ALBERT

Description: To determine whether the multidrug resistance gene MDR1could 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 theMDR1 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.

Description: Recently, there have been several reports using various superparamagnetic iron oxide (SPIO) nanoparticles to label mammalian cells for monitoring their temporal and spatial migration in vivo by magnetic resonance imaging (MRI). The purpose of this study was to evaluate the efficiency and toxicity of labeling cells using 2 commercially available Food and Drug Administration (FDA)-approved agents, ferumoxides, a suspension of dextran-coated SPIO used as an MRI contrast agent, and protamine sulfate, conventionally used to reverse heparin anticoagulation but also used ex vivo as a cationic transfection agent. After labeling of human mesenchymal stem cells (MSCs) and hematopoietic (CD34+) stem cells and other mammalian cells with ferumoxides-protamine sulfate complexes (FE-Pro), cellular toxicity, functional capacity, and quantitative cellular iron incorporation were determined. FE-Pro-labeled cells demonstrated no short- or long-term toxicity, changes in differentiation capacity of the stem cells, or changes in phenotype when compared with unlabeled cells. Efficient labeling with FE-Pro was observed with iron content per cell varying between 2.01 ± 0.1 pg for CD34+ cells and 10.94 ± 1.86 pg for MSCs with 100% of cells labeled. Cell labeling using these agents should facilitate the translation of this method to clinical trials for evaluation of trafficking of infused or transplanted cells by MRI. (Blood. 2004;104:1217-1223)

Description: We previously showed that removal of host-reactive donor T cells from allografts by anti-CD25 immunotoxin (IT) is clinically feasible and reduces the frequency of severe graft-versus-host-disease (GVHD) in a high-risk group of elderly patients undergoing matched-sibling transplantation (Blood, 2005,106:1123). However, it is possible that the concurrent removal of CD4+CD25+ regulatory T cells (Tregs) with alloactivated CD25+ T cells could precipitate more GVHD if depletion of alloreacting cells were incomplete. We, therefore, studied the recovery of Tregs in 16 patients receiving CD25-IT-treated stem cell grafts during the first 150 days after transplantation. Tregs were characterized by surface phenotyping for CD3, CD4, CD25 and CD27, intracellular staining for forkhead protein3 (foxp3) and quantitative real time PCR for foxp3 gene. We also measured Tregs in 13/16 donors (Don), 10/16 SD products and 13/16 patients pretransplant (Pre). Patients received a median of 1.0 (0.2–1.5) ×108/kg selectively depleted (CD25−) CD3+ T cells and a stem cell product containing 0.25 ×104/kg (0.1–5.0) residual unselected CD3+ T cells. Lymphocyte recovery was prompt with a median lymphocyte count of 613/μL (215–2883/μL) and median 97% donor T cell chimerism at day+30. Despite CD25-depletion, patients reconstituted sufficient levels of CD4+foxp3+ T cells at day+30. These CD4+foxp3+ T cells underwent further expansion and reached their highest median levels 150 days post transplantation (Figure A). Interestingly, all SD-products contained a significant fraction of CD4+foxp3+ Tregs that persisted after complete CD25-depletion. These cells were exclusively CD25− CD4+foxp3+ Tregs and represented 1.5–4.8% of the CD4+ cells in the SD product. Of note, the Treg content of the donor was associated with the risk of GVHD development posttransplant. Acute grade II GVHD was restricted to 5 patients whose donors had significantly fewer Tregs compared to those with no or grade I GVHD (Figure B). In this study we show efficient Treg reconstitution despite giving a CD25-depleted SD allograft. We identified a population of CD25− CD4+foxp3+ Tregs in the SD product. Such cells have been shown to exert regulatory function in mice. Our results suggest that Treg recovery following SD is derived from a major contribution of CD25− Tregs which escape the depletion process in addition to a residual fraction of CD25− and CD25+ Tregs delivered with the stem cell product. All these Treg fractions may undergo marked expansion post-transplant and provide additional GVHD protection if the removal of alloreacting T cells by the SD procedure were incomplete. Figure Figure

Description: Administration of mobilized peripheral blood progenitor cells (PBPCs) after high-dose chemotherapy rapidly restores multilineage hematopoiesis, but the ability of such products to restore lymphocyte populations remains unclear. In this report, we evaluated immune reconstitution in a series of patients treated with sequential cycles of high-dose chemotherapy, followed by autologous PBPC infusions (median CD34+ cell dose 7.2 × 106 cells/kg [range 2-29.3]). Although patients experienced rapid reconstitution of B cells and CD8+ T cells, we observed CD4 depletion and diminished immune responsiveness in all patients for several months after completion of therapy. Mature CD4+ T cells contained within the grafts did not appear to contribute substantially to immune reconstitution because CD4 counts did not differ between recipients of unmanipulated T-cell replete infusions versus CD34 selected, T-cell–depleted infusions. Rather, at 12 months after therapy, total CD4 count was inversely proportional to age (ρ = −0.78,P = .04), but showed no relationship to CD34 cell dose (ρ = −0.42, P = .26), suggesting that age-related changes within the host are largely responsible for the limited immune reconstitution observed. These results demonstrate that in the autologous setting, the infusion of large numbers of PBPCs is not sufficient to restore T-cell immune competence and emphasize that specific approaches to enhance immune reconstitution are necessary if immune-based therapy is to be used to eradicate minimal residual disease after autologous PBPC transplantation.

