Autologous Bone Marrow Transplantation - Blog Science Connections

94 Ex Vivo Marrow Purging
with marrow treated ex vivo with a combination of MAbs and complement with
mafosfamide.
PRECLINICAL STUDIES
Materials and Methods
Normal bone marrow cells were procured from the transplant donor. The
Reh6 leukemic cell line was continuously cultured in Iscove's medium with 20%
heat-inactivated fetal calf serum. The cell concentration was kept at 0.6-1.5 x
10 6 cells/ml. The cells were incubated at 37° C and 5% Co 2
in air. Before
treatment, equal volumes of bone marrow cells (3.8 x 10 7
cells/ml) and the
Reh6 cell line (2 x 10 6 cells/ml) were mixed to obtain a final concentration of 5%
Reh6 cells. The mixed cell suspension was adjusted to a concentration of 2 x
10 7 cells/ml.
The MAbs CD10/ALB2 and CD19/AB1 (Immunotech, Marseilles, France)
are lgG2a and IgM immunoglobulins, respectively, and have been previously
described (8,9). Complement from baby white rabbits (age, 23-28 days) was
prepared in our institution and stored in the gas phase of liquid nitrogen
(-150°C). The cis4-sulfoethylthiocyclophosphamide or mafosfamide-lysine
(provided by Asta-Werke, Bielefeld, Federal Republic of Germany) is a new type
of oxazaphosphorine (3). It is a white crystalline powder that in aqueous solution
is stable up to several hours at pH 4 but only 15-20 minutes at pH 7. Its
molecular weight is 548 daltons.
Normal human bone marrow or 5% of the Reh6 leukemia cell line mixed
with irradiated marrow cells was incubated with MAbs for 30 minutes at 4°C.
Rabbit complement was then added to a final concentration of 1:1 for 30
minutes at 25° C. Cells were pelleted, and the complement procedure was
repeated for a total of two cycles. After two washings, the cells were exposed to
30 jug/ml mafosfamide for 30 minutes at 37° C. After incubation, cells were
washed one time and were resuspended in Iscove's modification of Dulbecco's
medium (1MDM) for leukemic cell line clonogenic assay and granulocytemacrophage
colony-forming unit (CFCI-GM) assay (7 and 21 days) as previously
described (3).
Results
We summarize in Table 1 the results of a series of experiments that indicate
that a kill of greater than 4 logs of the Reh6 leukemic cell line can be achieved by
combining purging protocol with a chemical agent of acceptable toxicity for
myeloid progenitor cells detected in a middle-term liquid culture (21 days).
Combining CD 10, CD 19, and complement with mafosfamide is superior in
terms of log kill to using either the MAbs or the chemical agent alone.