GMP production and testing of Xcellerated T Cells for the treatment of patients with CLL.

BACKGROUND: Pre-clinical studies suggest Xcellerated T Cells have the potential to produce a potent anti-tumor effect, restore broad immune function and reduce the risk of infectious complications in patients with CLL. Unlike other cancer settings, T cells constitute only a small fraction of CLL patients' PBMC. To generate large numbers of Xcellerated T Cells of high purity from CLL patients' PBMC, a reproducible, streamlined and cost-effective good manufacturing process (GMP) is required. METHODS: The 10-L volume Wave Bioreactor-based Xcellerate III Process using Xcyte Dynabeads in a single custom 20-L Cellbag container was adapted, qualified and implemented for GMP operations. RESULTS: For n=17 CLL patients, starting with approximately 1.34 x 10(9) CD3+ T cells at 6.8+/-7.5% purity in the PBMC leukapheresis products, using the 10-L volume Wave Bioreactor-based Xcellerate III Process, it was feasible to manufacture 137.0+/-34.3 x 10(9) Xcellerated T Cells at 98.5+/-1.0% CD3+ T-cell purity. An average 400-fold clearance of malignant B cells was documented during the manufacturing process. The Xcellerated T Cells produced from the Xcellerate III Process exhibited high in vitro biologic activity and have their T-cell receptor repertoire restored to a normal diversity. In-process T-cell activation was reproducibly robust, as measured by increase in cell size, up-regulation of CD25 and CD154 expression and the secretion of IL-2, IFN-gamma and tumor necrosis factor (TNF)-alpha. DISCUSSION: A low-volume, high-yield bioreactor-based process has been developed, qualified and implemented for the reproducible, GMP manufacture of high purity, biologically active Xcellerated T Cells for the treatment of CLL patients in clinical trials.

Incidence of tumor-cell contamination in leukapheresis products of breast cancer patients mobilized with stem cell factor and granulocyte colony-stimulating factor (G-CSF) or with G-CSF alone.

We have assessed tumor contamination of peripheral blood progenitor cells (PBPC) in 203 high-risk breast cancer patients who were prospectively randomized to mobilization with stem cell factor (SCF) plus granulocyte colony-stimulating factor (G-CSF) versus G-CSF alone. The patients then received high-dose cyclophosphamide, cisplatin, and carmustine (BCNU) with PBPC support. One bone marrow aspirate obtained before treatment, one whole blood specimen obtained before cytokine infusion, and one to five leukapheresis products were tested for the presence of tumor cells by an alkaline phosphatase immunocytochemical technique with a targeted sensitivity of 1.7 tumor cells per 10(6) hematopoietic cells. Tumor cells were detected in the bone marrow, peripheral blood, and/or PBPC of 21 patients (10%). In 14 patients, bone marrow specimens were tumor-positive; in seven patients, premobilization whole blood specimens were tumor-positive, and in eight patients, leukapheresis products were tumor-positive. In five patients, repetitive or multiple specimens were tumor-positive, and in three cases, marrow, peripheral blood, and PBPC products were all tumor-positive. Nine of the patients in whom tumor cells were found in marrow or peripheral blood were clinical stage II to III and 12 were clinical stage IV. Nine of the tumor-positive patients were in the SCF + G-CSF arm and 12 were in the G-CSF arm. Tumor cells were detected in leukapheresis products of eight patients: three in the G-CSF + SCF arm and five in the G-CSF arm. We conclude that detectable tumor-cell contamination of bone marrow, peripheral blood, and/or PBPC occurred in approximately 10% of patients in this trial and was observed in stage II to III patients, as well as in stage IV patients. No significant difference could be found in the rate of PBPC tumor-cell contamination between patients who received SCF + G-CSF compared with those who received G-CSF alone. Neither mobilization regimen was found to increase the rate of tumor-cell contamination when control premobilization blood samples were compared with leukapheresis products.

CD19 selection improves the sensitivity of B cell lymphoma detection.

