Autologous Bone Marrow Transplantation - Blog Science Connections

366 Leukemia Cell Sensitivity to Immunotoxins
assay (DEA) that represents a simple and reliable procedure that can give
after standardization a semiquantitative indication of the sensitivity of bulk
tumor cells (3). The present study aimed to evaluate in vitro sensitivity of
various neoplastic blood cells expressing the T65 antigen to the antigenbinding
fragment (Fab) of T101 ricin A-chain immunotoxin (Fab T101-RTA)
and to explore possible correlations between efficacy and mean density of
T65 molecules on target cells. This study was performed with and without
NH4CI because NH 4
C1 (10 mM) has been found to be a safe and potent
enhancer agent suitable for ex vivo bone marrow treatment with ricin A-chain
immunotoxins (5,6) but inappropriate for in vivo use because of its general
toxicity at concentrations needed to obtain an optimal potentiating effect.
MATERIALS AND METHODS
A CEM III subclone was derived from CEM wild-type cell line by sorting
and selected on the basis of Tl antigen expression (30,000 Tl molecules/cell)
(3). Heparinized blood samples obtained from leukemia patients were separated
on a Ficoll-Hypaque gradient. Mononuclear cells were then washed and
resuspended in macroplate 2-ml wells at a final concentration of 10 6
cells/ml
in RPMI and 10% fetal bovine serum.
One million cells were incubated with Fab T101-RTA at a final concentration
of 10 8 M expressed in A-chain content with and without NH 4
C1 (10
mM) in a total volume of 1 ml. Cells were incubated at 37° C in 5% C0 2
in air
with gentle agitation during 24 hours. Controls consisted of treatment with
RPMI alone, ricin A-chain alone, and NH 4
C1 alone. All experiments were
performed in quadruplicate.
After incubation with immunotoxin, treated cells were washed and then a
DEA was performed as previously reported (3). Fluorescein diacetate (FDA)
staining propidium iodide (Pl)-negative living cells were enumerated at different
times after immunotoxin treatment—24,48, and 72 hours—in order to
improve the sensitivity of the test by counting cells after a long period of time
following the end of immunotoxin treatment. A 48-hour delay was found to be
a good compromise for serving the interests of the sensitivity of the assay and
viability of control cells. Sensitivity to immunotoxin was determined by the
ratio between the number of viable cells after immunotoxin treatment and the
number of viable cells in control samples, and it was expressed in a
percentage.
A cloning assay was performed on CEM cells as previously described (2)
and used to standardize the DEA by comparing the percentage of CEM living
cells evaluated with DEA 48 hours after treatment and the level of cytoreduction
as assessed by cloning assay at various doses of Fab Tl 01 -RTA (3).
T65 antigen mean density was measured by cytofluorometric quantification
according to the technique described by Poncelet and Carayon (7).