Harvested marrow or PBSC is transported to the Purging Laboratory where it is treated to remove any detectable neuroblastoma cells ^(1-2,5,6). For marrow, these ex vivo treatments consist of sedimentation ⁽²⁾, filtration ⁽²⁾, and monoclonal antibodies attached to magnetic immunobeads ⁽¹⁾, whereas for PBSC treatment consists of concentrating the stem cell product using carbonyl iron ⁽³⁾ depletion of phagocytic cells ⁽⁴⁾ followed by monoclonal antibodies attached to magnetic immunobeads ⁽¹⁾. An aliquot of the treated product is analyzed to determine if all detectable neuroblastoma cells were removed and to quantitate viable cells, CFU-G,M and CD34+ cells (before and after freezing). If no tumor cells are detectable and if adequate normal cells are present, the marrow or PBSC is suitable for reinfusion; if not, the product cannot be used for reinfusion and a second harvest or alternative therapy is necessary. Once the stem cells are cryopreserved they are stored in liquid nitrogen vapor at CHLA until a written request is received to ship the cryoprserved stem cells to the transplant center, at which time the stems cells will be shipped to the transplant center in a liquid nitrogen vapor cryogenic container.

The procedures for purging are described below. All processing is done in an isolated (GMP) laminar flow room using sterile technique; in a closed system. Bacterial and fungal cultures are collected prior to purging and at the time of cryopreservation and monitored for 14 days prior to certifying the product is free of microbial contamination.

Separation of marrow leukocytes and neuroblastoma cells
by sedimentation (bone marrow only).

Because neuroblastoma cells often grow in the marrow as clusters of 5-50 cells, they sediment with erythrocytes in hetastarch. To avoid break-up of tumor cell clumps, marrow is not filtered through wire mesh screens in the operating room. Upon receipt by the Purging Laboratory, it is mixed with hetastarch in L15 medium (final hetastarch concentration = 1.5%)and allowed to sediment in a 1 L Viaflex bag (Travenol, Deerfield, IL).

The sediment, which contains red cells and tumor clumps, is left behind when the supernatant of plasma and leukocytes is pumped out. This separation yields approximately 90% of marrow mononuclear leukocytes in the supernatant and removes up to 1 log of tumor cells.

Removal of neuroblastoma by filtration (bone marrow only).

The leukocyte/plasma supernatant in the Viaflex bag is pumped through a 40 µm multi-layer mesh filter (Pall Biomedical, Fajardo, PR) and then a 20 µm PDF-20 Pediatric Transfusion Filter (Travenol, Deerfield, IL); effluent cells are collected into a DuPont Stericell Processor and centrifuged at 400 x g (continuous flow).

The cells are then washed by the Stericell with L-15 and resuspended in L-15 + 5% human serum albumin. Filtration removes approximately 0.5 logs of tumor cells with recovery of 60% of marrow mononuclear cells and 60% of CFU-G,M.

Carbonyl Iron Fractionation of PBSC

To concentrate cells for purging, nucleated PBSC are placed in blood bags and 200-300 mg of carbonyl iron (CI) (C-3518, Sigma, St. Louis, MO) per 100 million cells is added in Leibovitz's L-15 medium (pre-warmed to 22°C) supplemented with 10% human serum albumin. Carbonyl iron is sterilized by dry heat (180°C for 8 hours).

Incubation in carbonyl iron is carried out by placing the bags horizontally at 37 deg C for 1 hour. The carbonyl iron and cells which attach to it are then attached to the sides of the bags using the magnetic field from 2 opposing samarium cobalt magnets as described in US Patent # 4,710,472 for 3-5 minutes.

Cells in suspension not adhering to the sides of the bags are then transferred to a new bag. The original bag is washed twice by gentle addition of 50 ml each to the same medium to recover all CI free cells if initial recovery of total cells is < 30%. Cells collected after separation are then used for purging with magnetic immunobeads.

Removal of neuroblastoma cells with
monoclonal antibodies and magnetic immunobeads

Cells passing through the filter (bone marrow) or after carbonyl iron fractionation (PBSC) are counted and appropriate cell numbers are dispensed into 600 ml transfer packs. The cells in the transfer packs are rotated for one-half hour at room temperature with magnetic immunobeads (1:1 bead to total cell ratio; 1 to 2 x 107 cells per ml in a 600 ml transfer pack); details of immunobead preparation are provided in Table 1.