Reinfusion of residual tumor cells into B cell non-Hodgkin's lymphoma (B-NHL) patients during autologous transplantation may be an important cause of disease relapse. Determining the extent to which B-NHL cells are present in autologous progenitor cell products and if the presence of residual B-NHL cells is predictive of relapse will require extremely sensitive methods of detecting rare B-NHL cells. We attempted to improve the sensitivity of polymerase chain reaction (PCR)-based detection of rare B-NHL cells by preselecting CD19+ cells using an immunomagnetic column. To measure detection sensitivity, we prepared samples containing different levels of B-NHL cell contamination by mixing B-NHL cell lines containing the chromosomal translocation t(14;18) bcl-2/JH) with control leukapheresis samples. DNA extracted from each CD19-selected sample and from each matched nonselected sample was added to a PCR to amplify the bcl-2/JH breakdown junction. CD19 preselection improved the sensitivity of detection of t(14;18)-positive B-NHL cells 115-fold, so that B-NHL cells at a concentration of 1 tumor cell per 1 x 10(6) hematopoietic cells were detected in every specimen evaluated. t(14;18)-positive cells were not detected in any of 13 control leukapheresis specimens. We conclude that a combination of CD19 preselection and PCR amplification may improve the sensitivity of detection of rare lymphoma cells by two orders of magnitude without a significant decrease in specificity.

Ex vivo expansion of megakaryocyte progenitors: effect of various growth factor combinations on CD34+ progenitor cells from bone marrow and G-CSF-mobilized peripheral blood.

Prolonged thrombocytopenia resulting from inadequate megakaryocyte (MK) progenitor cell reconstitution is a serious complication of hematopoietic cell-supported high-dose chemotherapy (HDC). In this situation, the infusion of MK progenitors that are expanded ex vivo could be clinically beneficial. In this study we investigated the ability of various growth factor combinations to generate MK progenitors. CD34+ cells derived from bone marrow (BM) and granulocyte colony-stimulating factor (G-CSF)-mobilized peripheral blood (PB) from 17 patients with breast cancer, lymphoma, or myeloma were cultured unpertubed for 10 days in a serum-free liquid culture system that contained recombinant growth factors. Five different growth factors combinations were evaluated: Stem cell factor (SCF), interleukin (IL)-3, IL-6 + G-CSF (combination 1); SCF, megakaryocyte growth and development factor (MGDF) + G-CSF (combination 2); SCF + MGDF (combination 3); MGDF alone (combination 4); and SCF, IL-3, IL-6, G-CSF + MGDF (combination 5). PB CD34+ cells yielded significantly higher numbers of CD41+ MK progenitors than BM CD34+ cells with any of the growth factor regimens assayed. PB CD34+ cells (2x10[5]) at day 0 generated 1.2 to 1.3x10(6) CD41+ cells by day 10 when cultured in the presence of growth factor combinations 1, 2, or 3. In contrast, 2x10(5) BM CD34+ cells produced 5x10(5) CD41+ cells after 9 days in the presence of combination 1, whereas lower numbers of CD41+ cells were generated in cultures with combinations 2 and 3 (2.3x10[5] and 4.2x10[4], respectively). The addition of MGDF to cultures that were grown with combination 1 for 5 days increased the number of CD41+ cells (1.7-fold increase in PB-derived cultures, 1.6-fold increase in BM-derived cultures). Treatment with MGDF alone resulted in higher frequencies of MK progenitors than those obtained in cultures with combined growth factors (79% in PB-derived cultures, 25% in BM-derived cultures), but because total cell growth was attenuated, absolute numbers of MK progenitors were lower (7x10(5) in PB-derived cultures, 7x10(4) in BM). Morphological analysis of immunocytochemically identified megakaryocytic cells revealed mononuclear cells as the predominant cell type in all of the cultures. During the 10-day culture period, PB-derived MK progenitors did not show notable maturation, even under the influence of MGDF, whereas in BM-derived cultures MGDF induced a significant shift to binuclear cells and stage I MK after day 5. Phenotypic analysis of cell surface markers showed that the majority of cultured megakaryocytic cells coexpressed CD34 and platelet glycoproteins (GPs), also indicating an immature stage of development. The ex vivo proliferative activity of CD34+ cells and their potential to develop into the megakaryocytic lineage demonstrated considerably high interpatient variations. There was no correlation between platelet recovery following HDC with hematopoietic cell support and the magnitude of GP+ cell expansion ex vivo, suggesting the feasibilty of MK expansion ex vivo in patients with prolonged thrombocytopenia posttransplantation. In summary, these data indicate that GCSF-mobilized CD34+ PBPCs are more effectively expanded ex vivo into the megakaryocytic lineage than are CD34+ BMPCs. CD34+/GP+ MK progenitors may be an appropiate cell population for transplantion as prophylaxis or treatment of prolonged thrombocytopenia. The efficacy of this procedure will be tested prospectively in a clinical trial.