Description: Selective depletion (SD) is a strategy to eliminate host-reactive donor lymphocytes from blood stem cell allografts to prevent GVHD and maintain GVL-effects. As an alternative to CD25-immunotoxin-based SD, we investigated a photodepletion (PD) process, whereby allo-activated donor cells are labeled with a photosensitizing rhodamine-based dye, 4,5-dibromorhodamide 123 (TH9402), and exposed to visible light, which preferentially eliminates allo-activated dye-retaining cells. Stimulator cells were prepared from recipient-derived leukapheresis mononuclear cells (MNCs) and cultured using anti-CD3 and 100 IU IL-2/ml. Responder cells (leukapheresis MNCs) from 3 random HLA-mismatched volunteers and 3 HLA-matched sibling donors were cocultured 1:1 with irradiated stimulators for 3 days. Cultured cells were incubated with 7.5 μM TH9402, followed by dye-extrusion and exposure to 5 Joule light energy in the PD light source (Celmed Bioscience Inc., Canada) at 5x106 cells/ml in FEP plastic bags. Flow cytometry was performed before and after PD and included surface phenotyping for CD25 and intracellular staining for forkhead protein3 (foxp3). Depletion efficacy was studied by mixed lymphocyte reactions (MLR) in mismatched pairs and by helper-T-lymphocyte precursor (HTLp) frequency assay in matched pairs. All six clinical-scale experiments provided sufficient reduction of allo-reactivity and retention of third party responses as measured against a pool of 5 donors. In mismatched pairs mean reduction of allo-reactivity was 703-fold (± 141) when compared to unmanipulated donors. Third-party responses were maintained, with a mean reduction of only 1.3 ± 0.15-fold (Figure A). In matched pairs alloreactivity was reduced below the “GVHD-threshold” of 1/100.000 whilst third party responses remained above 1/10.000 precursors (Figure B). Effective depletion was observed despite that fact that a small fraction of CD25+ T cells remained after PD. These were mainly CD4+CD25+ T cells (1.5 ± 1.1 % of CD4+) and very residual CD8+CD25+ T cells (0.3 ± 0.2 % of CD8+), and represented predominantly CD4+foxp3+ regulatory T cells (Tregs) which persisted after PD (1.4 ± 1.7 % of CD4+). This establishes a clinical scale PD process capable of highly efficient removal of alloreactive lymphocytes from mismatched and matched MLRs while maintaining desirable third party responses. As PD targets activation-based changes in MDR-1 that result in an altered dye efflux, the mechanism of action is distinct from surface-marker-based allodepletion (e.g. CD69, CD25). Thus, PD may overcome instability of activation-based surface marker expression resulting in more consistent and effective depletion. PD may further mitigate the risk of GVHD by conserving Tregs. This approach will now be tested in a clinical SD trial. Figure Figure.

