Development of a closed-system process for clinical-scale generation of DCs: evaluation of two monocyte-enrichment methods and two culture containers.

BACKGROUND: Clinical immunotherapy trials using DCs depend on large-scale methods for DC generation that fulfil current good manufacturing practice requirements. Our goal was to develop data on two variables, monocyte-enrichment method and culture container, which could be used to design a closed-system process for ex vivo generation of immature DCs. METHODS: Mononuclear cells were collected by leukapheresis and enriched for monocytes by either counterflow centrifugal elutriation, or immunomagnetic selection using Isolex, an automated closed-system device. Monocytes were cultured for 7 days in serum-free medium with GM-CSF and IL-4, using either plastic flasks or gas-permeable Stericell bags. Monocytes and cultured DCs were evaluated for yield, flow cytometric phenotype, and in vitro function in MLR, and autologous recall responses to tetanus toxoid and influenza virus. RESULTS: Enriched monocyte products from elutriation and immunomagnetic selection were equivalent in yield and purity, and were capable of generating immature DCs in either flasks or bags. DCs from all four culture conditions were equivalent in yield, phenotype, and in vitro function. Mean DC yield was 67-80% per seeding monocyte, and 11-13% per starting mononuclear cell (MNC). A leukapheresis product containing 5 x 10(9) MNCs processed by this method could therefore yield approximately 5 x 10(8) immature DCs. DISCUSSION: In this manufacturing process, the Isolex system was equivalent to elutriation, and Stericell bags were equivalent to flasks. Together, the Isolex system and Stericell bags can be incorporated into a closed-system process to generate immature DCs.

Development of a clinical-scale method for generation of dendritic cells from PBMC for use in cancer immunotherapy.

BACKGROUND: There is growing interest in the use of dendritic cells (DCs) for treatment of malignancy and infectious disease. Our goal was to develop a clinical scale method to prepare autologous DCs for cancer clinical trials. METHODS: PBMC were collected from normal donors or cancer patients by automated leukapheresis, purified by counterflow centrifugal elutriation and placed into culture in polystyrene flasks at 1 x 10(6) cells/mL for 5-7 days at 37 degrees C, with 5% CO(2), with IL-4 and GM-CSF. Conditions investigated included media formulation, supplementation with heat in activated allogeneic AB serum or autologous plasma and time to harvest (Day 5 or Day 7). DCs were evaluated for morphology, quantitative yield, viability, phenotype and function, including mixed leukocyte response and recall response to tetanus toxoid and influenza virus. RESULTS: DCs with a typical immature phenotype (CD14-negative, CD1a-positive, mannose receptor-positive, CD80-positive, CD83-negative) were generated most consistently in RPMI 1640 supplemented with 10% allogeneic AB serum or 10% autologous plasma. Cell yield was higher at Day 5 than Day 7, without detectable differences in phenotype or function. In pediatric sarcoma patients, autologous DCs had enhanced function compared with monocytes from which they were generated. In this patient group, starting with 8.0 +/- 3.7 x 10(8) fresh or cryopreserved autologous monocytes, DC yield was 2.1 +/- 1.0 x 10(8) cells, or 29% of the starting monocyte number. DISCUSSION: In the optimized clinical-scale method, purified peripheral monocytes are cultured for 5 days in flasks at 1 x 10(6) cells/mL in RPMI 1640, 10% allogeneic AB serum or autologous plasma, IL-4 and GM-CSF. This method avoids the use of FBS and results in immature DCs suitable for clinical trials.

Paclitaxel chemotherapy after autologous stem-cell transplantation and engraftment of hematopoietic cells transduced with a retrovirus containing the multidrug resistance complementary DNA (MDR1) in metastatic breast cancer patients.