HIV-gp120 induced cell death in hematopoietic progenitor CD34+ cells.

Haematologic abnormalities accompany the majority of HIV-1 infections. At present it is unclear whether this is due directly to HIV infection of hematopoietic progenitor cells, or whether this results from an indirect mechanism secondary to HIV infection. Here we provide evidence for an indirect mechanism, whereby hematopoietic progenitor cells undergo HIV gp120-induced apoptosis (programmed cell death) even in the absence of HIV infection. Freshly isolated, purified human hematopoietic progenitor CD34+ cells, derived from both umbilical cord blood and bone marrow, co-expressed the CD4 marker at low density on their surface. Although these CD34+CD4+ cells theoretically should be capable of productive infection by HIV, we found that HIV-IIIB could not establish productive infection in these cells. Nonetheless, gp120 from IIIB could bind the cells. Thus, binding of gp120 did not correlate with infectivity. Furthermore, binding of gp120 was a specific event, leading to apoptosis upon crosslinking with anti-gp120 through a fas-dependent mechanism. If apoptosis is also observed in vivo even in uninfected hematopoietic cells, this could contribute significantly to the impairment in hematopoietic cell number and function. Our data suggest a novel indirect mechanism for depletion of CD34+ and CD34+-derived cells even in the absence of productive viral infection of these cells.

Comparison of retroviral-mediated gene transfer into cultured human CD34+ hematopoietic progenitor cells derived from peripheral blood, bone marrow, and fetal umbilical cord blood.

Genetic alteration of stem cells ex vivo followed by bone marrow transplantation could potentially be used in the treatment of numerous diseases and malignancies. However, there are many unanswered questions as to the best source of hematopoietic cells for long-term reengraftment and the most effective way to introduce foreign genes into this target cell. We have compared retroviral-mediated gene transfer into CD34+-enriched cells derived from peripheral blood (PB), bone marrow (BM), or fetal umbilical cord blood (CB). Cells from all three sources that had been expanded ex vivo in the presence of stem cell factor (SCF), interleukin-3 (IL-3), IL-6, and granulocyte colony-stimulating factor (G-CSF) showed transduction efficiencies ranging from 5-45%, as measured by acquisition of G418 resistance. The average efficiencies of gene transfer from multiple experiments for PB, BM, and CB were not statistically different. To determine the effect of ex vivo expansion on gene transfer into CB CD34+ cells, we compared the transduction efficiencies of cells exposed to virus immediately after harvest and CD34 selection or after 6 days of culture CD34+ CB cells were more effectively transduced after expansion in culture, showing gene transfer efficiencies 3- to 5-fold higher on day 6 compared with day 0. Last, we examined retroviral transduction via spinoculation of CB CD34+ cells and found it to be approximately as effective as our standard transduction with no significant loss of cell viability as measured by colony formation in semi-solid medium.

Use of amifostine in bone marrow purging.

