A multicenter comparison study between the Endosafe PTS rapid-release testing system and traditional methods for detecting endotoxin in cell-therapy products.

BACKGROUND: Rapid-release testing reduces the waiting period for administration of time-sensitive cell-therapy products. Current assay systems are labor intensive and time consuming. The Endosafe portable test system (PTS) is a chromogenic Limulus amebocyte lysate (LAL) portable endotoxin detection system that provides quantitative results in approximately 15 min. To evaluate Endosafe performance with cell-therapy products, side-by-side testing of traditional LAL systems and the Endosafe system was conducted at the Production Assistance for Cellular Therapies (PACT) facilities and the National Institutes of Health's Department of Transfusion Medicine, USA. METHODS: Charles River Laboratories provided each center with a PTS reader and two commercially prepared lyophilized reference standard endotoxin (RSE) vials. All samples tested with the Endosafe system used 0.05-5.0 endotoxin unit/mL (EU/mL) sensitivity cartridges provided by Charles River. Each vial was reconstituted with LAL water and tested in triplicate using the Endosafe and in-house LAL methods. Subsequently, each center tested the endotoxin content of standard dilutions of cell-therapy products, thus creating paired test results for each sample. Additionally, fabricated endotoxin-positive samples containing varying concentrations of endotoxin were prepared and shipped to all centers to perform blinded testing. RESULTS: Valid paired results, based on each center's LAL method and the Endosafe system criteria, were analyzed. Endotoxin detection between paired results was equivalent in most cases. DISCUSSION: The Endosafe system provided reliable results with products typically produced in cell-therapy manufacturing facilities, and would be an appropriate test on which to base the release of time-sensitive cell-therapy products.

Purging tumor cells from bone marrow by use of antibody and complement: a critical appraisal.

This review highlighted several problems associated with the use of antibody and complement in the elimination of tumor cells from bone marrow that was to be used for transplantation, and it discussed some of the difficulties encountered in developing this approach in model systems. These problems should be seriously considered by any clinician contemplating this method for bone marrow purging.

Evidence for an anticomplementary factor associated with human bone marrow cells.

Complement (C)-mediated lysis of antibody-sensitized sheep erythrocytes was inhibited by the addition of human bone marrow cells. The anticomplementary activity could be attributed to a soluble factor that was released from the bone marrow cells. This factor inhibited at an early stage in the C-cascade and showed the characteristics of a factor that accelerates decay of C2. The release of such a factor by bone marrow cells would present an obstacle to the use of antibody and C to purge tumor cells from bone marrow that is to be used for autologous transplantation.

Selective loss of expression of a tumor-associated antigen on a human leukemia cell line induced by treatment with monoclonal antibody and complement.

The sensitivity of a common acute lymphoblastic leukemia-associated antigen (cALLa)-positive, human leukemia pre-B-cell line to killing by antibody and complement was studied. A stable subpopulation was selected by its ability to survive four sequential treatments with excess monoclonal antibody (MoAb) directed against an Mr 24,000 glycoprotein associated with human leukemia cells and excess rabbit complement. Analysis of the antigen expression by individual cells within the parental and the selected cell populations was achieved by flow cytometry and demonstrated a marked decrease of the leukemia-associated antigen expression on individual cells within the selected subpopulation. These low-antigen-density cells were stable in subculture, and the immunoglobulin heavy-chain gene rearrangement of the parent population and the low-antigen-density subpopulation were identical, indicating that they were derived from a single cell source. The selection of this subpopulation was specific in that the expression of a second antigen recognized by a cALLa-specific MoAb was not affected. The presence of subpopulations of tumor cells with low levels of surface antigen expression that are resistant to killing upon addition of excess antibody and complement will prove to be an obstacle to the use of this approach to eliminate tumor cells from bone marrow that is to be used for autologous transplantation.

Partially mismatched related-donor bone marrow transplantation for pediatric patients with acute leukemia: younger donors and absence of peripheral blasts improve outcome.