Description: Treatment of refractory or recurrent malignancy with donor lymphocyte infusion (DLI) after allogeneic hematopoietic stem cell transplantation (alloHSCT) is often not curative, with graft-vs-tumor (GVT) effects frequently accompanied by graft-versus-host disease (GVHD), requiring immune suppression that compromises efficacy. After alloHSCT, chimeric T lymphocytes infiltrating residual tumor (chimeric TIL) may provide enhanced antigen specificity and maintain tumor-specific homing. Compared with DLI, they may have a better GVT effect with less GVHD. Based on the success of autologous TIL therapy for melanoma, we tested the hypothesis that enhanced GVT with limited GVHD could be achieved through administration of ex-vivo activated chimeric TIL after alloHSCT. Preclinical TIL production carried out on several tumor types from non-transplanted patients demonstrated effective T cell isolation, expansion and activation using anti-CD3/CD28 bead co-stimulation, yielding a 10- to 30-fold expansion of CD3+ cells. Clinical evaluation of chimeric TIL therapy was initiated with a 51 year-old woman for metastatic breast cancer whose disease progressed after a T cell-depleted reduced-intensity alloHSCT with delayed DLI from a 6/6 HLA-matched sibling donor and subsequent conventional therapy plus DLI. Two weeks after administration of unmanipulated DLI, two thoracic metastases were surgically removed. T cells were liberated from 9.4 cm of tumor using enzymatic digestion and mechanical dispersion, lymphocyte-enriched by density gradient separation, and expanded for 14 days through co-stimulation with anti-CD3/CD28-coated magnetic beads (3:1 bead-to-total nucleated cell ratio) and media containing IL-2 (100 or 1000 IU/mL). This process yielded 42.5 x 106 cells, 33% expressing CD3, and generated 14.7 x 109 chimeric TIL, 85% expressing CD3 (a 3.1-log T cell expansion). There was no tumor contamination of the T cell product by immunohistochemistry. Flow cytometry demonstrated that the CD4/CD8 T cell ratio increased from 1.3 to 1.9 after expansion. Three infusions of the chimeric TIL product were given in a dose-escalating manner (5, 25 and 100 x 106 CD3+ cells/kg). A fourth infusion was given in conjunction with low-dose IL-2 (6mu SQ per day x 3D). CT scan performed after each infusion monitored disease response, with progressive disease the indication for the next dose administration. No infusion-related or delayed toxicities were observed, except for rigors following IL-2 administration. The patient shows no evidence of GVHD, even after the highest dose of 108 allogeneic T cells. Evaluation of the remaining thoracic lesion demonstrated progressive disease after the first two chimeric TIL dose-levels, transient disease stability one month after the third dose-level, and stable disease one month after the fourth dose with IL-2. This is the first clinical report of the application of chimeric TIL and represents a novel approach for developing new forms of adoptive immunotherapy in the setting of alloHSCT.

Description: One hundred and fifty-seven patients with leukemia (80 CML, 48 AML/MDS, 29 ALL) received a T cell depleted myeloablative allogeneic stem cell transplantation (SCT) from an HLA-matched sibling between 09/1993–09/2005. Conditioning consisted of TBI (12–13.6 Gy) + cyclophosphamide (96) or cyclophosphamide and fludarabine (61). The stem cell source was G-CSF mobilized peripheral blood stem cell (PBSC) in 129 and bone marrow in 28 patients. T cell dose with graft ranged from 0.2 – 2 × 105/kg CD3+ cells. GVHD prophylaxis was with low dose cyclosporine (level 100–200 ng/ml) in 103 and standard dose in 54. Patients without ≥ grade II acute GVHD received 1–2 donor lymphocyte infusion of 107 CD3+ cells/kg between days 45 and 100. Absolute lymphocytes on day 30 (LC30) was available in 154 patients (3 patients died before day 30) and 54 day +30 post-transplant samples were available for lymphocyte subset analysis. Median lymphocyte count on day +30 (LC30) was 400/μl, (range 10–3295) and 150/μl, (range 6–1005) for CD56+, CD16+ CD3− NK cells (NK30). Statistical analysis was performed on SPSS14 software. Median age of the group was 34 years (range 10–56). 78 patients had standard risk (SR) disease in first remission or first chronic phase of CML. The remaining 79 had high risk (HR) disease. At the time of analysis 85 patients are (51.5±4%) are alive with median follow-up of surviving patients 1392 days (range 147–4208). Only 9 patients (3 above median LC30) developed aGVHD before day+30. Patients with ≥ median LC30 had significantly better transplant outcome: survival 71±5 vs. 36 ±5.5%, p