The MDR1 multidrug resistance gene confers resistance to natural-product anticancer drugs including paclitaxel. We conducted a clinical gene therapy study to determine whether retroviral-mediated transfer of MDR1 in human hematopoietic cells would result in stable engraftment, and possibly expansion, of cells containing this gene after treatment with myelosuppressive doses of paclitaxel. Patients with metastatic breast cancer who achieved a complete or partial remission after standard chemotherapy were eligible for the study. Hematopoietic stem cells (HSCs) were collected by both peripheral blood apheresis and bone marrow harvest after mobilization with a single dose of cyclophosphamide (4 g/m2) and daily filgrastim therapy (10 microg/kg/day). After enrichment for CD34+ cells, one-third of each collection was incubated ex vivo for 72 h with a replication-incompetent retrovirus containing the MDR1 gene (G1MD) in the presence of stem-cell factor, interleukin 3, and interleukin 6. The remaining CD34+ cells were stored without further manipulation. All of the CD34+ cells were reinfused for hematopoietic rescue after conditioning chemotherapy with ifosfamide, carboplatin, and etoposide regimen. After hematopoietic recovery, patients received six cycles of paclitaxel (175 mg/m2 every 3 weeks). Bone marrow and serial peripheral blood samples were obtained and tested for the presence of the MDR1 transgene using a PCR assay. Six patients were enrolled in the study and four patients received infusion of genetically altered cells. The ex vivo transduction efficiency, estimated by the PCR assay, ranged from 0.1 to 0.5%. Three of the four patients demonstrated engraftment of cells containing the MDR1 transgene. The estimated percentage of granulocytes containing the MDR1 transgene ranged from a maximum of 9% of circulating nucleated cells down to the limit of detection of 0.01%. One patient remained positive for the MDR1 transgene throughout all six cycles of paclitaxel therapy, whereas the other 2 patients showed a decrease in the number of cells containing the transgene to undetectable levels. Despite the low level of engraftment of MDR1-marked cells, a correlation was observed between the relative number of granulocytes containing the MDR1 transgene and the granulocyte nadir after paclitaxel therapy. No adverse reactions to the genetic manipulation procedures were detected. Therefore, engraftment of human HSCs transduced with the MDR1 gene can be achieved. However, the overall transduction efficiency and stable engraftment of gene-modified HSCs must be improved before MDR1 gene therapy and in vivo selection with anticancer drugs can be reliably used to protect cancer patients from drug-related myelosuppression.

Engraftment of MDR1 and NeoR gene-transduced hematopoietic cells after breast cancer chemotherapy.

To determine whether the multidrug resistance gene MDR1 could act as a selectable marker in human subjects, we studied engraftment of peripheral blood progenitor cells (PBPCs) transduced with either MDR1 or the bacterial NeoR gene in six breast cancer patients. This study differed from previous MDR1 gene therapy studies in that patients received only PBPCs incubated in retroviral supernatants (no nonmanipulated PBPCs were infused), transduction of PBPCs was supported with autologous bone marrow stroma without additional cytokines, and a control gene (NeoR) was used for comparison with MDR1. Transduced PBPCs were infused after high-dose alkylating agent therapy and before chemotherapy with MDR-substrate drugs. We found that hematopoietic reconstitution can occur using only PBPCs incubated ex vivo, that the MDR1 gene product may play a role in engraftment, and that chemotherapy may selectively expand MDR1 gene-transduced hematopoietic cells relative to NeoR transduced cells in some patients.

Retroviral gene transduction of adult peripheral blood or marrow-derived CD34+ cells for six hours without growth factors or on autologous stroma does not improve marking efficiency assessed in vivo.

Our previous work in patients undergoing autologous transplant for multiple myeloma (MM) or breast cancer (BC) has shown that retroviral transduction of adult CD34+ cells for 72 hours in the presence of interleukin-3 (IL-3), IL-6, and stem cell factor (SCF) resulted in .01% to 1% long-term marking of peripheral blood and marrow cells (Blood 85:3948, 1995). In this study we compare these previous studies to transduction with no added growth factors, previously shown to result in higher levels of marking in children (Lancet 342:1134, 1993) or transduction in the presence of an autologous stromal layer. Peripheral blood (PB) mononuclear cells were collected via apheresis after high-dose cyclophosphamide and granulocyte colony-stimulating factor. Bone marrow (BM) was also harvested in all patients. One third of both BM and PB collections were enriched for CD34+ cells and transduced with one of two marking vectors containing the neomycin-resistance gene to distinguish cells originating from BM and PB posttransplantation. Cells from 3 MM and 2 BC patients were transduced without growth factors for 6 hours and cells from 2 MM and 2 BC patients were transduced in the presence of autologous marrow stroma. Immediately posttransduction, the percentage of Neo-resistant PB and BM progenitors (colony-forming units) were: 0% to 19% in the 6-hour no growth factor group and 0% to 36% in the autologous stroma group. After conditioning therapy, both transduced and untransduced PB and BM fractions were infused into the patients. Semi-quantitative nested DNA polymerase chain reaction was performed on total, mononuclear, and granulocyte fractions of PB and BM at 1, 3, 6, 9, 12, and 18 months. Poor marking has been observed in both groups, with no consistently positive patients. These results compare unfavorably with our prior experience using growth factors during transduction. Further optimization of transduction conditions and vectors needs to be developed to improve transduction efficiency of adult human repopulating hematopoietic cells.

Growth of tumor-infiltrating lymphocytes from human solid cancers: summary of a 5-year experience.

