Autologous Bone Marrow Transplantation - Blog Science Connections

Immunomagnetic Purging ofBurkitt's Cells 447
treated once only and residual cells detected by the Hoechst staining method,
two MAbs of the IgM subclass were tested, RFB 7
and SB 4
. These MAbs are
known to be very active in the complement lysis procedure and to recognize
high-density antigens in BL cells. When directly coupled to the beads they were
less efficient on cell line BLg 9
than was B, as used previously in the indirect
method. In addition, the last experiment suggests that IgM coupling on the bead
could be unstable. Such MAbs tested in an indirect method with a relevant
antimouse immunoglobulin (IgG + IgM) (KPL, Gaithersburg, MD) had an
activity similar to that of B,. The 1MD procedure has been tested in one case in
which the patient's bone marrow was pathological, and it allowed as much BL
cell elimination as the complement lysis procedure. Before purging 63% of the
sample was contaminated with BL cells. At day 10 after purging with B^, no BL
cells were detected.
These results prompt two comments. First, IMD is a very efficient
procedure that purges bone marrow with \% or less BL cell contamination.
Modifications of the procedure, initially described by J. Kemshead's group (3),
even if minimal when compared to the whole concept (e.g., the Ficoll
separation, the adjustment of the number of beads to the total number of cells
in the suspension, and finally the double treatment of the suspension),
significantly improved the BL cell depletion. Since such parameters are
physical, we assumed that they would be valid for the neuroblastoma model.
This assumption was confirmed by the results obtained by Seeger et al. (13) in
the neuroblastoma model. This refined methodology was then used to purge
grafts from 25 patients who had stage IV neuroblastoma, and in those samples
in which neuroblastoma cells were detected before the purging procedure, we
never noted any failure of the method as previously observed.
Second, the criteria for selecting relevant MAbs remain the main difficulty
in IMD, and a common rule can certainly not be applied for tumor cells of
different origins. In the model for Burkitt's lymphoma, results obtained with IgM
MAbs directly coupled to beads are less reproducible than those obtained with
IgG MAbs or the same IgM MAbs in the indirect method. Irrelevant MAbs can
inhibit the activity of an optimal one. This activity is not strictly linked to the
antigen density, even if B^ recognizes on BL cells an antigen with a higher
density than that recognized by ¥29/55 and J 5
. Indeed, these two MAbs, Y29/55
and J 5
, had a good reactivity with the three lines in all experiments (microscopic
analysis). It remains to be determined whether the antigen-antibody affinity is
constant in all malignant cells and is the only criterion for MAb selection. If not,
preclinical assays such as the one described here and elsewhere (see I. Philip et
al. in this volume) would have to be performed for each patient when possible.
We stress here, once again, the difficulty of transferring the experimental
model to the clinical situation, whatever purging method is chosen, and the
need for accurately quantifying residual malignant cells before and after the
purging for individual patients in clinical pilot trials before starting more
extensive controlled or even randomized multicenter studies.