Douglas J. Padley

Platelet lysate consisting of a natural repair proteome supports human mesenchymal stem cell proliferation and chromosomal stability

With favorable regenerative and immunotolerant profiles, patient-derived human mesenchymal stem cells (hMSCs) are increasingly considered in cell therapy. Derived from bone marrow (BM) and standardized with culture in fetal bovine serum (FBS), translation of hMSC-based approaches is impeded by protracted expansion times, risk of xenogenic response, and exposure to zoonoses. Here, human platelet lysate adherent to good manufacturing practices (GMP-hPL) provided a nonzoonotic adjuvant that enhanced the capacity of BM-hMSC to proliferate. The nurturing benefit of GMP-hPL was generalized to hMSC from adipose tissue evaluated as an alternative to bone marrow. Long-term culture in GMP-hPL maintained the multipotency of hMSC, while protecting against clonal chromosomal instability detected in the FBS milieu. Proteomic dissection identified TGF-β, VEGF, PDGF, FGF, and EGF as highly ranked effectors of hPL activity, revealing a paradigm of healing that underlies platelet lysate adjuvancy. Thus, GMP-adherent human platelet lysate accelerates hMSC proliferation with no chromosomal aberrancy, through an innate repair paradigm.

Infused peripheral blood autograft absolute lymphocyte count correlates with day 15 absolute lymphocyte count and clinical outcome after autologous peripheral hematopoietic stem cell transplantation in non-Hodgkin's lymphoma

Summary:Absolute lymphocyte count at day 15 (ALC-15) after autologous peripheral blood hematopoietic stem cell transplantation (APHSCT) is an independent prognostic factor for survival in non-Hodgkins lymphoma (NHL). Factors affecting ALC-15 remain unknown. We hypothesized that dose of infused autograft lymphocytes (A-ALC) directly impacts upon ALC-15. A total of 190 consecutive NHL patients received A-ALC between 1993 and 2001. The primary end point was correlation between A-ALC and ALC-15. A strong correlation was identified (r=0.71). A higher A-ALC was infused into patients achieving an ALC-15 ⩾500/μl vs ALC-15 <500/μl (median of 0.68 × 109/kg (0.04–2.21 × 109/kg), vs 0.34 × 109/kg (0.04–1.42 × 109/kg), P<0.0001). The median follow-up for all patients was 36 months (maximum of 109 months). The A-ALC threshold was determined at 0.5 × 109/kg. The median overall survival (OS) and progression-free survival (PFS) times were longer in patients who received an A-ALC ⩾0.5 × 109/kg vs A-ALC <0.5 × 109/kg (76 vs 17 months, P<0.0001; 49 vs 10 months, P<0.0001, respectively). Multivariate analysis demonstrated A-ALC to be an independent prognostic indicator for OS and PFS. These data support our hypothesis that ALC-15 and survival are dependent upon the dose of infused A-ALC in NHL.

The dose of infused lymphocytes in the autograft directly correlates with clinical outcome after autologous peripheral blood hematopoietic stem cell transplantation in multiple myeloma

Absolute lymphocyte count at day 15 (ALC-15) after autologous peripheral blood hematopoietic stem cell transplantation (APHSCT) is an independent prognostic factor for survival in multiple myeloma (MM); however, factors affecting ALC-15 in MM remain unknown. We hypothesized that the dose of infused peripheral blood autograft lymphocytes (autograft absolute lymphocyte count: A-ALC) impacts ALC-15 recovery. Between 1989 and 2001, 267 consecutive MM patients underwent APHSCT. We set out to determine the correlation between A-ALC and ALC-15 and the utility of A-ALC as a marker for ALC-15 recovery. A-ALC was found to be both a strong predictor for area under curve (AUC=0.93; P=0.0001) and strongly correlated with (rs=0.83; P=0.0001) ALC-15 recovery. Higher infused A-ALC was significantly correlated with an ALC-15⩾500/μl. In addition, median post-transplant overall survival (OS) and time to progression (TTP) were longer in patients who received an A-ALC⩾0.5 × 109 lymphocytes/kg versus A-ALC <0.5 × 109 lymphocytes/kg (58 vs 30 months, P=0.00022; 22 vs 15 months, P<0.00012, respectively). Multivariate analysis demonstrated A-ALC as an independent prognostic indicator for OS and TTP. These results indicate that an infused dose of autograft lymphocytes significantly impacts clinical outcome post-APHSCT in MM.