Between 1989 and 1993, 255 tumor biopsies representing 4 tumor histologies (melanoma, breast cancer, colon cancer and renal cell cancer) were received by the Surgery Branch of the National Cancer Institute. Tumor-infiltrating lymphocytes (TIL) were grown from single-cell suspensions of tumor biopsies over the course of 30-45 days. The TIL were grown in medium containing IL-2. To obtain numbers suitable for therapy (>10(11)), TIL were expanded using a large-scale system of cell culture and harvesting. While the largest number of biopsies was obtained from melanoma patients, TIL were successfully grown from 160 of 255 tumor biopsies representing all 4 histologies. Under the culture conditions employed, several characteristics of TIL expansion were observed. The cell surface phenotype of TIL which grew out from the tumor biopsies was generally a mix of CD3+/CD4+ or CD3+/CD8+ lymphocytes. Only TIL from melanoma biopsies were found to be consistently cytolytic and, in many cases, lysed autologous tumor cells preferentially. Interestingly, TIL derived from extra-nodal sites of metastatic melanoma biopsies (subcutaneous, lung, bowel; 36 of 67, 54%) were more likely to have these cytolytic characteristics than TIL derived from tumor-involved lymph node biopsies (7 of 39, 18%). The present study summarizes 5 years of laboratory effort and validates the technologies developed for the large-scale growth and harvesting of TIL. In addition, it summarizes the laboratory effort supporting previously published clinical reports on TIL from our group.

5-Azacytidine-induced 6-thioguanine resistance at the gpt locus in AS52 cells: cellular response.

Treatment of AS52 cells with 5-azacytidine resulted in an induction of 6-thioguanine-resistant [6TG] colonies, which reached a maximum by an expression time of 9 days. Dose responses for both cytotoxicity and mutation induction were determined following treatment with 5-azacytidine. At 20 microM treatment, 5-azacytidine exposure resulted in about 50% survival. Mutant frequency reached a maximum of 10 microM. At concentrations between 10 and 20 microM, 5-azacytidine was a potent mutagen but did not exhibit a dose response. Although many compounds both induce cell death and affect the growth rate of cells, 5-azacytidine specifically induced cell death and did not affect the doubling time of the surviving treated cell population.

Synthetic fibronectin peptides interrupt inflammatory cell infiltration in transforming growth factor beta 1 knockout mice.

Pronounced mononuclear leukocyte (MNL) infiltration occurs in multiple organs of mice homozygous for a transforming growth factor beta 1 (TGF-beta 1) loss-of-function gene mutation [TGF-beta 1 (-/-)], followed by cachexia and eventually death. Consistent with the increased leukocyte adhesion and tissue infiltration, MNLs isolated from spleen, thymus, and peripheral blood of symptomatic TGF-beta 1 (-/-) mice, as compared to littermate controls, exhibited increased adhesion to extracellular matrix proteins and to endothelial cells in vitro. Incubation of TGF-beta 1 (-/-) MNLs with selected synthetic peptides corresponding to cell- and heparin-binding sequences of fibronectin (FN) significantly attenuated adhesion of these cells not only to FN but also to endothelial cells in vitro. Based on these observations, mice were treated with the FN peptides in an attempt to rescue them from tissue inflammation and cardiopulmonary failure. Daily injections of a combination of four synthetic FN peptides that interact with beta 1-integrins and/or cell surface proteoglycans blocked the massive infiltration of MNLs into the heart and lungs of TGF-beta 1 (-/-) mice. Peptide treatment initiated on day 8, coincident with the first evidence of increased leukocyte-endothelial cell interactions, not only blocked tissue infiltration but also moderated the lethal wasting syndrome.

An in situ protocol for measuring the expression of chemically-induced mutations in mammalian cells.

The generation of expression curves and the evaluation of mutagenic responses of mammalian cells using standard mutagenesis assays can be inaccurate because mutant and wild-type cells are usually mixed during the expression phase. If some mutant progenitors or mutants grow more slowly than the wild-type cells during the expression period, there will be a decrease in the mutant to wild-type ratio with time and the mutant fraction will not accurately represent the number of mutational events that occurred. The mutant fraction may also inaccurately assess the number of mutations if these mutations are expressed over a number of generations during the time before selection. We previously showed that recovery of L5178Y mouse cell mutants is not complete when mutations are allowed to express in suspension because slowly growing mutants and/or mutant progenitors are diluted out during this time (Rudd et al., 1990). In order to more accurately quantitate the mutagenic response of the cells, we developed an in situ procedure which segregates and immobilizes cells during expression. Because of this immobilization, slowly growing mutant progenitors and mutants expressed at different times will have an equal probability of being scored as mutants. Thus, one mutation leads to one mutant colony and the measurement of the mutagenic response of the cells to the chemical accurately reflects the mutational events that occurred. We plated L5178Y tk+/- mouse cells in semisolid medium immediately after treatment. As the cells grew and formed microcolonies, the selective agent TFT was added as an overlay at specified times, permitting only TFTr cells to survive. In this procedure, each mutation was captured as an individual colony; consequently, the measured mutation fraction accurately reflected the mutational events that occurred at the selected locus. In addition, the induced mutant colonies arising in the agar are the result of independent mutational events. We previously described the in situ protocol for L5178Y cells and showed that the spontaneous mutation rate measured was 50-fold greater than when the cells expressed the phenotype in suspension (Rudd et al., 1990). From this we concluded that the slow growth phenotype was expressed before TFT resistance. In the present paper, we evaluate the effect of chemical treatment on the mutation fraction as a function of the time to TFT addition. Using the in situ protocol, we generated expression curves for three nucleotide analogs, 5-azacytidine, TFT and AraC. The numbers of TFTr colonies produced at various times after treatment indicated that chemically-treated cultures had higher mutation fractions than the solvent controls.(ABSTRACT TRUNCATED AT 400 WORDS)