Seven monoclonal antibodies (390, 459, HSAN 1.2, BA-1, NKH-1A, HNK-1 and 126) are attached to M-450 magnetic beads via GAM. For PBSC purging, 5 antibodies are used, with NKH1-A and 390 being omitted. An antibody "cocktail" is used to overcome the potential problem of heterogeneity in antigen expression by tumor cells. Bead-tumor cell conjugates are removed by applying "debulking" magnets to the outside of the 600 ml bag.

For the second cycle of purging, fresh magnetic immunobeads are added to marrow cells after they have been transferred to a second bag following debulking in the first bag. After debulking of the second bag twice, the marrow is passed through tubing compressed between 8 pairs of samarium cobalt magnets, and collected into fresh 600 ml transfer packs. Two cycles removes 3.6 to 4 logs of tumor cells with recovery of 35-40% of mononuclear leukocytes and 75-90% of CFU-G,M.

Our approach to the specificity of monoclonal antibodies is an operational one since it is difficult, if not impossible, to define absolutely tumor specific antigenic determinants. Thus, although monoclonal antibodies 459, 390, HSAN 1.2, and 126 react with some normal cells outside of the bone marrow, they are useful for this ex vivo treatment because they react with neuroblastoma but not normal marrow cells. BA-1 reacts with approximately 6% of normal marrow cells but does not react with pluripotent stem cells. Other antibodies used in this cocktail (HNK-1 and NKH-1A) provide increased efficacy in pre-clinical experiments because they bind to neuroblastoma cells, but do not decrease CFU-G,M. Our experience with > 300 patients using our current antibodies indicates that they do not compromise engraftment; thus, we do not routinely store a backup product when using this cocktail. If sufficient marrow is harvested, backup marrow or PBSC are stored for use in pilot studies (for those patients who relapse after myeloablative therapy).

Monoclonal antibodies and goat anti-mouse immunoglobulin (GAM) are prepared in sufficient quantities to meet our estimated needs for 6 months at a time, so that standardized, high quality reagents will be employed for each treatment. All antibodies are purified and passed through 0.22 µm filters for sterilization. Testing is carried out to demonstrate that the antibodies are functional, sterile, pyrogen-free, and free of 12 adventitious murine viruses (MAP test), ecotropic murine leukemia virus (XC plaque essay), and xenotropic murine leukemia virus (S+/L-assay). Purging with magnetic beads and monoclonal antibodies is carried out under FDA monitoring (BB-IDE 2259 "Murine Monoclonal Antibodies to Neuroblastoma").

TABLE 1

Method for Removing Neuroblastoma Cells from Marrow or PBSC with Monoclonal Antibodies Attached to Magnetic Beads

A. Suspend 4.5 µm polystyrene-magnetite immunobeads: Dynabeads M-450, uncoated, 30 mg/ml (w/v), 13.3 x 106 beads per mg in sterile water in Dulbecco's PBS-A, pH 7.4. Rotate with sterile, pyrogen-free goat anti-mouse antiserum (GAM) (50 µg/mg beads/0.1 ml) for 18 hr at 4°C, wash 2x, rotate 1 hr at 4°C, wash 2 times, then attach monoclonal antibodies to GAM coated beads:

*Not used for PBSC purging.

Rotate antibodies with beads for 180 min at 4°C, wash 2x, rotate in L15-10% fetal calf serum (FCS) at 4°C for 1 hr, wash 2x, resuspend in L15-2.5% human serum albumin (HSA). Beads are tested for bacterial or fungal contamination (two week incubation minimum).

B. Immunobeads may be used immediately or stored in L15-FCS for up to 3 months without losing activity. If beads are stored > 48 hours, wash 1x, suspend in L15-2.5% HSA sonicate, and count before using.

C. Add magnetic immunobeads to marrow or PBSC (bead to total cell ratio, 1:1) and rotate them together in a transfusion bag for 30 min at room temperature.

D. Remove tumor cells bound to magnetic immunobeads by placing transfusion bag with marrow in magnetic depletion device followed by draining cells into a second bag (for second cycle of purging) and pass cells from the second bag between eight double magnets at a flow rate 5\ml per min.

E. Collect cells that pass by the magnets for cryopreservation.

Purging of PBSC

PBSC fractionated by carbonyl iron are purged as described above for bone marrow using 5 monoclonal antibodies and magnetic beads. However, as tumor cells in PBSC are likely to be in smaller clumps then those found in bone marrow, the sedimentation and filtration steps in the purging process are omitted.