Idiotype-pulsed antigen presenting cells following autologous transplantation for multiple myeloma may be associated with prolonged survival

Vaccines are attractive as consolidation therapy after autologous stem cell transplantation (ASCT) for multiple myeloma (MM). We report the results of a phase II trial of the immunotherapeutic, APC8020 (Mylovenge™), given after ASCT for MM. We compared the results with that of other patients with MM who underwent ASCT at Mayo Clinic during the same time period. Twenty‐seven patients were enrolled on the trial between July, 1998 and June, 2001, and the outcomes were compared to that of 124 consecutive patients transplanted during the same period, but not enrolled on the trial. The median (range) follow‐up for patients still alive from the vaccine trial is 6.5 (2.9–8 years), and 7.1 (6–8 years) in the control group. The median age was 57.4 range (36.1–71.3) in the DB group and 56.4 (range, 30–69) in the trial group. Known prognostic factors including PCLI, B2M, and CRP were comparable between the groups. The median overall survival for the trial patients was 5.3 years (95% CI: 4.0 years—N/A) compared to 3.4 years (95% CI: 2.7–4.6 years) for the DB group (P = 0.02). The median time to progression and progression‐free survival for the trial group was similar to the DB group. Although not a controlled trial, the vaccines given after ASCT appear to be associated with improved overall survival compared to historical controls. This approach warrants further investigation to confirm this and define the role of vaccine therapy in myeloma. Am. J. Hematol. 2009.

Re-infused Autologous Graft Natural Killer Cells Correlates with Absolute Lymphocyte Count Recovery after Autologous Stem Cell Transplantation

Early absolute lymphocyte count (ALC) has been reported to be a powerful prognostic indicator of survival after autologous stem cell transplantation (ASCT). One possible source affecting ALC recovery includes the re-infused autologous graft lymphocytes (AGL). To assess if the re-infused AGL correlate with ALC recovery post-ASCT, we conducted a pilot study to identify which of the re-infused AGL subsets is most associated with day 15 ALC recovery in three patients with multiple myeloma and four patients with non-Hodgkins lymphoma. Using the Spearman rank correlation coefficient analysis (r ), we compared absolute numbers of CD3, CD4, CD8, CD19, and CD16+/CD56+ cells/kg of body weight from the apheresis product with ALC (cells/ µ l) at day 15 post-ASCT. The main lymphocyte subsets identified in the apheresis product were T cells and NK cells. There was no strong correlation between T or B cells from the apheresis product compared with the ALC at day 15 post-ASCT (CD3, r =0.21; CD4, r =0.32; CD8, r =0.39; and CD19, r =0.14 ). However, there was good correlation between NK cells from the apheresis product compared with ALC at day 15 post-ASCT (CD16+/CD56+/CD3 m, r =0.77 ). These data provide preliminary evidence that the number of re-infused autologous graft NK cells in the apheresis product significantly affect ALC recovery early post-ASCT. However, given the small sample size, our results are primarily hypothesis generating and subject of further research.

Infrastructure Development for Human Cell Therapy Translation

The common conception of a drug is that of a chemical with defined medicinal effect. However, cells used as drugs remain critical to patient care. Cell therapys origins began with the realization that complex tissues such as blood can retain function when transplanted to the patient. More complex transplantation followed, culminating with the understanding that transplantation of some tissues such as bone marrow may act medicinally. Administration of cells with an intended therapeutic effect is a hallmark of cellular therapy. While cells have been used as drugs for decades, testing a specific therapeutic effect of cells has begun clinical testing relatively recently. Lessons learned during the establishment of blood banking (including the importance of quality control, process control, sterility, and product tracking) are key components in the assurance of the safety and potency of cell therapy preparations. 1 , 2 As more academic medical centers and private companies move toward exploiting the full potential of cells as drugs, needs arise for the development of the infrastructure necessary to support these investigations. Careful consideration of the design of the structure used to manufacture is important in terms of the significant capital outlay involved and the facilitys role in achieving regulatory compliance. This development perspective describes the regulatory environment surrounding the infrastructure support for cell therapy and practical aspects for design consideration with particular focus on those activities associated with early clinical trials.