One of the main obstacles for the use of high-dose chemotherapy with autologous hematopoietic progenitor cell support in the treatment of malignancies is the possibility of reinfusing clonogenic tumor cells with the hematopoietic graft. Purging of the graft with chemicals can reduce the number of tumor cells but can also damage the normal hematopoietic progenitors. Preclinical studies showed that the phosphorylated sulfhydryl compound amifostine (WR-2721) can protect normal hematopoietic progenitors from damage from alkylating agents. We conducted a randomized clinical trial in patients with breast cancer, non-Hodgkin's lymphoma, and Hodgkin's disease undergoing autologous bone marrow transplant. In this study, patients were randomized to have their bone marrow purged with 4-hydroperoxycyclophosphamide (4-HC) with (arm A) or without (arm B) amifostine. The percentage of colony-forming unit granulocyte-macrophages recovered after purging was higher in the amifostine arm, both in patients with breast cancer and in those with lymphoma, although this difference was not statistically significant. In addition, the time to engraftment was significantly shorter in the amifostine arm in both cohorts. We showed that pretreatment of bone marrow with amifostine prior to purging with 4-HC can protect normal hematopoietic progenitors from damage by 4-HC. This resulted in shorter engraftment rates and less need for supportive care.

High-dose paclitaxel, cyclophosphamide, and cisplatin with autologous hematopoietic progenitor-cell support: a phase I trial.

PURPOSE: To determine the maximal-tolerated dose (MTD) of paclitaxel in combination with high-dose cyclophosphamide (CPA) and cisplatin (cDDP) followed by autologous hematopoietic progenitor-cell support (AHPCS). PATIENTS AND METHODS: Forty-nine patients with poor-prognosis breast cancer, non-Hodgkin's lymphoma (NHL), or ovarian cancer were treated with escalating doses of paclitaxel infused over 24 hours, followed by CPA (5,625 mg/m2 intravenously over 1 hour in three divided doses) and cDDP (165 mg/m2 intravenously as a continuous infusion over 72 hours) and AHPCS. Pharmacokinetic measurements for each drug were performed. RESULTS: Dose-limiting toxicities were encountered in two patients at 825 mg/m2 of paclitaxel; one patient died of multiorgan failure that involved the lung, CNS, and kidneys, and the other developed grade 3 respiratory, CNS, and renal toxicity, which resolved. The MTD of this combination was determined to be paclitaxel 775 mg/m2, CPA 5,625 mg/m2, and cDDP 165 mg/m2 followed by AHPCS. Sensory polyneuropathy and mucositis were prominent toxicities, but both were reversible and tolerable. The pharmacokinetics of paclitaxel correlated significantly with the severity of mucositis (P < .001) and peripheral neuropathy (P < .00004). Eighteen of 33 patients (54%) with measurable, heavily pretreated metastatic breast cancer achieved a partial response (PR). Responses were also observed in patients with NHL (four of five patients) and ovarian cancer (two of two). CONCLUSION: It is possible to escalate the dose of paclitaxel to 775 mg/m2 in combination with 5,625 mg/m2 of CPA, 165 mg/m2 of cDDP, and AHPCS. An encouraging response rate in poor-prognosis patients with breast cancer, NHL, and ovarian cancer warrants further study.

Bone marrow metastases.

This article discusses the clinical significance of bone marrow metastases and the current methods being used to detect tumor cells in marrow. The strategies being investigated for eradicating cancer cells from marrow in patients receiving hematopoietic cell autografts also are reviewed.

Immunocytochemical detection of breast cancer cells in marrow and peripheral blood of patients undergoing high dose chemotherapy with autologous stem cell support.