PURPOSE: To extend access to bone marrow transplantation (BMT), we used partially mismatched related donors (PMRD) for pediatric patients with acute leukemia. In this report we sought to determine pretransplantation factors that might predict outcome. PATIENTS AND METHODS: Of 67 such patients, 43 had acute lymphocytic leukemia and 24 had acute myelogenous leukemia. At the time of transplantation, 41 patients were in relapse. Donors included 40 parents, 24 siblings, and three cousins. HLA disparity of two to three major antigens was detected in two thirds of the donor-recipient pairs. Conditioning therapy, including total-body irradiation and chemotherapy followed by graft-versus-host disease (GvHD) prophylaxis with partial T-cell depletion of the graft using T10B9 or OKT3, was combined with posttransplantation immunosuppression. RESULTS: Estimated probability (EP) of engraftment was 0.96 and was not affected by donor-antigen mismatch (AgMM; P =.732). EP of grades 2 to 4 acute GvHD was 0.24 and was not affected by recipient AgMM (P =.796). EP of disease-free survival was 0.26 at 3 years but improved to 0.45 when donors were younger than 30 years (P<.001). EP of relapse at 3 years was 0.41 and reduced with younger donors' age. For patients who were in relapse at the time of transplantation, absence of blasts was associated with a lower relapse rate (0.46 v. 0.84; P =. 083), similar to that of patients in remission. CONCLUSION: PMRD-BMT in pediatric leukemia resulted in high engraftment and low GvHD rates. To improve outcomes, younger donors should be sought, and clinicians should attempt to reduce peripheral blasts in patients who are in relapse.

T-cell immunotherapy for adenoviral infections of stem-cell transplant recipients.

Human adenoviruses are ubiquitous lytic DNA viruses that can be divided into 51 different serotypes, grouped from A to F on the basis of genome size, composition, homology, and organization. Adenovirus infections, although frequent, are rarely fatal in immunocompetent individuals, due to potent innate and adaptive immune responses. By contrast, adenoviruses are a significant cause of morbidity and mortality in immunosuppressed individuals, for whom there are limited treatment options. Since antiviral drugs have variable efficacy in the treatment of severe adenovirus disease, iatrogenic reconstitution with in vitro expanded virus-specific cytotoxic T lymphocytes (CTLs) is an attractive option for prophylaxis and treatment, particularly because the endogenous recovery of adenovirus-specific T cells has proved important in controlling infection in vivo. Thus, we have characterized human T-cell responses to adenovirus in vitro and explored the potential of adoptive T-cell immunotherapy as a prophylactic or therapeutic strategy for adenovirus infections posttransplant.

The migration of hematopoietic cells: an in vitro study system.

An in vitro migration assay system for the study of hematopoietic cell migration was established. Using a large well modification of earlier described migration chambers, it was found that, in the presence of whole murine serum from either normal or aplastic mice, a heterogeneous population of cells was stimulated to migrate through limited size pores (8 mu) in a thin (5 mu) polycarbonate filter. In dilution studies, serum obtained from animals that had been rendered aplastic by total body irradiation provided a stronger migration stimulus than serum from normal animals. This observation is consistent with observations of hematopoietic cell migration in vivo. Primitive spleen colony-forming cells and in vitro granulocyte/macrophage colony-forming cells were present in the migrating population at a comparable fraction to that found in untreated bone marrow. These studies demonstrate the feasibility of studying hematopoietic stem cell migration under controlled experimental conditions.

Low antigen density leukemia cells: selection and comparative resistance to antibody-mediated marrow purging.

Low tumor-associated antigen (TAA) expressing tumor cells present an obstacle to effective antibody directed purging of tumor cells from bone marrow. In this study, a comparison was made of the efficiency with which low TAA expressing leukemia cells could be depleted using two monoclonal antibody (MoAb) directed purging techniques: 1) complement (C)-mediated cytolysis, and 2) physical separation using magnetic microspheres. Low TAA sublines were selected from a cultured human leukemia cell line by growing out cells remaining after treatment with anti-TAA and C, or after immunomagnetic (IM) purging. IM-selected sublines showed lower TAA expression than did C-selected sublines, and sublines resulting from multiple selections expressed less TAA than those that had only been through one selection. These sublines were then examined for sensitivity to C or IM purging. The highly selected, lowest TAA expressing sublines were markedly resistant to both IM and C. Less selected sublines were resistant to C, but not to IM. In both techniques, addition of MoAbs against a second TAA restored the efficiency of purging to that observed with the parental line. When low TAA subline cells were seeded into simulated bone marrow and subjected to purging, C-mediated lysis removed less than 40% of leukemia cells, whereas IM purging removed 85% of the cells. These results indicate that there are low antigen density cells that are resistant to C-mediated purging, but which retain sensitivity to IM removal.