Sterility testing of hematopoietic progenitor cell products: a single-institution series of culture-positive rates and successful infusion of culture-positive products

BACKGROUND: Administration of culture‐positive hematopoietic progenitor cells (HPCs) causing adverse events has been a hypothesized yet largely unmeasured risk of the clinical practice of HPC transplantation. To enhance patient safety, the FDA has issued regulations prohibiting the use of culture‐positive HPCs. Numerous studies have reported the infusion of culture‐positive HPCs; however, the low frequency of adverse events prevents accurate determination of this risk.

Preparing clinical-grade myeloid dendritic cells by electroporation-mediated transfection of in vitro amplified tumor-derived mRNA and safety testing in stage IV malignant melanoma

BackgroundDendritic cells (DCs) have been used as vaccines in clinical trials of immunotherapy of cancer and other diseases. Nonetheless, progress towards the use of DCs in the clinic has been slow due in part to the absence of standard methods for DC preparation and exposure to disease-associated antigens. Because different ex vivo exposure methods can affect DC phenotype and function differently, we studied whether electroporation-mediated transfection (electrotransfection) of myeloid DCs with in vitro expanded RNA isolated from tumor tissue might be feasible as a standard physical method in the preparation of clinical-grade DC vaccines.MethodsWe prepared immature DCs (IDCs) from CD14+ cells isolated from leukapheresis products and extracted total RNA from freshly resected melanoma tissue. We reversely transcribed the RNA while attaching a T7 promoter to the products that we subsequently amplified by PCR. We transcribed the amplified cDNA in vitro and introduced the expanded RNA into IDCs by electroporation followed by DC maturation and cryopreservation. Isolated and expanded mRNA was analyzed for the presence of melanoma-associated tumor antigens gp100, tyrosinase or MART1. To test product safety, we injected five million DCs subcutaneously at three-week intervals for up to four injections into six patients suffering from stage IV malignant melanoma.ResultsThree preparations contained all three transcripts, one isolate contained tyrosinase and gp100 and one contained none. Electrotransfection of DCs did not affect viability and phenotype of fresh mature DCs. However, post-thaw viability was lower (69 ± 12 percent) in comparison to non-electroporated cells (82 ± 12 percent; p = 0.001). No patient exhibited grade 3 or 4 toxicity upon DC injections.ConclusionStandardized preparation of viable clinical-grade DCs transfected with tumor-derived and in vitro amplified mRNA is feasible and their administration is safe.

Mature Myeloid Dendritic Cells for Clinical Use Prepared from CD14+ Cells Isolated by Immunomagnetic Adsorption