Detection of small numbers of breast cancer cells is important in staging the disease and can be helpful in assessing the efficacy of purging regimens prior to autologous stem cell infusion. Immunohistochemical methods are potentially useful and broadly applicable for this purpose since they are simple to perform, sensitive, and may be quite specific. We have used a combination of four monoclonal antibodies [260F9, 520C9, 317G5 (Baxter Corp); BrE-3 (Dr. R. Ceriani)] against tumor cell surface glycoproteins in a sensitive immunocytochemical assay to identify breast tumor cells in bone marrow and peripheral blood. Immunostained cytospin preparations were fixed prior to staining to preserve cytological details of immunopositive cells. After immunostaining, slides were counterstained with hematoxylin to confirm the identify of labeled cells. In cytocentrifuge experiments in which small numbers of CAMA human breast tumor cells were added to bone marrow mononuclear cells, a linear relationship between the number of tumor cells added and the number of tumor cells detected was obtained over a broad range of tumor cell concentrations. The probability of detecting tumor cells was dependent on the number of cytocentrifuge slides examined. When ten slides (5 million cells) were examined, the probability of detecting tumor at a concentration of 4 tumor cells per million bone marrow mononuclear cells was 98%. In clinical specimens, tumor cells were detected in marrow aspirates from 73 of 240 (30%) patients undergoing autologous transplantation, including 70 (37%) of 190 patients with clinical stage IV disease, 0 of 7 patients with clinical stage III disease, and 3 of 43 (7%) patients with clinical stage II disease. Seventy-three of 657 peripheral blood specimens from 26 of 155 patients (17%) contained breast cancer cells with counts ranging from 1 to 97 tumor cells per million leukocytes. Tumor cells were most frequently found in the blood of patients with stage IV disease [21 of 107 (20%)] but were also found in a substantial number [5 of 44 (11%)] of patients with stage II disease. Positive selection of CD34-positive hematopoietic progenitor cells as well as negative purging methods such as incubation with 4-hydroxyperoxy-cyclophosphamide (4-HC) were evaluated with respect to tumor cell depletion. Selection of CD34-positive progenitor cells from bone marrow or peripheral blood resulted in log reduction of 1 to > 4 tumor cells reinfused at autologous transplantation. A lesser log reduction (up to 1) was demonstrated following 4-HC purging. We conclude that properly performed and controlled immunocytochemical staining of bone marrow and peripheral blood cytospins is a sensitive and simple way to detect and quantitate breast cancer cells in hematopoietic specimens harvested for autotransplantation and that CD34-positive progenitor cell selection results in significant reduction in the number of breast cancer cells reinfused with marrow or peripheral blood stem cells.

Large volume ex vivo expansion of CD34-positive hematopoietic progenitor cells for transplantation.

A large volume culture system was developed for the ex vivo expansion of CD34 positive (+) hematopoietic progenitors, using cell donated by 15 patients receiving high-dose chemotherapy with autologous hematopoietic progenitor cell support (AHPCS). Substantial expansion of myeloid (181-fold) and megakaryocyte (41-fold) progenitors cells was demonstrated, using the conditions that we determined to be optimal: CD34+ progenitors cultured unperturbed for 7 (marrow) or 10 (blood) days in Teflon-coated bags with X-Vivo-10 medium containing 10% autologous plasma, 100 ng/ml, respectively, of recombinant stem cell factor (SCF), interleukin 3 (IL-3), interleukin 6 (IL-6), and granulocyte colony-stimulating factor (G-CSF). The studies demonstrated that (a) CD34 selection was necessary to obtain large, clinically relevant numbers of hematopoietic progenitors, (b) the addition of G-CSF to the baseline regimen of SCF/IL-3/IL-6 significantly enhanced the expansion of myeloid progenitors, (c) the addition of IL-1 to SCF/IL-3/IL-6 did not significantly enhance myeloid progenitor cell expansion, (d) CD34+ G-CSF-mobilized peripheral blood progenitor cells (PBPC) produced higher numbers of myeloid progenitors in culture than CD34+ marrow cells, and (e) long-term tissue culture (LTC) assays demonstrate the preservation of long-term initiating cells in ex vivo culture. The short-term and long-term reconstituting capability of CD34+ PBPC cultured in this system remains to be determined and will be evaluated in a clinical trial where they will be used as the sole source of AHPCS following high-dose therapy.

