INNOVATIVE 2-STEP NEGATIVE SELECTION OF GRANULOCYTE-COLONY-STIMULATING FACTOR-MOBILIZED CIRCULATING PROGENITOR CELLS - ADEQUACY FOR AUTOLOGOUS AND ALLOGENEIC TRANSPLANTATION

A major obstacle in purifying either autologous or allogeneic hematopo ietic stem cells from granulocyte colony-stimulating factor (G-CSF) mo bilized circulating progenitor cells (CPC) is represented by the huge cellularity present in each apheretic product. To obtain a significant debulking of unwanted cells from the leukapheresis, we developed a mo dified protocol of immune resetting whereby human ABO-Rh-compatible re d blood cells (RBCs) are treated with chromium chloride and then coate d with murine monoclonal antibodies (MoAbs) against leukocyte antigens . When experiments were performed with leukaphereses obtained from nor mal donors or from T-cell acute lymphoblastic leukemia (T-ALL) patient s, RBCs were coated with murine MoAbs against human mature myeloid cel ls (CD11b) and T cells (CD6); whereas, in the case of patients with B- precursor ALL, B-cell non-Hodgkin's lymphoma (B-NHL), or multiple myel oma (MM), RBCs were coated with anti-CD11b only. After incubation with CPC, resetting cells (myeloid precursor cells, granulocytes, monocyte s, and T cells) were removed by Ficoll-Hypaque density gradient centri fugation with a blood cell processor apparatus, COBE (Lakewood, CO) 29 91. After this step, a significant reduction of the initial cellularit y was consistently obtained (range, 72% to 97%), whereas the median ab solute recovery of the CD34(+) cells was above 85% (range, 64 to 100), with a 10-fold relative enrichment ranging from 3% to 41%. In a secon d step, CPC can be further purged of contaminating T or B cells by inc ubation with lymphoid-specific magnetic microbeads (anti-CD2 and -CD7 to remove T cells; anti-CD19 to remove B cells) and elution through a type-D depletion column (composed of ferromagnetic fiber) inserted wit hin a SuperMACS separator device (Miltenyi Biotech, Bergisch-Gladbach, Germany). By this approach, a highly effective (three to four logs) T -cell depletion was achieved in all experiments performed with normal donors or T-ALL patients (median loss of CD3(+) cells: 99.8% [range 99 .2 to 100]) and an equally efficient B-cell depletion was obtained fro m B-precursor ALL, B-NHL, or MM patients. At the end of the procedure the T- or B-cell depleted fraction retained a high proportion of the i nitial hematopoietic CD34(+) stem cells, with a median recovery above 70% (range 48% to 100%) and an unmodified clonogenic potential. In fiv e patients (two follicular NHL and three ALL) the purified fraction of stem cells was found disease free at the molecular level as assessed by polymerase chain reaction (PCR) analysis of the t(14;18) chromosome translocation or clone-specific DNA sequences of IgH or T-cell recept or gamma and delta chain genes. Purified autologous and allogeneic CPC s were transplanted in three and six patients, respectively, who showe d a prompt and sustained hematologic engraftment. In conclusion, this method represents a simple and reproducible two-step procedure to obta in a highly efficient purging of T or B cells from G-CSF expanded and mobilized CPCs. This approach might lead to the eradication of the neo plastic clone in the autologous stem cell inoculum as well as for T-ce ll depletion during allogeneic transplantation. (C) 1998 by The Americ an Society of Hematology.