Manufacturing genetically modified T cells for clinical trials.

Compliance with Food and Drug Administration regulations relating to initiating early phase clinical trials of new cellular therapy products often presents a hurdle to new investigators. One of the biggest obstacles is the requirement to manufacture the therapeutic products under current Good Manufacturing Practices-a system that is usually poorly understood by both basic researchers and clinicians. This article reviews the major points that must be addressed when manufacturing genetically modified T cells for therapeutic use.

Interaction between components of the human classical complement pathway and immobilized Cibacron Blue F3GA.

The interaction between the complement components in human serum and the dye, Cibacron Blue F3GA, immobilized on cross-linked agarose (Affi-Gel Blue) has been studied. All nine components of the classical complement pathway bound to the dye and could be recovered using a linear salt gradient. With the exception of C5 and C8, all the components were eluted over a narrow NaCl concentration range, with the following yields: C1, 17%; C2, 69%; C3, 92%; C4, 87%; C6, 105%; C7, 109%; C9, 128%. C5 and C8 eluted throughout the NaCl gradient with yields of 103% and 14%, respectively. Since all components could be eluted without substantial contamination by albumin or IgG, this procedure may prove valuable as an initial step in the purification of complement components. In addition, the ability of immobilized Cibacron Blue F3GA to physicallly remove complement components may prove useful for both the decomplementation of serum and in elucidating the role of complement in immunological reactions.

A rapid technique for measuring leukocyte chemotaxis in vivo.

A modification of connective tissue air bleb technique was used to develop a model system in inbred strains of mice for the study of chemoattractants in vivo. The method was developed using the well characterized n-formylated chemotactic peptide, f-Met-Leu-Phe, as the positive control. Injection of 0.1 ml of f-Met-Leu-Phe solutions from 10(-7) to 10(-10) M resulted in an influx of polymorphonuclear leukocytes within 2 h. Study of the kinetics of the response showed that the number of infiltrating cells reached a peak within 8 h and slowly declined over a 2-day period. The predominant infiltrating cell type during the first 24 h was the polymorphonuclear leukocyte. Between 24 and 48 h the polymorphonuclear leukocytes were replaced by monocytes. By utilizing an inbred mouse strain (DBA-1J and 2J) sufficient or deficient in C5 it was possible to distinguish compounds that were directly chemotactic from those that worked indirectly, or whose chemotactic potential could be enhanced by generation of the chemotactic complement split product C5a. The method was found to be technically simple, reproducible and semi-quantitative and represents a good model system to facilitate the comparison of chemotactic responses in vivo and in vitro.

Effects of target antigen density on the efficacy of immunomagnetic cell separation.

Immunomagnetic cell separation uses binding of an antibody to its epitope to identify the target cell, which is then removed by attachment to an anti-immunoglobulin-coated paramagnetic bead, and passage through a magnetic field. This method has previously been shown to be less sensitive to the effects of low target antigen density than are other cell elimination methods, such as complement-mediated lysis. In this paper we demonstrate that, with certain antibody/target cell combinations, the efficiency of immunomagnetic depletion can be adversely affected by high expression of the target antigen. This can occur by two non-mutually exclusive mechanisms. These are (i) steric hindrance of bead binding due to crowding of monoclonal antibodies on the cell surface; and (ii) binding of the monoclonal antibody molecule in a configuration that is poorly-accessible to the anti-immunoglobulin immobilized on the microspheres. The predominant effect operating in any system can be determined by analysis of the cells remaining after the separation procedure. In both cases pre-attachment of the monoclonal to the beads results in improved separation efficiency. These results emphasize the necessity of optimizing experimental conditions in each system that is investigated.

Generation of autologous Epstein-Barr virus-specific cytotoxic T cells for adoptive immunotherapy in solid organ transplant recipients.