427 RECENTLY, WE PREPARED highly pure and mature dendritic cells from the blood of patients suffering from chronic myeloid leukemia (CML) (1). We isolated dendritic cell precursors from peripheral blood mononuclear cells by CD14-specific immunoadsorption and cultured them for 7 days in granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4) followed by a 3-day incubation in the presence of tumor necrosis factor-a (TNF-a), IL-1b, IL-6, and prostaglandin E2 (PGE2) to differentiate the cells fully. The yield, phenotype, and function of CML-dendritic cells were indistinguishable from dendritic cells prepared from normal blood. Because the level of dendritic cell maturity relates positively to the induction of experimental (2) and clinical immunity (3), this result demonstrated that it was feasible to prepare CML-dendritic cells adequate for the use in clinical trials of CML immunotherapy. Now we report validation of this manufacturing method for a Phase I clinical trial of dendritic cells in CML. Peripheral blood mononuclear cells (PBMC) were collected from 4 CML patients and 6 normal volunteers using a Fenwal CS3000 apherersis machine at the flow rate of 50 ml/min for 4 h. Under these conditions, the mean mononuclear cell content is 966 8% (as determined in a series of 162 leukophereses; data not shown). Individual apheresis products were washed by two volumes of phosphate-buffered saline (PBS)/EDTA and centrifuged at 1200 rpm for 10 min. The cells were further incubated with cGMP-grade CD14-specific immunomagnetic reagent (Miltenyi Biotec, Bergisch Gladbach, Germany; 4 3 108 cells per 1.0 ml of reagent) for 30 min at room temperature. Excess reagent was removed by washing using a Cobe 2991 cell processor (Gambro BCT, Stockholm, Sweden). CD141 cells were isolated on a CliniMACS apparatus (Miltenyi Biotec, Auburn, CA) employing the “Enrichment 1.1” program. The resulting CD141 cells were counted, characterized, and used to generate dendritic cells. Cells were cultured according to our modification (1) of the method by Jonuleit et al. (4). They were plated at the density of 2 3 106 cells/ml in T175 flasks using XVIVO 15 medium (BioWhittaker, Walkersville, MD) containing 1.0% pooled human AB serum (HABS; C-6 Diagnostics, Mequon, WI), GM-CSF (800 IU/ml), and IL-4 (1000 IU/mL). Fresh medium containing the same components, but GM-CSF increased to 1600 IU/ml, was added in the amount of one-third of the original volume into the culture on the third and fifth days of incubation. On the seventh day, the cells were centrifuged and resuspended at 1.0 3 106/ml in the maturation medium (XVIVO 15, 1.0% HABS, 800 IU/ml GM-CSF, 1000 IU/ml IL-4, 1100 IU/ml TNF-a, 1870 IU/ml IL-1b, 1000 IU/ml IL-6, and 1.0 mg/ml PGE2). Nonadherent mature dendritic cells were collected 3 days later. We compared dendritic cells prepared from normal mononuclear cell apheresis products with dendritic cells prepared from apheresis products from CML patients. By the use of CD14-specific MicroBeads and a CliniMACS magnetic separator, we enriched CD141 cells from 15.86 2.3% (mean value6 standard deviation) in normal apheresis product to 98.16 1.4% and from 14.66 5.4% in CML apheresis product to 94.26 10.2% (p 5 0.42 for the difference between enriched normal and CML cells; calculated by two-sided Student’s t-test for independent samples). Enrichment in CD141 cells increased the level of Philadelphia chromosome-positive cells from 72.66 20.7% to 97.56 2.2% (measured by fluorescence in situ hybridization as in ref. 1). The final dendritic cell yield from CD141 cells was 30.36 6.4% for normal cells and 27.36 12.8% for CML cells (p 5

Testing the safety of clinical-grade mature autologous myeloid DC in a phase I clinical immunotherapy trial of CML

BACKGROUND We conducted a phase I clinical immunotherapy trial of CML to evaluate the safety of a clinical-grade leukemic DC product standardized for purity and mature phenotype. METHODS We injected autologous DC into patients in late chronic or accelerated phases of CML. The patients received mature CD83+ and bcr-abl+ DC prepared from CD14+ cells. Two cohorts of three patients received four injections each of 3 x 10(6) DC and 15 x 10(6) DC/injection, respectively. The first patient was studied before imatinib mesylate (IM) was available, four patients were treated concurrently with IM therapy and one did not tolerate the IM and was off the drug at the time of DC therapy. IM effects on WBC counts precluded DC preparation in numbers sufficient for further dose escalation. The first patient received DC s.c. and all subsequent patients received DC into a cervical lymph node under ultrasound guidance. RESULTS DC injections were well tolerated. We observed no clinical responses. T cells drawn later in the course of therapy were more sensitive to stimulation by CML DC in vitro. DISCUSSION The increase in T-cell sensitivity to CML-specific stimulation that accompanied active immunization by CML DC justifies further clinical studies, possibly with modifications such as an increased frequency and number of DC injections.