Autologous Bone Marrow Transplantation - Blog Science Connections

348 Chemoimmunoseparation of T Lymphoma Cells
(BFCIs-E), or bipotent granuloerythropoietic precursors (CFUs-GEMM) (3-5).
Antibodies reactive with cluster of differentiation 10 (CD 10), CD 9, and CD 20
proved particularly effective. Interestingly, the same monoclonal antibodies
conjugated with ricin A chain were remarkably ineffective in eliminating
malignant clonogenic Burkitt's cells in the absence of complement (6). Ricin
A-chain conjugates with anti-la and anti-CI monoclonal reagents could, however,
eliminate up to 4 logs of malignant clonogenic tumor cells.
In an attempt to improve elimination of malignant B cells, we treated
human bone marrow with a combination of monoclonal antibody, complement,
and the cyclophosphamide derivative mafosfamide (Asta Z 7557) (7). Using
this combination of chemoseparation and immunoseparation, we eliminated
approximately 4 logs of malignant cells while studying the effect of combined
chemoimmunoseparation on normal marrow precursors. Mafosfamide eliminated
most CFUs-GMwhen marrow was cultured immediately after treatment
(7) . Incubation of mafosfamide-treated marrow in long-term culture permitted
growth of new CFUs-GM, which was consistent with the possibility that more
primitive progenitors had been spared (7).
During the last 2 years we have asked whether a combination of
chemoseparation and immunoseparation would prove more effective than
either single modality for selectively eliminating T lymphoma cells from human
bone marrow. For chemoseparation we have used a combination of deoxycoformycin
and deoxyadenosine. The former is an adenosine deaminase
(ADA) inhibitor that has been shown to be selectively toxic for both normal and
malignant T cells. Incubation with the ADA substrate deoxyadenosine potentiates
deoxycoformycin toxicity. For immunoseparation we have utilized the
3A1 lgG2a murine monoclonal antibody that recognizes a CD 7 p40 cellsurface
antigen expressed by nearly all T-cell leukemias and lymphomas. CD 7
is also expressed on a bipotent lymphohematopoietic-myelohematopoietic
stem cell that can be isolated from human thymus, fetal liver, and bone marrow
(8) , but it is not expressed on the most primitive pluripotent stem cells. The 3A1
monoclonal antibody can lyse CD 7-positive cells in the presence of rabbit
complement.
A limiting dilution assay has been used to count clonogenic units before
and after chemoseparation and immunoseparation from human bone marrow
(2; A. Haleem et ai, unpublished data). Bone marrow mononuclear cells have
been separated over Ficoll gradients, irradiated with 50 Gy, and mixed in a
20-fold excess with 1 x 10 6
HSB-2 T lymphoma cells. Cell mixtures were then
incubated with deoxycoformycin for 1 hour to inhibit ADA and with a
combination of deoxycoformycin and deoxyadenosine for 4 additional hours.
When immunoseparation was attempted, mixtures were incubated with 3A1
plus rabbit complement three times for 30 minutes. Cells were plated in limiting
dilution assays. Plates were scored for clonogenic growth after 2 weeks'
incubation. Scores were converted to clonogenic units using a Spearman-
Karber estimator. In some experiments, T leukemia cells were cryopreserved