Improvement of tumor cell depletion by combining immunomagnetic positive selection of CD34-positive hematopoietic stem cells and negative selection (purging) of tumor cells.

One possible reason for relapse after high-dose chemotherapy is retransplantation of tumor cells contaminating autologous hematopoietic stem cell transplants. Residual tumor cells can be diminished by various purging methods. We studied tumor cell depletion by sequentially combining immunomagnetic positive selection of CD34+ hematopoietic stem cells using Isolex50 or Isolex300SA and negative tumor cell depletion using MACS, MaxSep or Isolex50 systems. Using these separation systems in different selection sequences, i.e. positive followed by negative selection (+/- selection) or vice versa, four groups of double selections (Isolex50/MACS, Isolex50/MaxSep, MaxSep/Isolex50, Isolex300SA/Isolex50) were studied. Testing these double-purging procedures mean additional tumor cell depletion (deltaTCD) achieved by the second selection step ranged from 1.1+/-0.58 log (n = 5, +/- Isolex50/MACS) to 2.0+/-1.1 log (n = 7, -/+ MaxSep/Isolex50). Loss of CD34+ cells during double selection sometimes was extensive and mean yield of CD34+ cells ranged from 12.8+/-11.5% (n = 6, +/- Isolex50/MaxSep) to 43.2% (n = 2, +/- Isolex300SA/Isolex50). Calculated values for mean yield-corrected deltaTCD ranged from 0.64+/-0.3 log (n = 5, +/- Isolex50/MACS) to 1.4+/-1.3 log (n = 7, -/+ MaxSep/Isolex50). During positive selection of -/+ selection (MaxSep/Isolex50) relative tumor cell enrichment was detectable leading to an increment of mean tumor cell contamination rate. Best results for total TCD were achieved by the combination of Isolex50/MaxSep (n = 6; TCD: 4.2 log; yield CD34+: 12.8%) and Isolex300/Isolex50 (n = 2; TCD: 3.8 log; yield CD34+: 43.2%). Furthermore, we have established and tested a new simultaneous +/- selection method by using CD34-specific releasing agent PR34+ in the Isolex300i. With this method we have obtained a mean total yield-corrected TCD of 4.7 log (n = 4; range: 4.1-6.0 log) with high CD34+ cell yield (mean: 69.8%) and CD34+ cell purity (mean: 92.8%). Since this new simultaneous +/- purging procedure is safe, applicable within a closed system (GMP-like) and most effective, we recommend it for further testing in a clinical setting.

The efficiency of tumor cell purging using immunomagnetic CD34+ cell separation systems.

Immunomagnetic separation with anti-CD34 monoclonal antibodies and paramagnetic microbeads has been used to enrich hematopoietic stem cells from human bone marrow (BM) or mobilized peripheral blood mononuclear cells (PBMNC). The introduction of this technique also constitutes a new principle of tumor cell purging. The efficiency in terms of purging tumor cells from PBMNC was evaluated in seven different experiments. Mobilized (chemotherapy and G-CSF) PBMNC were collected from patients with solid tumors (n = 6) and multiple myeloma (n = 1) by leukapheresis using an automated MNC separation system and contaminated with 1% (n = 5) or 10% (n = 2) tumor cells from different epithelial cell lines being CD34-negative. The cell mixture was sensitized with anti-CD34 (9C5) antibodies and sheep anti-mouse IgG1 paramagnetic microspheres and enriched for CD34+ cells using an Isolex 50 magnetic separator. Purify of CD34+ cells was studied by flow cytometry (FACScan) and tumor cell depletion was evaluated by comparative human tumor cloning assays (HTCA) containing methylcellulose and agar. We achieved a median purity of CD34+ cells of 85.9% (range 69.8-92.9%) and a median yield of 48.1% (range 21.0-85.2%). From these data in each case the estimated log depletion of tumor cells was calculated and compared with the experimentally achieved (HTCA) log depletion (log delta depletion = log experimental depletion--log calculated depletion). In our experiments we achieved a median depletion of 2.75 log (range 1.55-3.69 log). When corrected for CD34+ cell yield of each experiment we observed a median 'yield corrected depletion' of 2.38 log (range 1.48-3.15 log). The following delta depletion values were obtained: +0.32 log (HTB 129, breast), +0.21 log (HTB 26, breast), +0.04 log (HTB 26) for experiments with higher experimental depletion, and -0.23 log (HTB 26), -0.9 log (HTB 26, PBMNC from patient with multiple myeloma), -0.82 log (HTB 131, breast) and -1.66 log (HTB 131) for lower depletion efficacy than calculated. These data suggest that depletion may depend on specific cell surface characteristics of tumor cells. Moreover, plasma factors (eg paraprotein) may also have some impact. In summary, the Isolex 50 provides a high purity of CD34+ cells and depletion of tumor cells was efficient. However, calculated and experimental purging efficiencies are not necessarily identical.