BACKGROUND: Epstein-Barr virus (EBV)-driven posttransplant lymphoproliferative disorders (PTLD) affect 2%-27% of solid organ transplant (SOT) recipients. Adoptive immunotherapy may have therapeutic potential in this setting, but there is little experience in generating autologous EBV-specific cytotoxic T-cell lymphocytes (EBV-CTLs) from SOT recipients, and their efficacy and persistence in an immunosuppressed environment is unknown. METHODS: EBV-CTLs were generated from eight SOT recipients, using weekly stimulations with autologous lymphoblastoid cell lines (LCLs) and interleukin-2. CTL phenotype and function were evaluated in the presence of therapeutic concentration of cyclosporin A or FK506. RESULTS: In all cases, CTLs expanded with normal kinetics. The majority was CD3+CD8+ (mean, 76%), with less than 3% of natural killer cells. All ex vivo-generated CTLs produced significantly higher killing of autologous LCLs than of HLA-mismatched LCLs (mean, 56% vs. 14% at 20:1 ratio). No lysis of autologous or allogeneic PHA blasts was observed. The CTL expansion rate was reduced in a concentration-dependent manner in the presence of immunosuppressive drugs; however, neither lytic activity nor phenotype was affected. CONCLUSIONS: Using methods that are approved for clinical application, EBV-CTLs can be generated from SOT recipients, even those with frank lymphoma, or who are receiving immunosuppressive drugs. These CTLs retain their function in the presence of immunosuppressive agents. Although in vivo efficacy, safety, and persistence can be assessed only in clinical trials, our results suggest that CTLs can be effective for the treatment of PTLD, even when immunosuppression cannot be reduced because of the high risk of graft rejection.

Cyclosporine-induced autologous graft-versus-host disease following autologous blood stem cell transplantation for lymphoma.

Eight consecutive patients with relapsed/refractory non-Hodgkin's lymphoma or Hodgkin's disease received conditioning therapy with BCNU, etoposide, cytosine arabinoside and melphalan (BEAM) followed by autologous blood stem cell transplantation (ABSCT). Cyclosporine was administered from day +1 until day +28 post-ABSCT to induce autologous graft-versus-host disease (GVHD) for a possible antitumor effect. Three patients developed histologically documented grade II GVHD between 22-40 days post-transplant. GVHD resolved with local hydrocortisone 1% application in one patient and after a short course of steroid in the remaining two patients. Further studies are required to document any beneficial antitumor effect of such therapy following ABSCT.

T lymphocyte depletion of human peripheral blood and bone marrow using monoclonal antibodies and magnetic microspheres.

It has previously been demonstrated that graft-versus-host disease can be overcome in patients receiving HLA-mismatched bone marrow transplants by prior in vitro depletion of T lymphocytes from the marrow. In this report we describe the use of monoclonal antibodies and magnetic microspheres for the depletion of T cells from peripheral blood and bone marrow. The target cells are sensitized with antibodies directed against the CD2, CD3, CD4 and/or CD8 cell surface antigens, captured by magnetic beads coated with sheep anti-mouse IgG antibody and collected by placing the cell suspension in a magnetic field. This simple, rapid procedure results in the efficient removal of T cells from peripheral blood and from bone marrow without affecting the colony-forming potential of normal hematopoietic stem cells. The procedure is capable of being scaled up for the treatment of larger volumes of marrow that are required for clinical transplantation.

Acute rejection of marrow grafts in patients transplanted from a partially mismatched related donor: clinical and immunologic characteristics.

Bone marrow transplantation (BMT) from a partially mismatched related donor (PMRD) provides a treatment option for patients lacking a matched sibling donor. T lymphocyte depletion of the graft reduces the risk of severe graft-versus-host disease, but may increase the risk of graft failure. We evaluated the pattern of acute graft rejection in eight patients receiving PMRD BMT with respect to the conditioning therapy, diagnosis, age and sex of donor and recipient, degree of HLA mismatch, and peripheral blood immunophenotype at the time of graft failure. All grafts were partially depleted of T lymphocytes. Marrow grafts infused into patients who experienced acute rejection did not differ significantly in nucleated cell dose, degree of T lymphocyte depletion, T cell dose, or CFU-GM/kg infused, from those received by 31 patients who showed durable engraftment. In three of four patients who rejected their grafts, and had sufficient peripheral blood cells for immunophenotyping, a CD3+CD8+ T lymphocyte phenotype was predominant at the time of acute rejection. In one patient rejection was associated with a predominant population of CD3+CD4+ T lymphocytes. Rejection was significantly associated with chronic myelogeneous leukemia and in patients mismatched by more than two antigens.