Stem cell isolation.

High-dose chemotherapy with autologous hematopoietic progenitor cell support is increasingly used to treat a variety of malignant diseases. A drawback of this technique is the potential for infusing clonogenic tumor cells with the autograft, producing relapse of the disease in the patient. The use of positive selection techniques to isolate stem cells and thus reduce or eliminate tumor cell contamination has been extensively studied over the past few years. Preliminary clinical results have demonstrated that these procedures deplete 2 to 7 logs of tumor cells and do not impair engraftment. It is too early to assess the ultimate clinical benefit of this strategy. Additional applications of CD34-selection include ex vivo expansion of and gene transfer into hematopoietic progenitor cells and T-cell depletion of allogeneic grafts to reduce the incidence of graft-versus-host disease.

Amifostine (WR-2721) shortens the engraftment period of 4-hydroperoxycyclophosphamide-purged bone marrow in breast cancer patients receiving high-dose chemotherapy with autologous bone marrow support.

4-Hydroperoxycyclophosphamide (4-HC), a commonly used marrow-purging agent, is active against many tumors, but is also toxic to normal marrow progenitors. Amifostine (WR-2721) is a sulfhydryl compound with chemoprotectant activity. Preclinical studies using suspensions of bone marrow and breast cancer cells demonstrated that ex vivo treatment with amifostine followed by 4-HC resulted in protection of marrow progenitors, with no compromise in the antitumor effect of 4-HC. This fact stimulated the development of a clinical trial. Bone marrow was harvested from 15 poor-prognosis breast cancer patients and randomly assigned to ex vivo treatment with amifostine followed by 4-HC (amifostine + 4-HC), or treatment with 4-HC alone. High-dose chemotherapy was then administered followed by infusion of the purged autologous bone marrow support (ABMS). Leukocyte engraftment, defined as a white blood cell count > or = 1 x 10(9)/L, was achieved in an average of 26 days for patients whose marrow was purged with amifostine + 4-HC versus 36 days for patients whose marrow was purged with 4-HC alone (P = .032). The average number of platelet transfusions (12 v 29; P = .017) and days of antibiotic therapy (28 v 40; P = .012) were significantly less for patients whose marrow was exposed to amifostine + 4-HC, compared with 4-HC alone. Unpurged backup marrow fractions were infused into three patients whose marrow was purged with 4-HC alone, because of inadequate marrow recovery. None of the patients who received amifostine + 4-HC-purged marrow required a backup marrow fraction. Complete remissions were achieved in 83% of patients with measurable disease, with no difference between the two cohorts. Forty-three percent of patients remained alive and progression-free at a mean of 13 months posttransplant. There was no significant difference in the rate or pattern of relapse for patients whose marrow was purged with amifostine + 4-HC compared with those whose marrow was purged with 4-HC alone. Ex vivo treatment of marrow with amifostine significantly shortens the time to marrow recovery, thereby reducing the risk of myelosuppressive complications in breast cancer patients receiving high-dose chemotherapy and 4-HC-purged ABMS. Since supportive care requirements are also significantly decreased, amifostine may reduce the cost of such therapy.

Purging of autologous bone marrow for transplantation: the protection and selection of the hematopoietic progenitor cell.

Autologous bone marrow transplantation (ABMT) is the treatment of choice for selected patients with acute myelogenous leukemia, non-Hodgkin's lymphoma, and poor prognosis breast cancer. A possible limitation of this approach is that clonogenic tumor cells could be collected and infused back into the patient along with the normal bone marrow. The major emphasis in our laboratory has been the development of marrow purging regimens for breast cancer patients. This paper describes two investigative approaches hematopoietic progenitor cell protection and selection. We describe how the use of G-CSF in the patients who receive positively selected marrow shortens the rate of engraftment.