Description: Acute GVHD is a major contributor to morbidity and mortality following NST. A high incidence of grades II–IV GVHD occurs when cyclosporine A (CSA) alone is used as prophylaxis. Consequently, we investigated the effect of combining either mycophenolate mofetil (MMF) or methotrexate (MTX) with CSA on the incidence and severity of acute GVHD. Between 11/97 and 07/04, 185 consecutive patients (solid tumors n=116, hematologic malignancies n=48, non-malignant hematologic disorders n=21) underwent nonmyeloablative conditioning with fludarabine (125mg/m2) and cyclophosphamide (120mg/kg), followed by a G-CSF mobilized peripheral blood hematopoietic stem cell transplant from an HLA identical (n=177: 96%) or 5/6 antigen-matched (n=8: 4%) related donor. Twenty-four patients with a history of heavy RBC transfusions or those receiving a 5/6 HLA-matched transplant had anti-thymocyte globulin (40mg/kg/d x 4 days) added to their conditioning regimen. The initial cohort of patients (Group 1, n=66), received CSA alone (dose adjusted to maintain therapeutic serum levels) as GVHD prophylaxis. Due to the high incidence of severe acute GVHD in this group subsequent patients received CSA with either MMF (1 gram po bid; Group 2, n=82) or MTX (5mg/m2 days +1, +3, +6; Group 3, n=37). In all three groups, decisions regarding discontinuation of immunosuppression were based on the degree of donor T cell chimerism, presence of GVHD, and disease status in those with malignant diseases. In the absence of grade II–IV GVHD and disease progression, CSA (+/− MMF) was tapered slowly beginning on day +60. Baseline characteristics of patients in the three groups were compared using the Wilcoxon test for continuous variables and chi-squared tests for discrete variables. The three groups did not differ significantly in terms of age, sex, sex mismatch (female into male) and CD34 cell dose. Median follow-up in groups 1, 2 and 3 was 1901 days, 1248 days and 346 days respectively. The cumulative incidence of grades II–IV GVHD in these three groups was 56% (95% CI 44%–68%), 59% (95% CI, 48%–70%), and 34% (95% CI 18%–50%, p=0.11) respectively. The cumulative incidence of grades III–IV GVHD (30% vs. 34% vs. 16%, p=0.2) and the incidence of chronic GVHD (46% vs. 57% vs. 50%, p=0.49) were also similar in the three groups. Transplant related mortality was 15% (95% CI, 6%–24%) in group 1, 12% (95% CI, 5%–19%) in group 2 and 5% (95% CI, 0%–13%) in group 3 patients (p=0.44). The cumulative incidence of death from acute GVHD was 9% (95% CI, 0%–16%) and 2% (95% CI, 0–5%) respectively in groups 1 and 2, while no deaths from acute GVHD occurred in group 3. Overall survival in the three groups did not differ significantly (log-rank test, p=0.48), with medians 244 days (95% CI 196–402), 486 days (95% CI 306–620) and 438 days, (95% CI 210–662) respectively. The impact of adding MMF or MTX to CSA on disease-specific outcome in patients with malignant diseases was not assessed. Conclusion: There was a trend towards a lower incidence of grades II–IV GVHD in group 3 patients. However, despite the addition of either MMF or MTX to CSA, severe grade III–IV acute GVHD remains a major morbidity complicating NST. Additional strategies aimed at preventing GVHD and optimizing the management of established GVHD are needed to improve outcome following this approach.

Description: Selective allodepletion is a strategy to eliminate host-reactive donor T cells from hematopoietic stem cell allografts to prevent graft-versus-host disease while conserving useful donor immune functions. To overcome fluctuations in activation-based surface marker expression and achieve a more consistent and effective allodepletion, we investigated a photodepletion process targeting activation-based changes in p-glycoprotein that result in an altered efflux of the photosensitizer TH9402. Expanded lymphocytes, generated using anti-CD3 and IL-2, were cocultured with responder cells from HLA-matched or -mismatched donors. Optimal results were achieved when cocultured cells were incubated with 7.5 μM TH9402, followed by dye extrusion and exposure to 5 Joule/cm2 light energy at 5 × 106 cells/mL. In mismatched stimulator-responder pairs, the median reduction of alloreactivity was 474-fold (range, 43-fold to 864-fold) compared with the unmanipulated responder. Third-party responses were maintained with a median 1.4-fold (range, 0.9-fold to 3.3-fold) reduction. In matched pairs, alloreactive helper T-lymphocyte precursors were reduced to lower than 1:100 000, while third-party responses remained higher than 1:10 000. This establishes a clinical-scale process capable of highly efficient, reproducible, selective removal of alloreactive lymphocytes from lymphocyte transplant products performed under current Good Manufacturing Practice. This procedure is currently being investigated in a clinical trial of allotransplantation.

Description: Eighty patients with chronic myeloid leukemia (CML) underwent T cell-depleted stem cell transplantation from an HLA-identical sibling, with add-back of donor T cells on days 30 to 45 and days 60 to 100 in patients in whom grade 2 or greater acute graft-versus-host disease (GVHD) developed. The outcomes for 54 patients with chronic-phase (CP) and 26 with advanced-phase (AP) disease were as follows: overall survival, 85% ± 5% versus 36% ± 10%; transplantation-related mortality (TRM), 13% ± 5% versus 43% ± 11%; and current leukemia-free survival, 76% ± 6% versus 34% ± 9%. The day-30 lymphocyte count (LC30) was strongly associated with outcome. For patients in CP with counts greater than the median of 0.30 × 109/L, survival was 100% versus 70% ± 9% (P = .003); current LFS 100% versus 56% ± 9% (P = .002); and TRM 0% versus 26% ± 8% (P = .006). Higher-than-median LC30 correlated significantly with molecular remission (MR) at 3, 6, and 12 months and with higher CD34 doses. Lymphocyte subset analysis performed in 20 patients available for phenotyping showed that LC30 was highly correlated with absolute CD56+CD3- natural killer cell numbers (NK30), which also predicted for survival and MR. CD34 cell dose, LC30, and NK30, but not day-30 CD3+ cell count, were highly correlated and were significant predictors of transplantation outcome. These results suggest that transplanted CD34 cell doses greater than 5 × 106/kg may improve outcomes by increasing the early recovery of NK cells.