Use of multiple T cell-directed intact ricin immunotoxins for autologous
bone marrow transplantation
RC Stong, F Uckun, RJ Youle, JH Kersey and DA Vallera
The monoclonal antibodies (MoAb) T101, G3.7, 35.1, and TA-1 were conjugated
to intact ricin using a thioether linkage. These MoAb detect, respectively,
the CD5[gp67], CD7[p41], CD2[p50], and [gp95, 170] determinants that are
found in the vast majority of cases of T cell acute lymphocytic leukemia
(T-ALL). The resulting immunotoxins (ITs) and an equimolar mixture of these
ITs were evaluated as potential purgative reagents for autologous
transplantation in T-ALL. Leukemic cell lines were used to compare the
kinetics of protein synthesis inactivation mediated by each IT. The cells
were treated with IT in the presence of lactose in order to block the
native binding of ricin. The observed rates of protein synthesis
inactivation correlated with target antigen expression detected by
fluorescence-activated cell sorter analysis. Of the four ITs, T101-ricin
(T101-R) exhibited the fastest rate of inactivation, followed in order by
G3.7-ricin, TA-1-ricin, and 35.1-ricin. At concentrations greater than 300
ng/mL, a cocktail containing an equimolar amount of all four ITs (referred
to as the four- IT cocktail) exhibited kinetics that were as fast or faster
than those of T101-R. The long-term cytotoxic effects of individual ITs and
the four-IT cocktail were evaluated using a sensitive clonogenic assay.
Each IT was specifically cytotoxic and inhibited 1 to 4 logs of clonogenic
leukemic cells at doses (300 to 600 ng/mL) that can be used clinically. The
four-IT cocktail was highly cytotoxic; a concentration of 300 ng/mL
inhibited greater than 4 logs of leukemic cells while sparing the majority
of committed (CFU-GM, CFU-E) and pluripotent (CFU- GEMM) hematopoietic stem
cells. The determination of both short-term kinetics of protein synthesis
inactivation and longer-term inhibition of clonogenic growth allowed new
insight into cell killing by IT. Our results suggest that ITs continue to
act on clonogenic target cells for a period of three to five days.
Interestingly, the four-IT cocktail was not as potent against clonogenic
leukemic cells as T101-R alone, although it exhibited kinetics of protein
synthesis inhibition that were as fast as those of T101-R alone. This
finding suggests that internalized ITs may differ in the length of time
they remain active within the cell. Our results also demonstrate the
importance of using several different assays to evaluate IT reagents.
Volume 66,
Issue 3,
pp. 627-635,
09/01/1985
Copyright © 1985 by The American Society of Hematology
