistent with the observation that DAB4861L-2and rIL-2 compete for a common cellular receptor. Also of interest in this regard is the fact that the ICso for intoxication was characteristically 10-fold lower than the measured Kd . This suggests that the intoxication process mediated by DAB4861L-2is relatively efficient and may require only a small number of occupied receptor sites 403

ANNALS NEW YORK ACADEMY OF SCIENCES

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TABLE 1. DAB4861L-2Binding Parameters for High-Affinity IL-2

Receptor-Bearing Cells Cells HUT 102/6TG

Radioligand rIL-2 DAB486IL-2 rIL-2 DABaJL-2 rIL-2 DABaJL-2

C91/PL PHA-activated human PBMC

Binding Sites/Cell 19579 t 3079 9097 L 1815 3874 2 1274 4640 t 203 1730 t 470 1877 t 189

Kd (PMY 37 t 7 682 t 94 24 L 16 823 t 147 23 ? I 739 -C 16

"Average of Kd measurements for the three cell types above: rIL-2, 28 t 5 pM; DAB,s,IL-2, 748 41 pM.

*

in order for sufficient fusion toxin to be endocvtosed. Resting PBMC with natural killer cell activity, however, were found to be resistant to DAi34861L-2intoxication (IC~,, - 10-7 M).Z , In order to show that DAB486IL-2 mediated inhibition of protein synthesis was not merely due to steric blockade of the IL-2R, we examined the ability of activated PBMC to endocytose ('251)-DAB48JL-2.Using trypsin-resistance as an index of internalization, we determined that the tl/2 of DAB4861L-2internalization (1 1 min) was not significantly different from that of rIL-2 itself (-5-10 min). In earlier studies we also showed that the extent to which protein synthesis was impaired by DAB486IL-2 in IL-2R-expressing cells correlated with inactivation of elongation factor 2, the intracellular target of diphtheria toxin action.' These