In vitro expansion and characterization of dendritic cells derived from human bone marrow CD34+ cells.

Dendritic cells (DC), as professional antigen-presenting cells, play a major role in stimulating naive T cell responses in vivo and in vitro, and may exacerbate or modulate T lymphocyte-mediated reactions, such as interactions between a hematopoietic graft and the recipient, eg GVHD and graft-versus-leukemia. Here, we describe a two-stage cell culture system for expansion of functionally active human DC from CD34+ marrow precursors. Optimal outgrowth was achieved by initially culturing CD34+ cells for 5 days in medium containing GM-CSF, MGF and TNF-alpha. Substitution of CD40L and IL-4 for TNF-alpha during a subsequent 5-day subculture increased DC content, such that by 10 days the cultures contained approximately 40% DC as determined by immunophenotype and morphology. An increase in DC purity to 84% at 10 days was achieved by immunomagnetic separation for CD1a+ cells from 5-day cultures and subculturing these cells in medium with IL-4 and CD40L. Reversing the sequence of growth factors during culture and subculture decreased the yield and purity of DC. Expression of CD80 and CD86 was enhanced by adding CD40L and IL-4, and the DC showed stimulatory activity in MLC. In conclusion, we have described a simple two-stage culture system to generate functional DC from CD34+ marrow precursors.

Flow cytometric cell sorting combined with molecular chimerism analysis to detect minimal recurrent leukemia: good news and bad news.

Allogeneic BMT offers the possibility of cure for a variety of hematopoietic malignancies, but disease relapse remains a major cause of treatment failure. This report describes two cases in which flow cytometric cell sorting (FACS) and molecular chimerism analysis were combined to increase the sensitivity of minimal residual disease (MRD) detection. In the first case this approach was used to demonstrate that a suspicious phenotype was not recurrent leukemia, thus preventing the use of potentially toxic therapy. In the second case the recurrence of a leukemia which was undetectable by conventional analysis was confirmed. The potential benefits of combining these MRD detection methods are discussed.

Donor leukocyte infusion for treatment of graft rejection post partially mismatched related donor bone marrow transplant.

Graft rejection following bone marrow transplantation is more common in patients who receive their grafts from alternative donors and whose marrow is T cell depleted. Rejection in these patients is mediated by persistent host cells that interfere with successful establishment of donor-derived hematopoietic recovery. We describe a patient with chronic myelogenous leukemia in accelerated phase who rejected a T cell-depleted bone marrow graft, 2 months following partially mismatched related donor bone marrow transplant. Unmanipulated peripheral blood donor leukocyte infusion, without additional chemotherapy or immunosuppressive therapy resulted in complete hematopoietic recovery. Cytogenetics and RFLP demonstrated hematopoietic donor chimerism. The patient did not develop graft-versus-host disease.

Partially mismatched related donor transplants as salvage therapy for patients with refractory leukemia who relapse post-BMT.

Patients who relapse post-ABMT are usually resistant to conventional therapy, and a potentially curative therapy with allogeneic BMT is limited due to availability of a matched donor. To assess whether such patients can be salvaged using partially mismatched related donors (PMRD), eight patients age 6-50 years old underwent PMRD-BMT. All patients ALL (n = 3) and AML (n = 5) were in relapse 7-31 months after first BMT. Donors (1-3 Ag mismatch) were selected from family members. Conditioning included TBI, etoposide, Ara-C and cytoxan (n = 3), or busulfan, thiotepa, and etoposide (n = 5). GVHD prophylaxis consisted of partial T cell depletion followed by systemic immunosuppression. All evaluable patients established sustained engraftment by day 18. Severe regimen-related toxicity was evident in the gastrointestinal and hepatic systems (6/8 and 4/8, respectively), the latter associated with poor outcome (P < 0.014). Acute but not chronic GVHD, grade > or = II occurred in 3/7 patients. Four of eight patients are disease-free, maintaining longer remission than following their first BMT (14 vs 9 months). In conclusion, our data shows that PMRD-BMT is a feasible option for patients who relapse post-BMT and use of such alloreactive grafts may be appropriate earlier in the disease course of high risk patients.