Lysis of autologous melanoma cells by tumor-infiltrating lymphocytes: association with clinical response.

Tumor-infiltrating lymphocytes (TILs) can be grown in vitro in medium containing interleukin-2 (IL-2). In clinical trials at the Surgery Branch of the National Cancer Institute, patients with metastatic malignant melanomas were treated with IL-2 plus the adoptive transfer of autologous TILs. At the time of treatment, TILs were assayed for in vitro lysis of fresh autologous and allogeneic melanoma cells and Daudi cells. Patients were evaluated for clinical response 4-8 weeks later. Lysis of autologous tumor cells by TILs was significantly higher for responding than for nonresponding patients. Tumor cells from responding and nonresponding patients were equally sensitive to lysis by allogeneic lymphokine-activated killer (LAK) cells. There was no difference between TILs from responding and nonresponding patients for lysis of LAK-sensitive Daudi cells, which was low in most cases and demonstrated that TIL lysis of autologous tumor cells was not due to LAK cells. The observed association of autologous tumor cell lysis by TILs with clinical response suggests that the development of culture methods to optimize lysis of autologous tumors may lead to increased response rates using this TIL treatment regimen.

PAF increases hepatic vascular resistance and glycogenolysis in vivo.

Administration of platelet-activating factor (PAF) to portal venous circulation of anesthetized fed rats produced decreases in mean arterial pressure and increases in hepatic portal pressure and blood glucose concentration. These responses to PAF were dose dependent with ED50 values of 0.02-0.05 micrograms/kg and specific as lyso- and enantio-PAF did not reproduce effects of PAF. Specific PAF receptor antagonist SRI 63-675 (75 micrograms/kg) inhibited significantly these PAF (0.1 micrograms/kg)-induced responses in rats. Administration of prazosin (500 micrograms/kg) and propranolol (400 micrograms/kg) to rats abolished phenylephrine (50 micrograms/kg)-induced increases in mean arterial pressure, hepatic portal pressure, and blood glucose concentration but did not prevent PAF (1 microgram/kg)-induced alterations in these parameters. Glycogen phosphorylase alpha levels were increased significantly in livers of rats after administration of PAF (1 microgram/kg) or phenylephrine (50 micrograms/kg). Administration of prazosin and propranolol to rats inhibited phenylephrine- but not PAF-induced activation of hepatic glycogen phosphorylase. Hepatic adenosine 3',5'-cyclic monophosphate (cAMP) concentration was increased slightly by PAF, but these increases were eliminated by adrenergic blockade, suggesting that activation of hepatic glycogen phosphorylase by PAF is not dependent on increases in cAMP. Increases in hepatic portal pressure and blood glucose concentration in response to PAF (0.1 micrograms/kg) were not attenuated in adrenalectomized rats. Moreover, PAF (0.1 micrograms/kg) stimulated increases in hepatic portal pressure after administration of the ganglionic blocking agent chlorisondamine (2.5 mg/kg) to adrenalectomized rats. Administration of PAF (0.05 micrograms/kg) to rats instrumented with pulse Doppler flow probes produced decreases in hepatic arterial and portal vein blood flow and increases in hepatic arterial and portal vascular resistance. These observations provide direct evidence that PAF regulates hepatic hemodynamics and glycogenolysis in vivo. It is suggested that PAF plays an important role in regulating hepatic blood flow and supplying extrahepatic tissues with energy substrates by sympathetic-independent mechanism(s) after its release in acute pathophysiological situations.

Disaster procedures report. Report following the Moorgate train crash on 28 February, 1975.

There is no substitute for the use of intelligence and common sense both in the drawing up and interpretation of a disaster plan; for compromise in dealing with other rescue services; for ingenuity in filling the gaps in the equipment with which you find yourself provided; and, finally, perhaps most important, for self-discipline. None of us is indispensible--there are always others equally able to make decisions.